EMJ Microbiology & Infectious Disease 5.1 2024

Johnny ZhakhourNavigating the Complexities of Diagnostic Stewardship Interview

The Future of Infection Treatment and Prevention in Immunocompromised Patients Feature

46 Surveillance of Surgical Site Infections in Post-operative Patients and Bacterial Susceptibility in Tanzania

Karuhanga et al.

57 Occult Filariasis in a Case of Lymphocytic Thyroiditis

Diagnosed on Fine Needle Aspiration Cytology: An Unusual Finding of Dual Pathology

Shahab et al.

60 Virtual Discovery of Immune-Stimulating Epitopes in Chikungunya Virus for Vaccine Design

Ezediuno et al.

77 Hand Washing Practices and Antibiotic Susceptibility of Palmar Bacterial Flora in a Tertiary Education Institution in Nigeria

Akinwumi et al.

Editorial Board

Editor-in-Chief

Prof Rajeshwar Reddy Kasarla Universal College of Medical Sciences, Nepal

Editorial Board

Dr Ali Elbeddini University of Ottawa, Canada

Dr Emilio Bouza Hospital Gregorio Marañón, Spain

Dr Mohammad Nazish Farwaniya Hospital, Kuwait

Dr Muge Cevik University of St Andrews, UK

Dr Hisham Elkhayat Theodor Bilharz Research Institute, Egypt

Dr Oliver Grundmann University of Florida, USA

Dr Smita Shevade

Dr Rahul Garg

Millennium Path Lab, India

All India Institute of Medical Sciences-Raipur, India

Dr Poonam Gupta Kokilaben Dhirubhai Ambani Hospital, India

Prof Manisha Gupta Super Specialty Cancer Institute and Hospital, India

Dr Sanjay Bhattacharya Fakhruddin Medical College, India

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EMJ is an online only, peer-reviewed, open access general journal, targeted towards readers in the medical sciences. We aim to make all our articles accessible to readers from any medical discipline.

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This Publication

ISSN 2732-5326

EMJ Microbiology & Infectious Diseases is published once a year. For subscription details please visit: www.emjreviews.com

All information obtained by EMJ and each of the contributions from various sources is as current and accurate as possible. However, due to human or mechanical errors, EMJ and the contributors cannot guarantee the accuracy, adequacy, or completeness of any information, and cannot be held responsible for any errors or omissions. EMJ is completely independent of the review event (ESCMID 2024) and the use of the organisations does not constitute endorsement or media partnership in any form whatsoever.

Front cover and contents photograph: Barcelona, Spain home of the ESCMID 2024 © TTstudio / stock.adobe.com

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Welcome letter

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Dear Readers,

Evgenia Koutsouki Editor

I am happy to introduce the 2024 issue of EMJ Microbiology & Infectious Diseases, which brings you key highlights from the 34th European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Global Congress. With ESCMID Global being one of the largest and most engaging events on infectious diseases, it was a great pleasure for the EMJ team to attend and report on current points of interest such as the One Health approach, antimicrobial resistance, and the impact of climate change.

This issue’s highlights include data from studies on transplant infections, multi-drug resistant bacteria in elderly care centres, and outcomes of respiratory syncytial virus and influenza A in adults. Our selection of compelling articles includes a study focusing on the virtual identification of immune-stimulating epitopes within the chikungunya virus protein. These epitopes could be promising in helping the design and development of vaccines against chikungunya virus infection.

Finally, be sure not to miss our exclusive interview spotlighting key principles in antimicrobial and diagnostic stewardship, as well as novel avenues to tackle diagnostic pitfalls.

I would like to thank our authors, interviewees, reviewers, and Editorial Board for making this issue a captivating collection of content.

I also invite you, our valued readers, to actively engage with us by visiting our website and providing feedback on this content, utilising the opportunity to help shape our coverage and ensure that we continue to deliver relevant and timely content. We eagerly anticipate next year’s congress, so until then, we hope you enjoy reading through this journal!

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Foreword

Dear Colleagues,

It is with great joy that I present the latest edition of EMJ Microbiology & Infectious Diseases. In this issue, you will find peer-reviewed articles on the most current topics in the field, alongside interviews with esteemed experts, and a comprehensive overview of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Global Congress 2024, held in Barcelona, Spain, between the 27th–30th April. Microbiologists, epidemiologists, and statisticians from across the globe convened at ESCMID 2024 to share their research findings and insights, and to stay abreast of the latest advancements pioneered by their peers. From discussions on the ramifications of climate change on infectious disease spread, to innovative approaches for identifying and tackling antibiotic resistance, ESCMID 2024 emerged as the epicentre for global microbiology research, offering a comprehensive platform for knowledge exchange and collaboration.

For those who were unable to attend, you can also find a review of highlights from ESCMID Global 2024. This comprises the latest news, fascinating research abstracts, and comprehensive features on key sessions.

Reporting on hot-topic themes presented at ESCMID 2024, our two Congress features cover the future of genomic surveillance against antimicrobial resistance, and the evolving landscape of infection treatment and prevention for the immunocompromised. Do not miss our exclusive interview with expert Johnny Zakhour, who provides key insights into diagnostic stewardship for junior doctors.

In this issue, you will also find a range of peer-reviewed articles, with a research paper on the design of a multi-epitope vaccine against Chikungunya virus, a case report of Staphylococcus aureus bacteraemia in chronic kidney disease, and the importance of aetiology surveillance in surgical site infections amidst the antimicrobial resistance pandemic. Additionally, our selection of abstract reviews reflects current advancements in infectious disease treatment and control, including an abstract on first-time use of oritavancin for recurrent methicillinresistant S. aureus bone infection.

I would like to take this opportunity to thank all those who contributed to this publication. We hope you enjoy reading our 2024 issue.

Rajeshwar Reddy Kasarla

Professor and Head, Microbiology Department, Universal College of Medical Sciences, Bhairahawa, Nepal

ESCMID 2024

Review of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID)

Global Congress 2024

Location: Barcelona, Spain

Date: 27th–30th April 2024

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:10-21. https://doi.org/10.33590/emjmicrobiolinfectdis/DOOT5011.

“WE at the ESCMID continue to raise our game and lead the field to prevent, treat, and fight infections,” announced Annelies Zinkernagel, University Hospital Zürich, University of Zürich, Switzerland, and President of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Global. With 11,000 members and 45,000 affiliated members through their 77 national and internal affiliated societies, ESCMID Global is a renowned congress for all to attend. This year’s Global Congress took place in the lively city of Barcelona, Spain, from 27th–30th April 2024, packed with oral sessions, workshops, meet-the-expert sessions, and poster presentations.

"We

at the ESCMID continue to raise our game and lead the field to prevent, treat, and fight infections."

A vibrant musical performance started the opening ceremony on a high note. Zinkernagel subsequently spoke on the impressive growth of the society, prompting the name change from ‘ECCMID’ to ‘ESCMID Global’. She emphasised that the growing role of ESCMID Global now brings greater responsibility, and ESCMID can raise its game with two key approaches: prepare and educate.

Zinkernagel spotlighted recent initiatives to step up these efforts, such as the ‘Think Tank’ symposium, focusing on the importance and future of diagnostics in antimicrobial resistance (AMR), and the collaboration with the Global Leaders Group on AMR, forging partnerships between science and policy. An AMR event has additionally been developed, committed to accelerating political action on AMR based on a One Health approach. ESCMID will also contribute to the World Health Organization (WHO)-led World AMR Awareness Week in November; and, in the run-up to this year’s United Nations (UN) High Level Meeting on AMR at the UN General Assembly, will join forces with other organisations to convey the urgency of measures against AMR to global policy makers, demanding “ambitious commitments and concrete actions on AMR”.

In an exciting announcement, Zinkernagel then presented the new ESCMID Education Director: Mary Horgan, President of the Royal College of Physicians of Ireland, Dublin, Ireland. Horgan discussed several promising education initiatives, including the ESCMID Academy, a new online programme with a flexible, modular structure aimed to provide accessible education to the global community.

The ESCMID Global Programme Director, Jacob Moran-Gilad, Ben-Gurion University of

the Negev, Beersheba, Israel, followed with a clear breakdown of this year’s congress programme, and some interesting insights. This year, 112 countries were represented in the abstract submissions, with the top three presenting countries being Spain, the USA, and Italy. The congress additionally saw a 10% increase in participants, 23% increase in abstract submissions, 14% increase in faculty members, and 17% increase in industry sessions. These statistics are testament to the expansion and content quality provided at ESCMID Global.

Finally, the ceremony came to a close with the 2024 Awards. The ESCMID Young Investigator Awards were given to five promising, young

researchers: Nathan Brendish, University of Southampton, UK; Pieter de Cock, Universitair Ziekenhuis Gent, Belgium; Henning Grüll, University Hospital Cologne, Germany; Rasmus Leistner, Universitätsmedizin Berlin, Germany; and Ira Praharaj, Indian Council of Medical Research, India. Evelina Tacconelli, University of Verona, Italy, was honoured with the ESCMID Lifetime Achievements Award, in recognition of her ongoing, outstanding contributions to ESCMID’s mission.

Read on for key insights into ESCMID Global 2024, and don’t miss out on ESCMID Global 2025, taking place from April 11th–15th in Vienna, Austria. ●

Tackling Antimicrobial Resistance: Post-Pandemic Challenges

A RECENT study presented at the 2024 ESCMID Global Congress by Christina Yek, US National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA, and colleagues, examined antimicrobial resistance (AMR) trends in 120 American hospitals before, during, and after the COVID-19 pandemic. Despite strides in tackling AMR since the pandemic peak, hospitalacquired AMR infections persist well above prepandemic levels, particularly those from gramnegative resistant organisms. This underscores the pressing necessity to address the causes and remedies for challenging-to-treat gramnegative AMR infections.

The Centres for Disease Control and Prevention (CDC) report in 2022 noted a 15% rise in hospital-related AMR infections from 2019–2020. Despite this, the underlying causes and long-term impacts are not well understood. Researchers analysed data from 120 American hospitals before (January 2018–December 2019), during (March 2020–February 2022), and after (March–December 2022) the COVID-19 pandemic, focusing on infections by six pathogens. The study aimed to update the understanding of AMR trends in American hospitals, utilising the PINC-AI database representing 20% of hospitalisations in the USA.

Throughout the pandemic, the prevalence of AMR infections surged by 6.3%, reaching 193.3 cases per 10,000 hospitalisations compared to 181.9 pre-pandemic. The spike was predominantly driven by a 32% increase in hospital-acquired resistant infections, contrasting with only a 1.4% rise in communityacquired cases. Infections caused by gramnegative pathogens, known for their resistance to multiple drugs, surged by nearly 20%, while

gram-positive infections fell by 4.2%. The greatest increases were seen in hospitalacquired infections resistant to carbapenems, crucial antibiotics for severe cases. These included a 151% increase in Acinetobacter baumannii infections, a 62% rise in carbapenemresistant Enterobacterales cases, and a 54% increase in Pseudomonas aeruginosa incidents. Conversely, community-acquired Staphylococcus aureus infections dropped by 10% during the pandemic, and 19% afterwards; a trend potentially boosted by reduced societal interactions due to pandemic restrictions.

During the pandemic, hospitals experiencing high influxes of severely ill patients with COVID-19 saw significant increases in hospital-acquired AMR infections, particularly in larger facilities with higher bed capacity. These surges likely stemmed from increased antibiotic usage and challenges in adhering to infection control protocols. Additionally, shortages of personal protective equipment may have exacerbated the spread of infections. While overall AMR rates nearly reverted to pre-pandemic levels postpandemic, hospital-acquired infections remained 13% higher in December 2022. Gram-negative pathogen infections persisted, with hospitalacquired carbapenem-resistant organisms showing sustained elevated levels compared to pre-pandemic times. Between March–December 2022, hospital-acquired carbapenemresistant infections surged, with Acinetobacter baumannii up by 43%, carbapenem-resistant Enterobacterales by 81%, and Pseudomonas aeruginosa by 38%.

Yek emphasised the persistence of these high rates, stressing the need for further action to address challenging gram-negative infections. ●

"Throughout the pandemic, the prevalence of AMR infections surged by 6.3%, reaching 193.3 cases per 10,000 hospitalisations."

Global Measles Cases Almost Double in a Year

DESPITE substantial progress towards measles and rubella elimination over the past decade, recent data presented at the 2024 ESCMID Global Congress, held from April 27th–30th in Barcelona, Spain, revealed concerning trends in measles outbreaks worldwide. Discussions led by Patrick O’Connor, World Health Organisation (WHO) Headquarters, Geneva, Switzerland, and Hanna Nohynek, Finnish Institute for Health and Welfare, Helsinki, Finland, shed light on the current global measles situation, as well as strategies for elimination.

O’Connor’s presentation outlined a worrying trajectory, indicating that measles cases for 2024 are poised to match or exceed the provisional global total of 321,582 cases for 2023. Alarmingly, this 2023 total is almost double (88% higher) than the 2022 figure of 171,153. Furthermore, the number of countries suffering large or disruptive measles outbreaks (defined as 20 cases/million population continuously over a 12 month period) has tripled from 17 to 51 between December 2022–November 2023.

Nearly half of the 2024 cases (45%) have occurred in the WHO European Region, with Yemen, Azerbaijan, and Kyrgyzstan reporting the highest incidence rates globally. In absolute numbers, the percentage of global measles cases is dominated by low and lower-middle income countries, which have seen their proportion of all global cases increase from 80% in 2017 to 94% in 2022.

"The percentage of global measles cases is dominated by low and lower-middle income countries."

Highlighting the challenges faced in achieving and maintaining elimination, O’Connor emphasised the need for high, uniform, and equitable immunisation coverage. While vaccination coverage has been relatively high at the national level, any gaps in coverage pose a risk for outbreaks due to high infectivity of the virus. Nohynek drew attention to the impact of the COVID-19 pandemic on measles transmission, noting that reduced human contact during lockdowns led to a temporary decline in cases. However, with disruptions to vaccination programmes, many children remain unvaccinated, leading to a resurgence of cases in various regions worldwide.

Both speakers stressed the importance of leveraging successful strategies, such as those implemented in the WHO’s Americas region, where measles was successfully eliminated in 2016. They emphasised the need for robust immunisation programmes, strengthened by technical support, political will, and innovative approaches, such as the measles and rubella vaccine microarray patches, which do not require a needle and syringe.

Looking ahead, Nohynek underscored the critical role of measles vaccination in achieving the goals set forth by the Immunization Agenda 2030: preventing 50 million deaths from 2021–2030 using vaccination. ●

Host-Directed Therapies: A Novel Complement to Tuberculosis Treatment

NOVEL immune-enhancing therapies termed host-directed therapies (HDT) may hold the potential to target and treat drug-resistant forms of tuberculosis effectively. Findings presented by Susanna Brighenti, Karolinska Institutet, Stockholm, Sweden, at the 2024 ESCMID Global Congress, revealed how newly identified HDTs were able to reduce Mycobacterium tuberculosis growth by 50–70% in immune cells, even without antibiotic treatment.

Tuberculosis is a leading cause of illness and death globally, accounting for an estimated 7.5 million new diagnoses and 1.3 million deaths in 2022, of which 160,000 deaths are estimated to be a consequence of multi-drug-resistant tuberculosis. Mycobacterium tuberculosis’ extensive ability to manipulate host immune responses and subvert intrinsic antimicrobial effector functions of immune cells, in conjunction with frequent mutations conferring antibiotic resistance, underscores the need for novel treatments tackling tuberculosis.

Through the targeted restoration of multiple immune pathways or induction of antimicrobial activities in infected cells, HDTs are designed to strengthen immune responses to treat infections.

Brighenti showcased how several newly identified histone deacetylase inhibitors can be used to regulate the transcription of genes to enhance the expression of proteins associated with antibacterial host defence. Rather than serving as an independent therapy, HDTs could complement the activity, and protect the function, of existing antibiotics by operating as adjuvants, possibly reducing antibiotic dose and treatment length.

Immunotherapies that complement pre-existing treatments for cancer, asthma/allergy, and autoimmunity have already contributed to significant improvements in patient care. Readily available and approved HDT therapeutics, such as glucocorticoid treatment or cytokine neutralisation therapies, may represent an initial step and short-term option for patients with severe multi-drug-resistant forms of tuberculosis, or those with poor prognosis.

Brighenti emphasised the ever-growing call for personalised medicine, and concluded that the different sub-groups of tuberculosis mean that “tailored treatments that are optimised to individual patient needs” will be part of future tuberculosis treatment. ●

"Tailored treatments that are optimised to individual patient needs will be part of future tuberculosis treatment."

Variations by Age and Sex in Antimicrobial Resistance

LEVELS of antimicrobial resistance in bloodstream infections vary by age and sex, according to research presented by Gwen Knight, London School of Hygiene and Tropical Medicine, UK, at the 2024 ESCMID Global Congress. Available data on how prevalence of antimicrobial resistance varies with age and sex for different bacterial species and resistance phenotypes are lacking; however, better understanding of these associations would enable intervention targeting.

This study used routine surveillance data from 29 European countries, collected by the European Antimicrobial Resistance Surveillance Network (EARS-NET), and aimed to characterise the burden of antimicrobial resistance for bloodstream infections. A total of 6,862,577 susceptibility results between 2015–2019 were included, and resistance trends by age and sex were characterised.

The team noted substantial variation in antimicrobial resistance prevalence by age, and identified four main association forms: u-shaped with monotonic increase with age after infancy; constant; n-shaped with resistance peaking at intermediate ages; and monotonic decline with age. They further saw low association between sex and resistance, except for Klebsiella pneumoniae and Escherichia coli, as well as

Acinetobacter spp. at younger ages, with males more likely to have a resistant infection.

Data also showed more variation in trends within an antibiotic family than a bacterial species. For example, there was a clear increase in resistance prevalence by age for methicillin-resistant Staphylococcus aureus, whereas within Pseudomonas aeruginosa, researchers noted a peak to several antibiotics at around 30 years of age.

Knight stated: “Most experts assume that resistance prevalence would increase with age due to cumulative antibiotic exposure effects and contact with healthcare settings, but it was not the case with all pathogens.” She also noted that, surprisingly, females had lower prevalence of resistant bloodstream infections, despite having more risk factors, including urinary tract infection incidence and childbirth.

The team concluded that the prevalence of antimicrobial resistance in bloodstream infection varies by age and sex, and that patterns of association vary widely with resistance phenotype and bacterial species. These data reveal important gaps in our understanding of antimicrobial resistance drivers, and will affect intervention targeting. ●

"Females had lower prevalence of resistant bloodstream infections, despite having more risk factors."

Antibiotic Use in Patients with COVID-19

Offers No Benefit

IMPORTANT findings presented at this year’s ESCMID Global Congress shed light on the risks associated with antibiotic treatment for adults hospitalised with moderate COVID-19. Despite antibiotics being administered to over 40% of patients, a study led by Anette Friedrichs, University Hospital Schleswig-Holstein, Kiel, Germany, reveals a concerning trend of clinical deterioration among these individuals.

The study, conducted between March 2020–May 2023, analysed data from 1,317 hospitalised adults (median age: 59 years; 38% female) with confirmed SARS-CoV-2 infection. Researchers identified 1,149 patients with moderate disease, of whom 467 (41%) were treated with antibiotics commonly used for respiratory infections, such as β-lactams, macrolides, or moxifloxacin. Additionally, 70% of the 168 patients classified as having severe disease received antibiotic treatment. The team found that older, male patients with greater disease severity, a higher burden of comorbidities, and no prior COVID-19 vaccination had a significantly greater likelihood of being treated with antibiotics.

The researchers used the World Health Organization (WHO) Clinical Progression Scale to track patient’s trajectories over the course of their illness, incorporating factors such as hospital admission, O2 requirement, ventilator support, admission to intensive care unit, and

organ replacement therapy. The score was calculated for each patient at initial consultation, and again 14 days later to see whether the score had improved, remained stable, or worsened.

Microbiological investigations identified a mere 11 patients with bacterial superinfection, raising questions about the efficacy of widespread antibiotic use. Importantly, the study found that patients treated with antibiotics faced a five-fold greater risk of clinical deterioration after 14 days compared to those not treated with antibiotics. Furthermore, the team found that being aged ≥65 years tripled the likelihood of COVID-19 deterioration, compared to those aged 18–50 years; and younger, female, vaccinated patients showed significantly better clinical improvement.

The study, while observational and limited to patients in Germany, highlighted the critical need for rational antibiotic use, especially during viral pandemics like COVID-19. Friedrichs emphasised that antibiotics should only be administered for suspected or confirmed bacterial co- or superinfection, cautioning against indiscriminate prescribing. Antibiotic stewardship programmes must be strengthened to ensure that antibiotics are reserved for these cases, and diagnostic tests should always be used to confirm bacterial infection. ●

"Antibiotics should only be administered for suspected or confirmed bacterial co- or superinfection."

Making Transfusion-Transmitted

Malaria in Europe a Thing of the Past

IN THE BATTLE against transfusion-transmitted malaria (TTM) in Europe, Sophie Le Cam, representing the French transfusion blood service (Etablissement Français du Sang [EFS]), presented a comprehensive strategy at the 2024 ESCMID Global Congress, in Barcelona, Spain. Despite commendable progress, with only 10 reported cases of TTM in the last two decades, existing serological tests are insufficient to completely eradicate the risk.

