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Jeevanu Times Newsletter IAMM Delhi Chapter

Volume: 11

Number: 2

An official publication of Indian Association of Medical Microbiologists – Delhi Chapter


Editorial Committee

Chief Editor:

Dr. Raman Sardana Senior Consultant and Additional Director Medical Services Indraprastha Apollo Hospitals, New Delhi ramansardana@apollohospitals.com

Assistant Editors: Dr. Vikas Manchanda Chacha Nehru Bal Chikitsalaya Geeta Colony, Delhi microcnbc@gmail.com

Dr. Reetika Dawar Indraprastha Apollo Hospitals, New Delhi reetika_d@apollohospitals.com

Scientific Committee Dr. Anita Chakravarty Maulana Azad Medical College New Delhi Dr. Chand Wattal Sir Ganga Ram Hospital Delhi Dr. N P Singh University College of Medical Sciences Delhi Dr. Raman Sardana Indraprastha Apollo Hospitals, New Delhi

Dr. Sarman Singh All India Institute of Medical Sciences New Delhi Dr. Sonal Saxena Lady Hardinge Medical College New Delhi Dr. Vikas Manchanda Chacha Nehru Bal Chikitsalaya Delhi

Dr. Reetika Dawar Indraprastha Apollo Hospitals, New Delhi

Image on cover page: Carbapenemases Detection (Courtesy: Dr. Sumit Rai, UCMS, Delhi)

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Contents Probiotics- Recent Update ...................................................................................................................................................................... 4 Harneet Kaur , Rama Chaudhry. Department of Microbiology, All India Institute of Medical Science, New Delhi Hospital Acquired Pneumonia Including Ventilator Associated Pneumonia................................................................................. 6 Rushika Saksena, S Krishna Prakash. Department of Microbiology, Maulana Azad Medical College, New Delhi MALDI – TOF Assay: in Diagnosis and Research ............................................................................................................................ 13 Kaustuv De, Sumit Rai, Narender Pal Singh, Iqbal R Kaur. Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi Viral Hemorrhagic Fevers .................................................................................................................................................................... 16 Dr. Santosh Bairwa, Dr Sonal Saxena, Dr Renu Dutta. Dept of Microbiology, Lady Hardinge Medical College, New Delhi Carbapenem Resistance ........................................................................................................................................................................ 20 Dr Kanika Gupta, Dr Sonal Saxena, Dr Renu Dutta. Department of Microbiology, Lady Hardinge Medical College and KSCH, Delhi Infectious Diseases and Their HLA Association ............................................................................................................................... 25 Dipmala Das, Narender Pal Singh, Iqbal R Kaur. Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi Tuberculosis Assays: Past, Present and Future................................................................................................................................. 29 Ruchi Kotpal, CP Baveja, Preena Bhalla. Department of Microbiology, Maulana Azad Medical College, New Delhi Crossword puzzle no. 1102 Vikas Manchanda. Chacha Nehru Bal Chikitsalaya, Delhi…………………………………………………...……………………35

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Probiotics- Recent Update Harneet Kaur , Rama Chaudhry Department of Microbiology, All India Institute of Medical Sciences, New Delhi

Probiotics can be defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”. The lactic acid bacteria, particularly species of Lactobacillus, have formed the cornerstone of the probiotic movement to date. The mechanism of action of probiotics is related to their ability to compete with pathogenic microorganisms for adhesion sites, to antagonize these pathogens or to modulate the host’s immune response. Probiotics have shown to have significant clinical beneficial effects in the prevention and management of various infective and non infective disorders. Furthermore, probiotics may also become novel agents in dealing with major public health issues. Research in applications of probiotics in food products will rationalize product development, and health claims. As new frontiers continue to be explored the challenges to the basis of the hygiene hypothesis will influence further developments in the field of probiotics. __________________________________

A probiotic’, by the generally accepted definition, is a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance’ (Rasic,1983).1 The term probiotic was derived from the Greek, meaning “for life.” According to the currently adopted definition by FAO/WHO(2001), probiotics are: ‘Live microorganisms which when administered in adequate amounts confer a health benefit on the host.’2 The first microbe used specifically for this purpose was Lactobacillus bulgaricus, discovered in 1908 by Metchnikoff’s and promoted successfully as a life sustaining fermenting agent for soured milk products. The term probiotic, as an antonym to the term antibiotic, was originally proposed in 1965 by Lilley and Stillwell. The first probiotic species introduced into research were Lactobacillus acidophilus by Hull et al in 1984 and Bifidobacterium bifidum by Holcombh et al in 1991.3 Nowadays, new generation of Scientists and Physicians has identified and begun to evaluate several other micro organisms to have enormous therapeutic potential such as Bifidobacterium species, Enterococcus species and other Lactobacillus species. The lactic acid bacteria, particularly species of Lactobacillus, have formed the cornerstone of the probiotic movement to date. In modern era of medicine, the normal flora is always at risk due to the more and more usage of antibiotics, chemotherapeutic agents in cancers and surgical interventions. Therefore, there is a need to support the gut by friendly bacteria from external sources. Probiotics is one which can offer solution to this emerging problem. Natural sources of probiotics are milk and dairy products. At present, probiotics are almost exclusively consumed as fermented dairy products such as yoghurt or freeze-dried cultures. Probiotics can be added to fermented dairy products, fermented vegetables, fruit juices; muesli bars potato chips and many other food products. The only factor is their stability. Different products contain different strains of bacteria and the health benefits of probiotic bacteria are strain specific. Several factors have shown to pose challenges for probiotics such as water activity, the pH, storage

temperature, presence of oxygen and enzymatic activity. Higher sugar levels appear to improve survival of the probiotics, and incubation at slightly higher temperatures (43°C) may also improve survival, although these factors are usually strain-specific. Fruits, such as raspberry, may have detrimental effects on the survival of probiotics.4 The therapeutic and preventive effects of probiotics are mediated through multiple biological actions. One of the major mechanisms of probiotic action is through the regulation of host immune response. Probiotic genomic and proteomic studies have identified several genes and specific compounds derived from probiotics, which mediate immunoregulatory effects. Studies regarding the biological consequences of probiotics in host immunity suggest that they regulate the functions of systemic and mucosal immune cells and intestinal epithelial cells. This is due to stimulation of dendritic cells with heat inactivated Lactobacillus GG and stimulation of Caco-2 cells with heat-inactivated B. lactis 420, L. acidophilus NCFM or Streptococcus thermophilus St-21 that results in the production of IL-6, TNF-α and NFκB.4 Thus, probiotics have shown a therapeutic potential for several diseases, including several immune response-related diseases, such as allergy, eczema, viral infections, and potentiating vaccination responses. Studies performed in inflammatory bowel diseases (IBD) suggest that high doses of probiotics, preferably cocktails are more effective in decreasing inflammatory score and maintaining patients in remission, than a single probiotic strain. IBD is an inflammatory condition of the GI tract, which is widely considered to be the result of an inappropriate immune response against the GI microbiota. Certain probiotic strains may become effective therapy for inflammatory bowel syndrome (IBS) patients. Studies have demonstrated that GI-associated Clostridium species (predominantly species of Clostridium clusters IV and XIVa) facilitated anti-inflammatory immune responses in mice by promoting the accumulation and activity of regulatory T cells.5 Furthermore, they found that mice with an increased abundance of Clostridium species in the GI tract displayed resistance against experimentally induced IBD. These findings tally with reports that Clostridium

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clusters IV and XIVa are less abundant in fecal samples from IBD patients, compared with those from healthy individuals. Moreover, they suggest that probiotic administration of GI-associated Clostridium species may alleviate the symptoms of IBD and possibly other autoimmune diseases. Also, probiotics may decrease the fecal concentration of enzymes, mutagens and secondary bile salts which may be involved in colon carcinogenesis.6 It has been proven that after ingestion of the probiotic, the Lactobacillus detection rate increases, the total amount of Clostridium perfringens decreases, synthesis of fecal putrefaction products is inhibited, and there is an increase in short-chain fatty acid isobutyric acid.7 These findings suggest the possibility of preventing colorectal carcinoma with probiotics. The health promoting effects of probiotic bacteria are strain specific and results in different mechanism to produce beneficial health impacts. To summarize, probiotics may have at least five functions: 1. Reduction or elimination of potentially pathogenic micro-organism of various kinds; 2. Reduction or elimination of various toxins, mutagens, carcinogens, etc. 3. Modulation of the innate and adaptive immune defense mechanisms. 4. Promotion of apoptosis 5. Release of numerous nutrient, antioxidant, growth, coagulation and other factors

Conclusion Maintenance of the gut environment is a key factor in determining outcome in the care of patients. Probiotics have shown to have significant clinical beneficial effects in the prevention and management of various infective and non infective disorders. Research in applications of probiotics in food products will rationalize product development, and health claims. As new frontiers continue to be explored the challenges to the basis of the hygiene hypothesis will influence further developments in the field of probiotics. References 1.

2.

3.

4.

5.

A number of potential benefits arising from the use of probiotics have been proposed, including: 1. increased resistance to infectious diseases 2. Alleviate lactose intolerance 3. Prevention from diarrhea, gastritis, vaginal and urogenital infections 4. Reduction in blood pressure and regulation of hypertension, serum cholesterol concentration 5. Reduction in allergy, respiratory infections 6. Resistance to cancer chemotherapy and decreasing risk of colon cancer. Furthermore, probiotics may also become novel agents in dealing with major public health issues such as obesity, type I diabetes mellitus, and poor oral health.

6.

7.

Rasic JL (1983). The role of dairy foods containing bifido and acidophilus bacteria in nutrition and health. N Eur Dairy J 4: 80–88. FAO/WHO. Guidelines for the evaluation of probiotics in food. Report of a joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) working group on drafting guidelines for the evaluation of probiotics in food. World Health Organization (2002) ftp://ftp.fao.org/es/esn/food/wgreport2.pdf (Accessed 8 July 2011) Tanboga I, Caglar E, Kargul B (2003). Campaign of probiotic food consumption in Turkish children, oral perspectives-Probiotics for your child’. Int J Pediatr Dent 13(Suppl. 1): 59. Ouwehand C.(2011) Recent advances in probiotic research: a conference update. Future Microbiology.6(9):981-984. Atarashi K, Tanoue T, Shima T et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science331(6015),337–341 (2011). Marteau P R, de Vrese M, . Cellier C J, Schrezenmeir J.Protection from gastrointestinal diseases with the use of Probiotics. Am J Clin Nutr 2001;73(suppl):430S–6S. Iannitti T, Palmieri B. (2010) Therapeutical use of probiotic formulations in clinical practice. Clinical Nutrition 29; 701e725

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Hospital Acquired Pneumonia Including Ventilator Associated Pneumonia Rushika Saksena, S Krishna Prakash Department of Microbiology, Maulana Azad Medical College, New Delhi

Introduction Health care–associated pneumonia (HCAP) is defined as a pneumonia in any patient who was hospitalized in an acutecare hospital for 2 or more days within 90 days of the infection, resided in a nursing home or long-term care facility in the last 90 days, received recent intravenous antibiotic therapy, chemotherapy, or wound care within the past 30 days of the current infection or attended a hospital or hemodialysis clinic in the past 30 days. Hospital acquired pneumonia (HAP) is defined as an inflammatory condition of the lung parenchyma caused by infectious agents not present or incubating at the time of hospital admission; that is, conditions that develop more than 48 h after admission. HAP has been subdivided into pneumonias that occur in the ward and those that arise in the intensive care unit (ICU HAP). ‘Early-onset’ HAP occurs within the first 96 h of admission to the hospital. ‘Late-onset’ HAP arises beyond this time. Ventilator-associated pneumonia (VAP) is a subset of HAP and includes pneumonia that arises more than 48–72 hours after endotracheal intubation. VAP occurs almost exclusively in the ICU and represents approximately 86% of all ICU HAP. Epidemiology Incidence - HAP is the second most common nosocomial infection with a crude overall rate of 6.1 per 1000 discharges. The incidence of HAP is greater among patients

in the ICU with approximately 30% of HAP occurs in critical care settings. Various Asian health care facilities have reported incidences ranging from 1 to 21 per 1000 hospital admissions. The incidence of VAP from the National Nosocomial Infections Surveillance (NNIS) data is 7.6 cases per 1000 ventilator-days with highest incidence for trauma ICUs (15.2 per 1000 ventilator-days). The risk for VAP peaks at day 5 of mechanical ventilation. Mortality - HAP has been shown to have the highest mortality rate of all nosocomial infections. In case of nonICU HAP, mortality rate from HAP varied from 7% in patients in general wards to as high as 62% in patients in bone marrow transplant units. The mortality rate for VAP ranges from 24% to 50%, and can reach as high as 76% in specific settings or when lung infection is caused by highrisk pathogens. Etiology Etiology of HAP and VAP is same in general, except Acinetobacter spp and Stenotrophomonas maltophilia is found predominantly in VAP. Source of infection can be either endogenous or exogenous (Figure 1) Unusual pathogens such as Aspergillus species, Candida species, Legionella pneumophila, Pneumocystis jiroveci, Nocardia species and viruses are causes of HAP and VAP in patients who are immunosuppressed.