TTM occurs when a recipient receives infected blood or its components, leading to accidental Plasmodium infection. Even minimal parasite presence can cause infection, posing a significant threat, especially in non-endemic regions where immunity is scarce. Le Cam highlighted the dire consequences of TTM, particularly for immunocompromised patients, and those unexposed to malaria.

Europe mandates precautionary measures, such as deferral periods for donors returning from endemic regions. However, existing diagnostic methods lack the necessary sensitivity and specificity. Microscopic examination, the gold standard for malaria diagnosis, is not feasible for blood bank operations, while serologic tests fall short of expectations.

A critical challenge lies in detecting infections in donors with immune tolerance, who may harbour low parasite densities for extended periods. Le Cam advocated for advanced molecular methods to enhance sensitivity. Yet, cost remains a barrier to widespread adoption.

"A critical challenge lies in detecting infections in donors with immune tolerance."

“The key to transfusion security remains the deferral period after the return of donors from endemic countries, but we really need to develop new testing strategies. The parasite inactivation of blood using new technologies that are able to selectively inactivate pathogens without damaging cells or plasma could also be a good option, but the technology is not completely reliable for packed red blood cells, and is very expensive,” explained Le Cam. She suggested combining various strategies, including molecular testing, to safeguard blood transfusions effectively. In essence, eliminating TTM in Europe demands a concerted effort. While donor deferral remains pivotal, investment in innovative diagnostic tools and technologies is equally crucial. ●

CRISPR for Antimicrobial Resistance:

Too Good to Be True?

CRISPR has been highlighted in recent years as a promising measure against antimicrobial resistance (AMR), but is it the answer to this growing health pandemic? Recent research by Ibrahim Bitar, Department of Microbiology, Faculty of Medicine in Plzeň, Charles University, Prague, Czechia, presented at this year’s ESCMID Global Congress, underscores the challenges of harnessing CRISPR to combat AMR.

Clustered regularly interspaced short palindromic repeats, better known as CRISPRs, are repetitive lengths of DNA that are separated by fragments called spacers. Spacers account for adaptive immunity in prokaryotes, originating from an identical heterologous nucleic acid sequence that previously invaded the host bacterium. The CRISPR-Cas system employs a short CRISPR RNA (crRNA)-Cas effector complex. Guide crRNAs contain spacers which enable identification of repeat infectious agents with matching genetic material. This effector complex base pairs to the complementary strand of the invasive RNA, and induces sequence-specific cleavage, inhibiting the replication of pathogenic genetic material and, therefore, the proliferation of invasive species.

The hypothetical use of CRISPR in the fight against AMR hinges, therefore, on harnessing Cas proteins belonging to the CRISPR-Cas system, and delivering them via vectors such as bacteriophages or conjugative bacterial strains, to selectively target and remove AMR genes.

Likewise, it may be used to develop antimicrobial agents by introducing self-targeting crRNAs that produce double-stranded breaks within the bacterial DNA.

While taking advantage of this system boasts great promise, there are some potential obstacles to its utilisation in clinical practice. The CRISPRCas system, as with most biological systems, is regulated by several, highly complex pathways that are yet to be fully characterised. Equally, as the mechanism relies on horizontal gene transfer via conjugation, a process also regulated by numerous highly complex pathways, there is no guarantee that the percentage of successful delivery within a bacterial population will be sufficient for successful resensitisation to antibiotics.

Furthermore, bacteria encode anti-CRISPR systems that serve to repair any damage that arises from CRISPR-Cas activity. For example, the gene acr, which aids in the inhibition of horizontal gene transfer, clusters alongside antagonists that similarly contribute to host defence, producing defence islands. As a result, resensitisation efforts may be impeded by innate defence systems.

Commenting on the challenges of introducing CRISPR-Cas-mediated resensitisation, Bitar wrote: “These regulated pathways must be studied in depth in order to avoid selective pressure favouring anti-CRISPR systems activation, hence prevalence of resistance in a more aggressive manner”. ●

"While taking advantage of this system boasts great promise, there are some potential obstacles to its utilisation in clinical practice."

Artificial Intelligence Versus Doctors: Fact Meets Practice

AN ARTIFICIAL intelligence (AI) chatbot proficiently answered factual queries, sometimes surpassing doctors; however, it struggled with applying theory to clinical situations, found a study conducted by Andrea De Vito, University of Sassari, Italy, and colleagues. They cautioned against using the technology for patient treatment decisions, and highlighted ChatGPT’s accessibility and potential for medical advice. The study aimed to evaluate ChatGPT’s accuracy in antibiotic treatment guidance compared to medical professionals.

Experts designed 72 questions across various formats to assess knowledge in treating specific infections. The questions covered true/false statements, open-ended inquiries, and clinical cases with antibiograms. They explored scenarios like necrotising pancreatitis and ruptured aortic aneurysm, along with challenges and therapeutic strategies for infections like those caused by multidrug-resistant Acinetobacter baumannii Clinical cases provided detailed patient scenarios, including medical history and bacterial sensitivity, requiring identification of resistance mechanisms and appropriate treatment options.

The study, presented at the 2024 ESCMID Global Congress, administered questions to four senior residents, four infectious disease specialists, and two versions of ChatGPT-4. Responses, totalling 720, were evaluated by blinded antibiotic treatment experts for accuracy and appropriateness. All groups performed similarly

on true/false queries, with around 70% accuracy. ChatGPT-4 excelled in open-ended responses but slightly lagged in clinical case accuracy and antibiotic selection compared to doctors. It favoured older antibiotics over newer, potentially more suitable options due to challenges accessing and comprehending complex, updated medical data and guidelines, explained Vito.

He emphasised that understanding ChatGPT’s response generation process is vital, as it constructs sentences based on context rather than comprehension, potentially hindering its accuracy with the latest medical guidelines. Both ChatGPT-4 versions tended towards longer treatment durations than necessary. While proficient in answering factual queries, ChatGPT struggled with interpretation and application, especially with new treatments, compared to doctors. The team warned against relying on ChatGPT for clinical decisions, highlighting its limitations and stressing the importance of professional judgment. While AI advances offer promise, challenges like accuracy, reliability, and patient privacy persist, delaying chatbot integration into medical practice. Public reliance on chatbots for infection treatment advice is discouraged.

Vito highlighted that, while ChatGPT excels with factual questions, it lacks the ability to gather crucial diagnostic information from patients or conduct physical exams, emphasising that AI tools should not substitute for medical professionals. ●

"The questions covered true/false statements, open-ended inquiries, and clinical cases with antibiograms."

Surveillance Breakthrough: Tracking Global Antimicrobial Resistance Prevalence

ANTIMICROBIAL resistance (AMR) is on the rise globally, according to new estimates presented at the 2024 ESCMID Global Congress in Barcelona, Spain. AMR poses a significant threat to global health; however, traditional surveillance methods struggle to provide comprehensive data due to limited resources and diagnostic capacity in many countries, causing the true scale of the problem to remain unknown.

A research team led by Sneha Kotian, Amsterdam Institute for Global Health & Development, The Netherlands, has estimated AMR levels in countries lacking data with a novel approach using sociodemographic and health-related drivers of AMR, greatly improving the global coverage of AMR prevalence rates. New estimates have revealed that AMR prevalence of World Health Organization (WHO) priority pathogens is expected to be substantial in countries lacking current data.

The study utilised data from 2004–2021, sourced from the Vivli AMR Register and ResistanceMap, for nine key gram-negative pathogen-antibiotic combinations. Sociodemographic and healthrelated factors were extracted from the WHO Health Inequality Data Repository and the Global Data Lab. Advanced statistical analyses, including a stacked ensemble model comprising various regression models, were employed to elucidate the relationship between indirect predictors and AMR rates.

This approach increased global coverage of AMR prevalence rates from 24% to 85%. It indicated that resistance to third-generation cephalosporins (one of the most widely used

antibiotic classes) in Escherichia coli, and resistance to carbapenems (antibiotics of last resort) in Acinetobacter baumannii, are both on the rise; with a notably sharp increase observed in Sub-Saharan Africa and South Asia between 2004–2021.

"This approach increased global coverage of AMR prevalence rates from 24% to 85%."

Kotian explained: “Understanding leading pathogen-drug combinations contributing to AMR in specific regions is crucial to making informed decisions about how to prioritise surveillance activities, and underscores the urgent need for collaborative efforts to address and mitigate this escalating challenge in global public health.”

This study demonstrates the effectiveness of sociodemographic and health-related indirect predictors in supplementing national surveillance data for estimating AMR prevalence. These findings serve as a foundation for further refinement and improvement of predictive models.

However, the model performed more effectively for certain pathogen-antibiotic combinations over others; thus, future steps will involve refining the approach for these combinations. The researchers also noted that, given the approach uses laboratory-based surveillance which has associated bias, the results should be interpreted as an approximation of the true prevalence in a country. ●

Drastic

Disparities in Global Coverage of Human Papillomavirus Vaccination

WORLD Health Organization (WHO) Dashboard data have revealed that, despite the rollout of human papillomavirus (HPV) vaccination, contributing to a significant reduction in cervical cancer and HPV-related disease incidence and mortality worldwide, there exists a dramatic disparity in global coverage.

The advent of safe and efficacious HPV vaccines at the beginning of the 21st century prompted the WHO to call for an ambitious 30% reduction in cervical cancer incidence and mortality by 2030. To achieve this target, each country must aim to vaccinate 90% of females by age 15 years, administer high precision screening testing to at least 70% of females by age 35, and ensure that 90% of all females requiring treatment receive it. However, while evidence accumulates in support of HPV vaccination, there remains wide variation in coverage between high-income countries and low- and middle-income countries.

Data presented by Suzanne Garland, University of Melbourne, Australia, at the 2024 ESCMID Congress, demonstrated that 137 countries have a total/partial HPV national programme, with average vaccine coverage highest at 60% in WHO’s European region, lowest at 31% in the Americas Region, and a global average coverage

of 44%. Australia has the world’s leading HPV vaccine rollout, with coverage in males and females of 15 years of age in 2021 at 84.4% and 86.2%, respectively. The extensive rollout of HPV vaccines in Australia has led to a 92% reduction in the prevalence of HPV type, and the national public health programme aims to be the first to eliminate cervical cancer.

Garland emphasised that the main barriers for countries to improve coverage are competing vaccines, price and delivery, interruptions from global events, political will, and the number of doses required. In spite of these challenges, Malaysia represents a low- or middle-income country that was able to ensure high coverage.

Focus should not be diverted from other cervical cancer and HPV-related disease prevention measures, including condom use, sex education, male circumcision, cervical disease screening, and tobacco control strategies. Nevertheless, Garland concluded that there is strong evidence of HPV vaccination effectiveness against cervical cancer, and stressed: “Scaling up vaccine access and coverage globally is critical to reduce inequities between and within countries.” ●

"Scaling up vaccine access and coverage globally is critical to reduce inequities between and within countries."

Implementation of Genomic Surveillance for Antimicrobial Resistance: Typhoid Fever and Klebsiella pneumoniae

Authors: Ellen Charge, EMJ, London, UK

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:22-25. https://doi.org/10.33590/emjmicrobiolinfectdis/OUWR9161.

AN ILLUMINATING session on the implementation of genomic surveillance for monitoring of antimicrobial resistance (AMR) patterns globally, and its role in public health, was held at the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Global Congress in Barcelona, Spain, from 27th–30th April 2024. Chaired by Sylvain Brisse, Institut Pasteur, Paris, France; and Maurizio Sanguinetti, Catholic University of the Sacred Heart, Milan, Italy, the session explored the various digital resources, and advancements in knowledge, underpinning the application of genomic surveillance for multidrug-resistant pathogens Salmonella typhi and Klebsiella pneumoniae (Kpn) as a promising measure against the threat of AMR to public health.

THE NEED FOR GENOMIC SURVEILLANCE

Kathryn Holt, London School of Hygiene & Tropical Medicine, UK, opened the session by highlighting the growing integration of genomic surveillance in public health, commenting: “The last 5 years has seen a real maturation of pathogen genomics, and its role in public health.”

AMR, the so-called ‘silent pandemic’, is a significant and growing threat to public health. With the increasing prevalence of multidrugresistant bacterial pathogens, the ability to track the genetic evolution of implicated strains with reference to key virulence factors, AMR determinants, and serotype, provides a clearcut avenue for infection prevention, and for monitoring the spread and impact of multidrugresistant pathogens on a local, national, and global scale.

Despite the promising uses of genomic surveillance in tackling AMR, its implementation in clinical settings presents a host of challenges,

as underscored by Holt: “The path from genomics data to actual knowledge we can act on is a very complex one.” Indeed, even if sufficient sourcing and sequencing of data can be achieved, genomic surveillance cannot be accomplished without a sophisticated digital infrastructure, and data presentation that can be readily interpreted by epidemiologists, microbiologists, and healthcare professionals alike. During the session, Holt elaborated on her team’s efforts to delineate the essential criteria for such an infrastructure, and the progress they have made in rendering genomic surveillance actionable.

Generating common lineage nomenclature for various resistant strains is one of the foremost requirements for making genomic surveillance a workable reality. When terminology differs by region, it invites the possibility for confusion and misunderstanding. Holt highlighted the need for universal nomenclature that can be recognised by all experts and healthcare professionals across the world.

TYPHOID FEVER

Typhoid fever (typhoid) is a systemic bacterial infection with Salmonella enterica serovar typhimurium. When treatment is delayed, it can result in serious complications, and ultimately prove fatal. At present, there are 11 million cases of typhoid globally, and 116,000 deaths per annum. Typhoid is most common in low-income countries where sanitation is poor, and clean water is not readily available. The infection is endemic in South Asia, most notably in Pakistan, India, and Bangladesh. Considering the importance of prompt, effective treatment, AMR represents a significant health risk for those infected.

In the absence of diagnosis, the majority of antibiotic treatment for typhoid fever is empirical therapy. Treatment guidelines are determined according to local resistance patterns, and increasingly the only option for complicated cases is admission of intravenous antibiotics, giving rise to more hospital admissions, increased economic burden, and morbidity.

"The path from genomics data to actual knowledge we can act on is a very complex one."

The S. typhi genome was first sequenced at the Wellcome Sanger Institute, Cambridgeshire, UK, in 2001.1 Since then, our understanding of the population structure, resistance patterns, and strain diversity of S. typhi has significantly advanced, leading to the establishment of the Global Typhoid Genomics Consortium. This consortium aims to facilitate the sharing of typhoid genome data to enhance public health, enable the extraction and reporting of essential data, and promote the dissemination of genomic information for monitoring AMR and disease spread. These efforts intend to inform treatment guidelines and immunisation strategies, and ultimately reduce transmission.

Advancements in digital resources, in particular, have enabled the development of TyphiNET,2 a multi-institutional global genomic surveillance network, supporting the visualisation of the S. typhi genotype and AMR data. Most notably, TyphiNET allows for the rapid integration of genomes into the global phylogeny without requiring extensive phylogenetic or comparative analysis.

A key goal of Holt and team is to generate common nomenclature, specifically that relating to S. Typhi strains. To achieve this, the team have produced an AMR dictionary that

highlights genetic determinants to look for, and how to interpret them with relevance to AMR. Sequenced genomes are uploaded following a defined metadata standard that specifies the purpose of sampling and the country of travel. These metadata help distinguish between routine sequencing and sequencing conducted in response to an outbreak or other specific events. Likewise, the inclusion of the country of travel provides global location data and information about the prevalence of specific strains and lineages per area, and local resistance patterns.

KLEBSIELLA PNEUMONIAE

Kpn was first described by Carl Friedlander in 1882,3 after being isolated from the lungs of patients who had died of pneumonia, thus giving it its name. Kpn is an opportunistic pathogen, naturally found among the microbiota of the mouth, skin, and gastrointestinal tract, but proving highly virulent when it infects other parts of the body. Its extensive virulence is, concerningly, accompanied by extensive AMR, making it a desirable target for genomic surveillance.

Kpn is a leading cause of multidrug-resistant hospital-acquired infections and neonatal sepsis.

It contributes significantly to disease burden and mortality, particularly in low- and middleincome countries. Its impact on neonatal sepsis has spurred considerable interest in maternal immunisation to protect neonates, the group most vulnerable to this disease. This interest led to the publication of the vaccine value profile for Kpn in March of this year, highlighting the potential to prevent approximately 400,000 cases of neonatal sepsis, and 80,000 deaths.4 To support these efforts, Holt and his team have developed various modelling techniques to account for outbreak clusters in the data, and to generate regional and global estimates of antigen distributions. They have also created theoretical coverage estimates for different vaccine compositions to aid in the development of effective vaccines.

With the goal of monitoring the emergence and spread of AMR, and the appearance of hypervirulent lineages, alongside an investigation into nosocomial outbreaks and serotype distribution, Holt and colleagues contributed to the development of the Klebsiella pneumoniae Genomic Surveillance Platform (KlebNET-GSP),5 which incorporates four key tools for genomic typing and analysis: Kleborate,6 Kaptive,7 Pathogenwatch,8 and BIGSdb-Pasteur.9 Kleborate and Kaptive were

"Holt

highlighted the need for universal nomenclature that can be recognised by all experts."

specifically developed by Holt and colleagues. The former provides accurate identification of species, lineage, resistance determinants, and virulence genotypes. The latter, conversely, enables bacterial surface polysaccharide locus typing and variant evaluation. BIGSdb-Pasteur9 is a collection of databases of bacterial isolates, genomes, and genotypes based on multilocus sequence typing and whole genome-based typing. Pathogenwatch8 specifically compares pathogen genome assemblies from different global regions, utilising data from BIGSdbPasteur9 and Kaptive.7

Holt and team will host the first international symposium focused on Kpn from the 20th–22nd November 2024, at the Institut Pasteur in Paris, France. This symposium will feature workshops on the previously mentioned tools, as well as online drop-in sessions. Additionally, the conference will include lectures by leading experts from around the globe, covering various aspects of Kpn, including epidemiology, ecology, genomics, therapeutics, and surveillance.

References

1. Parkhill J et al. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature. 2024;413(6858):848-52.

2. TyphiNET. Available at: https:// www.typhi.net/. Last accessed: 16 May 2024.

3. Ashurst JV, Dawson A. Klebsiella pneumonia [Internet] (2023) Treasure Island: StatPearls.

CONCLUSION

Multidrug resistance represents a formidable public health challenge, evoking concerns about a regression to the pre-antibiotic era, where once easily treatable diseases give rise to excessive disease burden and high rates of mortality. Particularly vulnerable to these effects are populations in low- and middleincome countries, lacking sufficient access to healthcare, and facing heightened exposure to communicable disease risk factors. As the threat of AMR looms large, highly virulent superbugs like S. typhi and Kpn, still endemic in less developed regions, are sources of great concern. The digital infrastructure to which Holt and colleagues have contributed, and their evident potential in monitoring the spread of AMR and tailoring treatment guidelines according to the geographical prevalence of specific strains, presents a clear and promising pathway for mitigating the proliferation and impact of antimicrobial-resistant pathogens. ●

Available at: https://www.ncbi.nlm. nih.gov/books/NBK519004/. Last accessed: 16 May 2024.

4. Dangor et al. Vaccine value profile for Klebsiella pneumoniae. Vaccine. 2024:S0264-410X(24)00248-2.

5. KlebNET-GSP. Available at: https:// klebnet.org/. Last accessed: 16 May 2024.

6. Kleborate. Available at: https:// kleborate.erc.monash.edu/. Last

accessed: 16 May 2024.

7. Kaptive. Available at: https:// kaptive-web.erc.monash.edu/. Last accessed: 16 May 2024.

8. Pathogenwatch. Available at: https://pathogen.watch/. Last accessed: 16 May 2024.

9. BIGSdb-Pasteur. Available at: https://bigsdb.pasteur.fr/. Last accessed : 21 May 2024.

Authors:

The Future of Infection Treatment and Prevention in Immunocompromised Patients

Laith Gergi, EMJ, London, UK

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:26-29. https://doi.org/10.33590/emjmicrobiolinfectdis/ZATI1178.

THE emerging role of biological therapies in treating and preventing infection in patients who are immunocompromised was the primary focus of a compelling symposium, exploring ‘the future of infection treatment and prevention in the immunocompromised’. The session was hosted during the 34th Annual European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Global Congress, held in Barcelona, Spain, between 27th–30th April 2024, and co-chaired by Michael Ison, National Institutes of Health (NIH), USA; and Elda Righi, Verona University, Italy.

BIOLOGICAL THERAPIES IN MODERN MEDICINE

Isabel Ruiz-Camps, University Hospital Vall d’Hebron, Barcelona, Spain, commenced the session by discussing the role of biologic therapies in patients who are immunocompromised. The use of biologic therapies or ‘biologicals’ has transformed the treatment landscape and outcomes for a myriad of diseases, including solid cancers, haematological malignancies, rheumatoid arthritis, and inflammatory bowel disease. Biologicals encompass monoclonal antibodies, receptor analogues, and chimeric small molecules, often targeting specific receptors, cytokines, or cell types, directly or indirectly intervening in immunological pathways, to modulate innate and adaptive immune responses. Ruiz-Camps noted how biologic agents are increasingly used daily due to their potency advantages and reduced side effects. However, Ruiz-Camps called to attention the association between the application of biologicals and infectious complications, especially in patients with a history of

autoimmune and inflammatory diseases. She stated that this association represents a significant hurdle in their safe implementation. Therefore, a nuanced understanding of the factors that govern infection risk with biologicals is required to improve patient outcomes.

RISK PROFILE OF PATIENTS

Ruiz-Camps discussed that the risk of infectious complications in patients who are immunocompromised, and receiving biologicals, is dynamic. The risks fluctuate over the patient’s lifetime, and are influenced by three key factors: specific treatment regimen, underlying disease, and host health status. While biologicals may lead to an increased risk of infection to all microorganism origins, specific infection risk is affected by the cytokine receptor, or immune cell targeted for inhibition. For example, there is a well-established relationship between infliximab, a chimeric monoclonal antibody targeting TNF, and the reactivation of latent tuberculosis. Nevertheless, Ruiz-Camps emphasised the significance of host factors in infection risk.

"The use of biologic therapies or ‘biologicals’ has transformed the treatment landscape and outcomes for a myriad of diseases."

She expanded on this by discussing a study examining the infection risk in patients with juvenile idiopathic arthritis from a prospective, observational registry receiving different biologic therapies.1 The study’s main findings were that relative to TNFα inhibitors, IL-1 inhibitors and IL-6 inhibitors had a higher impact on increasing infection risk.1 However, the most striking risk factors for infectious complications were the use of corticosteroids, younger age, cardiac comorbidities, and lack of disease control in the host. Accordingly, a similar study evaluating the risk of infection in patients with inflammatory bowel disease found that other than the type of biologic agent, the most consistent risk factors for opportunistic infection included advanced age, severe disease activity, and use of corticosteroids.2

PREVENTION METHODS OF INFECTIOUS COMPLICATIONS

Although there is a growing understanding of infection risk factors associated with biologic agents, Ruiz-Camps highlighted the absence of universal standards for prevention methods. Therefore, to implement prophylaxis treatments, it is essential to fully understand

the immunological effect of a biologic agent and relevant host factors; hence, a complete clinical history should be carried out. For a patient undergoing treatment, Ruiz-Camps listed personal comorbidities, history, place of origin, travels, sexual behaviour, contact with pets, and pharmacological history as important details of personal history to be collected. Additionally, vaccination and screening for patients are essential, and proactive prevention strategies should ideally be performed before the start of the treatment. Some adverse events mimic infection; therefore, if an infection is suspected, accurate and aggressive diagnosis is required to prevent severe infection.

Ruiz-Camps explained that infection is frequent in patients who are immunocompromised and undergoing treatment with biologicals, and the risk is dependent on the specific biologic therapy, underlying disease, and host health status. Comprehensive risk assessment, proactive prophylaxis treatment, timely diagnosis, and as always, antimicrobial stewardship are required to optimise patient outcomes.

CELLULAR THERAPIES FOR IMMUNOCOMPROMISED DIFFICULT-TO-TREAT INFECTIONS

In the second talk of the session, Nina Khanna, University Hospital Basel, Switzerland, introduced the potential of cellular therapies, specifically virus-specific T cell therapy (VST), in strengthening the immune response in patients who are immunocompromised. Khanna emphasised that the number of patients who are immunosuppressed is increasing, and due to impaired immunity, these patients are at a high risk of viral infections. Allogeneic haematopoietic stem cell (HSCT) and solid organ transplant recipients have a high incidence of doublestranded DNA viral infections in the immunedeficient post-transplant period. Infectious complications represent a leading cause of death and morbidity in HSCT and solid organ transplant recipients. Typically, patients with infectious complications will be treated with anti-infectives; however, because of ongoing immune deficiency, patients have a high risk of persistence and resistance development. Hence, Khanna indicated that the best approach is employing cell-based therapy to strengthen and improve immunity.

ADOPTIVE VIRUS-SPECIFIC T CELL THERAPY

Khanna explained how virus-specific T cells can be obtained from partially HLA-matched seropositive donors. Following this, peripheral blood mononuclear cells are extracted, stimulated with viral antigens, and specific cells are isolated

and expanded. Infusion of VST into allogeneic haematopoietic stem cells and solid organ transplant recipients allows the reconstitution of antiviral immunity, as there is self-renewal of this new T cell repertoire, providing host protection. However, Khanna pointed out some of the challenges in VST application, including a lack of indications for when VST is recommended, and that use is based mainly on Phase I/II trial data. Additionally, for stem cell transplant recipients, the best donor for T cells is the stem cell donor in HSCT, but the donor is not always eligible because they are seronegative.

EFFICACY AND SAFETY OF VST

Discussing the outcomes of over 1,500 patients who have so far received VST post-HSCT, Khanna explained that based on data from all studies, 70% of these patients experienced either partial or complete resolution of symptoms, while the risk for graft-versus-host disease remained low, at around 10%. Furthermore, in a Swiss-wide Phase I/II trial initiated in 2015 to treat refractory virus infection with VST, 14 patients were treated; seven with cytomegalovirus and seven with Epstein–Barr virus (EBV).3 The outcome was similar to the literature, with 85% and 57% partial/complete resolution for cytomegalovirus and EBV, respectively.3

Khanna discussed an open-label Phase II trial evaluating the safety outcomes of posoleucel, an allogenic, off-the-shelf multi-virus-specific T cell therapy, used to treat refractory viral infection in transplant recipients.4 The study included 58 patients with persistent or recurrent infection

"Infectious

complications represent a leading cause of death and morbidity in HSCT and solid organ transplant recipients."

despite 14 days of standard treatment, or after treatment failure after 7 days.4 Patients received an intervention of a single infusion of VST. Results showed partial/complete resolution in 95% of patients, increased immunological response, and clinical safety within tolerable levels with 22% acute graft-versus-host disease.4 Moreover, another VST immunotherapy called tabelecleucel was approved by the European Medicines Agency (EMA) in 2023, for adults and children from 2 years of age with EBV-positive post-transplant lymphoproliferative disorder. These studies serve as evidence for, and provide support to, the utilisation of VST in clinical settings.

CHALLENGES AND OUTLOOKS

Although promising data are emerging regarding using VST to protect stem cell and solid organ transplant recipients, Khanna stressed that the problem of impaired long-term persistence, due to an immune suppressive microenvironment and ongoing systemic immune suppression, compromises VST. Increased T cell exhaustion and impaired functional capacity of T cells are often observed in treated patients. In addition, the rejection of infused cells often means that serial VST infusions are required in clinical settings. Therefore, with these challenges in mind, Khanna’s research group investigated whether T cell exhaustion can be countered by utilising early differentiated T cells.

Early differentiated T cells show high proliferation and self-renewable capacity. Khanna’s group pursued a hypothesis exploring whether enrichment of VST product with early differentiated T cells could induce higher persistence and longevity. A rapid expansion protocol was used to generate a new immunotherapy product to test this.

References

1. Thiele F et al. Comparative risk of infections among real-world users of biologics for juvenile idiopathic arthritis: data from the German BIKER registry. Rheumatol Int. 2021;41:751-62.

2. Singh S et al. Comparative risk of serious infections with biologic and/or immunosuppressive therapy in patients with inflammatory

The Khanna group tested the product on immunodeficient mice infected with EBV. The mice were treated after 3 days with the traditional VST product, and the newly generated T cell product. Both therapies showed similar macroscopic tumour control, but the rapid protocol T cells demonstrated higher proliferation and persistence capacity, and lower residual tumour cells. Hence, VST enriched for earlydifferentiated therapy may represent the future of virus-specific cell therapy.

CONCLUDING REMARKS

VST is an emerging immunotherapy for immunocompromised difficult-to-treat infections, with increasing evidence supporting the introduction of VST in clinics. However, Khanna underscored how unanswered questions regarding the optimal indication and timing of the T cell product for treatment needed to be addressed.

Khanna concluded her presentation by highlighting novel immune therapeutics for immunocompromised patients, such as soluble T cell receptors acting as antibody-like agents, and genetically modified T cells used for individuals without suitable T cell donors.

A common theme throughout the session revolved around the challenges and opportunities introduced by the new age of biologic therapies. While promising data continue to accumulate, demonstrating the potential of biologicals in transforming patient outcomes, continued research evaluating how doses or specific applications can be optimised to confer effectivity while minimising complications is needed. ●

bowel diseases: a systematic review and meta-analysis. Clin Gastroenterol and Hepatol. 2020;18(1):69-81.

3. University Hospital, Basel, Switzerland. A phase I/II single-center study to assess safety and feasibility of direct infusions of donor-derived virusspecific T-cells in recipients of hematopoietic stem cell transplantation with post-

transplant viral infections using the cytokine capture system. NCT02007356. https://classic. clinicaltrials.gov/ct2/show/ NCT02007356.

4. Thomas P et al. Posoleucel, an allogeneic, off-the-shelf multivirus-specific T-cell therapy, for the treatment of refractory viral infections in the post-HCT setting. Clin Cancer Res. 2023;29(2):32430.

Abstract Review

Showcasing the latest research in the fields of microbiology and infectious diseases from abstracts presented at the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Global Congress 2024.

Oritavancin Use in Patients with Recurrent Bone Infections by Methicillin-Resistant Staphylococcus aureus and Role of Therapeutic Drug Monitoring

Authors: *Marco Bongiovanni,1 Paul Thoueille,2 Beatrice Barda,1 Thomas Mercier,2 Marzolini Catia,2,3 Eva Choong,2 Marco Cantu,4 Laurent A. Decosterd1,5,6

1. Division of Infectious Diseases, Ente Ospedaliero Cantonale, Lugano, Switzerland

2. Service and Laboratory of Clinical Pharmacology, Lausanne University Hospital, University of Lausanne, Switzerland

3. Division of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Switzerland

4. Istituto di Medicina di Laboratorio EOLAB Ente Ospedaliero Cantonale, Bellinzona, Switzerland

5. University of Geneva, Switzerland

6. University of Southern Switzerland, Lugano, Switzerland

*Correspondence to marco.bongiovanni@eoc.ch

Disclosure: The authors have declared no conflicts of interest.

Keywords: Bone infections, long-acting, oritavancin, osteomyelitis.

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:30-31. https://doi.org/10.33590/emjmicrobiolinfectdis/EBCU9574.

BACKGROUND

Oritavancin is a novel long-acting lipoglycopeptide that is active against grampositive micro-organisms, including methicillinresistant Staphylococcus aureus (MRSA) and enterococci 1 The pivotal clinical trials SOLO I and SOLO II demonstrated non-inferiority of oritavancin compared to vancomycin for the treatment of acute bacterial skin and skin structure infections.2,3 Due to its favourable pharmacokinetic/pharmacodynamic properties and safety profile, oritavancin is considered a possible option for beyond-thelabel infections that need longer treatments, such as osteomyelitis.4 Its use in the context of recurrent bone infections by MRSA not responding to conventional antibiotics has not yet been evaluated, nor the potential benefit of therapeutic drug monitoring (TDM).

CASE 1

An 84-year-old male with MRSA spondylodiscitis (L4) was treated with surgical drainage and vancomycin, which was subsequently replaced by daptomycin (8 mg/ kg) due to kidney failure. Daptomycin was administered for 6 weeks, but 1 week after stopping, the patient was hospitalised again for septic shock due to MRSA. MRI showed a progression of the abscess with L5 involvement. Despite surgical drainage and targeted antibiotic treatment, the fever persisted for more than 2 weeks, which prompted the switch to oritavancin, administered at a dose of 1,200 mg every 10 days, four times (minimum inhibitory concentration [MIC] for oritavancin: 0.125 mg/L ). Researchers followed the patient for 10 months and MRI showed a complete regression of spondylodiscitis. The trough

plasma concentrations (Cmin) of oritavancin measured 1 hour prior to each administration were 2.2, 1.9, and 2.3 mg/L.

CASE 2

A 61-year-old male with osteomyelitis due to diabetic foot involving the right III, IV, and V metatarsal bones was repeatedly hospitalised for MRSA sepsis. The first episode was treated with vancomycin for 4 weeks; the second, which occurred 2 weeks later, was treated with daptomycin (8 mg/kg) for 4 additional weeks. One month later, the patient presented a third septic episode and refused the proposed amputation. Therefore, oritavancin at a dose of 1,200 mg every 10 days was initiated (MIC for oritavancin: 0.060 mg/L), and administered five times. Six months after oritavancin discontinuation, the MRI showed complete regression of the osteomyelitis. The Cmin values of oritavancin measured 1 hour prior to each administration were 2.1, 2.5, 3.3, and 4.4 mg/L.

CONCLUSION

Despite the high rate of clinical success and the bactericidal activity in vitro of oritavancin, definitive recommendations on optimal dose, number of doses, and dosing interval for the treatment of osteomyelitis are lacking.5 The strains isolated from the patients had different MICs; nevertheless, the team used the same loading dose and the same dosing interval, but

different maintenance doses. Patient 2 had a lower MIC value, a renal impairment, and a progressive increase of Cmin levels; however, the team decided to administer 1,200 mg for loading and maintenance doses to minimise the risk of amputation, considering the absence of toxicity. TDM was useful to ensure that oritavancin concentrations were maintained above the MIC after correction for protein binding. However, larger studies are warranted to determine the optimal area under the curve/MIC target for the treatment of bone infections. In conclusion, oritavancin represents a valid therapeutic option for MRSA osteomyelitis. TDM could be an essential tool to individualise and tailor oritavancin dose and dosing intervals in order to achieve appropriate concentration-time curve and area under the curve/MIC ratio in each patient. ●

References

1. McKay GA et al. Time-kill kinetics of oritavancin and comparator agents against Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. J Antimicrob Chemother. 2009;6(3):1191-9.

2. Corey GR et al. Single-dose oritavancin in the treatment of acute bacterial skin infections. N Engl J Med. 2014;370(23):2180-90.

3. Corey GR et al. Single-dose oritavancin versus 7–10 days of vancomycin in the treatment of gram-positive acute bacterial skin and skin structure infections: the SOLO II noninferiority study. Clin Infect Dis. 2015;60(2):254-62.

4. Lupia T et al. Role of oritavancin in the treatment of infective endocarditis, catheter- or device-related infections, bloodstream infections, and bone and prosthetic joint infections in humans: narrative review and possible developments. Life (Basel). 2023;13(4):959.

5. Chastain DB, Davis A. Treatment of chronic osteomyelitis with multidose oritavancin: a case series and literature review. Int J Antimicrob Agents 2019;53(4):429-34.

Abstract Highlights

The following highlights showcase selected abstracts presented at this year’s European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Global Congress, held in Barcelona, Spain. The chosen pieces shed light on current and exciting areas of the field, from the alarming extent of antibiotic loss in intensive care infusion practices, to the influence of antibiotic types on the risk of chronic urinary tract infections.

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:32-40. https://doi.org/10.33590/emjmicrobiolinfectdis/WLZX1153.

Unveiling the Tales of Transplant Infection

OVER the last 30 years, there has been a significant rise in both the number of solid organ transplant recipients, and the success rates of transplants. However, infections continue to pose a challenge to long-term outcomes, particularly those occurring more than 1 year after transplantation, which have not been well understood. The goal of this study, which was presented at the 2024 ESCMID Global Congress, was to provide a detailed overview of the occurrence and impact of late-onset infections in the Swiss Transplant Cohort Study.

Since 2008, the Swiss Transplant Cohort Study has been systematically enrolling over 95% of all solid organ transplant recipients in Switzerland. This involves gathering pre-determined data, including information on infections, at specific intervals. The study includes adult recipients of heart, liver, lung, kidney, and kidneypancreas transplants conducted between 1st May 2008–December 2020, who have been followed up for at least 12 months. All types of clinically significant infections, such as proven bacterial, fungal, parasitic, and viral infections; as well as probable fungal, parasitic, viral, and mycobacterial infections; along with viral syndromes, were analysed.

The study included 4,378 patients who underwent various organ transplants, with a median follow-up duration of 6.3 years. Among them, 64.9% experienced at least one clinically relevant infection, with an incidence rate of 0.69 infections per 1,000 transplantation days.

"Infections persisted at high rates throughout the observation period."

Notably, lung, kidney-pancreas, and kidney transplant recipients had the highest incidence rates. Bacterial infections were the most common (57.8%), followed by viral (39.8%), fungal (4.4%), and parasitic (0.9%) infections. Enterobacterales and respiratory viruses were the primary pathogens identified. Viruses were the main cause of infections in heart and lung recipients, while bacteria prevailed in kidney, liver, and kidney-pancreas transplant recipients. These infections persisted at high rates throughout the observation period.

In conclusion, this study highlights a continued significant burden of late-onset infections following transplantation. ●

Minimising Antibiotic Loss in Intensive Care Infusion Practices

EFFECTIVE antibiotic administration is critical in intensive care settings, where precise dosing can have an effect on recovery and resistance. Research increasingly suggests that achieving optimal pharmacokinetic/pharmacodynamic targets in the intensive care unit (ICU) may be compromised by physical losses of the drug before it even reaches the patient. A recent study, presented at the 34th ESCMID Global Congress, showed significant loss of antibiotic doses due to remnants in vials and intravenous lines after reconstitution.

The researchers performed an observational prospective study in the ICUs at the University Hospitals Leuven, Belgium, in November 2023, where they evaluated the residual volume and antibiotic loss in infusion sets from ICU patients. The study assessed 40 intermittent antibiotic infusions to determine the residual volume and antibiotic loss in the infusion sets used at the hospital. The team focused on common antibiotics such as amoxicillin-clavulanate, piperacillin-tazobactam, and vancomycin, using the 110.1569 Dialex Biomedica (Dialex Biomedica, Bilzen, Belgium) infusion sets.

Results showed that the majority of infusion sets were re-used to administer the same antibiotic (55%), and another 25% were used for different drugs. Most notably, antibiotic loss occurred in 85% of administrations, with a median loss

of 24%, while considering factors such as flushing, antibiotic stability, and time interval until subsequent administration through the same infusion set.

"To mitigate these losses, it is recommended to flush infusion lines post-administration."

The results implicate that the combined loss of antibiotic doses through residuals in vials and infusion sets may have significant influence on pharmacokinetic/pharmacodynamic target attainment and clinical outcomes in the ICU. Additionally, data showed that substantial amounts of prescribed antibiotic doses do not reach the patients with intermittent antibiotic infusions. This issue is particularly acute with antibiotics that have limited stability, such as meropenem or amoxicillin-clavulanate. To mitigate these losses, it is recommended to flush infusion lines post-administration, and to consider switching intermittent infusion to bolus injection to minimise residual volumes.

The findings emphasise the need for new strategies and methods to ensure complete and effective delivery of prescribed doses to patients, particularly in ICU settings. ●

Varying Effects of β-Lactams on the Gut Microbiota

NEW RESEARCH presented at the ESCMID Global Congress 2024, which took place from 27th–30th April, compared the effects of ceftriaxone (CRO), piperacillin-tazobactam (TZP), and ceftazidimeavibactam (CZA) on the gut microbiota. Previous research has confirmed that antibiotics affect the composition of the microbiota, but the strength of the effect of each antibiotic remains unclear. The research team, based in Paris, France, compared these three β-lactams in order to begin to understand their effect.

"Previous research has confirmed that antibiotics affect the composition of the microbiota."

Healthy volunteers were exposed to 1 g/day CRO, 4 g/0.5 g three times/day TZP, 2 g/0.5 g three times/day CZA, or no antibiotic (control group) for 7 days. Their faeces were collected before, at the end, and 30 days after treatment, and the metagenomes were then sequenced (minimum 20 M paired-end reads per sample). After cleaning, reads were assembled using MEGAHIT, and metagenome-assembled genomes were obtained using Metabat2.

Richness was calculated at the species level. β-lactamase-encoding genes were identified using pair-wise comparative modelling with a final expertise validation.

Ultimately, 130 metagenomes out of 144 could be sequenced. From baseline, CRO, TZP, and CZA induced a significant richness drop at the end of treatment (-18, -22 and -37; P<0.01, respectively, for each). Thirty days after treatment, only subjects exposed to CZA had a lower richness compared to baseline (-17; P<0.001). A total of 149 β-lactamase-encoding genes were predicted on the whole dataset, including 21 and 37 whose relative abundance increased from baseline to the end of treatment, and 30 days after treatment, respectively.

The team found that subjects exposed to CZA had a greater loss of richness after 7 days of exposure compared to subjects exposed to CRO and TZP. Moreover, they still had a richness loss 30 days after exposure. Though the study continues as the team carry on with synthesising and characterising the β-lactamase-encoding genes, this research highlights the impact of various β-lactams on the gut microbiota. ●

Prevalence of Multi-drug Resistant Bacteria in Elderly Care Centres

THE World Health Organization (WHO) has identified multi-drug-resistant (MDR) extended-spectrum β-lactamase producing Enterobacterales (ESBL-PE), carbapenemaseproducing Enterobacterales (CPE), vancomycinresistant enterococci (VRE), and toxigenic Clostridioides difficile (tCD) as significant threats to human health. Moreover, the prevalence of carriage of MDR micro-organisms in elderly care centres (ECC) is not well-established. Therefore, researchers conducted a worldwide systematic review examining MDR carriage prevalence and risk factors in ECCs. Data from this study were presented at the 2024 ESCMID Global Congress.