Epidemiology in India Study

Joseph et al (2009) (JIPMER, Pondicherry) Rakshit et al (2005) (Grant Medical College, Mumbai) Mukhopadhyay et al (2003) (SGPGI, Lucknow)

Incidence of HAP/VAP

22.94 per 1000 ventilator days

26 per 1000 ventilator days

HAP - 53.9/100 ventilated patients VAP - 81.7/100 ventilated patients

Crude Mortality Rate 16.2%

37%

47.3%

Tarsheen Kaur Sethi, M.K. Daga, S. Krishna Prakash, R Kumar (LNH, Delhi) (2005 – 06)

VAP – 30% of patients

80%

Kaur et al (2003)

VAP – 30% of patients

-

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FIGURE 1

The following etiological agents are commonly isolated:

ORGANISM ISOLATED

FREQUENCY OF ISOLATION (% OF PATIENTS)

Gram-negative bacilli Pseudomonas aeruginosa Acinetobacter species Escherichia coli Klebsiella species Enterobacter species Proteus species Serratia marcescens Stenotrophomonas maltophilia

35-80%

Gram-positive cocci Streptococcus pneumoniae Streptococcus species Staphylococcus aureus (MSSA and MRSA)

9–46%

Polymicrobial

9-80%

No growth

2-54%

Pathogenesis In the normal nonsmoking host, multiple host defence mechanisms play an essential role in prevention of pneumonia. These include anatomy of airways, cough reflex, mucus, mucociliary clearance mechanism. In the lower respiratory tract below the terminal bronchioles, alveolar macrophages, leukocytes, immunoglobulin, complement, lactoferrin, basement membrane plays a protective role.

In the mechanically ventilated patient, a number of factors conspire to compromise host defences such as critical illness, comorbidities, and malnutrition impair the immune system. Endotracheal intubation thwarts the cough reflex, compromises mucociliary clearance, injures the tracheal epithelial surface and provides a direct conduit for rapid access of bacteria from above into the lower respiratory tract. Invasive devices and procedures and antimicrobial therapy helps create a favorable milieu for antimicrobial-

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resistant nosocomial aerodigestive tract.

pathogens

to

colonize

the 4)

Routes of colonization/infection in mechanically ventilated patients (Figure 2) Endogenous Routes 1) Oropharyngeal Colonization - Normal flora of the oropharynx in the nonintubated patient without critical illness is composed predominantly of viridans streptococci, Haemophilus species, and anaerobes. During critical illness, especially in ICU patients, the oral flora shifts dramatically to a predominance of aerobic Gram-negative bacilli and Staphylococcus aureus. Bacterial adherence to the orotracheal mucosa is further facilitated by reduced mucosal IgA and increased protease production, exposed and denuded mucous membranes, elevated airway pH, increased numbers of airway receptors for bacteria, due to acute illness and antimicrobial use. The leakage of endotracheal secretions around the endotracheal cuff leads to microaspiration of oropharyngeal contents containing a large bacterial inoculum that overwhelms host defences which are already compromised leading to development of VAP. 2)

3)

Gastric Colonization and Aspiration - In healthy persons, few bacteria entering the stomach survive in the presence of gastric acid. Conditions that reduce the gastric pH, such as achlorhydria, treatment with H2 antagonists or proton-pump inhibitors, or enteral nutrition, predispose to bacterial proliferation in the stomach. Gastric microorganisms reflux up the esophagus, abetted by recumbency and the ever-present naso- or orogastric tube which is aspirated into the trachea leading to development of VAP. This is not a major pathogenic route for development of VAP. Hematogenous spread from distant sites of infection, although not a common cause, may also occur in postoperative patients as well as in patients with intravenous or urinary catheters.

Exogenous routes Exogenous infection with nosocomial pathogens acquired from the hospital environment is less common and generally occurs late in the ICU admission. 1) Biofilms of the Endotracheal Tube (ETT) - The ETT acts as a reservoir for infecting microorganisms, which adhere to the surface of the foreign body, producing a biofilm. 2) Health care workers or medical equipment may harbour pathogenic flora that can prompt colonization of the tracheobronchial tree. 3) Devices used on the respiratory tract for respiratory therapy (e.g., nebulizers, endotracheal tubes), diagnostic examination (e.g., bronchoscopes or spirometers), or administration

5) 6) 7)

of anesthesia are potential reservoirs or vehicles for microbes Contaminated humidification reservoirs during mechanical ventilation may lead to aerosolization of pathogens and subsequent colonization and infection. Condensates of ventilator circuits can also be a potential source of microorganisms Contaminated hospital water may lead to infection with P. aeruginosa, Legionella spp. Contaminated ambient air may cause invasion of filamentous fungi, Mycobacterium tuberculosis, SARS corona virus.

FIGURE 2: Routes of colonization/infection mechanically ventilated patients

in

Risk factors for HAP Host factors - Factors enhancing airway colonization - previous and continuing antibiotic therapy, endotracheal intubation, smoking, malnutrition, general surgery, dental plaque, renal dysfunction, diabetes, coma, shock, advanced age and underlying lung disease - Factors related to surgery - preoperative smoking, longer preoperative admissions, longer surgical procedure times, and thoracic or upper abdominal surgery, cardiothoracic surgery and head trauma Other independent factors include male sex, ICU admission for trauma, intermediate severity of underlying disease, longer time on ventilation > 7 days, reintubation, body position during ventilation â&#x20AC;&#x201C; supine position increases risk, enteral feeding , Glasgow coma score of < 9, chronic obstructive pulmonary disease, extensive burns, neurosurgical conditions, ARDS also implicated. Environmental factors -In-dwelling nasogastric tubes increase risk of gastroesophageal reflux. -Nasopharyngeal intubation increase risk of concomitant sinusitis.

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- the movement of ICU patients for diagnostic and surgical procedures out of the ICU is an independent risk factor for VAP. Pharmacological factors Drugs raising gastric pH and administration of a paralytic drug predispose to HAP. The role of prophylactic antibiotics is controversial. Some experts suggest that prophylactic antibiotics in the ICU encourage the risk of superinfection by multiresistant bacteria but delay the onset of nosocomial infection. Clinical criteria used for diagnosing VAP 1) CPIS Criteria was proposed by Pugin et al in 1991. A score of ≥ 6 of the maximum 12 correlates well with high bacterial counts isolated from lower respiratory tract. Modified CPIS score given by Fartoukh et al where microbiology is not relevant. Only positive Gram-stain of a BAL specimen or Protected Specimen Brushing (PSB) was taken into consideration. Microbiological diagnosis The bacteriologic strategy uses quantitative cultures of lower respiratory secretions (endotracheal aspirates, BAL or PSB specimens collected with or without a bronchoscope) to define both the presence of pneumonia and the etiologic pathogen. Growth above a threshold concentration is required to diagnose VAP/ HAP and to determine the causative microorganism(s) whereas below the threshold is assumed to be due to colonization or contamination. The results are used to guide decisions about whether to start antibiotic therapy, which pathogens are responsible for infection, which antimicrobial agents to use, and whether to continue therapy. Procedures to quantitatively identify likely pathogens include 1) Endobronchial aspirates - The results of quantitative cultures on specimens obtained by endotracheal aspiration vary depending on the bacterial load, the duration of mechanical ventilation and the prior administration of antibiotics. Sensitivity and specificity can range from 38% to 100% and 14% to 100%, respectively. The threshold for infection is taken as 10 cfu/ml.

2) Bronchoscopic techniques – i. Bronchoscopic BAL – Atleast 140 ml of sample should be taken. A cut-off of 10 cfu/ml is taken as a positive result but 10³cfu/ml – 105 cfu/ml also taken as positive in various studies, with sensitivity inversely proportional to the cutoff. Samples contaminated by upper airway secretions, as reflected by a high percentage of squamous epithelial cells, should be used with caution. The sensitivity and specificity is 73% and 82%, respectively. Detection of intracellular organisms is highly specific (89-100%) and has a high positive predictive value. ii. Protected Sample Brushing (PSB) – 10³ cfu/ml is taken as positive. It is more specific (95%) than sensitive (67%). The quality of sample is difficult to measure and reproducibility is not exact. 3) Nonbronchoscopic techniques i. Blinded bronchial sampling (BBS) – In this technique, a catheter is blindly wedged into a distal bronchus, and secretions are aspirated without instillation of fluid. ii. Mini-BAL - A sterile, single sheathed, 50 cm, plugging, telescoping catheter is used and 20 mL to 150 mL of lavage fluid is instilled. An unprotected catheter can also be used, sometimes. iii. Blinded Protected Specimen Brushing - a sterile brush that is protected from contamination is used. Qualitative cultures of endotracheal secretions used in lieu of invasive diagnostic testing, because health care workers can perform the aspiration procedure at the bedside with minimal training. Typically, qualitative cultures identify pathogenic organisms found by invasive tests and thereby suggest high sensitivity. Such tests frequently identify nonpathogenic organisms as well, thereby reducing the positive predictive value of this procedure.

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2) CDC Criteria Radiological signs (Atleast 1 in ≥2 serial CxR)

Microbiological criteria (Atleast 1 of the following)

Other criteria (Atleast 2 of the following)

Fever (temperature > 38˚C)

Positive growth in blood culture not related to another source of infection

New onset of purulent sputum, or change in character of sputum

Consolidation

Leukopenia (< 4000 WBC) or leukocytosis (>12000 WBC)

Positive growth in culture of pleural fluid

Increased resp. Secretions, or increased suctioning requirements

Cavitation

Altered mental status, for adults 70 yrs or older, with no other recognized cause

Positive quantitative culture from BAL (> 10CFU/ml) or protected specimen brushing (> 10³ CFU/ml)

New-onset or worsening cough or dyspnea or tachypnea

5% or more of cells with i/c bacteria on Gram-stain of BAL fluid

Rales or bronchial sounds

Histopathological evidence of pneumonia

Worsening gas exchange

New or progressive persistent infilterate

and

Clinical signs (Atleast 1 of the following)

Increased oxygen requirements

Recommendations for Diagnostic Strategies (American Thoracic Society, 2005) 1. A patients with suspected VAP should have a lower respiratory tract sample sent for culture, and extrapulmonary infection should be excluded, as part of the evaluation before administration of antibiotic therapy 2. If there is a high pretest probability of pneumonia, or in the 10% of patients with evidence of sepsis, prompt therapy is required, regardless of whether bacteria are found on microscopic examination of lower respiratory tract samples 3. Diagnostic techniques that identify etiologic pathogens on the basis of qualitative cultures will lead to therapy for more organisms than diagnostic techniques based on quantitative cultures 4. Semiquantitative cultures of tracheal aspirates cannot be used as reliably as quantitative cultures to define the presence of pneumonia and the need for antibiotic therapy 5. If bronchoscopic sampling is not immediately available, nonbronchoscopic sampling can reliably obtain lower respiratory tract secretions for quantitative cultures, which can be used to guide antibiotic therapy decisions 6. Delays in the initiation of appropriate antibiotic therapy can increase the mortality of VAP and thus therapy should not be postponed for the purpose of performing diagnostic studies in patients who are clinically unstable

Antimicrobial selection and resistance In various studies, it has been seen that MRSA accounts for 40-70% of all S. aureus isolated from HAP patients. According to the NNIS, 21.1% P. aeruginosa isolates were resistant to imipenem, 31.9% to 3rd generation cephalosporins, 29.5% to quinolones and 17.5% to piperacillin. Resistance to 3rd generation cephalosporins was seen in 20.6% of K. pneumonia and 5.8% of E. coli. Selection of appropriate antibiotics for the initial management of HAP, VAP, and HCAP is done on the basis of time of onset of disease and risk for MDR pathogens. Broad-spectrum empiric antibiotic therapy should be accompanied by a commitment to deescalate antibiotics, on the basis of serial clinical and microbiologic data, to limit the emergence of resistance in the hospital. Thus, following strategies can be followed for the empirical treatment of VAP. Initial empiric antibiotic therapy for hap or vap in patients with no known risk factors for mdr pathogens, early onset, and any disease severity Potential Pathogen

Recommended Antibiotic

Streptococcus pneumoniae Haemophilus influenzae Methicillin-sensitive Staphylococcus aureus Antibiotic-sensitive enteric gram-negative bacilli Escherichia coli Klebsiella pneumoniae Enterobacter species Proteus species Serratia marcescens

Ceftriaxone or Levofloxacin, moxifloxacin, or ciprofloxacin or Ampicillin+sulbactam or Ertapenem

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Initial empiric therapy for hap, vap and hcap in patients with late-onset disease or risk factors for mdr pathogens and all disease severity.

equipment or devices used for respiratory therapy, pulmonary-function testing, or delivery of inhalation anesthesia.

Potential Pathogens

Combination Antibiotic Therapy

Pathogens listed above and MDR pathogens

Antipseudomonal cephalosporin (cefepime, ceftazidime)

Pseudomonas aeruginosa

Or

Klebsiella pneumoniae (ESBL)

Antipseudomonal carbepenem (imipenem or meropenem) or β-Lactam+β-lactamase inhibitor (piperacillin–tazobactam) AND Antipseudomonal fluoroquinolone (ciprofloxacin or levofloxacin)

Acinetobacter species Methicillin-resistant Staphylococcus aureus (MRSA) Legionella pneumophila

Or Aminoglycoside (amikacin, gentamicin, tobramycin) AND Linezolid or vancomycin

or

If an ESBL strain, such as K. pneumoniae, or an Acinetobacter species is suspected, a carbepenem is a reliable choice. If L. pneumophila is suspected, the combination antibiotic regimen should include a macrolide (e.g., azithromycin) or a fluoroquinolone (e.g., ciprofloxacin or levofloxacin) should be used rather than an aminoglycoside. In selecting empiric therapy for patients who have recently received an antibiotic, an effort should be made to use an agent from a different antibiotic class. Initial antibiotic therapy should be given promptly. Antibiotic restriction can limit epidemics of infection with specific resistant pathogens. Heterogeneity of antibiotic prescriptions, including formal antibiotic cycling, may be able to reduce the overall frequency of antibiotic resistance. Surveillance of VAP Active surveillance is required to accurately identify patients with VAP. Case finding by review of administrative data alone, such as discharge diagnosis codes, is inaccurate and lacks both sensitivity and specificity. Surveillance for bacterial pneumonia should be conducted in ICU patients who are at high risk for HCAP (e.g., patients with mechanically assisted ventilation or selected postoperative patients) to determine trends and help identify outbreaks and other potential infection-control problem. The following guidelines should be kept in mind: • Include data on the causative microorganisms and their antimicrobial susceptibility patterns • Express data as rates (e.g., number of infected patients or infections per 100 ICU days or per 1,000 ventilator days) to facilitate intrahospital comparisons and trend determination • In the absence of specific clinical, epidemiologic, or infection-control objectives, do not routinely perform surveillance cultures of patients or of

Prevention and control measures These measures are designed to interrupt the three most common mechanisms by which HAP develops: 1) Colonization of aerodigestive tract 2) Aspiration of secretions 3) Use of contaminated equipment General Strategies i. Conduct active surveillance for VAP. ii. Adhere to hand-hygiene guidelines published by the Centers for Disease Control and Prevention or the World Health Organization. iii. Use noninvasive ventilation whenever possible. iv. Minimize the duration of ventilation. v. Perform daily assessments of readiness to wean and use weaning protocols. vi. Educate healthcare personnel who care for patients undergoing ventilation about VAP Strategies to reduce colonization of aerodigestive tract i. Orotracheal intubation is preferable to nasotracheal intubation ii. Avoid H2–blocking agents and proton pump inhibitors for patients who are not at high risk for developing a stress ulcer or stress gastritis iii. Perform regular oral care with an antiseptic solution. Strategies to prevent aspiration i. Maintain patients in a semirecumbent position (30˚- 45˚ elevation of the head of the bed) unless there are contraindications ii. Avoid gastric overdistention. iii. Avoid unplanned extubation and reintubation. iv. Use a cuffed endotracheal tube with in-line or subglottic suctioning. v. Maintain an endotracheal cuff pressure of at least 20 cm H2O. Strategies to minimize contamination of equipment used to care for patients receiving mechanical ventilation - Most devices are classified as semicritical in Spaulding Classification. - Thus, after they are thoroughly cleaned, they can be subjected to high-level disinfection by either using liquid chemical disinfectants or by pasteurization at >70˚C for 30 minutes. -Sterile water is preferred to tap or unsterilized distilled water for rinsing off residual liquid chemical disinfectant from a respiratory device that has been chemically disinfected for reuse. - If this is not feasible, rinse the device with filtered water or tap water, and then rinse with isopropyl alcohol and dry with forced air or in a drying cabinet.

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References 1.

2.

3.

4.

American Thoracic Society: Guidelines for the management of adults with hospital-acquired, ventilator-associated, and health-care associated pneumonia. Am J Respir Crit Care Med 2005. C Rotstein, G Evans, A Born, et al. Clinical practice guidelines for hospital-acquired pneumonia and ventilator-associated pneumonia in adults. Can J Infect Dis Med Microbiol 2008;19(1):19-53. Safdar N, Crnich CJ, Maki DG. The Pathogenesis of Ventilator-Associated Pneumonia: Its Relevance to Developing Effective Strategies for Prevention. Respir Care 2005;50(6):725â&#x20AC;&#x201C;739. CDC. Guidelines for Preventing Health-CareAssociated Pneumonia, 2003. Recommendations of the

5.

6.

7.

CDC and the Healthcare Infection Control Practices Advisory Committee. MMWR 2004. Coffin SE, Klompas M, Classen D. Strategies to Prevent Ventilator-Associated Pneumonia in Acute Care Hospitals. Infect Con Hos Epid 2008;29(1):31-40. Chastre J, Trouillet J-L, Combes A. Diagnostic Techniques and Procedures for Establishing the Microbial Etiology of Ventilator-Associated Pneumonia for Clinical Trials: The Pros for Quantitative Cultures. Clin Infect Dis 2010; 51(S1):S88â&#x20AC;&#x201C;S92. Joseph NM, Sistla S, Dutta TK. Ventilator-associated pneumonia in a tertiary care hospital in India: incidence and risk factors. J Infect Dev Ctries 2009; 3(10):771777.

Solution to the crossword puzzle 1202 (page 35 this issue)

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MALDI – TOF Assay: in Diagnosis and Research Kaustuv De, Sumit Rai, Narender Pal Singh, Iqbal R Kaur Department of Microbiology, University College of Medical Sciences, Delhi

Introduction Conventional culture and diagnostic methods have been surpassed by many newer methods. Among the many developed and validated methods, mass spectrometry (MS) based methods are one of the most nascent methods. Among the various MS methods, Matrix Ass Assisted Laser Desorption Ionization – Time of Flight (MALDI – TOF) MS is one of the most upcoming techniques increasingly being used in microbiology. MS is an analytical technique that measures the mass-tocharge ratio of charged particles. It is used for determining d masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds unds to generate charged molecules or molecule fragments and measuring their mass-to-charge charge (e/z) ratios. Ions produced from the target analyte are moved using the charge dipole (cathode / anode). A strong magnetic field is used to deflect these ionized particles. rticles. Depending upon the charge to mass ratio, the particles are separately detected and converted into graphic representation and compared with prefed data for analysis. Largest e/z represents the singly charged intact analyte while subsequent lower e/ e/z ratio represents different fragmented ions. Matrix Assisted Laser Desorption/Ionization MALDI – TOF is a technique based on the afore mentioned MS technique. It is a soft ionization technique used for analysis of biomolecules and large organic molecules molecule which are fragile and tend to fragment with conventional ionization. MALDI is a two step process: The first includes a Desorption procedure that is triggered by a UV laser beam. The matrix material heavily absorbs UV laser light with ablation of upper layer ayer of the matrix material. A hot plume (ionic cloud) produced during the ablation contains many species: neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters and nanodroplets. The second step is ionization oor more accurately protonation or deprotonation. Protonation (deprotonation) of analyte molecules takes place in the hot plume. Some of the ablated species also participate in protonation (deprotonation) of analyte molecules. The exact mechanism of MALDI is still debated. Matrix of MALDI The matrix material used in MALDI consists of crystallized molecules used in a solution of mixture of highly purified water and organic solvent (Acetonitrile [ACN] or Ethanol). Trifluoroacetic acid [TFA] may be added as a stabilizer. A good example may be a 20 mg/ml of matrix in ACN: water: TFA (50:50:0.1).

A matrix material must be of fairly low molecular weight (to allow facile vaporization) but large enough (ie with a low enough vapour pressure) so as not to evaporate dduring sample preparation or while standing in the spectrometer. It is often acidic and acts as a proton source, though basic matrices are used. It also must have a strong optical absorption in either the UV or IR range to rapidly and efficiently absorb the laser irradiation, a property attributed to their conjugated double bonds. They are functionalized with polar groups allowing preparation of aqueous solutions. Table 1: Showing the various matrix compounds and compatible solvents

Different lasers have been used in MALDI assays. Some of these include UV lasers such as nitrogen lasers (337 nm), Nd:YAG lasers with their frequency tripled or quadrupled Loading a Sample The sample preferably should be a purified growth on solid media, suspended in a suita suitable broth (Trypticase Soy Broth). But currently numerous studies are being conducted for identifying organisms directly from the clinical samples. The matrix solution is mixed with the analyte. A mixture of water and organic solvent allows both hydrophobic and hydrophilic molecules to dissolve into the solution. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize leaving only the recrystallized matrix with analyte molecules embedded into MALDI crystals. The matrix and the analyte are said to be co-crystallized. co Ionization Method The laser is fired at the matrix crystals in the dried-droplet dried spot. The matrix absorbs the laser energy and it is thought that primarily the matrix is desorbed and ionized (by 13


addition of a proton) by this event. The matrix transfer proton to the analyte molecules (e.g., protein molecules) charging the analyte. Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na]+ in the case of an added sodium ion, or [M-H]- in the case of a removed proton. MALDI is capable of creating singly charged ions, but multiply charged ions ([M+nH]n+) can also be created. The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer) mainly due to its large mass range. The TOF measurement procedure is also ideally suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation. Time of Flight (TOF) Time-of-flight mass spectrometry (TOF MS) is a method of mass spectrometry in which an ion's mass-to-charge (e/z) ratio is determined using time measurement. Ions are accelerated by an electric field of known strength resulting in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach the detector at a known distance is measured. This time will depend on the mass-tocharge ratio of the particle with heavier particles reaching lower speeds. Mathematics of the Calculation: Using this time and the known experimental parameters one can find the mass-tocharge ratio of the ion. Potential Energy (Ep) of a charged particle Ep = q x U (q – charge, U – electric potential) Kinectic Energy (Ek) of an accelerated charged particle Ek = ½ m v2 (m – mass, v – velocity) Ep = EK or qU = ½ m v2 Velocity of the charged particle after acceleration does not change as it moves through a field free TOF tube. If ‘d’ is the tube length and t is the time to digital converter (detector), then, V = d/t So, qU = ½ m (d/t)2 Or t = k √m / √q where k is a constant d/√2U (The kind of ionization of peptides produced by MALDI is typically +1 ions, so q = e) Different techniques may be used to enhance TOF MS like: Delayed Extraction, Reflectron, Orthogonal acceleration, TOF / TOF Tandem MS etc. The explanation of these variations of TOF is beyond the scope of this review.

Surface Enhanced LDI (SELDI): A Modification of MALDI Surface-enhanced laser desorption/ionization (SELDI) ionization method in mass spectrometry is used for the analysis of protein mixtures. SELDI is typically used with TOF mass spectrometers and is used to detect proteins in tissue samples, blood, urine, or other clinical samples. The protein mixture is spotted on a surface modified with a pre – determined chemical functionality. Some proteins in the sample bind to the surface, while the others are removed by washing. Matrix is now applied to the surface and allowed to crystallize. Binding to the SELDI surface acts as a separation step with subsets of proteins being easier to analyze. Some common surfaces include CM10 (weakpositive ion exchange), H50 (hydrophobic surface), IMAC30 (metal-binding surface). Surfaces can also be functionalized with antibodies, other proteins, or DNA. Detector The ion detector typically consists of microchannel plate detector or a fast secondary emission multiplier (SEM) where first converter plate (dynode) is flat. The electrical signal from the detector is recorded by means of a time to digital converter (TDC) or a fast analog-to-digital converter (ADC). Uses of MALDI-TOF-MS MALDI TOF MS has been widely used in proteomic based studies, organic and polymer chemistry and clinical microbiology. However a consolidated, validated, comprehensive, uniform and an easily accessible database for microbial detection is yet to be made available. Direct extraction of bacterial vegetative cells or spores followed by MALDI TOF MS analysis has become popular for bacterial identification, since it is simple to perform and mass spectra are readily interpreted. However, only highabundance peptides that are of low mass and ionize readily are observed (e.g., those in the 2,000 to 10,000-Da mass range). Generally the spectra are plotted as the amount of each protein present (as defined by its molecular weight [MW]). Unfortunately, MW alone is not sufficient to identify a characteristic biomarker, and one must rely on the entire spectrum - often referred to as mass profiling or fingerprinting. Recently a study was performed that used the MALDI TOF MS based rapid method for detecting carbapenem resistance within 1 – 2.5 hours. This has a major clinical significance considering the upsurge in carbapenem resistance especially in members of family Enterobacteriaceae. Another study was performed for identification of Aspergillus spp using MALDI TOF MS from the A database that included the reference spectra of 28 clinically relevant species from seven Aspergillus sections Pitfalls and Shortcomings Raw spectral data from MALDI-TOF with MS profiling techniques usually contains complex information not readily providing biological insight into disease. The association of identified features within raw data to a known peptide is extremely difficult. Data pre – processing to remove uncertainty characteristics in the data is normally 14


required before performing any further analysis. The first high throughput screening (HTS) applications which have proven to be the gold standard and are currently being used for database update are genotyping of single nucleotide polymorphisms. The MALDI database requires a continuous up gradation using such genetic methods. Until and unless that has not been standardized, it’s universal use and as a back up to replace conventional techniques remains uncertain. Mass spectrometry based methods are expected to enable a very early diagnosis of diseases with minimally invasive methods of investigation. This type of high end screening application has the potential to revolutionize the early diagnosis of many diseases