The study employed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology to search PubMed, Web of Science, and Cochrane databases for all studies until 2022, evaluating the prevalence of specific MDR bacterial species. Essential information, including prevalence, risk factors, sample size, sampling period, and study design, was extracted from articles included in the study. Through electronic database searches, the researchers identified 2,015 articles, of which a total of 25 articles involving 12,790 participants

across 14 countries were selected for the systematic review.

Of the 25 articles reviewed, the global prevalence of MDR species, including ESBLPE, CPE, VRE, or tCD in ECCs, was estimated at 13.1%, 1.1%, 3.3%, and 16.5%, respectively. Moreover, the most significant risk factors for MDR carriage identified consistently across studies included incontinence, the presence of an invasive medical device, and a history of antibiotic use.

In ECC environments, colonisation of residents with ESBL-PE or tCD is common, affecting one resident in six, while CPE and VRE colonisation rates are significantly lower. The identified risk factors of MDR carriage in ECCs are aligned with the risk factors in the literature. An improved understanding of the prevalence and risk factors for MDR colonisation in ECCs could lead to improvements in prevention. Nevertheless, complete adherence to infection control measures and appropriate antibiotic stewardship remains necessary to protect populations from MDR microorganisms. ●

"In ECC environments, colonisation of residents with ESBL-PE or tCD is common, affecting one resident in six."

Understanding Antimicrobial Resistance at the Healthcare Facility Level

INCIDENCE of hospital-acquired or resistant gram-negative bacilli bloodstream infections (GNB-BSI) was measured in a recent study benchmarking hospitals in a large public healthcare consortium in the Parisian areas of France. Data were presented at the 2024 ESCMID Global Congress in Barcelona, Spain. Benchmarking can be employed to identify priorities for hospitals, and in this study, it was used to produce indicators for understanding differences in antimicrobial resistance (AMR) and healthcare-associated infections (HAI) between hospitals. The increasing frequency of AMR in bacteria, driving the rise in HAIs worldwide, poses a serious threat to healthcare systems, increasing the cost, length of hospitalisation, and mortality and morbidity of patients. Therefore, monitoring the incidence of GNB-BSIs as a marker of HAI and AMR burden is essential to survey and control the spread of these phenomena.

The study was composed of a cohort of 28 hospitals from a network of 38 hospitals belonging to the largest hospital group in France, Assistance Publique-Hôpitaux de Paris. All data analysed in this study were from 2019 due to the impact of COVID-19 on organisation of hospitals and downstream effect on AMR. The use of the same laboratory information system in the bacteriology laboratories across the hospitals in the study meant that all data extraction and analysis were standardised. Spearman’s rank

correlation coefficient was used to determine correlation between incidence density rates for resistance patterns of concern. Principal component analysis of different micro-organisms isolated from BSIs was performed to identify correlations between incidence rates of GNBBSIs due to micro-organism species, patterns of resistance, and scores for subsequent analysis. Finally, multivariate linear regression analysis was conducted to examine associations between AMR and HAI scores and various explanatory factors.

Results revealed that within each hospital there is a strong positive correlation between the incidence of GNB-BSI and resistant GNBBSI, and hospital-acquired GNB-BSI. Two scores for AMR and HAI rates were created by combining different GNB-BSI, and multivariate analysis demonstrated significant associations between the proportion of surgical beds, use of carbapenems, staff absenteeism, and alcoholbased hand rub consumption. The researchers acknowledged that carbapenem association with AMR may be a result of carbapenems being the preferred antibiotic drug for resistant infections.

The researchers concluded that the identification of factors associated with HAIs and AMR, particularly staff absenteeism and consumption of alcohol-based hand rub, are amenable to targeted intervention at the healthcare facility level. ●

"Monitoring the incidence of GNB-BSIs as a marker of HAI and AMR burden is essential to survey and control the spread of these phenomena."

Comparative Outcomes of Respiratory Syncytial Virus and Influenza A in Adults

ANNUAL incidence rates and hospital admissions of respiratory syncytial virus (RSV) are higher than those for influenza A. RSV infection in older patients is associated with poorer outcomes compared to influenza A.

RSV is associated with major causes of influenza-like morbidity worldwide, especially in infants and the elderly. Research presented at the 34th ESCMID Global Congress emphasises the importance of investigating the incidence rates, risk factors, clinical characteristics, and outcomes of hospitalised patients with RSV versus influenza A.

The study, performed at the Tel Aviv Sourasky Medical Center, Israel, comprised two parts. Firstly, the researchers collected incidence data of hospitalised adults with RSV and influenza A, between January 2018–April 2023. Secondly, they conducted a case-control study of hospitalised adults with RSV versus influenza A between October 2021–April 2023 throughout the winter seasons. They gathered information on several clinical characteristics like age, pre-existing medical conditions, the Charlson Comorbidity Score (CCS), and 30-day mortality.

Results showed that incidence rates and hospitalisations were higher in patients with RSV than influenza A. Additionally, multivariate analysis showed older patients with RSV had higher CCS and 16% 30-day mortality rate, nearly double that of patients with influenza A.

"RSV infection in older patients is associated with poorer outcomes compared to influenza A."

This study highlights key differences between RSV and influenza A infections, and indicates that RSV infection leads to increased hospitalisation and mortality rates compared to influenza A, especially in elderly patients. This underscores the significant burden that RSV places on adult populations. Future research should aim to replicate these findings across multiple medical centres, and explore the efficacy of RSV vaccines and antivirals, particularly in populations with high comorbidity scores. ●

Urinary Tract Infections Recurrence: Play of Treatment Choice

URINARY tract infections (UTI) are the leading reason for doctors in general practice to prescribe antibiotics in Denmark. For general practitioners (GP), the recommended initial treatment for UTIs includes pivmecillinam as the first choice, with nitrofurantoin or trimethoprim recommended as alternative options for those allergic to the first-line treatment. However, despite receiving appropriate treatment, over 30% of females with UTIs experience another infection within a year. This study aimed to evaluate the selection of initial UTI treatments by GPs across various age groups, and determine their effectiveness in preventing recurrent UTIs (rUTI).

"This study aimed to evaluate the selection of initial UTI treatments by GPs across various age groups."

In this retrospective cohort study, researchers analysed data from 95,722 adult females with findings of uropathogenic organisms in urine cultures conducted by GPs. From this pool, 12,058 females were selected and categorised into three age groups: 18–22, 41–45, and 61–65 years. Data were collected from the Department of Clinical Microbiology laboratory information system and The Danish National Health and Prescription Registers. UTI episodes were identified based on significant uropathogenic laboratory findings and subsequent antibiotic prescriptions. rUTI was defined as experiencing ≥2 UTIs within 6 months, or ≥3 within 12 months. The appropriateness of empirical UTI (EUT) treatment was assessed

based on antibiotic resistance. Informed treatment referred to the use of antibiotics after receiving test results. Logistic regression was employed to calculate odds ratios for rUTI onset, adjusting for factors such as timing of treatment, type of antibiotic, death, age, and uropathogen.

The study's results revealed that EUT treatment was linked to a higher rate of rUTI debuts, particularly in the 61–65 age group. There was no significant disparity in rUTI odds between appropriate EUT and informed treatment, but inappropriate EUT was associated with increased odds of rUTI. The likelihood of rUTI was notably higher in older age groups, particularly in the 61–65 cohort. Among the antibiotics recommended for UTI treatment, only ciprofloxacin demonstrated a reduction in rUTI odds, while oral ampicillin, which has limited efficacy for oral treatment, was associated with increased odds of rUTI. Comparatively, Staphylococcus saprophyticus lowered the odds of rUTI, whereas Klebsiella pneumoniae increased the odds. The study's strengths were its large cohort size, diverse species variety, and high quality of data from the Danish registers. However, limitations included reliance on samples analysed by the Department of Clinical Microbiology and assumptions regarding patient compliance.

This study concludes that the choice of treatment for UTIs plays a significant role in affecting the risk of recurrent UTIs, with age and certain bacterial species being particularly influential factors. ●

Closing Knowledge Gaps: Online Education in Female HIV Care

ONLINE medical education tools substantially enhance physicians’ knowledge of the biological considerations and comorbidities in females living with HIV, according to new research presented at the ESCMID 2024 Global Congress. Fifty-three percent of people living with HIV in 2022 were females, who face unique challenges throughout various life stages, including pregnancy, menopause, and ageing. Moreover, virological, immunological, and socio-behavioural disparities exist among females living with HIV, compared to their male counterparts and HIVnegative females.

This study by Julia Duffey, Medscape Education Global, London, UK, and team, aimed to evaluate the effectiveness of online independent medical education in enhancing the knowledge and confidence of HIV and infectious disease (ID) physicians concerning female-specific aspects of living with HIV, and associated comorbidities. The researchers conducted two online, independent medical education activities comprised of an expert-led 30-minute lecture on biological and immunological aspects of HIV infection that are unique to females, and a 30-minute discussion in which three experts exchange viewpoints about increasing awareness and supporting best practice in the management for this group. Each participant completed both pre- and

post-education questions to determine if their knowledge was improved, reinforced, or if they need further education.

For the first activity, which included 632 HIV/ ID specialists, there was an overall improvement of correct answers from 30 to 63% (P<0.001). In addition, 48% (n=45) of participants reported improved confidence in managing non-HIVrelated comorbidities specific to females. A similar trend could be seen after the second activity, which included 87 specialists, where correct answers improved from 28 to 56% (P<0.001). However, there was a high proportion of specialists (49%) who still required education regarding human papillomavirus vaccination and testing in females living with HIV.

This study has demonstrated that online education can substantially improve specialists’ knowledge and confidence concerning health issues disproportionately affecting females with HIV. The baseline knowledge regarding comorbidity risk and prevalence in this population was low, indicating an unmet need for education in prevention and management. This shows the need for education on this topic to be a priority for HIV and ID specialists to meet the unique challenges faced by this demographic. ●

"48% of participants reported improved confidence in managing non-HIVrelated comorbidities specific to females."

Congress Interview

In this exclusive conversation with EMJ, Johnny Zakhour delves into the critical role of diagnostic stewardship and the myriad challenges of its implementation in clinical practice. He also shares key insights on the ESCMID Global 2024 Congress, and the fundamental role it plays in the continuous education of healthcare professionals.

Johnny Zakhour

Internal Medicine Department, Henry Ford Hospital, Detroit, Michigan, USA

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:41-44. https://doi. org/10.33590/emjmicrobiolinfectdis/LATS3322.

Q1What inspired you to focus your medical career on infectious diseases and antimicrobial resistance (AMR)?

I was born and raised in Beirut, Lebanon, where I attended medical school. I was fortunate to train at one of the largest teaching hospitals in the country, where I encountered many attendings who cultivated my interest in infectious diseases. I have always been fascinated by the pathophysiology of infectious diseases, and the concept of antimicrobial stewardship as a means to reduce the burden of AMR. After graduating from medical school, I joined Souha Kanj at the American University of Beirut Medical Center for a post-doctoral research fellowship.

Kanj is a global pioneer in infectious diseases research, and has played a vital role in advancing knowledge about AMR and stewardship in the Middle East. She further cultivated my interest in infectious diseases. Throughout my year with her, I gained a deep understanding of the development of AMR, and the determinants that can affect it. She fostered a sense of stewardship in me, and I actively involved myself in multiple research projects and initiatives focusing on AMR and stewardship. I believe that stewardship is one of the most important qualities a physician could have, as it reflects a sense of responsibility and commitment to patient care.

"Educators and mentors should empower their trainees with the evidence behind

diagnostic stewardship."

Q2

In the talk you gave at The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) 2024, ‘Empowering junior doctors with diagnostic stewardship principles’, what were the key messages you wished to convey?

After starting my residency in internal medicine at Henry Ford Hospital, Detroit, USA, I gained further knowledge about ways to combat AMR in a setting with more available resources compared to my home country. I witnessed firsthand the stewardship strategies that I had previously researched.

I further discovered the key role that residents play in stewardship, whether in terms of diagnostic or antimicrobial stewardship. As a resident, it can sometimes be frustrating to encounter a hard stop on the electronic medical record. However, with residents being at the forefront of ordering diagnostics, they hold the highest level of responsibility regarding the judicious use of diagnostics, and developing this sense of stewardship is very important for their training. Early-career physician hesitancy plays a significant role in excessive prescribing. If I had to choose a single message from my talk, it would be that educators and mentors should empower their trainees with the evidence behind diagnostic stewardship, and reiterate the importance of physical examination and historytaking, as diagnostics alone are insufficient.

Q3

How has ESCMID used its position this year to educate healthcare professionals about AMR and stewardship? Are there any sessions you found particularly insightful?

With the rising tension around the world, I was particularly impressed by Kanj’s presentation regarding AMR in displaced populations. I believe this presentation shed light on a topic that is rarely discussed; the severe impact of human and natural disasters on AMR. This serves to further enlighten healthcare professionals who are fortunate to practice in high-income settings about the struggles of both patients and providers in areas with a high prevalence of displaced populations, and the global impact of these struggles.

Q4

What steps can junior doctors take in their practice to ensure faster, more accurate detection of pathogens? What criteria should be used when deciding whether to order blood cultures?

To ensure faster and more accurate detection of pathogens, junior doctors should consider the pre-test probability of any diagnostic test before ordering it. This requires careful history-taking and physical examination. Subsequently, the fastest and most accurate detection is feasible only when collaboration among multiple teams is at the forefront, including the nursing, microbiology, and infection prevention and control teams. Although novel diagnostics are tempting with their extremely high sensitivities, junior doctors need to be aware that speed can sometimes compromise accuracy, and that finding the right balance between the two is crucial.

False positive results in infectious diseases greatly contribute to overtreatment and adverse events in patients. This is particularly important for blood cultures, as ordering them has become reflexive in response to any unexplained elevation in white blood cell count or fever. However, junior doctors need to be aware of the pre-test probability of blood cultures in different clinical scenarios. They should ask themselves, "what will I do with this test if it is negative?" and "what will I do with it if it is positive?" Furthermore, if it is positive, they should consider how the isolated organism and the duration to positivity will affect their management. Beyond their provider role, they should also be mindful of the possibility of contamination during the process of drawing blood cultures.

For example, while positive blood cultures can be helpful in isolating the causative organism of infective endocarditis or vertebral osteomyelitis, they are much less useful in cases of uncomplicated cellulitis or post-operative fevers. Unfortunately, there are still no clear indications for blood cultures. Ultimately, ordering blood cultures relies on the clinical scenario, differential diagnoses, the pre-test probability of a certain diagnosis, and the changes to management that a blood culture result will bring.

"Adopting diagnostic stewardship principles in immunocompromised populations is also challenging."

Q5

What are some common diagnostic pitfalls to watch out for, as a clinician?

The yield of a diagnostic tool, especially diagnostics for infectious diseases, should be carefully considered before ordering it. This serves largely to reduce the incidence of false positives, healthcare costs, length of stay, and patient discomfort.

In infectious diseases, urinary tract infections can represent significant diagnostic challenges. This is most relevant in cases of catheterassociated urinary tract infections and asymptomatic bacteriuria which are associated with a high risk of unnecessary antimicrobial prescriptions with consequential adverse events and emergence of resistance. It is very important to be aware that asymptomatic bacteriuria and non-infectious pyuria are very common, and that treatment is not warranted in the majority of those cases.

Clostridioides difficile colitis poses another diagnostic challenge to clinicians, especially early-career clinicians. This is largely due to the reflexive approach of ordering C. difficile testing for patients presenting with watery diarrhoea. Interestingly, over half of the admitted patients who test positive for C. difficile are only colonised rather than infected and do not require treatment. Careful history taking, clinical exam, and review of the medication administration record are essential to identify other causes of watery diarrhoea, and avoid false positive results.

Q6 Are there any novel diagnostic tools with enhanced sensitivity and specificity that you find particularly promising?

Although novel microbiologic diagnostics, particularly molecular diagnostics, have been shown to be highly sensitive, they carry a higher risk of false positives than other conventional diagnostics as they are more likely to detect colonisation or contamination. Interpreting these

diagnostics in collaboration with the microbiology team and the antimicrobial stewardship team can help spare the risk of false positives.

Although most diagnostics for infectious diseases focus on detecting a causative pathogen, new diagnostics are currently based on exploring the host’s response to an infection. Host-response-based diagnostics further explore the response of hosts to a certain pathogen, and may be more appropriate to differentiate infection from colonisation or contamination. However, most of these diagnostics are still in the research stage and are not yet available for clinical use.

Q7

In your opinion, what are currently the main challenges to diagnostic stewardship implementation, and how can these issues be tackled?

The benefits of implementing diagnostic stewardship in the inpatient setting have been well-proven. For instance, implementing hard

Johnny Zakhour and Souha Kanj

stops in the electronic medical record to prevent providers from ordering C. difficile testing for patients with watery diarrhoea in the absence of certain criteria was able to reduce the incidence of hospital-onset C. difficile by two-fold, without any reported missed diagnoses, and no impact on patient outcomes. Similar applications have been used for catheter-associated urinary tract infections and ventilator-associated pneumonia. However, little evidence exists regarding diagnostic stewardship in the outpatient setting. Hence, areas where diagnostic stewardship can be applied in the outpatient setting needs to be explored.

Adopting diagnostic stewardship principles in immunocompromised populations is also challenging. This is largely due to the lack of patients who are immunocompromised, and who are at a higher risk of severe infections, but also due to physician hesitancy when caring for such high-risk patients. However, some evidence is starting to emerge. For instance, at our institution, Henry Ford Hospital, we were able to show that routine microbiologic testing ordered for lung transplant recipients does not have a high diagnostic yield, and can expose patients to unnecessary treatment and interrupt life-saving immunosuppressive regimens.

Additional challenges include the unavailability of novel diagnostics in low-resource settings, lack of knowledge regarding diagnostic stewardship principles, physician hesitancy, and lack of evidence regarding the diagnostic yield of some novel diagnostics. Further research is warranted in these areas to optimise patient outcomes.

Q8

Where can we see your research lie in the near future? Are there any exciting projects on the horizon?

Diagnostic stewardship is definitely a hot topic in infectious diseases and microbiology right now. We are slowly moving from focusing on creating more diagnostics to optimising the yield of our existing ones. Exploring the use of diagnostic stewardship in patients who are immunocompromised is one area that I would like to look into, as well as ways to implement diagnostic stewardship in low-resource settings. Currently, we are looking into ways to improve data reporting on AMR in low-resource settings, in particular the Middle East, as this would be the foundation for any future projects pertaining to diagnostic stewardship in that area. ●

www.emjreviews.com

Surveillance of Surgical Site Infections in Post-operative Patients and Bacterial Susceptibility in Tanzania

Authors:

Disclosure:

*Theresia A Karuhanga,1,2 Philbert B Madoshi,3 Robert S Machang’u4

1. Department of General Surgery, Faculty of Medicine, St. Francis University of Health and Allied Sciences, Ifakara, Tanzania

2. Department of Surgery, St. Francis Referral Hospital, Ifakara, Tanzania

3. Community Infection Control, Department of Public Health Faculty of Medicine, St. Francis University College of Health and Allied Sciences, Ifakara, Tanzania

4. Department of Microbiology, Faculty of Medicine, St. Francis University College of Health and Allied Sciences, Ifakara, Tanzania

*Correspondence to drkaruhangat@gmail.com

The authors have declared no conflicts of interest. This study was funded by the Royal Society of Tropical Medicine and Hygiene (RSTMH) under the early career small grant for 2021–2022 in infectious disease and antimicrobial resistance for low- and middle-income countries. The aim of this grant is research capacity-building for health professionals. The received fund was 4,700 GBP. There were no personal salaries, payment for services, or honoraria payment; the fund was used for cost of logistics and laboratory reagents. Travel and publication costs were not among the included items.

Received: 17.10.23

Accepted: 15.05.24

Keywords: Antimicrobial resistance, hospital post-surgical infections, Pseudomonas aeruginosa, Staphylococcus aureus.

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:46-56. https://doi.org/10.33590/emjmicrobiolinfectdis/EPYJ6316.

Abstract

Surgical site infections (SSI) are infections occurring within 30 days of the postoperative procedure. They are common post-operative morbid complications that may cause death if not treated timely. The common causes of SSI include infectious bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and some Enterobacteriaceae.

This was a cross-sectional study conducted at St. Francis Referral Hospital, Ifakatra, Tanzania over a period of 12 months to investigate the causes of SSI and antimicrobial susceptibility of the causal agents. The study included consenting patients who developed post-operative wound infections during the study period. Identification of infecting micro-organisms and their antimicrobial susceptibility was done at St Francis Referral Hospital Laboratory. Antibiotic susceptibility tests

of the isolates were performed by the Kirby–Bauer (K–B 1966) disc diffusion test, and extended spectrum β-lactamase producing Gram-negative species were tested by using the modified double disc synergy test.

A total of 130 patients developed post-operative wound infection. Third and fourth decades were the most affected age groups; females were the dominant group with a 1:1.4 male: female ratio. Out of the 130 specimens, 121 isolates were obtained, and nine specimens were negative for culture. P. aeruginosa was the most commonly isolated agent (42.1%), followed by S. aureus (19.8%), while the least were Streptococcus spp. at 0.8%. The isolates showed the highest resistance to ampicillin (91.7%), and least to ciprofloxacin (1.7%). P. aeruginosa was highly resistant to both amoxicillin + clavulanic acid (98%), and to ampicillin (98.0%).

Extended spectrum β-lactamase E. coli producers were 68.4%.

The bacteria causing SSI require continuous monitoring to obtain data that will support local and national guidelines in the battle against antimicrobial resistance, and improve therapeutic outcomes following surgical interventions.

Key Points

1. Surgical site infection is a surgical challenge resulting in unfavourable surgical outcomes. The condition is more pronounced in low- and middle-income countries, where surveillance of diseases (including surgical site infection) is low. The major aim of this article is to meet the Global Sustainable Development Goal 3: ensuring health and well-being for all. In order to comply to the World Health Organization (WHO) antimicrobial resistance reduction target, the use of surveillance systems should be advocated for at an institutional level.

2. The manuscript explains the cause of wound infection in post-operative patients, and the profile of bacterial susceptibility. The study results showed that Gram-negative bacteria more frequently cause surgical site infection, and are also highly resistant to commonly used antimicrobial agents at the authors’ health institution.

3. The surgeon and clinicians should be aware of the presence of highly antimicrobial-resistant bacteria. The presence of extended spectrum β-lactamase-producing Escherichia coli indicates the need for evidence-based infection prevention and control at a local and national level.

INTRODUCTION

Surgical site infections (SSI) are defined as infections occurring within 30 days after simple skin incisions, or complex interventions involving subcutaneous tissue, organs, or manipulated space during primary operation.1-3 SSIs are common post-operative complications which may cause significant morbidity.4–7 Globally, the incidence of patients developing SSI postsurgery ranges from 15.45–23%, as reported by the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC).8 Approximately 77% of post-operative

deaths are reported to be related to postoperative infections.9 The condition is probably high in developing countries, but it is still difficult to quantify the burden due to the limited availability of data and publications.10,11

Causes of SSI differ from one region of the body to another. The most commonly reported bacteria in SSI are hospital-acquired infections, involving Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and other Enterobacteriaceae.1,8,12-15 SSI prevention and control are currently challenged by the emergence of antimicrobial resistance, which in developing countries is aggravated by

underlying poor health systems.15-17 Irrational antibiotic use and unavailability of SSI data are among the areas which need to be critically addressed to improve surgical services.18 SSI can occur in any surgical procedure ranging from obstetrics and gynaecology, to general surgery and orthopedics.19-22 SSIs complicate therapeutic outcomes associated with prolonged length of hospitalisation, and increased hospital costs.6,8,19

Multidrug resistant bacteria commonly include methicillin resistant S. aureus and extended spectrum β-lactamase (ESBL)-producing Gram-negative bacteria.23-26 The ESBLproducing Enterobacteriaceae, particularly E. coli, Klebsiella spp., and P. aeruginosa, have shown high levels of resistance, ranging from 50–100% for commonly used antimicrobial drugs, such as ampicillin, chloramphenicol, and third generation cephalosporin; while the same bacteria have been reported to have resistance below 25% to piperacillin-tazobactam and imipenem.9,27,28 Specific therapeutic options for patients with SSIs depend on data from antimicrobial sensitivity tests done by clinical laboratories. One zonal hospital in Tanzania reported an SSI rate of about 10.9% involving mostly S. aureus and Enterobacteriaceae.2,29 Tanzania, like most Sub Saharan African countries, lacks reliable evidence-based data on SSIs, particularly in rural health facilities, including referral hospitals.3,8 The aim of this study was to evaluate the SSI causative agents and their antimicrobial susceptibility at a local health institute.

METHODOLOGY

Study Site

A prospective hospital-based cross-sectional study was conducted from January 2022–December 2022 at St. Francis Referral Hospital, Ifakara, Tanzania, which is a rural faith-based private referral hospital. The hospital is also a teaching hospital for medical students from St. Francis University College and Allied Sciences (SFUCHAS). The hospital provides general and specialised surgical services, including orthopaedic, urologic, obstetric, and gynaecologic, and also offers haemodialysis.

Sampling Techniques

Non-probability sampling method was used for all patients who were operated on during the study period at St. Francis Referral Hospital. The patient particulars, diagnosis, and procedures were collected from the individual patient’s file after they developed a wound infection. There was a clinician who was responsible for data collection from the respective department, particularly general surgery, orthopaedics, obstetrics and gynaecology, outpatient clinics, and urology. Therefore, post-operative patients were subjected to a routine daily wound review. Early infections were identified during ward rounds, while late infections were identified after discharge. At the time of discharge from hospital admission, the patients were requested to attend outpatient clinic follow-up, weekly, for 4 consecutive weeks. The patients were also requested to provide their contact details for easy follow-up after discharge. In case of signs of wound infection while at home, the patients were requested to return to the hospital for clinical evaluation. Each patient was discharged from post-surgical follow-up 30 days later, when free of wound infection and clinically stable. The patient who developed post-operative wound infection was informed about the study, and was requested to participate in the study. Two patients were excluded from the study: one refused to sign the consent form, and the second was lost from the follow-up after discharge. Identification of SSI was according to the clinical criteria for SSIs, and was classified according to the CDC for SSI Classification System (superficial incision SSI, deep incision SSI, and organ/ space SSI).24,30,31 Specimens were collected by swabbing the infected wound before antiseptic dressing to avoid skin contamination. The swabs were then immersed in a container with peptone water pH transport medium,32,33 and sent to the bacteriology laboratory.

Processing of Specimens Culture and Bacterial Identification

The specimens were inoculated by streaking on McConkey, and mannitol salt agar with egg yolk media, then sub-cultured on blood agar and incubated at 35–37°C for 24 hours. Isolates were identified by colonial morphology, Gram staining, and conventional biochemical tests, including catalase, coagulase, mannitol fermentation, and haemolysis for Gram-positive

bacteria; and for Gram-negative bacteria, colony morphological features and biochemical tests, including urease, citrate, oxidase, indole, and sugar utilisation on triple sugar iron.

Antimicrobial Susceptibility Testing

Antibiotic susceptibility tests of the isolates were performed by the Kirby–Bauer (K–B 1966) disc diffusion test.32 From a pure culture, three to five colonies of bacteria were picked using a sterile loop, and soaked into a tube with 5 mL of normal physiological saline, to obtain a density equal to 0.5 MacFarland scale, and then seeded culture evenly over the entire surface of sterile Mueller–Hinton agar plate (Oxoid, UK). The plates were then kept at room temperature to dry, and then antibiotic discs (Oxoid, UK) were placed and incubated at 35–37°C for 18–24 hours. The discs tested were from the commonly used antibiotics at the hospital, including ampicillin (10 μg), clindamycin (15 μg), vancomycin (30 μg), amoxicillin-clavulanic acid (20/10 μg), gentamicin (10 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), meropenem (10 μg), and piperacillin-tazobactam (100/10 µg).34

ESBL-producing Gram-negative species were tested by using the Modified Double Disc Synergy Test (MDDST) using a disc of amoxicillin-clavulanate (20/10 μg) along with meropenem (10 μg), ciprofloxacin (5 μg), ceftriaxone (30 μg), and piperacillin-tazobactam (100/10 μg). The use of fourth generation of cephalosporins was not included in this study as it is recommended for MDDST, because these group of drugs are not commonly used in the authors clinic set-up.35 Any increase in the zone towards the disc of amoxicillin-clavulanate was considered positive for ESBL production.12,25,36

The quality control of the laboratory works was maintained through aseptic and sterility conditions, labelling, and storage in recommended environmental conditions.19,32 International control bacteria strains, E. coli ATCC25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 25923 were used.12,32,37

Inclusion Criteria

All patients who signed the consent form to participate in the study voluntarily were included.

All patients who were operated on during the study period, and developed a wound infection during the study period, were included.

Exclusion Criteria

The exclusion in this study was based on involuntary consent of the patient, with congenital or acquired speaking impairment such as aphasia autism (due to a language barrier and lack of special group language interpreters in the authors’ area).

All patients who withdrew from the study were also excluded.

Other exclusion criteria were ophthalmological, ear, nose, and throat surgical procedures, as these particular procedures were not commonly performed at the authors' set-up during the study period due to a lack of specialised clinicians in these fields.

Data Management and Analysis

Data was collected from the patient after follow-up during post-surgical care for wound progression, and laboratory results by using data-collecting tools. Data were entered into a statistical package for the Social Sciences 26 version. After this data entry, cleaning was computed by running frequency. Nominal variables including isolate names were compared with antibiotics to determine sensitivity and resistance patterns. Multivariate logistic regression was used to analyse the factors associated with post-operative wound infection. The odds ratio test was used to verify the correlations between variables and the SSI rate. In the logistic regression analyses, the variables included age, sex, department, procedure, and wound. Estimation of odds ratio and 95% confidence interval (CI) was performed for selected variables. A P-value <0.05 was considered statistically significant.

STUDY LIMITATIONS

The study was performed within a rural hospital where advanced clinical laboratory diagnostic facilities are limited. There was a time limitation of 12 months depending on the project period.

RESULTS

A total of 1,763 patients were operated on during the study period, and out of this cohort, 130 patients developed post-operative wound infection (7.4%). Among them, 41.5% (54/130) were male, and 58.5% (76/130) were female, with a 1:1.4 male-to-female ratio. The most affected age groups were within the third and fourth decades, making up 57% of all who developed SSIs. The overall mean age was 34.0±15.7 years. Most of the patients were from general surgery (43.08%; 56/130), whereas 37.0% (44/130) were from the obstetric ward. Laparotomy was the most common procedure at 44.8% (58/130); other procedures were caesarean section at 37.0% (48/130); open reduction and fixation

at 8.5% (11/130); and other various procedures made up 11.54% of these patients (15/130), including herniorrhaphy (4), thyroidectomy (1), Mayo’s repair of umbilical hernia (3), hydrocelectomy (2), donor site for skin grafting (2), subcutaneous cystic excision (1), open prostatectomy (1), and plate removal (1). Most of the wounds were clean (50.0%; 65/130) 26.15% of wounds (34/130) were dirty, and 19.23% (25/130) were contaminated.

Out of 130 cultured specimens, 121 were positive and nine were negative. Most of the patients who developed SSI were post-operative patients from general surgery, accounting for 44.63%, followed by obstetric-caesarean surgical cases accounting for 38.84% (Table 1).

N/A: not applicable; Obs & Gyn: obstetrics and gynaecology; ORIF: open reduction and internal fixation.

Table 1: Association of clinical and demographic characteristics of patients who developed wound infection.

Gram-negative bacteria, particularly P. aeruginosa, was the predominant isolate at 42.1% (51/121) followed by S. aureus at 19.8% (24/121), E. coli at 15.7% (19/121), and K. pneumonia at 13.2% (16/121). Other isolates were Proteus spp at 5.0% (6/121), Bacillus spp at 3.3% (4/121), and Streptococcus spp at 0.8% (1/121) (Table 2).

The overall resistance to ampicillin was 91.7%, followed by amoxicillin + clavulanic acid at 86.8%. The least resistance was against ciprofloxacin at 1.7%, while antimicrobial resistance rate against piperacillin + tazobactam was 4.13%, gentamycin 4.9%, meropenem 6.6%, and ceftriaxone 10.0%. On multivariate logistic regression analyses, history of laparotomy and dirty wound were significantly associated with development of wound infection (Table 3).

Antimicrobial Susceptibility Testing

Staphylococcus was significantly resistant to amoxicillin + clavulanic acid (79.2%), and to ampicillin (87.5%), but sensitive to ciprofloxacin (91.6%), piperacillin + tazobactam (95.8%), meropenem (100%), and gentamycin (87.5%). Pseudomonas was highly resistant to amoxicillin + clavulanic acid (98.0%) and ampicillin (98.0%), but less resistant to piperacillin + tazobactam (5.8%), meropenem (7.8%), and gentamycin (1.2%). Other antimicrobial susceptibility patterns are summarised in Table 4

ESBL Testing

Of the 19 E. coli isolates, 68.4% (13/19) were found to be ESBL producers by MDDST. Among these, seven isolates showed a clear edge of inhibition of ceftriaxone towards amoxicillinclavulanic acid. Otherwise, all 13 E. coli strains were sensitive to ciprofloxacin and meropenem.

DISCUSSION

In this study, the reported prevalence of SSIs (7.4%) is higher than previously reported in a number of developing countries, which ranged from 2.3–4.5%.1,3,6,26,38,39 The results of this study agree with those of other researchers.18,37,40 This study has further shown that Gram-negative bacteria are the major causes of SSI, with

P. aerugenisa being the predominant pathogen, accounting for 42.1% of all SSIs, consistent with other studies.7,40,41 Other causes are S. aureus (19.8%), E. coli (15.7%), K. pneumoniae (13.2%), Proteus spp (6.6%), and Bacillus spp (3.3%). The Bacillus spp described in this study should not be considered as a contaminant, since they were isolated from the infected wound, as reported in other studies as well.13,28,42-44

The sources of the pathogens shown by this study include the patient’s own indigenous flora, the hospital environment, health workers, and visitors, as reported elsewhere.4 However, the findings of this study differ from others which reported E. coli and S. aureus as the major causes of SSI.2,6,14,15,29,41,45,46

In the authors’ study, all patients received pre-operative antibiotic prophylaxis, consisting of ampicillin and cloxacillin, or ceftriaxone with metronidazole; however, 7.4% of patients developed a SSI, which is lower than other studies, which report a SSI prevalence of 14.8–59.0%.8,9

Despite the national program on quality improvement in Tanzania’s health facilities, post-operative wound infections are a significant challenge for surgeons.9,19,47 Introducing infection prevention and a control pre-operative check list, and pre-operative antibiotic prophylaxis in surgical practice would be helpful to reduce further post-operative adverse outcomes.16,17,24,29-31,48,49

SSIs still occur at high rates in low- and highincome countries alike. For instance, 0.7 infections occur per 100 open procedures in the USA.21,38,50 Therefore, there is a need to review prophylaxis regimes locally, including the timing. According to the CDC guidelines, administration of antibiotic prophylaxis should be within 60–120 minutes before the skin incision is made.5,17,49,51,52 However, repeated dosage is recommended in case of prolonged procedures, and depending on the antimicrobial agent’s half-life.4,17,53 An overwhelming number of resistance cases to ampicillin and amoxicillin + clavulanic acid was observed in this study. This may be attributed to the irrational use of these drugs by the community, since these drugs are commonly dispensed by drug sellers and dealers over the counter. This is

Table 2: Distribution table of bacterial growth and antimicrobial resistance.

Table 3: Multivariate logistic regression analysis for factors associated with bacterial isolation from surgical site infections.

Table 4: Pattern of bacterial isolates and antibiotic resistance profile among isolates from postoperative wounds.

STAPH N=24

PSEUD N=51

AMCL: amoxicillin + clavulanic acid; AMP: ampicillin; BACL: Bacillus spp; CFTR: ceftriaxone; CIP: ciprofloxacin; ECOLI: E. coli; GNT: gentamycin; I: intermediate; KLPN: K. pneumoniae; MRP: meropenem; ND: not done; No. IST: number of isolates; P+T: piperacillin+ tazobactam; PRMR: P. mirabilis; PRVL: P. vulgaris; PSEUD: Pseudomonas; R: resistant; S: sensitive; STAPH: Staphylococcus; STRP: Streptococcus spp.

a regulatory challenge which needs to be seriously addressed in Tanzania.

The study also showed that there is a moderate bacterial resistance to piperacillin + tazobactam (4.13%), gentamicin (5.00%), meropenem (6.60%), and ceftriaxone (10.00%). Similar observations have been reported by other authors.15,16,25,26,40 This may be due to the reduced prescription of these drugs, because of their relatively higher price, and restriction of their availability in the essential drug shops (as known in Swahili words “Maduka ya dawa muhimu”).

Regardless of the low rate of bacterial resistance to piperacillin + tazobactam, meropenem, and ceftriaxone shown in this study, this remains a ‘red flag’, with a possibility of therapeutic failure of these drugs in the future.

It is important that clinicians perform a thorough identification of pathogens, and choose the right antimicrobial agent for the management of bacterial infections. Antibiotics are fundamental in clinical practice, whether prophylactic or therapeutic. Drug availability, selection, and rational use constitute a triangle of global efforts against antimicrobial resistance.7,12,14,17 The correct antimicrobial selection must be based on correct bacterial identification and its susceptibility rather than the clinician’s choice.5,34,49,53

This study has shown a significantly high level of quinolones efficacy, particularly ciprofloxacin, which encountered an overall resistance of 1.7% by SSI pathogens. This has been shown by other studies to be the most powerful drug, with high antimicrobial potential.2,15,19,37 However, it should be noted that its level of resistance is increasing, and precautions are needed.26,29,34,41

From this study, the authors observed antimicrobial resistance to the so-called ‘stable drugs’, including meropenem (6.6%) and piperacillin + tazobactam (4.13%). This has also been demonstrated in other studies.1,19 Further studies are required on emerging carbapenemase-producing Gram-negative strains,28,54 particularly K. pneumoniae, P. aeruginosa, and some Enteobactericiae.40,49,55,56

The ESBL-producing E. coli (68.4%; 13/19), observed in this study correlate with reports from previous studies.13,15,24,25,45,57 However, ESBL-producing bacteria have been reported to

exhibit resistance to non-β-lactam antimicrobial drugs.25,28,49,58 They counter the therapeutic effect, thus narrowing the range of treatment of choice.15,18,21 However, the high frequency of ESBL among E. coli requires additional research focusing on factors contributing to this emergence, which possibly could be the inappropriate use of antimicrobials, ill-equipped diagnostic laboratories, or non-adherence to guidelines on diagnosis and therapeutic approach for infectious diseases.

ETHICAL CONSIDERATIONS

The study was approved by the St. Francis University of Health and Allied Sciences Internal Reviewer Board (IRB), and ethical clearance was obtained from the National Institute of Medical Research (NIMR). Confidentiality was guaranteed by using identity numbers rather than the names of patients.

CONCLUSION

Conclusively, there is an increase in postoperative wound infections caused by antimicrobial-resistant pathogens. The most commonly identified organisms were P. aeruginosa, S. aureus, E. coli, and K. pneumoniae. Resistance to the most commonly used antibiotics, particularly third-generation cephalosporin, ampicillin, amoxicillin + clavulanic, and meropenem, is an alarming sign which should be responded to, in order to tackle this effect. In the authors’ study, ciprofloxacin and gentamycin showed effect in post-operative infection-causing agents. There is a significant need for adherence to the ‘reserve drug’ concept to reduce misuse of available antimicrobial drugs.

RECOMMENDATIONS

The SSI bacterial diversity demonstrated by this study should be a matter of concern, and it calls for strict adherence to infection prevention and control in hospital surgical wards. This study also calls for continuous interpretation of antimicrobial sensitivity tests at a local level to guide the choice of effective drug prescriptions. Furthermore, ‘over-the-counter’ dispensing should be strongly condemned and punishable

by the regulatory bodies in the country. The National Treatment Guidelines for Antimicrobial Use in Infectious Diseases, and the Surgery Safety Checklist should be reviewed periodically for adherence and applicability at all levels of health facilities where surgery is commonly practised. This also includes pre-operative

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Occult Filariasis in

a Case of Lymphocytic Thyroiditis Diagnosed on Fine Needle Aspiration

Cytology: An Unusual Finding of Dual Pathology

Authors:

Juhi Shahab,1 Adil Aziz Khan,1 *Sana Ahuja,1 Mukul Singh1

1. Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India *Correspondence to sanaahuja11@yahoo.com

Disclosure: The authors have declared no conflicts of interest. Each author has participated sufficiently in the work and takes public responsibility for appropriate portions of the content of this article. Written, informed consent was obtained from the patient for publication of this case report.

Received: 12.02.23

Accepted: 18.10.23

Keywords: Brugia malayi, filariasis, lymphocytic thyroiditis, microfilaremia, Wuchereria bancrofti

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:57-59. DOI/10.33590/emjmicrobiolinfectdis/10303849. https://doi.org/10.33590/emjmicrobiolinfectdis/10303849.

Abstract

Filariasis is a common public health problem in Southeast Asia. The two species of worms most commonly associated with this disease are Wuchereria bancrofti and Brugia malayi

Cases of microfilaraemia have also been reported from atypical sites in the past. None of the previously reported cases of microfilaria in the thyroid gland have described occult filariasis against a background of lymphocytic thyroiditis. The authors present a case of incidental microfilaria in a case of lymphocytic thyroiditis, diagnosed on fine needle aspiration cytology of thyroid swelling in a female, a coexistence of dual pathology. There has been no case in literature where filariasis was reported against a background of lymphocytic thyroiditis. Peripheral blood examination also did not show any evidence of filariasis.

Key Points

1. Filariasis is a common public health problem in Southeast Asia. There are as many as 60 million people infected in the region, and approximately 31 million people have the clinical manifestation of this disease.

2. This article presents a case of microfilaria in a case of lymphocytic thyroiditis diagnosed on fine needle aspiration cytology (FNAC) of thyroid swelling in a female, a coexistence of dual pathology.