References 1. 2. 3. 4. 5.

http://en.wikipedia.org/wiki/Matrixassisted_laser_desorption/ionization http://en.wikipedia.org/wiki/Time-offlight_mass_spectrometry http://en.wikipedia.org/wiki/Mass_spectrometry Book on Advanced Techniques in Microbiology Mazzeo MF, Sorrentino A, Gaita M, Cacace G, Stasio MD, Facchiano A et al. Matrix-Assisted Laser Desorption

Ionization-Time of Flight Mass Spectrometry for the Discrimination of Food-Borne Microorganisms. Appl. Environ. Microbiol. February 2006 72:1180-1189; doi:10.1128/AEM.72.2.1180-1189.2006 6. http://www.bartsandthelondon.nhs.uk/assets/docs/OurServices/Pathology/Introduction-of-MALDI-TOF-arevolution-in-diagnostic-microbiology.pdf 7. Fox A. Mass Spectrometry for Species or Strain Identification after Culture or without Culture: Past, Present, and Future J. Clin. Microbiol 2006; 44:2677-80 8. http://cadth.ca/media/pdf/htis/april-2011/L0259_MALDITOF_Final.pdf 9. Burckhardt I, Zimmermann S. Using Matrix Assissted Laser Desorption Ionization- Time of Flight Mass Spectroscopy to detect carbapenem resistance within 1 to 2.5 hours. J Clin Microbiol 2011; 49: 3321–4 10. Alanio A, Beretti JL, Dauphon B, Mellado E, Quesne G, Lacroix C. Matrix assisted laser desorption and ionization time id flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species. Clin Microbiol Infect 2010; 17: 750-5 11. Application of MALDI – TOF Mass Spectrometry in screening and diagnostic research. Curr Pharma Design 2005; 11: 2577-91.

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Viral Hemorrhagic Fevers Dr. Santosh Bairwa, Dr Sonal Saxena, Dr Renu Dutta Department of Microbiology, Lady Hardinge Medical College, New Delhi

Introduction In general, the term "viral hemorrhagic fever" is used to describe a severe multisystem syndrome (multisystem in that multiple organ systems in the body are affected). Characteristically, the overall vascular system is damaged, and the body's ability to regulate itself is impaired. These symptoms are often accompanied by hemorrhage (bleeding); however, the bleeding is itself rarely lifethreatening. While some types of hemorrhagic fever viruses can cause relatively mild illnesses, many of these viruses cause severe, life-threatening disease. Viral hemorrhagic fever (VHF) refers to a group of illnesses caused by 4 distinct families of RNA viruses that effect humans and non-human primates. VHF viruses are members of four distinct families: Arenaviruses, Filoviruses, Bunyaviruses and Flaviviruses. These families share a number of features: • They are all RNA viruses, and all are covered, or enveloped, in a fatty (lipid) coating. • Their survival is dependent on an animal or insect host, called the natural reservoir. • The viruses are geographically restricted to the areas where their host species live. • Humans are not the natural reservoir for any of these viruses. Humans are infected when they come into contact with infected hosts. However, with some viruses, after the accidental transmission from the host, humans can transmit the virus to one another. • Human cases or outbreaks of hemorrhagic fevers caused by these viruses occur sporadically and irregularly. The occurrence of outbreaks cannot be easily predicted. • With a few noteworthy exceptions, there is no cure or established drug treatment for VHFs. In rare cases, other viral and bacterial infections can cause a hemorrhagic fever; scrub typhus is a good example. Transmission of Viruses causing VHF Viruses associated with most VHFs are zoonotic. This means that these viruses naturally reside in an animal reservoir host or arthropod vector. They are totally dependent on their hosts for replication and overall survival. For the most part, rodents and arthropods are the main reservoirs for viruses causing VHFs. The multimammate rat, cotton rat, deer mouse, house mouse, and other field rodents are examples of reservoir hosts. Arthropod ticks and mosquitoes serve as vectors for some of the illnesses. However, the hosts of some viruses remain unknown -- Ebola and Marburg viruses are well-known examples.

Viruses causing hemorrhagic fever are initially transmitted to humans when the activities of infected reservoir hosts or vectors and humans overlap. The viruses carried in rodent reservoirs are transmitted when humans have contact with urine, fecal matter, saliva, or other body excretions from infected rodents. The viruses associated with arthropod vectors are spread most often when the vector mosquito or tick bites a human, or when a human crushes a tick. However, some of these vectors may spread virus to animals, livestock, for example. Humans then become infected when they care for or slaughter the animals. Some viruses that cause hemorrhagic fever can spread from one person to another, once an initial person has become infected. Ebola, Marburg, Lassa and Crimean-Congo hemorrhagic fever viruses are examples. This type of secondary transmission of the virus can occur directly, through close contact with infected people or their body fluids. It can also occur indirectly, through contact with objects contaminated with infected body fluids. For example, contaminated syringes and needles have played an important role in spreading infection in outbreaks of Ebola hemorrhagic fever and Lassa fever. Clinical manifestations of VHF VHF is a severe multi-system syndrome characterized by diffuse vascular damage. Bleeding often occurs and depending on the virus may or may not be life threatening. Some VHF’s cause mild disease presenting onlt with fever, myalgia, malaise, fatigue, bradycardia, conjunctival injection and mild gastrointestinal symptoms. Severe cases of VHF show bleeding under the skin (petechiae), in internal organs or from body orifices like mouth, eyes, ears and vagina. Jaundice, oliguria and decreased renal function may be present. ARENAVIRIDAE The Arenaviridae family contains the following viruses: Junin, Machupo, Sabia, Guanarito, and Lassa. History:In 1958, the Junin virus was isolated in the plains of Argentina in agricultural workers. It was the first arenavirus found to cause hemorrhagic fever. Others soon followed including Machupo virus in Bolivia in 1963 and Lassa virus in Nigeria in 1969. Since 1956, a new arenavirus has been discovered every one to three years, but not all cause hemorrhagic fever. BUNYAVIRIDAE This is the largest family of viruses contains more than 300 species. The Bunyaviridae family include: Crimean-Congo hemorrhagic fever, Hantavirus, and Rift Valley fever. History:RVF virus was first isolated in 1930 from an infected newborn lamb, as part of investigation of a large epizootic of disease causing abortion and high mortality in 16


sheep in Egypt. Crimean-Congo Hemorrhagic Fever virus was first recognized in the Crimean peninsula located in southeastern Europe on the northern coast of the Black Sea in the mid-1940s, when a large outbreak of severe hemorrhagic fever among agricultural workers was identified. The outbreak included more than 200 cases and a case fatality of about 10%. CCHF is found in Eastern Europe, Western China, Central Asia Southern Europe, Middle Africa, Middle East and Indian subcontinents. Recently, 3 CCHF cases have been reported from Ahmadabad district of Gujarat state. The discovery of Hantaviruses traces back to 1951 to 1953 when United Nations troops were deployed during the border conflict between North and South Korea. More than 3,000 cases of an acute febrile illness were seen among the troops, about one third of which exhibited hemorrhagic manifestations, and an overall mortality of 5% to 10% was seen.

are long thread like viruses (filum means thread) and can appear in shapes like a U, figure of six or spiraled. History: Marburg virus was first isolated in 1967 from several cases of hemorrhagic fever in European laboratory workers in Germany and former Yugoslavia working with tissues and blood from African green monkeys imported from Uganda. Ebola virus was first reported simultaneously in Zaire and Sudan in 1976 when two distinct subtypes were isolated in two hemorrhagic fever epidemics. Both subypes later named Zaire and Sudan caused severe disease and mortality rates greater than 50%. A third subtype of Ebola (Reston) was later found in macaques imported from the Philippines into the US in 1989 and Italy in 1992. Four humans were asymptomatically infected and recovered without any signs of hemorrhagic fever. In 1994, a fourth subtype of Ebola was isolated from a animal worker in Côte d'Ivoire who had preformed a necropsy on an infected chimpanzee. Scattered outbreaks have occurred periodically with latest being an outbreak of Ebola in the Republic of the Congo in 2003.

FILOVIRIDAE This family contains two morphologically identical but antigenically different viruses Marburg and Ebola. These

Table:1 Family: Arenaviridae Virus Lassa Junin

Geography West Africa Argentine, Pampas

Reservoir Rodents Rodent

Vector None None

Machupo

Disease Lassa fever Argentine viral fever Bolivian fever

Bolivia

Rodent

None

Guanarito

Venezuelan H F

Venezuela

Rodent

None

Treatment i.v. Ribavarin Convalescent plasma & Ribavarin-suggested Convalescent plasma & Ribavarin-suggested Unknown

Sabia

Brazilian HF

Brazil

unknown

None

Unknown, Ribavarin Suggested

Table.2 Family: Bunyaviridae Virus

Disease

Geography

Reservoir

Vector

Treatment

Phlebovirus

Rift Valley fever

Sub-Saharan Africa

Livestock

Aedes Mcintoshi

Unknown

Nairovirus

Crimean-Congo fever

Soviet Union, Middle East, Africa

Livestock, Hares

Hyalomma Ticks

Unknown

Hantaviruses -Hantaan -Dobrava -Seol -Puumala

Hemorrhagic fever with Renal Syndrome(HFRS)

Former Soviet Union,Pakistan, China(Korea)

Mice & Rats

None

Ribavarin for Korean HF

Table.3: Family: Filoviridae Virus

Disease

Geography

Reservoir

Vector

Treatment

Marburg

Marburg Hemorrhagic fever

Unknown

Unknown

Unknown

Ebolavirus (4 subtype) Zaire Sudan Reston Cote d’Ivoire

Ebola Hemorrhagic Fever

Human cases from Uganda , Kenya, Zimbabwe Outbreaks in Zaire, Sudan, Ivory cost, Gabon

Unknown

Unknown

Unknown

17


FLAVIVIRIDAE The Flaviridae family includes: Kyasanur Forest Disease, Omsk hemorrhagic fever, Yellow fever and Dengue. History:Yellow fever virus was first flavivirus isolated in 1927 and the first virus to be proved to be transmitted by an arthropod vector. Dengue virus which was also found to be transmitted by an arthropod was isolated in 1943. Major outbreaks of dengue with hemorrhagic fever have occurred in Australia in 1897, Greece in 1928, and Formosa 1931. Omsk hemorrhagic fever virus was first isolated in 1947 from the blood of a patient with hemorrhagic fever during an epidemic in Omsk and Novosibirsk Oblasts of the former Soviet Union. Kyasanur Forest virus was isolated from a sick monkey in the Kyasanur Forest in India in 1957. Since its recognition 400 to 500 cases a year have been reported. Diagnostic Considerations for VHF:The diagnosis of viral hemorrhagic fever should be considered in three group of patients 1. Those who have been in an endemic area within three weeks of onset of fever. 2. Those who have had contact with blood and other body fluids of persons or animal with VHF. 3. Those who worked in a laboratory that handles hemorrhagic viruses. All suspected cases of VHF should be reported immediately to National Reference Centers and to CDC, Atlanta. Laboratory tests for these agents are performed only in bio safety level-4 (BSL-4) laboratories. Samples should be sent to NCDC, Delhi ,CDC, Atlanta and U.S Army Medical Research Institute of Infectious Diseases or any such National Reference Centre.. Blood is usually the best specimen for diagnosis of VHF. - Cell culture or suckling mouse inoculation can be used for many of these viruses. - Electron microscopy is useful in filovirus infections. -Antigen detection by Immunohistochemistry can be performed on tissue obtained from fatal cases. - Serology directed at detection of virus specific immunoglobin M (IgM) is often useful. - Reverse- transcription polymerase chain reaction (RTPCR) can be done as a rapid means of testing samples for evidence of specific viral RNA.

Treatment The mainstay of treatment in VHF is supportive in nature with careful maintenance of fluid and electrolyte balance, circulatory volume, and blood pressure. management of DIC, sepsis, shock based on the established guidelines should be undertaken. Treatment options specific to the disease are limited. There have been reports of possible benefits with treatment of patients using serum prepared from the blood of recovered VHF patients or gammaglobulin obtained from immunization of horses. Patients receive supportive therapy, but generally speaking, there is no other treatment or established cure for VHFs. Treatment with convalescent-phase plasma has been used with success in some patients with Argentine hemorrhagic fever. Recently immunotherapy has also been attempted via passive transfer of CCHF survivor convalescent plasma. But these results are based on uncontrolled experiments and definitive evidence regarding their effectiveness is lacking. The antiviral drug Ribavirin has shown benefits in in-vitro studies. It has been found effective in treating some individuals with Lassa fever or HFRS. although it has not been approved by US FDA for VHF patients. Prevention and control: • Avoid contact with host species -Rodents Control rodent populations. Discourage rodents from entering or living in human populations. Safe clean up of rodent nests and droppings. -Insects Use insect repellents. Proper clothing and bed nets. Window screens and other barriers to insects. • Vaccine available for Yellow fever .Experimental vaccines under study are Argentine HF, Rift Valley Fever, Hantavirus and Dengue HF. • If human case occurs − Decrease person-to-person transmission. − Isolation of infected individuals.

Table:4 Family: Flaviviridae Virus Dengue (4 subtypes) -DEN1, DEN-2, DEN3, DEN-4

Disease Dengue fever & DHF

Geography Central & South America, SouthEast Asia

Reservoir None

Vector Aedes aegypti & A.albopictus

Yellow Fever

Yellow fever

Nonhuman primates

Aedes mosquitoes

Kyasanur Forest

Kyasanur Forest Disease

Sub-Saharan Africa, South America India

Omsk HF

Omsk H F

Nonhuman primates, Birds, livestock Small mammals

Haemophysalis spinigera, H. turtura (Hard Ticks) Dermacentor ticks (Ixodid ticks)

Siberia (former Soviet Union)

Treatment I.V. fluids(supportive therapy) Vaccine under trials: -Tetravalent Live attenuated vaccines -Chimeric -Recombinant subunit vaccines -DNA vaccines 17-D vaccine for control

Supportive therapy

Unknown, Supportive therapy

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Postexposure Prophylaxis Postexposure prophylaxis should be considered potentially for those exposed to haemorrhagic fever viruses in a bioterrorist attack and all known high-risk individuals such as those who have mucous membrane contact (kissing or sexual contact with a patient) or have percutaneous injury in contact with the patients’ secretions, excretions, or blood. Prophylaxis should also be considered for those with close contacts such as living or shaking hands with the patients and those who process laboratory specimens. Such people are placed under medical surveillance and made to observe themselves with temperature monitoring twice daily. If a temperature of 38.3°Cor higher develops, treatment with ribavirin should be initiated promptly as presumptive treatment of CCHF. The dose for PEP recommended is oral ribavirin 200 mgs twice daily for 5 days. Conclusion Scientists and researchers are challenged with developing containment, treatment, and vaccine strategies for these diseases. Another goal is to develop immunologic and molecular tools for more rapid disease diagnosis, and to study how the viruses are transmitted and exactly how the disease affects the body (pathogenesis). A third goal is to understand the ecology of these viruses and their hosts in

order to offer preventive public health advice for avoiding infection References 1.Knipe MD, Howley MP. Field’s Virology. In: Peter JC. Emerging Viral Diseases. Lippincot William & Wilkins, 2007; 5th ed.(1): 605-622. 2. Park K. Park’s Textbook of Preventive and Social Medicine.19th ed. 2007.206-242. 3. Gubler JD. Dengue and Dengue hemorrhagic fever. Clin. Microbiol Rev 1998; 11(3):480-496. 4. Chaturvedi UC, Shrivastava R, Nagar R. Dengue vaccines: Problems & prospectus. Ind J Med Res 121; 2005:639-652. 5.Buchy P, Yoksan S, Peeling WR, Hunsperger. Laboratory Tests For The Diagnosis Of Dengue Virus Infection. Available from: http:/www.who.int/tdr/publications/swg dengue 2.htm. 6. Bajpai S, Nadkar YM. Crimean Congo Hemorrhagic Fever: Requires Vigilance and Not Panic. JAPI, 2011;59:164-67. 7. Times of India website. http://articles.timesofindia.indiatimes.com/2011-0312/ahmedabad/28683512_1_congo-fever-crimean-congohemorrhagic-congo-deaths. March 12, 2011. Accessed on March 13, 2011. 8.CDC — MMWR Weekly, June 30, 1995.144(25);475-479. 9.CDC — Disease Information Viral Hemorrhagic Fevers: Fact Sheet, dated 02/07/2002. Available from: http://www.cdc.gov/ncidod/dvrd/spb/mnpages/vhfmanual.htm#con tent.

19


Carbapenem Resistance Dr Kanika Gupta, Dr Sonal Saxena, Dr Renu Dutta Department of Microbiology, Lady Hardinge Medical College and KSCH, Delhi

Introduction: Microorganisms can adapt to environmental pressures in a variety of effective ways, and their response to antibiotic pressure is no exception. An inevitable consequence of antimicrobial usage is the selection of resistant microorganisms, perhaps the most obvious example of evolution in action. Overuse and inappropriate use of antibiotics has fuelled a major increase in prevalence of multidrug- resistant pathogens, leading some to speculate that we are nearing the end of antibiotic era. Carbapenems: Carbapenems are structurally related to β lactam antibiotics.1 The carbapenems can be divided into three groups based on their spectrum: 1. Broad-spectrum Carbapenems, with limited activity against non-fermentative Gram-negative bacilli, particularly suitable for community acquired infections. E.g. ertapenem. 2. Broad-spectrum Carbapenems, with activity against non-fermentative Gram-negative bacilli that are particularly suitable for nosocomial infections. E.g. imipenem and meropenem. 3. Carbapenems with clinical activity methicillinresistant Staphylococcus aureus, e.g. Razupenem. Imipenem/cilastatin was the first carbapenem to be approved in 1987, followed by meropenem in 1996, ertapenem in 2001 and then, most recently, doripenem in 2007. Razupenem is still under clinical trial. The carbapenems act by inhibiting the bacterial growth by interfering with the transpeptidation reaction of bacterial cell wall synthesis.1 These are broad spectrum antibiotics indicated for use in infections caused by susceptible organisms that are resistant to other available drugs, eg Pseudomonas aeruginosa (except ertapenem which has a limited activity against Ps. aeruginosa) and in mixed aerobic and anaerobic infections. These are drugs of choice for extended spectrum β lactamase (ESBL) producing organisms.1 Carbapenems are one of the newer but most powerful members of the antimicrobial world. Resistance to

carbapenems is often associated with extensive, sometimes total, antibiotic resistance. Mechanisms of resistance Intrinsic resistance to carbapenems is seen in methicillinresistant Staphylococcus aureus (MRSA) and Enterococcus faecium. All the carbapenems (with the exception of the newer carbapenems like razupenem) have poor binding affinity for PBP 2a (found on MRSA) and PBP 5 (found on E. faecium).2 Extrinsic resistance to carbapenems is caused by (i) hydrolysis of the β lactam ring by carbapenemases which is the most common mechanism of resistance against carbapenems; (ii) impaired penetration of carbapenems to target penicillin binding proteins (PBPs) due to loss of porin channels like OprD, which, for example, is associated with imipenem resistance and reduced susceptibility to meropenem in Ps.aeruginosa; and (iii) efflux pump which consists of cytoplasmic and periplasmic protein components that efficiently transport β lactams from the periplasm back across the outer membrane.2 Carbapenemases (Table 1): Carbapenemases, better known as carbapenem hydrolyzing enzymes, represent the most versatile family of β lactamases. Many of these recognize almost all hydrolysable β lactams, and are most resilient against inhibition by all commercially viable β lactamase inhibitors.3 MBLs, in gram positive bacilli, were amongst the first carbapenemases to be described. In mid- to late 1980s, another set of carbapenemases emerged in Enterobacteriaceae, which unlike the MBLs, were not inhibited by EDTA. Until early 1990s, all carbapenemases described were chromosomally encoded, species specific. With identification of plasmid encoded (IMP-1, ARI-1, KPC-1), pattern of carbapenemase dissemination has changed from clonal spread to interspecies dispersion.3

Table 1: Classification of carbapenem hydrolyzing enzymes2: Ambler system A (Serine penicillinases) B (Metallo β lactamases) D (Oxacillinases)

Bush Jacoby Medeiros system 2f

Inhibited by clavulanate +

3

-

2d

+/-

Examples SME-1,2, NMC-A, IMI-1, GES-2, KPC-1-3 IMP-1, VIM-2

OXA 23-27, OXA-40 and OXA-48

Remarks

Most prevalent carbapenemases Inhibited by EDTA Characterized from Acinetobacter baumannii only.

20


Detection of carbapenem resistance4 Carbapenem resistance is detected by the standard disk diffusion and broth dilution methods described by CLSI. The break points are indicated in table 2. Once the resistance to carbapenems has been established, further tests can be undertaken to detect the carbapenemase production.

clover leaf appearance) indicates carbapenemase production. For tests with positive MHT, perform MIC test before reporting any carbapenem results as the clinical interpretation is based solely on the MIC. 2. Re-Modified Hodge test 5 It is based on the fact that the metallo- β-lactamases have Zn 2+ in their active sites so the catalytic activity of these enzymes enhanced by addition of zinc to the antibiotic disk. The procedure is similar to MHT. The indicator strain is inoculated on MHA plate as described in MHT. Two points are marked on the Mueller Hinton agar plate ~ 3 cm apart. On one, imipenem (10mcg) disk is placed and on the other point, a disk containing10mcg of 50mM (140mcg) ZnSO4 + imipenem (10mcg) is placed. The test strains are inoculated as in MHT. Enhancement of growth of the indicator strain in presence of zinc indicates presence of MBLs.

Detection methods for carbapenemase activity (A) phenotypic tests for detection of carbapenemases: 1. Modified hodge test (MHT)4: In 2009, CLSI incorporated the Modified Hodge test for detection of carbapenemases, especially KPC’s, from Enterobacteriaceae. It can detect all carbapenemases and is simple to perform. It is used for testing isolates for epidemiological or infection control purposes only. No change in the interpretation of carbapenem susceptible test results is required for MHT-positive isolates. Procedure (fig 1): A 0.5 McFarland standard suspension of Escherichia coli (ATCC 25922) is prepared in broth or saline and a 1:10 dilution is prepared. This suspension is inoculated onto a Mueller Hinton agar (MHA) plate and is allowed to dry for 3-5 minutes. Ertapenem (10mcg) or Meropenem (10mcg) disc is place on the agar plate. The test strain is inoculated in a straight line out (radially) from the edge of the disk and incubate at 37 ˚C in ambient air for 16-20hrs. the plate is examined for the enhanced growth of E.coli at the intersection of the streak of the test organism and the zone of inhibition of E.coli.Enhanced growth (or

3. Disc potentiation test for MBL The basis for this test is that in presence of EDTA, MBL gets inhibited and the diameter of inhibition zone increases. A lawn culture of the test strain is prepared on MHA and two disks are placed on it containing imipenem (10mcg) and imipenem (10mcg) + EDTA (750mcg) respectively (fig.2). An increase in zone diameter ≥7mm around the disk containing EDTA indicates presence of MBL.

Table 2: Zone diameters and MIC interpretive standards as per CLSI (2011) Agent

Organism tested

Enterobacteriaceae

Disk content (mcg) 10

Zone diameter break point (in mm) S I R ≥ 23 20≤ 19 22

MIC interpretative standard (mcg/mL) S I R ≤1 2 ≥4

Doripenem

Ertapenem

Enterobacteriaceae

10

≥23

2022

≤19

≤ 0.25

0.5

≥1

Imipenem and Meropenem

Enterobacteriaceae

10

≥23

2022

≤19

≤1

2

≥4

Pseudomonas aeruginosa, Acinetobacter spp

10

≥16

1415

≤13

≤4

8

≥16

Comments

Interpretative criteria based on a dosage regimen of 500mg every 8hrs Interpretative criteria based on a dosage regimen of 1g every 24hrs Interpretative criteria based on a dosage regimen of 500 mg every 6hrs or 1g every 8hrs for Imipenem and 1g every 8hrs for meropenem

21


Fig. 1: Modified Hodge test

Fig 2: Disk potentiation test for MBL

Fig.3 Double disk synergy test

22


4. Double disk synergy test A 6 mm EDTA disks is prepared by incorporating 10 µl of 0.5mM solution of EDTA on each disk (750mcg). A suspension 0.5 McFarland equivalent of the test organism is prepared and inoculated onto the MHA plate (fig 3). EDTA disk is placed 20mm apart (edge to edge) from the imipenem (10 µg) disk and incubated overnight. A zone of synergy (even if small zone) between the antibiotic disc and EDTA is taken as positive for MBL production. 5. E-Test for MBLs6 One half of the E- Test strip is impregnated with an imipenem gradient across seven dilutions and the other half with another imipenem gradient overlaid with a constant concentration of EDTA. A reduction of Imipenem MICs by more than or equal to 3 fold dilutions in the presence of EDTA is interpreted as suggestive of MBLs production. 6. Broth microdilution test: for MBLs A four fold reduction in MIC in the presence of EDTA is indicative of MBL producing organism. 7. Rapid test using chromogenic media 7,8: Various chromogenic media are now available for rapid detection of carbapenem resistance in enterobacteriaceae such as Oxoid Brilliance CRE Agar by Thermo Fisher Scientific and CHROMagar KPC. Color and morphological characteristics permit easy identification of carbapenem resistant bacterial colonies within 16-24 hours of inoculation of the clinical sample.