3. This case report reiterates the importance of FNAC in the diagnosis of occult filariasis in an uncommon site like the thyroid gland. Thus, it is worth noting that with a simple procedure of FNAC, a coexistence of dual pathology was identified in an asymptomatic case. It also emphasises the significance of careful screening of smears in an endemic area.

INTRODUCTION

Filariasis is an infectious disease caused by any one of various parasitic roundworms. Wuchereria bancrofti and Brugia malayi are the two most frequent causative organisms of filariasis.1 Filariasis is a public health problem in Southeast Asia, with as many as 60 million people infected, and 31 million people exhibiting clinical manifestation of this disease.1 The disease is endemic in all parts of India, particularly in Bihar, Jharkhand, Uttar Pradesh, Andhra Pradesh, Orissa, and Tamil Nadu.1, 2

There have been few cases of microfilaraemia reported in uncommon sites, such as in breast lumps, pleural/pericardial fluid, lymph nodes, and bone marrow. The authors present a case of microfilaria in a case of lymphocytic thyroiditis diagnosed on fine needle aspiration cytology (FNAC) of thyroid swelling in a female, a coexistence of dual pathology.

CASE REPORT

A 40-year-old female, resident of Bihar, first presented to the outpatient department with complaints of painful anterior neck swelling for 2 months. The patient was of average build with stable vitals and no lymphadenopathy.

Thyroid examination revealed a Grade 3 asymmetrical goiter (right lobe>left lobe) with firm consistency and smooth surface. However,

it was non-tender on palpation. The patient was further advised for radiological examination and a thyroid profile test.

A neck ultrasound revealed enlargement of both lobes of the thyroid, with a few Thyroid Imaging Reporting and Data Systems (TIRADS) 3 nodules of size 5x6 mm in the left lobe, suggestive of thyroiditis. The thyroid profile test showed elevated thyroid stimulating hormone (Table 1).

FNAC was done and yielded a blood mixed aspirate. The Giemsa-stained smears were cellular, and showed thyroid follicular cells with lymphocyte impingement along with focal Hürthle cell change (Figure 1A, 1B). Smears also showed the presence of sheathed, coiled, and slightly curved microfilaria (Figure 1C) against a background of scant colloid. A final diagnosis of filariasis in a case of lymphocytic thyroiditis was made.

The patient was advised serological testing for anti-thyroid peroxidase (anti-TPO) antibodies and peripheral blood film examination. Post-FNAC, serological investigations revealed an elevated level of anti-TPO antibodies, which favoured the coexistence of Hashimoto thyroiditis with occult filariasis. However, the peripheral blood smears were unremarkable, but showed mild eosinophilia.

The patient was treated with diethyl carbamazepine for 6 months, which led to a mild reduction in size of the swelling.

Table 1: Thyroid profile of patient.

A B C

A) Thyroid follicular cells with microfilaria marked with red arrow (40x magnification; Giemsa stain).

B) Thyroid follicular cells with lymphocytic impingement with Hürthle cell change (200x; Giemsa stain).

C) The sheathed curved microfilaria against a background of scattered follicular cells (200x; Giemsa stain).

DISCUSSION

Filariasis is a major public health problem in Southeast Asian countries. It is endemic in a few regions of India, particularly in Bihar, Jharkhand, Uttar Pradesh, Andhra Pradesh, Tamil Nadu, Gujarat, Orissa, and Kerala.1

Most infected individuals in filarial endemic areas are asymptomatic. Adult worms are known to reside in the lymphatic vessels of the definitive host, from where microfilaria are released to circulate in the peripheral blood. The disease is transmitted by the larval form through mosquito bite. Early symptoms include fever with chills, headache, and skin lesions.3,4

The presence of microfilaria against a background of lymphocytic thyroiditis is a rare finding, with very limited data available. None of the cases which have reported filariasis in the thyroid observed a background of lymphocytic thyroiditis.

References

1. Gangopadhyay M et al. Microfilaria in thyroid aspirate - an unexpected finding. J Cytol. 2011;28(4):240-1.

The present case is an uncommon presentation, since the patient presented with diffuse anterior neck swelling, which showed presence of microfilaria with evidence of lymphocytic thyroiditis with elevated anti-TPO antibodies, leading to a diagnosis of occult filariasis in a case of Hashimoto thyroiditis. However, in the present case, the patient was an asymptomatic carrier, with larvae present in the microvasculature of the thyroid gland. Few studies have proposed a possible mechanism of rupture of vessels, leading to haemorrhage and release of microfilaria into the thyroid. However, the presence of microfilaria against a background of lymphocytic thyroiditis is a rare finding in itself.1,2,5 This case report reiterates the importance of FNAC in the diagnosis of an occult filariasis in an uncommon site like the thyroid gland. Thus, it is worth noting that with a simple procedure of FNAC, a coexistence of dual pathology was identified in an asymptomatic case. It also emphasises the importance of careful screening of smears, particularly in endemic areas.

2. Chowdhary M et al. Microfilaria in thyroid gland nodule. Indian J Pathol Microbiol. 2008;51(1):94-6.

3. Mondal RK et al. A rare case of microfilaria in thyroid aspirate. Bangladesh J Med Sci. 2014;13(1):99-100.

4. Maheshwari V et al. Microfilaria in thyroid aspiration smear –an unexpected finding. Diagn Cytopathol. 2007;36(1):40-1.

5. Bhartiya R et al. Microfilaria in fine needle aspirate of thyroid nodule. Thyroid Res Pract. 2016;13(2): 86-8.

Figure 1: Cytomorphological findings.

Virtual Discovery of Immune-Stimulating Epitopes in Chikungunya Virus for Vaccine Design

Authors:

*Louis O. Ezediuno,1-4 Michael A. Ockiya,5 Kehinde B. David,6 Luqman O. Awoniyi,7 Faith O. Robert,8 Elijah K. Oladipo,9 Olusola N. Majolagbe2,3

1. Department of Microbiology, Faculty of Life Science, University of Benin, Nigeria

2. Department of Microbiology, University of Ilorin, Nigeria

3. Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomosho, Nigeria

4. Biodata Analytical Laboratory, Abeokuta, Nigeria

5. Department of Animal Science, Faculty of Agriculture, Niger Delta University, Wilberforce Island, Nigeria

6. Department of Physiology, Ladoke Akintola University of Technology, Ogbomosho, Nigeria

7. Institute of Biomedicine and MedCity Research Laboratories, University of Turku, Finland

8. Department of Medical Biochemistry, Rivers State University, Port Harcourt, Nigeria

9. Laboratory of Molecular Biology, Immunology and Bioinformatics, Department of Microbiology, Adeleke University, Ede, Nigeria

*Correspondence to ezediunolouis@gmail.com

Disclosure: The authors have declared no conflicts of interest.

Received: 18.09.23

Accepted: 30.04.24

Keywords: Allergenicity, antigenicity, bioinformatics, chikungunya fever (CHIKF), chikungunya virus (CHIKV), docking, epitopes, physicochemical analysis.

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:60-76. https://doi.org/10.33590/emjmicrobiolinfectdis/RZNV7904.

Abstract

Epitope identification is a key step in vaccine development, and this can be achieved much faster and less expensively with in silico methods, compared to traditional methods for vaccine production. In silico methods applied in this research utilised both bioinformatics and immunoinformatics approaches for chikungunya virus vaccine design, which involved the retrieval of sequences from databases, and identification of conserved regions within the sequences by multiple sequence alignment on the MEGA X software (Pennsylvania State University, State College, USA). The epitopes in the conserved regions were selected, and various immunological predictions and screenings were carried out by employing immunological databases and tools. This process identifies epitopes such as conservation of cytotoxic T lymphocyte, helper T lymphocytes, and B cell epitopes. The primary, secondary, and tertiary structure of the vaccine was also predicted using structure predicting servers, and finally, the vaccine candidate was docked to toll-like receptor 4 to study its binding affinity and configuration.

A total of 125 conserved antigenic epitopes were selected from capsid, 6K, and E1 proteins, which were found to be non-allergens and conform to acceptable

physicochemical standards, as reported by other authors with similar work. The epitopes were predicted to be capable of inducing cytotoxic T lymphocytes, helper T lymphocytes, and B cell production. Construction of secondary structure was done using the Self-Optimized Prediction Method with Alignment (SOPMA), which predicted 17.96% α-helices, and 4.69% β-turns, among others. Predicting the tertiary structure provided five models, of which Model 1 was selected on the bases of its confidential score of 0.59, estimated TM-score of 0.79±0.09, and root mean square deviation of 8.0±4.4Å. Validity analysis revealed a Ramachandran plot where 97.2% of the vaccine residue was within the favoured region, and the peptide showed a Z-score of -1.52. The predicted peptide effectively docked with toll-like receptor 4 with a binding energy of -1,072.8. From the data obtained, it was revealed that the selected epitopes are highly immunogenic, non-allergenic, conform to native protein, and form a peptide capable of vaccine application. The authors can conclude this is a promising candidate for vaccine design and development.

Key Points

1. Chikungunya fever, first reported in East Africa, now affects over 110 countries globally. Without a cure, emphasis on prevention is crucial to combat the spread of this debilitating and neglected tropical disease.

2. The research focuses on virtual identification of immune-stimulating epitopes within the chikungunya virus protein, which have promising properties for the design and development of vaccines against infection.

3. Preventive measures are crucial in controlling infections such as chikungunya fever, given its gradual progression towards pandemic status. Identifying and implementing preventive strategies is key to effective management.

INTRODUCTION

Chikungunya fever (CHIKF) is a viral disease that is similar to dengue fever. It is a key public health concern in tropical and sub-tropical countries such as Kenya, where it re-emerged in 2004 and caused one of the largest epidemics on record.1 The causative pathogen is the chikungunya virus (CHIKV), belonging to the genus Alphavirus of the family Togaviridae. It is an enveloped virus transmitted between humans by two species of mosquitoes (Aedes aegypti and Aedes albopictus).2 CHIKV is comprised of a positivestrand RNA virus genome that is approximately 11.8 kb long with two open reading frames (ORF). The first ORF codes for four non-structural peptides responsible for genomic RNA replication and RNA transcription to structural proteins; and the second ORF codes for the structural protein C-E3-E2-6K-E1, which cleaves to form structural proteins such as capsid (Cp) and E3-E2/TF-E1.3

CHIKV replicates within the fibroblast or macrophages at the site of inoculation after it has been deposited into the skin or blood of a host by an infected mosquito’s bite. The virus spreads from the inoculation site through the lymphatic system and blood stream to other sites of replication, such as the lymphoid organs, muscles, peripheral joints, and tendons, within 2–4 incubation days. After this, a sudden manifestation of clinical symptoms that include headaches, rigors, incapacitating joint pain, high fever, photophobia, and petechial rash occurs. CHIKV has been reported to reach the brain and liver in very severe cases, leading to a more life-threatening condition.4

Acute phase symptoms of CHIKV infection can typically last from a few days to weeks, while the duration of symptoms for chronic phase disease has not been extensively studied. Report of recurring joint pain lasting for years has been recorded among 30–40% of infected patients; but this is suggested to be mediated by immune response and not as a result of chronic infection.5

Currently, there are no drugs for treating CHIKV infection, despite the fact it was declared an epidemic in over 14 countries by the World Health Organization (WHO) by the end of April 2014.6 In the absence of any known therapeutic, preventive measures such as a vaccine should be considered the best alternative to controlling the spread of the disease, as the virus is limited in its variety of antigen, and lifelong immunity is impacted for individuals who have suffered infection.7

The search for a CHIKF vaccine started just after the first outbreak in the 1960s, and this has resulted in the development of various types of vaccines, including live-attenuated vaccines, inactivated vaccines, chimeric vaccines, recombinant virus-vectored vaccines, and subunit vaccines.8

Subunit vaccines are vaccines that primarily contain a specific antigenic epitope from the pathogen of interest; therefore, they do not contain components of the pathogens that could cause harm when introduced to a host. This means that risks like virulence recovery or incomplete inactivation and pre-existing immunity are significantly eliminated.9 Subunit vaccines are known to cause less adverse reactions compared to inactivated or live vaccines; thus, they are generally safe and are believed to be able to target specific, welldefined neutralising antibodies with enhanced immunogenic efficacy.10 However, due to their purification process, they may be less immunogenic and may also lose their innate immune properties because of reduced antigenic components.11 To deal with these disadvantages, subunit vaccines are usually linked to adjuvants to increase their immunogenic potential.

In this study, the authors try to propose a subunit vaccine by identifying a vaccinable complex that comprises components from CHIKV structural proteins with antigenic potentials. The viral antigens selected for the study include 6K, E1, and Cp proteins, which are known parts of CHIKV structural protein,12,13 and play vital roles in the virus pathogenicity.

CHIKV Cps are involved in the virus RNA binding and particle assembly after replication. The Cp is a small-sized monomeric peptide of approximately 30 kDa, and as such, it can

passively transport through nuclear pores. There are reports of Cp directly interacting with host cell transport receptors such as the CRM1 receptors to transport CHIKV proteins.14. The Cps are also known antigens capable of inducing the production of Cp-specific antibodies within the host.14

E1 proteins are part of the envelope protein E1-E2 heterodimer triplets that form the spike glycoprotein found on the surface of CHIKV. CHIKV uses the E1-E2 heterodimer to interact with several host receptors, such as the glycosaminoglycans and Mxra8, to achieve cell attachment and entry.15 Once E1-E2 heterodimer attachment is complete, pH-dependent membrane fusion and endocytosis mediated by clathrin bring about the intake of the particle. The low pH of the host cell causes the E1-E2 heterodimer to dissociate, thereby exposing the fusion residues located at the E1 cd loop. The fusion residues then bind to the target receptor, which causes E1 to conform by back folding upon itself and trimerising, resulting in viral entry.16

Alphavirus 6K proteins are small peptides, typically 58–61 amino acids long. They are hydrophobic and membrane-linked. These proteins act as a stop-transfer sequence for the insertion of E1 proteins into the endoplasmic reticulum membrane. 6K protein expression also boosts membrane permeability and viral budding in the cells of eukaryotes. The alphavirus 6K protein is suggested to be a viroporin (a viral ion channel) due to its characteristic features, such as being an integral membrane protein, its short length (50–120 amino acids long), its role in virus assembly and release, and its function in membrane permeability of infected cells.17

The structural glycoprotein E1 has been reported to cause neutralising antibody production by the host cells, which are capable of clearing CHIKV and thus protect vaccinated hosts. E1 glycoprotein is reported to be more antigenic when linked with an adjuvant.8 It has been used in vaccinating experimental animals along with other immune-inducing glycoproteins, such as E2 glycoprotein, in combination with adjuvants. The results obtained show that vaccinable complexes, like those developed from glycoproteins such as E1 and E2, can actively neutralise CHIKV.18

There is very little information on the application of chikungunya Cps and 6K glycoprotein in the development of a vaccine against the virus, but they were selected in this in silico study due to their high antigenic score on the antigenic prediction tools ANTIGENpro.19

With the selected CHIKV peptides E1, 6K, and Cps, the use of in silico strategies was focused on identifying epitopes within the conserved regions of these immune-stimulating peptides that can be recognised by B cells, as well as being able to bind to toll-like receptors during host immune recognition for the development of a possible subunit vaccine. This was achieved by first retrieving the peptides of interest from available databases; confirming their antigenic propensity on antigen-predicting tools; determining the conserved regions within the selected peptides; confirming the antigenic potential of the selected conserved regions; and determining the presence of antigenic epitopes that can sensitise B cells, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) production. Next up was constructing a primary structure of the possible vaccine by linking the CTL, HTL, and B cell antigenic epitopes with appropriate linkers; predicting the possible secondary and tertiary structure of the proposed vaccine; validating tertiary structure; and finally, docking the proposed vaccine with a toll-like receptor 4 (TLR4).

METHODOLOGY

Retrieval of Chikungunya Virus Structural Protein Sequences From UniprotKB FASTA format sequences of CHIKV structural proteins were downloaded from the UniprotKB database (National Institutes of Health, Bethesda, Maryland, USA),20,21 which is a curated database that acts as a central access point for integrated protein information. The database provides a comprehensive compendium of all reported protein sequences obtained from laboratory experiments or computational predictions. The structural proteins retrieved for this research were Cp proteins, 6K glycoproteins, and E1 glycoproteins. The selections of these proteins was informed on the basis of their antigenic properties, as previously explained.22, 23

Multiple Sequence Alignment

Selected sequences were aligned on the multiple sequence alignment tool using the ClustalW algorithm on the MEGA X software (Pennsylvania State University, State College, USA). MEGA X carries out statistical analysis for molecular evolution. The alignment explorer of MEGA X is a built-in tool for viewing, editing, and creating alignments through the ClustalW and MUSCLE programs to identify conserved regions common among sequences. The ClustalW algorithm globally aligns the strings of the sequence and compares the whole length of the sequences with each other to identify rejoins of similarity and identity.13,14 Identifying the conserved regions within the selected proteins is important for the identification of a universal vaccine capable of inducing immunological response for all variants of the virus.6

Antigenic Screening of Conserved Epitopes

The conserved sequences, selected after multiple alignments, were subjected to antigenic screening on the ANTIGENpro server.19 This server is a sequence-based prediction tool capable of predicting the antigenicity of whole proteins and providing the probability score for the predicted antigenicity. The tool is trained with reactivity data obtained by protein microarray analysis of five pathogens. Epitopes with an antigenic score of 0.8, which is the cutoff mark for antigenicity on the ANTIGENpro, are accepted as immunogens.24

Screening for Cytotoxic T Lymphocytes From Conserved Epitopes

Since antigens bind to major histocompatibility complex (MHC) Class I molecules to initiate immune responses, the conserved epitopes that passed antigenic screening were analysed on the NetCTL version 1.2 web server25 to confirm their CTL activating potential. NetCTL version 1.2 web server is a human CTL epitope prediction tool that integrates MHC Class I affinity, proteasomal cleavages, and transporter associated with antigen processing efficiency predictions to identify human CTL epitopes.26

Screening for Helper T Lymphocytes from Conserved Epitopes

While CTL epitopes initiate an immune response by binding to the molecule of MHC Class I, MHCs Class II initiate an immune response and memory cell formation by presenting surface antigens to T cells and B cells. Researchers used the Immune Epitope Database (IEDB)27 to predict the HTL epitopes in the conserved sequences of CHIKV structural protein.28

Screening for B Cells from Conserved Epitopes

Another immune-stimulating epitope of interest, which was predicted on the BCPred web server29 from the conserved sequences, is the B cell epitope. B cell epitopes are essential for peptide-based vaccines, as they recognise and bind to B cell epitopes to initiate an immune response and memory cell, which is a key component to initiating an immune response. At the default setting, epitopes with a score of 1 are selected as B cell epitopes.30

Constructing Possible Primary Structure of Proposed Vaccine

A potential primary vaccine construct was developed by linking the CTL, HTL, and B cell epitopes with appropriate linkers, such as Ala-Ala-Tyr (AAY), which was chosen on the basis of its function. The AAY linker is the mammalian cell proteasome cleavage site, known to increase immunogenicity of multiepitope vaccines. Epitopes linked with AAY result in effective separation within the cell, thereby reducing junctional immunogenicity. HTL and B cell epitopes were also linked by GPGPG, which has been shown to induce HTL responses, an important multi-epitope vaccine property. Like AAY, it is also able to prevent junctional immunogenicity.

Following the respective linking of HTL and B cell epitopes, the linked CTL, HTL, and B cell epitopes were further joined to each other to form a long chain of polypeptide by the GPGPG linker in the format: [CTLAAYCTL] nGPGPG[HTLGPGPGHTL]nGPGPG[BcellsGPGPGB-cells]n

To complete the construction of the primary vaccine structure, the team attached an

adjuvant with the linker EAAAK to the N-terminal of the long chain of polypeptides, which were linked together by AAY and GPGPG linkers. This is because, for a multi-epitope vaccine to be strongly immunogenic, adjuvants are required, as they catalyse innate immune responses by inducing a localised inflammatory reaction initiated by interactions between TLRs and antigens. However, most synthetic adjuvants, such as aluminium-based adjuvants, result in adverse effects. So, in an effort to mitigate these side effects, natural candidates like the TLR agonist (RS09) were developed. In this study, RS09 was selected as the adjuvant of choice, as it is capable of stimulating and activating TLR4 and NF-kB, which is reported to result in an increased antibody production.31 The EAAAK linker used is a solid α-helix-forming protein, which contains intramolecular hydrogen bonds and a closely packed backbone. EAAAK maintains a fixed distance and, hence, reduces interference between the linked epitopes. This ensures that the epitopes keep their individual functionality within the functional domain. With this, the final primary construct of the vaccine now has a structural format: RS09EAAAK[CTLAAYCTL] nGPGPG[HTLGPGPGHTL]nGPGPG[BcellsGPGPGB-cells].32

Scoring the Allergenic and Antigenic Potential of the Primary Construct and Predicting its Physicochemical Properties

Allergenic screening

Vaccines are designed to be an immunogen without causing allergic reactions. The primary structure of this vaccinable complex was assessed on the AllerTOP v.2.0 server33 to confirm its non-allergenic properties. AllerTOP v.2.0 predicts allergenicity by using the K nearest neighbours (KNN) model, and is reported to have an 85.9% accuracy.34

Antigenic screening

Predicting the antigenicity of the vaccinable complex was achieved on the ANTIGENpro server: a complex with an antigenic score ≥0.8 is considered an antigen. To confirm the antigenic potential of the complex after ANTIGENpro prediction, the complex was subjected to the VaxiJen online server set

at a threshold of 0.4 and selecting virus as the source of the complex. A score ≥0.4 is considered an antigen and confirms the antigenicity of the vaccine complex.