Surveillance and carbapenem resistance control guidelines9 In light of the clinical and infection control challenges posed by carbapenem resistant enterobacteriaceae (CRE) and advances in the ability to detect these pathogens, CDC and HICPAC have developed new guidance for CRE infection prevention and control in an effort to limit the further emergence of these organisms (box next page). Conclusions Carbapenems are one of the last in the spectrum of antimicrobials for members of the family Enterobacteriaceae. Resistance to carbapenems is often associated with resistance to most of the other antibiotics. Hence, the detection of carbapenem resistance, its surveillance and prevention is of utmost importance in todays world. References: 1.

2.

3. 4.

5. 6

(B) Genotypic methods of carbapenem resistance These include molecular methods like PCR for genes for IMP, VIM etc, DNA probes and DNA sequencing. PCR and DNA probing, while sensitive, do not indicate the type of variant that is present and require specific DNA primers and probes respectively. DNA sequencing is the molecular gold standard test but is labour intensive and requires experience.

6.

7. 8. 9.

Chambers HF, Deck DH. Beta – lactam & other cell wall- & membrane- active antibiotics. In: Katzung BG, Masters SB, Trevor AJ. Basic & clinical pharmacology. 11th ed. New York: The McGrowHill Companies Inc.; 2009. 773-93. Rice LB, Bonomo RA. Mechanisms of resistance to antibacterial agents. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. Manual of clinical microbiology. 9th ed. Washington DC: ASM Press; 2007. 1114-46. Queenan AM, Bush K. Carbapenemases: the versatile β- lactamases. Clin Microbiol Reviews. 2007 Jul; 20(3): 440-58. Cockerill FR, Wikler MA, Bush K, Dudley MN, Eliopoulos GM, Hardy DJ, et al. Performance standards for antimicrobial susceptibility testing; twenty first information supplement (M100- S21). Pennsylvania: Clinical Laboratory Standards Institute; 2011 Jan. Vol. 31, No.1. Rai S, Manchanda V, Singh NP, Kaur IR. Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae. Indian J Med Microbiol. 2011; 29(3): 275-79. Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo- βlactamases: the quite before the storm?. Clin Microbiol Reviews. 2005 Apr; 18(2): 306-25. CHROMagar KPC in clinical microbiology. [Cited on 2011 Oct 25]. Available at: http://www.chromagar.com/ OXOID: Brilliance CRE AGAR.[Cited on 2011 Oct 25] Available at: http://www.oxoid.com/ MMWR Weekly. Guidance for control of infections with carbapenem-resistant or carbapenemase producing Enterobacteriaceae in acute care facilities. CDC. 2009 Mar; 58 (10): 256-60.

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Infectious Diseases and Their HLA Association Dipmala Das, Narender Pal Singh, Iqbal R Kaur Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi

Introduction Susceptibility to infections is through a complex interaction of environmental and host genetic factors. Many genetic loci make modest contributions to human disease susceptibility. Recent focus in this field has been on the identification of functional variants at these loci and their effects on infection and other conditions. Among these loci, human leukocytic antigen (HLA) loci have stood out as the leading candidates for infectious disease susceptibility. In mid 1930s, Peter Gorger identified blood group antigens (Antigen I, II,III,IV) using inbred strains of mice. Few years later George Snell established that antigens encoded by genes in group II took part in rejection of transplanted tumor or tissue. He called these genes controlling tissue rejection as histocompatibility genes. In mid 1950’s Jean Dausset discovered the human MHC by demonstrating leukoagglutinating antibodies in sera of patients who received multiple transfusions. These antibodies were the first antibodies to define human histocompatibility antigen which is now termed as human leukocytic antigen (HLA) and in the 1970’s, immunological role of MHC was brought to focus. In 1975, Zinkernagel and Doherty found that the T cell responses were restricted not only to antigen but also to MHC molecule. HLA-genetics The MHC is an extended region of the genome that spans for about 4000 kb and is located on 6p21.3 region of short arm of human chromosome 6 (Figure 1). The human map reveals clusters of genes grouped roughly into a MHC class II region covering about 1,000 kb, a MHC class III region 1000kb , and a MHC I region of 2000kb. The MHC is polygenic, that is it contains several different MHC class I and MHC class II genes, so that every individual possesses a set of MHC molecules with different ranges of peptide-binding specificities. It is also highly polymorphic implying that there are multiple variants of each gene within the population as a whole. The MHC genes are, in fact, the most polymorphic genes known. MHC Class I α-chain genes include HLA –A, HLA-B, HLA-C (classical Ia) and HLA-E, HLA-F, HLA-G (non classical Ib) and are located at telomeric side. MHC Class II α and β chains genes (HLA-D) designated with three letter code (eg HLA DRB) indicates class D, family (M, O, P, Q or R) and chain (α or β) are clustered at centromeric end. Between DM β and DO β genes lie LMP genes (for low-molecular-weight proteins) and TAP (for transporter associated with antigen- processing) genes. LMP and TAP genes encode molecules that are involved in peptide generation in the cytosol and peptide transport across the endoplasmic reticulum and mutations in these genes may cause Some class IB genes, for example the members of the MIC gene family, are under a different regulatory control from the classical MHC class I genes and are induced in response to cellular stress (such as heat shock). Among five MIC genes, only two—MICA and MICB—are expressed and produce protein products. They are expressed in fibroblasts and epithelial cells, particularly in intestinal epithelial cells, and may play a part in innate immunity or in

the induction of immune responses in circumstances where interferons are not produced.

Figure-1: Showing the location of the MHC genes on Chromosome 6 HLA as an Allogenic System: Two unrelated humans will share individual set of MHC antigens distinguishing individual in given species. HLA haplotypes: Each antigenic specificity of any given MHC locus is determined by one structural gene. At each MHC locus individual carries two genes , one from maternal chromosome and one from paternal chromosome Set of alleles that an individual carries at each locus on single chromosome forms haplotype. Genetic relationship among members of a family are important for disease inheritance. Co-Dominance of the MHC Alleles: For each MHC locus, a given individual may be homozygous or heterozygous. Most individuals are heterozygous for any given locus and will express the two specificities inherited from each parent that are coded by the two DNA strands of the same chromosome .(e.g. a heterozygous B8/B27 will have a gene coding for B8 in one chromosome and a gene coding for a B27 in the other). Both specificities for each locus will be expressed by every individual cell of this heterozygous individual. Therefore,the MHC genes are co-dominant at the cellular level, and there is no allelic exclusion in their expression.

25


Linkage disequilibrium: Certain combinations of alleles (i.e., certain haplotypes) occur with a higher frequency than expected. Thus, many HLA antigens occur together on the same chromosome more often than is expected by chance. As an example, The HLA –A1 allele is found in the Caucasian population with a frequency of 0.158, and the HLA-B8 allele is found with a frequencyof 0.092. The A1, B8 haplotype should therefore be found with a frequency of 0.158 × 0.092 = 0.015. In reality, it is found with a frequency of 0.072. The linkage disequilibrium is expressed as the difference (D) between the observed and expected frequencies of the alleles (i.e., 0.072 0.015 = 0.057) MHC and Disease susceptibility: HLA alleles occur at a much higher frequency in people suffering from certain diseases than general population The association between an HLA allele and a given disease may be quantified by determining the frequency of that HLA allele expressed by individuals afflicted with disease and comparing these with frequency of same allele in general population. (Ag+/Ag-)disease group Relative Risk= -------------------------------------(Ag+/Ag-)control group

Infectious diseases and HLA associations Genetically controlled differences exist in the magnitude of immune responses to a disease. Several HLA-linked examples of infectious diseases are available and this provides a attractive mechanism to account for disease susceptibility. A large number of candidate based studies are required to establish genetic basis of diseases. The extent of HLA polymorphism observed in population is maintained by balancing selection and specifically pathogen driven selection. Different populations tend to exhibit frequency distributions of allele or haplotypes particular to that group. This can potentially confound analysis of individual allele or haplotypes between various studies with respect to ethnic groups. HLA and viral diseases Effective host responses against viruses that breach early innate immune response is HLA restricted T cell responses. CD4+ T cells help in induction of CD8 + CTL and recruitment and activation of macrophages at site of infection. CD4 + T lymphocyte provide T cell help to the generation of antiviral antibodies. CD8+Tcells kill infected cells through the release of perforin and granzymes or through Fas-Fas L interaction. MHC class I and class II gene products are critical in regulation of immunity against viral infection. In HIV, controlling of CD4+ depletion by virus specific cytotixic T-cell lympocytes(CTL) is an immunogenic responses towards protecting individual both from infection and progression to AIDS . HIV-1 virus mutates rapidly and effectively generating extreme diversity which remarkable between even within individual variability. Extreme viral diversity within an individual may allow the virus to evade HLA TCR restriction at two levels (peptide binding in HLA molecule and TCR recognition). An

activating KIR allele (KIR3DS1) in combination with subset of the HLA-Bw4 cluster (HLA Bw4lle80) is associated with delayed progression. If KIR3DS1 is absent, the HLA-B allele is not protective and if specific HLA-B alleles are absent KIR3DS1 is associated with more rapid progression to AIDS defining illness. Hypersensitivity reaction to abacavir found to be associated with HLA B*5701. HLA B*5701 screening has now become part of standard HIV clinical care in resource rich countries and probably represents the most convincing translational success of genetic association data in infectious diseases. HLA class II heterozygote advantage has been demonstrated for clearance of Hepatitis B virus infection and for progression of end stage liver diseases in hepatitis. HLA associations with hepatitis B and C infection are largely inconsistant among different ethnic groups. HLA DRB1* 07 is associated with viral persistence in both hepatitis C and B in European and Asian populations. HLA DRB1*1301 is consistently associated with hepatitis B clearance across no of diverse population. Hepatitis B and C with HLA HLA class II heterozygote advantage has been demonstrated for clearance of Hepatitis B virus infection and for progression of end stage liver diseases in hepatitis. HLA associations with hepatitis B and C infection are largely inconsistant among different ethnic groups. HLA DRB1* 07 is associated with viral persistence in both hepatitis C and B in European and Asian populations. HLA DRB1*1301 is consistently associated with hepatitis B clearance across no of diverse population. HLADRB1*11/*12 alleles and DQB1*0301 are associated with HBV persistence but with HCV clearance worldwide. Consistent association of DRB1*03 and *07 is observed with HCV susceptibility and non-responsiveness to HBV vaccination across the population. HLA DR13 is protective for vertical HBV and HCV transmission in Chinese and Italian neonates, but different alleles are associated with their susceptibility in these populations. HLA classⅠmolecule interactions with Killer cell immunoglobulin like receptors (KIR) of natural killer (NK) cells modulate HCV infection outcome via regulating immune regulatory cells and molecules. Genetic influences on dengue virus infection Hyperendemic transmission of multiple DEN serotypes and apparent absence of DHF and DSS was observed in Haitian population . In Cuba ,black people were hospitalised less frequently with DHF and DSS than whites during epidemics. These led to hypothesis that human genetic factors ,gene polymorphism may contribute to variable susceptibility. Studies have confirmed that some genetic polymorphism in HLA loci may protect or predispose an individual to DHF and DSS.HLA B 51 alleles are associated with development of haemorrhagic fever in dengue as well as hantaan virus infection. HPV and HLA In HPV induced cervical neoplasia, an activating KIR allele (KIR3DS1) in combination with subset of the HLA homozygous associated with increased progression to CIN. 26


On the other hand , no KIR3DS1 and HLA C 2 and/or HLA Bw4 is associated with delayed progression of CIN.

Association were also observed between TT and HLA class I chain related genes A and B (MIC A and MICB)

CMV and HLA HLA DQ 3 is frequently associated with CMV infection in high risk kidney transplant patients.

HLA, Bacterial infection and autoimmune diseases

HLA association with Mycobacterial Infection: Tuberculosis: Studies in non-Asian countries One of the first reports of an association between HLA and tuberculosis showed an increased frequency of HLA-B8 in Canada. Other studies showed an increased frequency of HLA-B5, B15and -DR5 in the North American black. HLA-A2and -B5 in the Egyptian population and -B27 in the Greek population. A negative association has been reported for -DR6 in American blacks. Studies in Asian populations A significantly increased frequency of HLA-DR2 was seen in the major studies which have revealed HLA-DR2 association with higher susceptibility to tuberculosis in Russia , China and Canada. An increased frequency of HLA-DR2 and –DQ1 was shown to be associated with the susceptibility to pulmonary tuberculosis in Indian population. Molecular study has revealed that the allele DRB1 *1501 of HLA-DR2 was higher compared with DRB1 *1502 in north Indian patients. HLA-DRB1 *1501,HLA-DQB1 *0601 (a subtype of HLADQ1) and-DPB1 *02 were found to be positively associated with susceptibility to pulmonary tuberculosis in south Indian patients. Leprosy: Spectrum of leprosy varies from multibacillary lepromatous leprosy(LL) to paucibacillary tuberculoid leprosy(TT). Between two poles patients with intermediate features are seen in borderline lepromatous and borderline,borderline tuberculoid form. Multibacillary patients have numerous sensitive anaesthetic skin lesions with high bacterial load and associated with IL4, IL10,CD4+ Th 2 immune response. Paucibacillary leprosy patients have limited no of hypopigmented sensitive anaesthetic skin lesions with few bacilli associated with IFN γ, IL2,CD4+Th1. Borderline form of diseases present with intermediate phenotypes with mixed Th1 and Th2 immune response and are not stable. Association with classical HLA II molecules could be major genetic determinant of disease phenotype. Large no of studies documented associations of DR and DQ alleles and different clinical subtypes of leprosy. Increased frequency of DR2 and DQ1 in LL patients and DR3 in TT patients observed. Molecular typing method of DR2 showed that majority of the allele in patients and controls was DRB1*1501 and DRB1*1502. Allele DRB1*1501 showed stronger asociation with LL patients than TT patients. Allele DQ B1 *0601 ad DQ A 1 *0103 were also more frequently observed in LL patients. Allele DQA1*0120 was frequently observed in borderline lepromatous leprosy. Allele DRB1*0701, DQB1*0201 and DQA1*0201 decreased in LL than controls.

Most autoimmune diseases are associated with HLA types. RA(Reactive arthritis) and AS (ankylosing spondylitis) are classical examples of the association of HLA with rheumatoid conditions. More than 90% of RA patients possess HLA-DR1 or other sub-type and >96% of AS patients reportedly possess HLA-B27. The involvement of HLA antigens in the pathogenesis of autoimmune diseases has been suggested to be due to the molecular similarities between certain bacterial antigens and HLA antigens. Organisms involved in reactive arthritis are: Campylobacter jejuni Chlamydia trachomatis Salmonella enterica serovars Typhimurium enteritidis,paratyphi B and C, and others Shigella flexneri, S. sonnei and S. dysenteriae Ureaplasma urealyticum Yersinia enterocolitica O3, O8, and O9 and Y. Pseudotuberculosis

HLA and parasitic infections Malaria Allele HLA- B53 in West African population is associated with protection against severe malaria. (HLA -B53 restricted CTL recognised a conserved epitope from liver stage specific antigen) . HLA class II haplotype (DRB1*1302–DQB1*0501) common in West Africans also associated with protection against severe malaria. Association between DR /DQ phenotypes and immune response to circumsporozoite protein were investigated in Thai adults.HLA-DRB1*04 Allele is Associated with Severe Malaria in Northern Ghana. In Thailand,HLA-B46, -B56 & HLA-DRB1*1001 are found in patients with severe noncerebral & cerebral malaria. 27


Leismaniasis A statistically significant association has been observed between HLA A11, B5, B7 and diffuse cutaneous leismaniasis in global population . HLA A 26 is commonly associated with visceral leishmaniasis. HLA A 28 and HLA DR 11 are frequently observed with localised cutaneous leishmaniasis in American population. A recent study in Bihar found HLA-A *02010101 and HLADRB1 *150201 alleles associated with protection against visceral leishmaniasis, while HLA-A *020601 & *24020101 and HLA-DRB1 *150101 alleles are associated with susceptibility to visceral leishmaniasis. A recent study in Bihar found HLA-A *02010101 and HLADRB1 *150201 alleles associated with protection against visceral leishmaniasis, while HLA-A *020601 & *24020101 and HLA-DRB1 *150101 alleles are associated with susceptibility to visceral leishmaniasis. Schistosomiasis: Severe pathology of schistosomiasis is due to immune response to eggs deposited in host tissue .A number of studies have looked for associations with HLA I and HLA II gene polymorphism and various clinical phenotypes (hepatosplenomegaly, cirrhosis, fibrosis). Two HLA-DRB1 alleles, HLA-DRB1*0901 and *1302 and two HLA-DQB1 alleles, HLA-DQB1*0303 and *0609 were found to be significantly associated with susceptibility to fibrosis due S japonicum infection in Chinese population . HLA-Al and B5,B8 significantly associated with hepatosplenomegaly due to S mansoni infection in Egyptian population.

Reversed immunogenetics: Genetic variants that are associated with resistance to infection or severe form clinical phenotypes can be used for design of vaccines. Protective HLA allele associations can allow the identification of pathogen epitopes that are restricted by the specific HLA allele. These epitopes may be incorporated into vaccine design in the expectation that the natural resistance can be replicated by immunisation (HLA restricted CTL response). eg Among malaria immune Africans HLA B 53 restricted CTL recognised a conserved epitope from liver stage specific antigen and this support the candidacy of liver stage specific antigen as malaria vaccine component. References: 1.Singh N , Agarwal S, Rastogi A.K.Infectious Diseases and Immunity: Special Reference to Major Histocompatibility Complex .Emerging Infectious Diseases.1999;3:41-49. 2.Blackwell J, Jamieson S, Burgner David. HLA and Infectious Diseases. Clinical Microbiology Reviews.2009;22:370-385. 3. Wagenaar J. Genetic Influences on Dengue Virus Infections. Dengue Bulletin 2004;28:126-134. 4. Singh R, Kaul R, Kaul R, Khan K. A comparative review of HLA associations with hepatitis B and C viral infections across global populationsWorld J Gastroenterol 2007 March 28; 13(12): 1770-1787. 5. Selvaraj P. Host genetics and tuberculosis susceptibility. Current Science 2004 . 86:115-121 6. J Fitness1, K Tosh1 and AVS Hill1. Genetics of susceptibility to leprosy. Genes and Immunity (2002) 3, 441â&#x20AC;&#x201C;453.

Chicken soup for the Indian Doctor;s Soul records 101 personal experiences some penned by doctors and some by patients, to reveal an extraordinary world of healthcare. Look at this book as a global clinic peopled by cases of triumph, tragedies, challenges, miracles and life changing event. One such experience is being shared by Dr Sonal Saxena, Professor, Microbiology, Lady Hardinge Medical College. Her story entitled "Selfless love" talks about love of a man for his wife. The story had a life changing effect on author's career choice.

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Tuberculosis Assays: Past, Present and Future Ruchi Kotpal, CP Baveja, Preena Bhalla Maulana Azad Medical College, New Delhi

Introduction Despite the discovery of the tubercle bacilli more than a 100 years ago, and all the advances in our knowledge of the disease made since then, tuberculosis still remains one of the major public health problems facing mankind, particularly in developing countries. It is estimated that about one third of the current global population is infected with Mycobacterium tuberculosis. Early diagnosis of tuberculosis and initiating optimal treatment will not only enable a cure but also curb the transmission of infection. For over 100 years, since Robert Koch’s discovery of Mycobacterium tuberculosis, sputum smear microscopy has been the cornerstone of tuberculosis diagnosis, especially in the developing countries. Recently the growing acknowledgement – particularly with the HIV pandemic and global spread of MDR-TB - that this will be insufficient to control TB, has driven development of a new generation of tests. There are three core diagnostic needs in TB control – i) detection of active tuberculosis, ii) identification of drug resistance and iii) detection of latent tuberculosis infection. Detection of Active tuberculosis Active (post-primary or secondary or adult) tuberculosis develops in previously infected people either due to reactivation of latent infection (post-primary progression, endogenous reactivation) or due to exogenous reinfection. The characteristic feature of such disease is extensive tissue necrosis and cavity formation. Bacilli escaping from cavities may enter sputum and expectorated, thereby infecting other people. 1. Microscopy The presumptive diagnosis of active tuberculosis depends on demonstration of acid-fast bacilli in sputum smears by microscopy. Microscopy is the simplest and most rapid procedure currently available for detection of acid fast bacilli in sputum smear after staining by Ziehl-Neelsen (ZN) method. Fluorescent dyes like Auramine, rhodamine also bind to the mycolic acid of and the bacilli can be detected by fluorescent microscope as bright yellow in colour against the dark background. Fluorochrome stains offers an advantage of greater sensitivity compared with carbolfuschin method since larger area of the smear can be scanned per unit of time. Advantages of smear microscopy i. It is inexpensive ii. It is relatively easy to perform iii. Results can be reported within hours of receipt of sample. Provides reliable epidemiological indicators needed for evaluation of National tuberculosis Control Programme. Limitation of Acid Fast stains 1. Not specific for M. tuberculosis as other non mycobacterial organisms are also stained with

acid fast stains like Nocardia, Rhodococcus, cysts of Cryptosporidium and Isospora spp. 2. Cannot differentiate M. tuberculosis from other nontuberculous mycobacteria 3. Acid fast smears have very low sensitivity 22 to 78% 4. Atleast 10,000 bacilli/ml must be present in the sputum to be seen on microscopy. 2.

Culture The gold standard for TB diagnosis is the cultivation of M. tuberculosis. It can detect as few as 100 viable bacilli/ml of specimen. At present mycobacterial culture can be performed on following media.

i. Solid culture media Egg-based Media which includes the conventional Lowenstein-Jensen media without starch, as recommended by International Union Against Tuberculosis (IUAT). Egg bases media have i) good buffering capacity, ii) long shelf life, iii) support good growth of mycobacteria and iv) materials in the inoculum or medium toxic to mycobacteria are neutralized. But the media is easily liquefied when contaminated and growth occurs in 3-8 weeks . Agar-based Media includes Middlebrook’s 7H10 and Middlebrook’s 7H11 medium. Growth can be detected within 10 to 12 days by microscopic examination. They do not readily support the growth of contaminants. Disadvantages include that they are i) expensive to prepare, ii) have a relatively short half-life, and iii) exposure to excessive heat or light may result in deterioration and release of formaldehyde, which is toxic to mycobacteria. ii) Liquid culture media Liquid media commonly used are, Middlebrook’s 7H9 and Middlebrook’s 7H12. Liquid media have few advantages i) growth occurs early within 7-10 days, ii) can be used for antigen preparation, iii) used for sensitivity testing and iv) used for vaccine preparation. 3. Automated Detection Systems The introduction of broth based growth systems, has significantly reduced the time to detection and increased the total number of positive cultures. i)

Radiometric BACTEC 460 TB method : This technique is specific for mycobacterial growth, wherein 14C labelled substrate present in the medium is metabolized, 14CO2 is produced proportional to the amount of growth in the media and measured by the BACTEC system in terms of growth index (GI) value. It takes much lesser time as compared to Lowenstein-Jensen medium (12days). But has a i) high cost, ii)

29


inability to observe colony morphology, iii) overgrowth by contaminants and iv) need for disposal of radioactive materials. But for smear (+) specimens culture yield was 96.5% by BACTEC and only 90.4% by LJ ii)

MGIT 960 mycobacteria detection system: Growth detection is based on the AFB metabolic O2 utilization and subsequent intensification of an O2 quenched fluorescent dye contained in a tube of modified MGIT. A series of algorithms are used to determine presumptive positivity and alert the operator to the presence and location of positive tubes. It is a non-radiometric system, accurate with mean time of detection is 15.5 days. It has a sensitivity of 95.4% and a specificity of 100% with a 100% positive predictive value and 89.3% negative predictive value.

iii)

MB/BacT system: This is a non-radiometric continuous monitoring system based on colorimetric detection of CO2. The bottom of each broth bottle is fitted with a gas-permeable sensor that changes from dark green to bright yellow when CO2 is produced by the metabolizing mycobacteria. It has minor disadvantages such as increase contamination rate and longer time for detection of growth (16 days).

• iv)

ESP culture system II: This is a fully automated continuous monitoring system based on the detection of pressure changes within the headspace above the broth culture medium in a sealed bottle, i.e. either gas production or gas consumption due to microbial growth. Mean time of detection for M. tuberculosis is 15.5days. It is a reliable, non radiometric, labour intensive alternative to BACTEC. 4.

Manual Detecting System

Septi-check AFB method: The septi-check AFB system consists of a bottle with enriched Middlebrook’s 7H9 broth with a paddle with agar media One side of the paddle is covered with nonselective Middlebrook’s 7H11 agar, the reverse side is divided into two sections: one contains 7H11 agar with para-nitro-á-acetylamino-ßhydroxypropiophenone (NAP) for differentiation of M. tuberculosis from other mycobacteria, the other section contains chocolate agar for detection of contaminants. This non-radiometric approach and facilitates early detection of positive cultures within 3 weeks. 5.