Prediction of the physicochemical properties of the primary construct

Predicting the physicochemical properties of the primary construct is indicative of the physicochemical behaviour of the complex. Predicting the physicochemical parameters involves predicting properties of the complex, such as the grand average of hydropathy (GRAVY) score, isoelectric point, instability index, aliphatic index, etc. Physicochemical prediction of the vaccine primary structure was done on the ProtParam web server.35

Secondary

structure prediction

Secondary structure of a polypeptide refers to the spatial arrangement of said polypeptide, consisting of α-helices, β-sheets, and turns. Predicting the secondary structure of a vaccine gives information on how the vaccine polypeptides are folded, and assists in determining how the vaccine will interact with the host immune system, such as whether the vaccine possesses the right conformation. Predicting secondary structure affords a chance to optimise the vaccine to achieve stability and enhanced antigenicity for an efficacious vaccine. The secondary structure of the vaccinable complex was predicted on the Self Optimized Prediction Method (SOPMA) web server.36 This web server is an improved secondary structure prediction tool capable of predicting secondary structure with a higher success rate. SOPMA has been reported to have 69.5% accuracy for predicting the α-helix, β-sheet, and coils of protein secondary structure in a database holding 126 chains of non-homologous polypeptides.34,37

Tertiary structure prediction

The 3D model of the proposed vaccinable complex was predicted on the I-TASSER server.38 The server predicts the 3D structure of polypeptides by assessing quantitative scores. The server provides five outputs of full length for every query input, along with other parameters, such as standard deviation, TMscores, confidence score, and root mean square deviation.39 I-TASSER was selected as the best server for protein structure and function prediction. It predicts proteins by first identifying

structure templates from Protein Data Base (PDB) using multiple threading methods and fulllength atomic models built by iterative templatebased fragments.

Linear and Discontinuous B Cell Conformational Epitopes Mapping

B cell epitopes are comprised of either linear (continuous) or conformational (discontinuous) epitopes. Linear B cell epitopes are sequential epitopes and make up a lesser percentage of the B cells, while conformational B cell epitopes are composed of fragments of solvent-exposed nonsequential epitopes, which make up the larger percentage of B cells. It becomes important to map both the linear and discontinuous B cell epitopes, because antibodies that recognise linear B cell epitopes may not be able to recognise conformational B cell epitopes. To map the B cell epitopes of vaccine constructs, Ellipro suite is used.40 The web tool is set at a minimum threshold of 0.5, and a maximum distance of 6. Ellipro suite uses both the MODELLER program and Jmol viewer to evaluate and visualise the 3D structure of B cell epitopes of vaccine constructs.24

Refinement of the Predicted Tertiary Structure

The tertiary structure of a protein is dependent on the peptides it is composed of; both the peptide composition and structure of the protein determine the protein’s function. If a protein does not conform to the native structure, it may lose its natural function. To ensure that the 3D model of the study’s predicted protein is as structurally similar as possible to the native protein structure to which it was modelled, refinement needs to be done. 3D structure refinement of the vaccine candidate was done on the GalaxyWEB server.41 Refinement increases the number of residues in the favoured region. GalaxyRefine uses CASP 10 and functions by reconstructing the protein side chains, relaxing the structure by repacking, and molecular dynamics simulation.34

Tertiary Structure Validation

Once refinement was done on the authors’ vaccinable complex, they validated the refined structure on the ProSA web tool,42 which is used for checking potential errors in the 3D model of experimentally determined protein structures,

theoretical models, and engineered protein. Validation was obtained as Z-score and local model quality. PROCHEK was also used for the Ramachandran plot analysis, which validates and verifies the complex functionality.24

Molecular Docking of Vaccinable Complex With TLR4

Visually studying the interaction of a ligandreceptor bonding, like that of an antigenantibody interaction, requires an in silico simulation of the interaction. The authors studied the interaction of their complex with TLR4, the tool ClusPro 2.0 server.43 ClusPro 2.0 predicted the binding domain between the vaccine construct and TLR4 in their most stable complex form and lowest binding energy.44

RESULTS

Retrieval, Multiple Sequence Alignment, and Antigenic Screen of the Conserved Epitopes of CHIKV Structural Protein Sequences

A total of 125 CHIKV structural protein sequences, comprising 13 Cps, 22 6K glycoproteins, and 90 E1 glycoproteins, were retrieved from UniprotKB database in FASTA format. The retrieved sequences were analysed to identify epitopes of shared conserved regions. These epitopes were selected and screened for their antigenic properties, which revealed that all the conserved epitopes were antigenic as they had antigenic scores ≥0.8.

Screening

for Cytotoxic T Lymphocytes, Helper T Lymphocytes, and B Cells From the Conserved Epitopes

Seventeen 9mers CTL epitopes with high comb scores were selected among the predicted epitopes from the conserved CHIKV structural epitopes. A total of 32 15mers HTL epitopes were also selected from the total predicted HTL epitopes on the basis of their low percentile rank score, and 16 B cell epitopes were selected from the total B cell epitopes predicted from the virus structural protein based on their high score. These are shown in Table 1.

Construction of Primary Vaccine Candidate

A total of 1,236 amino acid residues, comprising the selected CTL, HTL, and B cells linked with appropriate linkers, were used to construct the primary vaccine candidate. An adjuvant was linked to the N-terminal of the vaccine candidate to improve its antigenicity. Figure 1A shows a schematic presentation of the primary vaccine structure.

Assessment of Allergenic, Antigenic, and Physicochemical Properties of the Vaccine Primary Construct

The primary vaccine construct was predicted to be non-allergenic on the allergen predicting tool AllerTOP v.2.0. It was also confirmed to be antigenic with a high score of 0.946655 on the ANTIGENpro server; and a score of 0.4621 on the VaxiJen online server confirmed the antigenicity of the proposed vaccine, indicating it to be antigen with potential to induce immune response.15 Physicochemical assessment of the primary structure revealed that the sequence has a molecular weight of 125060.86, instability index of 23.96, aliphatic index of 65.11, and GRAVY of -0.121.

Vaccine Secondary Structure Prediction

Secondary structure prediction of the vaccine candidate on the SOPMA server predicts the composition of the secondary structure of the vaccine candidate. SOPMA predicted a 17.96% α-helix, 27.67% extended strand, 49.68% random coil, and 4.69% β-turns for the vaccine construct.

Vaccine Tertiary Structure Prediction

Predicting tertiary structure of the vaccine candidate was carried out on the I-TASSER server. Five models were predicted as having favourable properties, but Model 1 was selected on the basis of its confidential score of 0.59, estimated TM-score of 0.79±0.09, and root mean square deviation of 8.0±4.4Å. The 3D structure is shown in Figure 1B

Linear and Discontinuous B Cell Conformational Epitopes Mapping

Ellipro suite predicted a total of 244 residues for the linear B cell epitopes, which were between

Table 1: Predicted cytotoxic T lymphocytes, helper T lymphocytes, and B cells.

1 Conserved epitopes of glycoprotein E1

2 Conserved epitopes

3

Table 1 continued.

Table 1 continued.

residue numbers 993–1,236; the predicted score for the linear B cell epitope was 0.855. A 206 residue for discontinuous B cell epitope was also predicted for the vaccine construct with a score of 0.856. The linear and discontinuous B cell epitopes were visualised with the Jmol viewer, as seen on Figure 2

Refinement of Tertiary Structure Prediction

The refinement of the vaccine tertiary structure provided five models; Model 1 (Figure 1B) was selected as the best model.

Tertiary Structure Validation

Validating the final structure of the vaccine candidate requires that the PDB file of the vaccine be subjected to a few statistical validation softwares. The result obtained revealed the vaccine’s structure Z-score, graph of the vaccine local model quality, and Ramachandran plot analysis. These are shown in Figures 1C and 1D

Molecular Docking of Vaccine Construct with TLR4

In silico molecular docking of the vaccine candidate with TLR4 on the ClusPro server predicted 10 possible models at the balance setting. Model 0 was selected based on the binding energy (-1,072.8). This is shown in Figure 1

DISCUSSION

Outbreaks of CHIKF have been reported over the years across many countries within Africa, Asia, and Polynesia. The largest outbreak of the disease on record was in 2004–2007 in the Indian Ocean Island and India, where a serological survey suggests that more than 60% of the population in the region was exposed to the virus.32 The first recorded outbreak of the disease occurred in the Newalla and Masisi districts in the southern province of former Tanganyika Territory, now Tanzania, between 1952–1953, with a 23% incidence rate.46,47 Since the first outbreak, the disease has spread from East Africa to Asia and Europe.

For an infectious disease with no known treatment and high potential to spread, the most realistic means to manage it is preventive measures; only with prevention can there be a chance for control.

This research focuses on identifying viral epitopes with potential for vaccine development. In this study, CHIVK structural proteins capable of causing sensitisation of the immune system were selected, and conserved epitopes from these selected protein were analysed for their CTL, HTL, and B cell recognition.8, 18

To determine the conserved regions of the E1, 6K, and Cp proteins, each protein type was respectively aligned on the Mega X tool, and their conserved regions identified. Identified regions were selected for further analysis. Identifying conserved regions ensures that

IEDB: Immune Epitope Database.

only regions within sequences shared among all available variants of CHIKV are selected and used for vaccine design. Using conserved regions makes sure that all variants of the virus found on the database at the time of this research were represented, guaranteeing the vaccine efficacy to confer protection against all variants of CHIKV.48 Nevertheless, using conserved regions for vaccine design comes with its drawbacks. One of the most important challenges of using conserved regions for vaccines is the risk of altered immunogenic potential of the sequence. When this happens, it is usually a result of parts, or all, of the active sites being lost because they are located in the region identified as non-conserved. To ensure that the selected conserved sequences maintain their antigenic potential, the sequences should be screened for antigenicity.

In this study, the researchers screened for the antigenicity of the conserved regions of the selected epitopes using the ANTIGENpro online tool. All of the authors’ selections were predicted to have antigen scores >0.8 (Table 1). These are good indications of their immune stimulating properties, which make them promising epitopes for vaccine development against CHIKV infection considering that the antigen of CHIKV is limited and lifelong immunity usually occurs after infection.18 Hence, vaccinating a host with this candidate vaccine is expected to produce similar results.

After constructing the primary structure of the vaccinable complex, researchers identified epitopes that can be recognised by HTLs, CTLs, and B cells. Identification of HTL epitopes is an important step in in silico vaccine development, because HTLs are responsible for activating CTLs, B cells, and macrophages in adaptive immunity.49 The team predicted CHIKV epitopes recognisable by HTLs on the IEDB platform.27 The tool predicted 32 15mers HTL epitopes from the E1, 6k, and Cp protein. CLT and B cell epitopes were predicted, as earlier stated, to identify epitopes that can initiate cellular and humoral immunity. This is shown on Table 1. Predicting these epitopes was important because vaccines are required to induce antibodies and memory cell production.50 Mahmoodi et al.51 and Zakaria et al.52 reported to have identified CT, HTL, and B cell epitopes from CHIKV structural protein. In their research, Mahmoodi et al.51 reported

that the CTL, HTL, and B cell epitopes identified showed high immunogenic potentials and conferred immunity against CHIKV infection. Zakaria et al.52 also reported the activation of both humoral and adaptive immune cells, and production of various cytokines by the structural epitopes capable of controlling CHIKV. On the premise of the reports of these two authors, the team selected epitopes of CHIKV structural protein with high antigenic scores that qualified them as an active component for an effective CHIKV multi-epitope vaccine candidate.51,52

An active multi-epitope vaccine should contain all the epitopes that can induce all various immune cells. In this research, the immune cells of interest are the HTL, CTL, and B cells. The epitopes with the potential to induce these cells were predicted and are reported in Table 1 Designing a vaccine using the in silico method requires the predicted epitopes to be linked to form the primary vaccine structure. To achieve this, the HTL epitopes were linked by GPGPG. AAY was used to link CTLs together, and B cells were also linked together with GPGPG. The linked epitopes were further linked by GPGPG, with the HTLs at the N-terminal and the B cells at the C-terminal. At the N-terminal, an adjuvant is connected to the vaccine polypeptide chain by a specific linker, EAAAAK, to create a primary vaccine structure: RS09EAAAK[CTLAAYCTL] nGPGPG[HTLGPGPGHTL]nGPGPG[BcellsGPGPGB-cells (Figure 1).

The primary structure of the vaccinable complex was further assessed to determine its allergenic propensity, antigenic potential, and physicochemical properties. It was found that the vaccine molecule is a non-allergen with a high antigenic potential, scoring an antigenic score of 0.946655 on the ANTIGENpro server and a score of 0.4621 on the VaxiJen server. Owing to the molecular weight (125060.86 Da) of the vaccine complex, it is confirmed that the vaccine consists of multiple antigenic regions, which makes it capable of stimulating a robust immune response. Its instability index score was 23.96; this meets the <40 threshold, indicating the vaccine’s structural stability. The aliphatic index of 65.11 also provides information on the vaccine’s thermo-stability. The negative GRAVY score of the vaccine molecule indicates its hydrophilic nature, providing information on its water-soluble property. A similar approach was

The Ramachandran plot analysis for the candidate vaccine shows that 97.2% of the residue is in the most favoured region, 2.8% of the residue is in the allowed region, and 0.0% in the disallowed region.

CTL: cytotoxic T lymphocyte; HTL: helper T lymphocyte.

used by Foroutan et al.53 to develop a vaccine candidate against Toxoplasma gondii. Reporting results with comparable physicochemical properties to these, Forountan et al.53 went further to validate their vaccine by carrying out animal trials using laboratory mice. The team reported that their vaccine induced a strong humoral and cellular immune response in the mice. Findings corresponding to this were also reported by Sumera et al.54 in their publication in 2022. By comparing results with the results from these, and other papers, it was established that this vaccine’s primary structure physicochemical properties were within an accepted threshold.55

The secondary and tertiary structure of the vaccine were predicted following the construction of the primary structure. These were done on the SOPMA and I-TASSER servers, respectively. The linear B cell epitopes were also predicted before refinement of the tertiary vaccinable complex was done on the Galaxy

refine server. Refinement is necessary to ensure the vaccine tertiary structure conforms to the native protein.

The team structurally validated the vaccinable complex to ensure that the sequence and structure used behave like those of native proteins from which it is predicted. From the structural validation analysis, an overall model quality graph and Z-score (–1.52) were obtained, which validate the vaccine tertiary structure as conforming to native structure. The graph of the Z-score revealed that the protein structure is determined by NMR spectroscopy (Figure 1C). The Z-score value and the contour line under the 0.0 threshold of the overall model quality graph both indicate the good quality of the predicted candidate vaccine tertiary structure. This is because the authors’ tertiary structure has very little 3D structural error. Ramachandran analysis carried out revealed that the structure possesses proteins with 97% of its residue in

Figure 1: A) Schematic representation of the primary vaccine structure; B) Predicted tertiary structure of vaccine candidate; and C) Ramachandran plot analysis for the candidate vaccine.

the favoured region, while none of the residue was in the disallowed region. All of these data validate the quality of this vaccine as a potential vaccine candidate.

TLR4 is expressed in various immune cells in humans, such as macrophages, monocytes, and granulocytes. TLR4 binding prediction is vital in vaccine design and development because TLR4s recognise pathogen-associated molecular patterns and induce immune response. Activating TLR4 improves antigen presentation, adaptive immune response, cytokine production, and vaccine effectiveness.56 It is because of these functions of TLR4, to both innate and adaptive immune activation, that a TLR4 agonist such as RSO9 is used as adjuvant in epitope vaccines to enhance their immunogenicity.57 To evaluate the binding affinity of the candidate vaccine tertiary structure to the antigen recognising receptors, a TLR4 molecule was docked with the PDB structure of the vaccine candidate on the Cluspro server. Ten models were predicted,

and Model 0 was selected on the basis of its low binding energy of -1,072.8. Low binding energy is significant in vaccine design and development, as it is indicative of the strength of the proteinligand binding complex. The lower the binding energy, the stronger the complex, hence, the more robust the immune response. Binding energy also gives insights into the structure of the protein-ligand complex. With that information, decisions for more effective vaccines can be made. From this study, the low binding energy obtained for Model 0 informed the selection. Model 0 is predicted to have the strongest and most stable binding interaction. The structure of the docked complex is shown on Figure 2.

Multi-epitope vaccines against CHIKV are predicted to be advantageous in many ways, including broader protection against diverse strains of the virus, capturing the risk of escape mutant by enhanced cross-reactivity;58 and reducing host immune evasion, their potential for long-lasting immunity, their flexibility in design,

Figure 2: A) Conformational linear B cell structure (yellow) attached to the vaccine structure (grey-blue); and B) 3D representation of docked vaccine candidate with toll-like receptor 4.

and their higher level of safety. Regardless, developing a multi-epitope vaccine for CHIKV poses several challenges and limitations, such as the challenge of antigenic diversity of CHIKV and the possibility of low immunogenicity of some of the epitopes. T cell response, which has always been an important part of multiepitope vaccine design, and the long-term protection they confer on the host, is also a vital research area for improving multi-epitope vaccines.59 To correct these challenges, a CHIKV vaccine must consist of various epitopes for it to have the immune coverage that accounts for all the diversity of CHIKV. It is also imperative to identify immunologically viable epitopes to use as component for a multi-epitope vaccine.60 Attaching adjuvants to the CHIKV vaccine is an important process in its design and development; this is because it greatly improves the immunogenicity of the vaccine. In the in silico design and development of this vaccine candidate, the adjuvant RS09 was attached to the N-terminal to improve its immune-stimulating properties. There are many known adjuvants, such as the RS09 and CpG oligodeoxynucleotides, that could be used to improve CHIKV vaccine effectiveness. Selecting an adjuvant with maximum efficiency among all the available adjuvants, and developing new and novel adjuvants, should be another area of research that is focused on.61 Apart from deep adjuvant reach, optimising T cell activation by CHIKV multi-epitope vaccines should also be a vital point in future CHIKV multi-epitope vaccine investigations.62 Clinical trials are also necessary step in vaccine development. This is vital in order to validate the safety, effectiveness, and durability of CHIKV multi-epitope vaccines in diverse populations, especially those in endemic regions.

CONCLUSION

The growing scourge of diseases across the globe, including CHIKV, requires immediate response. Preventive medicine has proven to be the most effective strategy for controlling infectious diseases, especially when performed properly. CHIKV, which was first discovered in

the United Republic of Tanzania in 1952, has spread to become a global threat and there is no approved treatment for the infection. The symptoms of CHIKF are self-limiting, but the risk of eye, neurological, and heart complications; severe joint pain; or even death is ever looming, resulting in the need for a means to control infection rate.

This study focused on designing a multi-epitope vaccine capable of reducing the spread of CHIKV infection by sensitising the immune system to the infection. This in silico vaccine construct was built from antigenic epitopes from CHIKV 6K, E1, and Cp proteins. The proteins were predicted to be able to activate CTLs, HTLs, and B cells, and bind effectively to TLR4, which is indicative of an efficacious vaccine candidate. The primary, secondary, and tertiary structures were predicted to be composed of structurally stable molecules that interact to create a thermal and chemically durable compound. This was validated by physicochemical and statistical analyses done on various online servers. In conclusion, the candidate vaccine is expected to be effective and capable of inducing antibody production by way of sensitising the host’s humoral and cell-mediated immunity against future exposure to CHIKV. The vaccine candidate would be capable of improving the health of people, especially those in endemic communities, as the vaccine was designed with conserved regions from CHIKV antigenic epitopes, granting it a wide coverage for many variants of the CHIKV. The vaccine being a multi-epitope is safe, as issues such as virulence recovery or incomplete inactivation and pre-existing immunity are eliminated. From the data gathered, the researchers could make an educated suggestion that a vaccine developed using E1, 6K, and Cp proteins would be a promising vaccine that could be effective in the control of the spread of CHIKF. The method applied in this research provides necessary data indicating that the candidate vaccine will be effective in inducing immunological response of the host immune system, inducing production of B cells that can be stored as memory cells and bind perfectly to antigenic receptors of the immune system.

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Hand Washing Practices and Antibiotic Susceptibility of Palmar Bacterial Flora in a Tertiary Education Institution in Nigeria

Authors: Florence Akinwumi,1 *Chiamaka Ifeyinwa Okorafor,1 Lawrencia Ukatu,1 Ezekiel Akinkunmi1

1. Obafemi Awolowo University Faculty of Pharmacy, Ile-Ife, Osun, Nigeria

*Correspondence to okoraforchiamakaifeyinwa@gmail.com

Disclosure: The authors have declared no conflicts of interest.

Received: 06.03.24

Accepted: 24.05.24

Keywords: Antimicrobial resistance, hand hygiene, microbiology, palmar bacterial flora.

Citation: EMJ Microbiol Infect Dis. 2024;5[1]:77-88. https://doi.org/10.33590/emjmicrobiolinfectdis/EPHS3736.

Abstract

Introduction: Hand hygiene is one of the most effective and inexpensive means of preventing the spread of communicable diseases. Rates of handwashing worldwide are low, and poor handwashing practices in universities remain a public health challenge.

Objective: The objective of this study was to examine the practice of handwashing, the microbial communities, and the susceptibility pattern of micro-organisms isolated from the palms of students of the Obafemi Awolowo University in Ile-Ife, Osun, Nigeria.

Methods: A combination of qualitative and quantitative methods was used to retrieve data. A self-administered questionnaire was utilised to gather socio-demographic characteristics and the practice scale of handwashing from the respondents. Isolation and identification were carried out by culture-based surveys and biochemical tests. Disk diffusion was used to determine susceptibility.

Results: The majority of respondents were between ages 21–25 years, with 54.3% of them being female. About half of individuals never used an alcohol-based hand sanitiser, while only 4.9% of respondents admitted to always using soap to wash their hands. Presumptive identification of the organisms showed 38.1% of organisms as Staphylococcus epidermidis and 17.58% as Micrococcus spp. None of the antibiotics showed total efficacy. The resistance to chloramphenicol was low, and <50% of the isolates showed resistance to tetracycline, novobiocin, and sulphonamide. Resistance to nalidixic acid was seen in 58.4% of organisms tested, and 77.75% were susceptible to tetracycline.

Conclusion: Instances of improper hand hygiene were high, and this may increase the spread of micro-organisms through hand carriage. High resistance to the antibiotics tested was prevalent. Local trends of antimicrobial resistance must be robustly studied and proper interventions developed.

Key Points

1. Misuse of antibiotics has led to increasing antibiotic resistance. A significant strategy to reduce the misuse of antibiotics, and thus reduce antibiotic resistance, is to decrease the transmission of microorganisms via hand carriage. Therefore, it is important to assess handwashing practices of individuals, as well as the microbial spectrum and susceptibility of micro-organisms isolated from the palmar surface.

2. This study was a cross-sectional study targeting the undergraduate students of a tertiary institution in Nigeria to assess their handwashing habits, and to determine the susceptibility of isolated palmar micro-organisms to selected antibiotics.

3. Results indicated poor hand hygiene practices and high resistance to the antibiotics tested. Local patterns of antibiotic resistance must be studied, and programmes for antibiotic stewardship put in place to deal with the increasing prevalence of antimicrobial resistance.

INTRODUCTION

Resistance of bacteria to antibiotics is an issue that has plagued clinicians since the discovery of antibiotics. Antibiotic resistance is the ability of bacteria to resist the toxic effects of drugs, and grow in the presence of a concentration that will normally kill or inhibit its growth.1 This leads to higher medical costs, prolonged hospital stays, and increased mortality. The World Health Organization (WHO) cautions: “Without urgent action, we are heading for a pre-antibiotic era, in which common infections and minor injuries can once again kill.”2

In the golden age of antibiotics, this problem was easily solved by the discovery, or synthesis, of new antibiotics. In recent years, however, the production of novel antibiotics has slowed down immensely, and the introduction of novel therapeutic agents is outpaced by the everevolving microbes.3 The development of new antibiotics takes years and millions of dollars, only for the drug to be active for a relatively short period of time, and its activity reduced or eliminated by microbial resistance. Pharmaceutical companies have thereby diverted funds into more rewarding areas such as the development of drugs for chronic illnesses.4

Antibiotic stewardship is an important part of controlling the improper use of antibiotics and thus, extending their lifespan. The WHO suggests the establishment of antimicrobial stewardship committees in healthcare facilities, and the dedicated collection of data to “assess the extent and quality of antibiotic use, identify

problematic prescribing practices, and compare appropriate use […] over time.”5

Another significant strategy in reducing the misuse of antibiotics is reducing the rate of pathogen transmission via hand carriage. Pathogen transmission is broken down into five steps: 1) organisms must be present on the skin or inanimate objects in the immediate vicinity; 2) organisms must be transferred to the hands; 3) organisms must be able to survive on the carrier’s hands; 4) failure to carry out proper hand antisepsis; and 5) the carrier's contaminated hand deposits organisms on another individual or inanimate object that will come in contact with another individual. Hand hygiene, when practised at any of these stages, will drastically reduce the hand carriage of micro-organisms.6

Proper hand hygiene involves the use of an alcohol-based sanitiser containing at least 60% alcohol for 20–30 seconds, or the washing of hands with soap and water for a minimum of 40 seconds. To ensure the hands have been thoroughly cleansed when washing, the Africa Centres for Disease Control and Prevention (Africa CDC)7 suggests wetting the entire surface of the hands with warm or cold running water, lathering the backs of the hands, between the fingers and under the nails, with soap, rubbing the hands together for at least 20 seconds, and rinsing completely with clean, running water.

Several studies have been conducted on handwashing knowledge, attitudes, and practices of individuals. It was shown by Duong et al.8 that, while most participants knew the

usefulness of handwashing in curbing the spread of infection, less than half regularly washed their hands daily. This study further noted that community education would emphasise proper handwashing behaviour.

It is not in all situations that awareness of the usefulness of handwashing was found to be sufficient. A study conducted in Saudi Arabia showed that only 46% of students were of the opinion that handwashing could protect against disease, and 34% of the correspondents in that study thought handwashing was only useful for removing dirt.9 The study also showed that many students received handwashing information from their parents.

In the results published by Sultana et al.,10 it was shown that the practice of handwashing could be improved by the removal of obstacles to hand hygiene, such as low availability of soap and adequate water supply within the university.

Due to the major role the hand carriage of micro-organisms plays in the dissemination and selection of resistant micro-organisms, the hand washing awareness of the public must be assessed. If the degree of awareness is high, the spread of micro-organisms reduces, and vice versa. Once the degree of hand washing awareness has been assessed, then crucial steps towards the improvement of public health knowledge and sanitation can be taken.

The WHO states that the “failure to perform appropriate hand hygiene is considered the leading cause of hospital-acquired infections (HAI) and spread of multidrug-resistant organisms, and has been recognised as a significant contributor to outbreaks.”6 Therefore, it is important that awareness of the usefulness of hand washing by the populace be evaluated and the microbial community present on the hands identified and characterised. This study assesses the hand washing practices of a selected student population of the Obafemi Awolowo University, Ile-Ife, Osun, Nigeria, and determines the microbial spectrum as well as the susceptibility of the microorganisms isolated from the palmar surface of those students.

MATERIALS AND METHODS

Study Location

This study was conducted at the Obafemi Awolowo University (OAU), Ile-Ife, Osun State, Nigeria. The University is one of the largest tertiary institutions in Nigeria, with about 35,000 students.

Study Design

This was a cross-sectional study of male and female students between the ages of 18–40 years at Obafemi Awolowo University. The study comprised both the filling of questionnaires and laboratory experiments. The questionnaire was adapted and developed from a previous study,11 and included close-ended questions on the attitude and practices of students when washing their hands.

Study Population

The sample size was calculated using Cochran’s population proportion formula with the following assumptions: proportion of individuals who have transient bacterium present on their palms (p)=95% from a similar study;12 95% confidence interval; z=the standard normal tabulated value; and desired level of precision (margin of error; d)=5%.

Cochran’s formula: N= z2p(1-p) d2

The calculated sample size was approximately 72 participants. To make allowance for nonresponse, a total number of 81 pretested closedended questionnaires were administered.

The criteria for inclusion were participants within the specified age of 18–40 years, participants in classes 100–500 levels, and participants registered full-time in the university. Students were excluded if they did not meet the inclusion criteria, had physical disabilities, had visual and hearing impairments, and were post-graduate students.

Data Collection

The students were approached, and the purpose of the study, as well as what was required of them, were explained thoroughly. Students who volunteered for the study were introduced to the questionnaire, its purpose, and how to fill it. Confidentiality was assured to enable students to fill out the questionnaire accurately and without bias. The questionnaire was self-administered by the students.

Sample Collection and Processing

The various samples were collected over a period of 14 days (15th–28th June 2021) at various sites in the Obafemi Awolowo University.

The palmar surfaces of both hands of the students were swabbed with a sterile swab stick moistened with a solution of sterile 0.1% Tween™ 80 (Croda International, Snaith, UK). The entire palm surface was swabbed in two perpendicular directions to ensure that the maximum surface area of each palm was represented in the sample. The swabs were then suspended in 2.5 mL of the same solution in universal bottles, and shaken on a wrist action shaker for 5 minutes.13

The samples were streaked onto the surface of plates of four different agars: nutrient agar, which serves as a general-purpose growth medium for the isolation and cultivation of bacteria; mannitol salt agar, which selectively allows for the growth of gram-positive bacteria such as Staphylococcus and Micrococcaceae; eosin-methylene blue agar, which serves as a growth medium for the selective growth of gram-negative bacteria; and MacConkey agar, which allows for the selective growth of gram-negative bacteria and differentiates the bacteria based on their ability to ferment lactose. These plates were incubated for 48 hours at 37 ºC. The grown colonies from the nutrient agar plates were then sub-cultured on other freshly prepared nutrient agar plates and incubated at 37 ºC for 48 hours. The various morphological characteristics of the colonies such as size, shape, colour, elevation, surface, and margin were noted.14 The isolated colonies were then stored on nutrient agar slants and cryopreservative medium for further use.

The isolates were gram-stained and subjected to several biochemical tests (catalase, coagulase, indole production, blood agar, biofilm formation, and triple sugar iron tests) to identify them.15

Antimicrobial Susceptibility Testing

Antibiotic susceptibility testing was carried out on all isolates and the disk diffusion method was employed (Kirby–Bauer technique) with Muller–Hinton Agar (Oxoid Ltd, Hampshire, UK).16 The antibiotics tested include tetracycline (30 µg), chloramphenicol (30 µg), novobiocin (5 µg) for 247 of the isolates; and nalidixic acid (30 µg), trimethoprim (2.5 µg), compound sulphonamide (300 µg) for 113 of the isolates. The results were obtained by measuring the zone of inhibition and comparing it with the Clinical and Laboratory Standards Institute (CLSI) 2016 interpretative performance standard for antimicrobial disk susceptibility testing.17,18

Only six antibiotics were used in the susceptibility testing, due to the financial limits of the project. This study was conducted after trade in Nigeria commenced after the COVID-19 pandemic and there was a shortage of antibiotic discs. Due to this, cephalosporins, carbapenems, vancomycin, metronidazole, and combinatorial antibiotics were not available for purchase.

Data Analysis

Data collation was done using Microsoft Excel 365 (Microsoft, Redmond, Washington, USA). Analysis was carried out using SPSS Version 21 (IBM, Armonk, New York, USA). The descriptive statistics used include the mean, standard deviation, frequency, and mean weighted averages. Bar charts were used where necessary.

RESULTS

Sociodemographic Characteristics of Participants

All 81 questionnaires administered were retrieved. Table 1 provides details on the sociodemographic characteristics of the study population. On the sex of the respondents, 54.3% of the respondents were female. The age of the respondents ranged from 16–34 years, with a mean of 21.2±0.9 years.

Table 1: Attitude to hygiene and hand washing practices.

Table 1 continued.

Table 1 continued.

If yes, how?

Attitude to Hand Hygiene and Hand Washing Practices

Concerning their attitude to hand hygiene, the majority of students (34.6%) washed their hands frequently, and 22.2% of the students washed their hands twice a day. Table 2 shows that a slight majority of respondents washed their hands for about 20 seconds to 1 minute, while 40.7% of the respondents washed their hands for less than 20 seconds. The calculated average length of time spent hand washing was 27.8±2.4 seconds.

As shown in Table 1, when questioned about how long ago they practised hand hygiene, 82.7% responded that they last washed their hands over half an hour ago, 9.9% responded less than 15 minutes ago, 4.9% of the respondents responded 5–15 minutes ago, and 2.5% of the respondents responded 15–30 minutes ago. Many of the respondents (39.5%) sometimes washed their hands after using the toilet, and 39.5% of the respondents sometimes washed their hands after eating. Most of the respondents (66.7%) sometimes washed their hands after returning to their room or house after school, with a similar proportion of the respondents never using soap to wash their hands. Most of the respondents (54.3%) never used a hand

sanitiser (alcohol-based) to cleanse their hands, and a vast majority (95.1%) of respondents never used hot water when washing their hands. About half of the students (51.9%) wiped their hands after washing, with 39.8% of the respondents drying by shaking the water droplets off their hands. A majority of respondents noted that the coronavirus outbreak affected their handwashing practices, and acknowledged that their frequency of handwashing increased.

Distribution and Presumptive Identification of the Isolates

Regarding the number of micro-organisms isolated from the palmar surface of the study participants and the presumptive identification of micro-organisms isolated, Staphylococcus epidermidis was the predominant isolate, with 38.10% of the micro-organisms present on the respondents’ palms; Micrococcus spp was a distant second, with 17.58% of the isolates; a total of 11.72% were Staphylococcus aureus; 8.43% were Corynebacterium spp; 5.86% were Streptococcus spp; and 5.13% were from the Bacillus genus.

Table 2: Antibacterial susceptibility pattern of palmar isolates.

Chloramphenicol

The organisms present in the smallest numbers were Listeria monocytogenes (2.56%), Neisseria spp (1.88%), Klebsiella/Enterobacter spp (1.47%), Haemophilus spp (1.10%), and Pseudomonas/ Proteus spp (1.10%), with Staphylococcus saprophyticus and Escherichia coli being the rarest isolates (0.73%).

Figure 1 depicts the distribution of the microorganisms isolated from the palmar surfaces of the study population.

Susceptibility and Resistance Pattern of Isolates

In Table 2, the general susceptibility and resistance pattern of the isolates are described. The isolates were most sensitive to chloramphenicol, regardless of whether they were gram-positive or gramnegative. For the gram-positive organisms, over 70% of the isolates were susceptible to tetracycline and compound sulphonamide. Nalidixic acid and

trimethoprim showed the least efficacy, with only 39.8% of the gram-positive isolates being susceptible. The gram-negative isolates were the least susceptible to trimethoprim and compound sulphonamide (41.7%).

DISCUSSION

The objectives of this study were to describe the handwashing practices of students of Obafemi Awolowo University, to characterise the hand flora in terms of bacteria present, and to determine the patterns of resistance of the isolated micro-organisms to selected antibiotics.

Regarding the hand-washing practices of participants in the study, results indicate that most respondents maintained a form of hand hygiene, including washing hands with or without soap, but most respondents failed to meet the WHO standards for proper hand hygiene, with

Figure 1: Presumptive identification of isolates.

the majority washing their hands for the sufficient length of time, but without soap or warm water.9

It was also noteworthy that most individuals did not use an alcohol-based hand sanitiser. Many students also agreed to washing their hands rarely, once or twice a day, or whenever necessary. This is an insufficient number for proper hand hygiene and is not sufficient to prevent the spread of infection through hand carriage.19,20

On culturing in nutrient agar, a total of 273 colony-forming units (CFU) were isolated from the palmar surfaces of the 81 students, with the majority of individuals having more than 3 CFU on their hands. Out of a total of 273 CFU, a large majority (93.77%) were grampositive, and only 6.23% were gram-negative. Staphylococcus spp. was the most abundant genus to be isolated from the students’ palmar surfaces, with S. epidermidis having the highest

occurrence. Micrococcus spp. was the second most abundant organism isolated, followed by Corynebacterium spp., Streptococcus spp., Bacillus spp., and Listeria spp.

These results were in accordance with literature that states that S. epidermidis is one of the most abundant micro-organisms present on the skin.21 The identity of the other organisms isolated is also in line with previous studies on skin bacteria, although in different population numbers.22,23 This confirms that while the bacterial flora present on the skin is diverse, there are a few constant genera that are considered to be residential bacteria. Transient bacteria have been shown to often be a reflection of the environment.

It has, however, been observed that the skin genus Staphylococcus is more readily cultivated than organisms like Corynebacterium spp., and this difference in growth might lead to potential erroneous results from culture-based surveys. To

overcome this shortcoming, sequencing methods should be henceforth applied to identify members of microbial communities.24

Many of the resident bacteria do not cause disease but some transient micro-organisms are found on the skin that have high pathogenicity and can cause diseases in immunosuppressed individuals.25 There is also a possibility that transient microbe exposure could result in tissue damage and allergic, inflammatory, and autoimmune responses, even after the causative organism is no longer present.26 An important example is S. aureus. The CDC estimated that about 33% of individuals carry S. aureus. S aureus can produce clinically important enzymes such as haemolysins and leucocidins that aid in its pathogenicity.27

Most of the bacteria that inhabit the skin are gram-positive, but there are a few gram-negative bacteria that are found, although infrequently, on the skin. An important example is Pseudomonas aeruginosa (which 1.10% of the isolates in this study were identified as), a gram-negative rod that is a known pathogen. In addition to being associated with leg ulcers, P. aeruginosa is one of the leading causes of morbidity and mortality in burn victims.28

The antimicrobial resistance and susceptibility pattern of the organisms isolated on the palmar surfaces is also crucial, as these organisms can cause serious infections upon entering the bloodstream. Listeriosis, a rare but severe foodborne disease, is caused by Listeria monocytogenes, another organism isolated in this study. Previous studies in the region have shown a high level of resistance of L. monocytogenes isolates to commonly used antibiotics.29 Palmar isolates can also aggravate wounds, infecting them, increasing the time it takes to heal, and reducing the effectiveness of antibacterial agents used. In general, grampositive bacteria showed more sensitivity to the antibacterial agents tested.30

In the interpretation of the results, the number of organisms that displayed an intermediate reaction to the antibiotics will be grouped as resistant to give a clearer picture of the resistance/ susceptibility pattern.

Tetracycline was selected for inclusion in the study because it is one of the most commonly

used (and misused) antimicrobials in the country.31,32 Only 20.2% of gram-positive isolates showed resistance to tetracycline, but that proportion increased to 41.7% in the gramnegative isolates tested.

A study done by Mama et al.33 found that the majority of organisms isolated displayed resistance to nalidixic acid, while a minority showed resistance to chloramphenicol. This is comparable with the results obtained in this study. The resistance of the isolates to chloramphenicol was low in this study (12.96%). Although resistance to this antibiotic is well documented, the low figures of resistance obtained can be attributed to the fact that chloramphenicol is not commonly prescribed due to its side effects.34

Generally, less than 50% of the isolates showed resistance to tetracycline, novobiocin, and sulphonamide. The majority of the isolates tested displayed resistance to trimethoprim. No antibiotic was 100% effective against the isolates.

The high resistance to trimethoprim could be related to its bacteriostatic mode of action and the fact that it is a competitive inhibitor, whose effectiveness will reduce in higher concentrations of dihydrofolate (the substrate it inhibits). When combined with sulphonamides, it is expected that the resistance would reduce, since a bactericidal effect would result.35

Trimethoprim and sulphonamide combination drugs are one of the most prescribed antibiotics in low- and middle-income countries like Nigeria, and this leads to the high levels of resistance seen in this study and in similar studies conducted in the region.36,37

A significant proportion of the gram-positive cocci isolated from the study participants were coagulase-negative Staphylococci (CoNS). CoNS, including S. epidermidis, are often referred to as 'accidental pathogens'. However, in recent years, CoNS have been reported to cause clinically significant ocular symptoms.38 Antibiotic resistance in CoNS is of utmost importance because CoNS, such as S. epidermidis, inhabit the same ecological niche as the pathogenic S. aureus, and may serve as reservoirs of genes that facilitate methicillin-resistant S. aureus (MRSA) infection after horizontal gene transfer.39

As indicated in the results, CoNS are more susceptible to antibiotic action, but susceptibility to nalidixic acid, trimethoprim, and compound sulphonamide is low for both species in the Staphylococci genus, while susceptibility to tetracycline is relatively high. This is in contrast to other studies, such as by Petrillo et al.,38 which showed high susceptibility to trimethoprim and sulphonamide, whilst recording high resistance to tetracycline. Differences in prescriber habits and over-the-counter use may be the reason for the variation in antibiotic resistance trends. Similar studies in Nigeria40 show moderate resistance to chloramphenicol at 45%.

Novobiocin was mainly used to differentiate between S. saprophyticus and other coagulasenegative Staphylococci. This test exploits the intrinsic resistance of S. saprophyticus to the antibiotic. The basis of this inherent resistance is, however, unknown.41 Catalase-positive and coagulase-negative Staphylococci that present with little or no zone of inhibition for novobiocin were presumptively identified as S. saprophyticus. Although simple, a limitation of this test is the fact that occasional human isolates that are not S. saprophyticus may also be resistant to novobiocin. It is therefore recommended that other biochemical, molecular, or immunological methods of testing be employed for confirmatory identification.42

CONCLUSION

The results obtained in this study showed the handwashing practices, spectrum, and susceptibility pattern of the microbial

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This study did not make use of genotypic testing to conclusively identify the organisms isolated. As such, the isolates could only be identified phenotypically. Additionally, the use of culturebased surveys might produce isolates that are more easily cultivated and not a true reflection of the palmar community.

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