Genotypic methods

Polymerase chain reaction: PCR allows of specific sequences of M. tuberculosis to be amplified in vitro, which can be visualized and

identified. If appropriate sequences specific for M. tuberculosis is selected, 10-1000 organisms can be identified. It is a rapid method and results are available within a day. PCR assays could be based on conventional DNA based PCR, nested PCR and RT-PCR. The most common target used in PCR is IS6110 which is specific for M. tuberculosis. Other targets like MPB64, repetitive sequences, GC repeats, IS 1081. PCR when compared to culture has 83.5% sensitivity and 99% specificity. RT-PCR has been investigated for rapid and specific detection of M. tuberculosis in clinical specimen. Ligase chain Reaction: It is a variant of PCR, in which pair of oligonucleotides are made to bind to one of the DNA target strands, so that they are adjacent to each other. The second pair of oligonucleotides is designed to hybridized the same regions on the complementary DNA. The action of polymerase and ligase in the presence of nucleotides result in the gap between adjacent primers to be filled with appropriate nucleotides. The LCX M. tuberculosis assay kit mainly used for respiratory specimen with high sensitivity and specificity. Transcription mediated amplication (TMA) and nucleic acid amplification (NAA) identifies the presence of genetic information unique to M. tuberculosis from pre processed sample. FDA approved molecular methods for direct detection of tuberculosis from sputum samples 1. Amplicor test: The technique detects the presence of mycobacterial 16S RNA dene by PCR amplification followed by an ELISA reaction. The complete process takes 6.5hours and an automated version is known as Cobas Amplicor test. The test has the sensitivity between 80-92% and specificity of 99% among smear positive patients. 2. Enhanced Mycobacterium tuberculosis direct test (E-MTD): It is based on Transcription mediated amplification system.

Serological Test WHO has recommended that serological test for antibody detection in case of tuberculosis should be phased out because in the endemic country like India baseline seropositivity is very high, giving false positive result. Drug Susceptibility Testing Multi-drug resistant tuberculosis is defined as TB caused by strains of Mycobacterium tuberculosis resistant to atleast isoniazid and rifampicin. MDR-TB should be suspected if the patient has persistently positive sputum smear, radiological deterioration, or clinical deterioration despite adequate treatment. To add to the problem came another drug resistant form of tuberculosis which had to be classified as Extensively Drug Resistant Tuberculosis (XDR-TB) , a very serious form of TB against which is resistant not only to isoniazid and rifampicin (the definition 30


of multidrug-resistant TB, MDR-TB), but also to any fluoroquinolone and to at least one of the three following injectable drugs used to treat TB- capreomycin, kanamycin and amikacin. Drug resistant tuberculosis often goes undetected and untreated in many countries. Early choice of appropriate treatment is an essential determinant of favourable outcome and thus rapid determination of drug resistance can allow a customized approach to treatment early in the course of the disease. Drug susceptibility can be performed by the following methods: 1.

2.

Phenotypic methods: Among the conventional phenotypic methods of drug susceptibility testing, include the proportion method, the absolute concentration method and the resistance ratio method. Proportion method has been considered the most reliable and is taken as a reference for comparing any method. The proportion of bacilli resistant to a given drug is then determined by comparing these numbers and expressing the resistance proportion as a percentage of the total population tested. The proportion method is currently the method of choice for estimating drug resistance and this principle has been applied to the following rapid testing methods: i. BACTEC 460 ii. MGIT 960 iii. MB/BacT system iv. ESP II system Newer phenotypic methods : Several new phenotypic methods have been developed which are easy to perform, rapid and reliable. Most methods are also relatively less expensive and thus suited to resource limited nations like India. Also used for identification, drug susceptibility or both. i.

ii.

iii.

E-Test indirect DST The E-test is an MIC method in which a strip containing an exponential gradient of antimicrobial is placed on an agar surface onto which M. tuberculosis has been inoculated. TK colorimetric media TK Medium is a novel colorimetric system that indicates growth of mycobacteria by changing the colour of the growth medium. It also permits susceptibility testing for drug resistance. Microscopic Observation Drugsusceptibility Assay (MODS) The MODS assay is based on the observation of the characteristic cord formation of M tuberculosis that is visualized microscopically in liquid

medium with the use of an inverted microscope within 7 days. iv.

Bacteriophage-based Assays This technology uses bacteriophages to infect live M. tuberculosis and detect the bacilli using either phageamplification method or detection of light.

1) Pha B assay based on the ability of viable M. tuberculosis to support the replication of an infecting mycobacteriophage. In the case of drug-resistant M. tuberculosis, bacilli will remain viable and protect the mycobacteriophage. For rapid detection, the released mycobacteriophages are mixed with rapidly growing M. smegmatis host in which they undergo rapid cycle of infection, replication and lysis. Lysis is easily seen as clear areas or plaques in a lawn culture of M. smegmatis. 2) Luciferase reporter phage assay: In this technique, viable mycobacteria are infected with reporter phages expressing firefly luciferase gene. Easily detectable signals are seen a few minutes after the infection of M.tuberculosis with reporter phages.

v.

vi.

Mycolic acid index susceptibility testing This is a modification of the original mycolic acid analysis by HPLC where a coumarin compound is used as a fluorescent derivatizing agent of mycolic acid instead of pbromophenacyl bromide. The drug sensitivity is assessed by measuring the total area under mycolic acid (TAMA) chromatographic peaks of a culture of M.tuberculosis, and this area has a very good correlation with log CFU per ml. Colourimetric Methods This technique uses colourimetric detection of mycobacterial growth. This method either make use of an oxidation-reduction indicator that changes colour in response to the metabolic products associated with MTB growth, or nitrate reduction which is revealed by an added indicator. Indicators used are Alamar blue, Tetrazolium dye etc.

31


Figure summarizing bacteriophage plaque assay

3.

Genotypic Methods The identification of specific mutations responsible for drug resistance has facilitated the development of novel, rapid molecular tools for DST. The following are the important genotypic drug susceptibility testing methods: i. DNA sequencing Sequencing of PCR amplified products is the most accurate and reliable method for mutation detection, and it is used as the gold standard technique. This technique is being mainly used for Rifampicin, because it requires several sequencing reaction per isolate, which become costly and labour intensive. ii. Line Probe Assay (Solid phase hybridization assay) Commercial assay used to identify M.tuberculosis species and presence of mutation in rpoB core region. This region is amplified and biotin labeled by PCR. This amplicor is detected by hybridization with a strip with five probes for wild type rpoB sequence, four for specific rpoB mutation, a conjugate control and M. tuberculosis control probe is immobilized. Bound amplicons

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are detected by a colored reaction as depicted by band formation. Commercially available INNO Lipa test is currently not FDA approved. DNA Microarrays It is based on the principle of hybridization. In this PCR amplicons are labelled with fluorophore moieties are generated from sample to to be hybridized to large collection of probes on the solid surface. These hybridize with the probe to emit fluorescent signals. It is used for species identification and detection of RIF mutations, but use is limited as it is under research, requires expertize and is costly. Molecular beacons Molecular beacons are singlestranded oligonucleotide hybridization probes that form a stem-and-loop structure. The loop contains a probe sequence that is complementary to a target sequence of interest (either wildtype or mutant-type) and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence. A quenching moiety is attached to the end of 32


one arm and a fluorescent moiety is attached to the other. When the target sequence is absent, the probe cannot fluoresce, as the stem places the fluorophore very close to the quencher. When the target is present, the probe and the target hybridize, and the beacon undergoes a spontaneous conformation change that forces the fluorophore and the quencher to dissociate and move away from each other, causing fluorescence that can be detected in a real-time PCR.58 Beacons are highly sensitive and specific,allowing the detection of point mutations.

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PCR Single-strand conformation polymorphism (SSCP) PCR SSCP is based on the property of single stranded DNA to fold into a tertiary structure whose shape depends on its sequence. Single strands of DNA differing by only one or a few bases will fold into different conformations with different mobilities on a gel, producing what is called a single strand conformation polymorphism. In combination with PCR, SSCP has been applied for the detection of resistance to Rifampicin and Isoniazid. FRET probes Fluoroscence Resonance Energy Transfer probes also known as light cycler probes are highly specific probes to detect mutations

vii.

in real time reactions. It consists of two hybridization probes- one labelled with flouroscene dye and other with flourophore Light Cycler Red 640. When they both hybridize with target molecule, dye release energy with is transferred to the cycler which emits red light. FRET probe assays for rapid detection of resistance to INH and RMP have been developed. The Xpert MTB/RIF assay It detects M. tuberculosis and RIF (Rifampicin) resistance by PCR amplification of the rifampin resistance determining region (RRDR) of the M. tuberculosis rpoB gene. Xpert MTB/RIF uses 3 specific primers and 5 unique molecular probes for identification and determination of resistance with results obtained in 2 hours. It is an integrated diagnostic device that performs sample processing and real-time PCR analysis in a single hands-free step. WHO has adopted Xpert MTB/RIF assay in the RNTCP programme.

Detection of Latent Tuberculosis WHO has estimated one third of the world’s population is infected with M. tuberculosis. Infection in most patients with M. tuberculosis is contained by host cellular response and remain latent. Due to the risk of progression from latent infection to active disease, testing for latent TB infection (LTBI) is important. 1. Mantoux test It is a delayed or type IV hypersensitivity reaction. Originally described by Robert Koch in which old tuberculin (OT) was used which was a crude preparation of 6 to 8 week culture filtrate of tubercle bacilli. Now a purified preparation of the active tuberculoprotein (PPD) is used which gives fewer non specific reaction. 1 TU of PPD is injected intradermally in the forearm and read after 72 hours. Induration of 10mm or more is positive i.e the person is infected with tuberculosis. This test helps to measure the prevalence of infection in a community, diagnosing active infection in young children. It is also an indicator of successful BCG vaccination. 2. Interferon γ release assays (IGRAs): It is an indirect test for M. tuberculosis complex infection (tuberculosis disease or latent tuberculosis infection LTBI). It is based on the principle that T cells in the heparinized whole blood are stimulated by M. tuberculosis specific antigen (EAST-6 and CFP10) in vitro, which results in release of γ interferon 33


(INF- γ) which is detected and quantified. Commercially available kits are T Spot TB test and QuantiFERON TB Gold. Implication of latent TB • Latent TB can be reactivated when the immune system is depressed as in HIV infection resulting in active infection. HIV infected individual should be tested for latent TB as soon as possible and LTBI people should be treated immediately to prevent them from developing active TB disease. • All laboratory personnel should be screened for LTBI and those found to be infected should not be kept away from the laboratory.

Detection of TB among HIV infected To add to the existing burden of TB, the situation is compounded by large scale increase of new TB cases associated with increasing HIV infection. Of the 8.8 million incident cases of TB globally in 2010, an estimated 1.1 million were people with HIV. The immune defects produced by HIV influence the natural history of TB infection. In most people with early stages of HIV infection, symptoms of tuberculosis are similar as in people without HIV. In such cases tuberculosis programme should continue to focus on microscopy. However diagnosis of tuberculosis is more difficult if the individual has advanced HIV infection because: • HIV positive patients with pulmonary tuberculosis have a higher frequency of negative sputum smears. Confirming the diagnosis may require culture. • HIV positive people with tuberculosis have a higher frequency of false negative tuberculin skin test. In other words, TST is a poor indicator of latent TB in HIV infected cases. • Chest radiograph may be less useful in people with HIV as they have less cavitation. • Cases of extra pulmonary tuberculosis seems to be more common in people who are co-infected.

In short, screen for tuberculosis using sputum smear microscopy, if the result is positive start the treatment but if it is negative, sputum culture should be carried out along with one of the automated and molecular methods wherever possible. Conclusion Although, today many new techniques are available for diagnosis of tuberculosis and testing for drug susceptibility, detection of AFB by direct microscopy is the only feasible method recommended for tuberculosis control programme of India in detecting infectious pulmonary tuberculosis cases and monitoring the response to therapy. Wherever facilities are available, isolation of mycobacteria by culture and drug sensitivity by conventional methods till remain the recommended methods in disease endemic countries. The new techniques described, though have a high sensitivity and specificity and rapid time of detection but involves prohibitive expenditure in terms of instrumentation, expertise and reagents, making them out of reach of many laboratories in developing countries like India. References 1.

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Ramachandran R, Paramasivan CN. What is new in the diagnosis of Tuberculosis? Part I: Techniques for diagnosis of Tuberculosis. Ind J Tub 2003; 50:133-42 Ramachandran R, Paramasivan CN. What is new in the diagnosis of Tuberculosis? Part II: Techniques for drug susceptibility. Ind J Tub 2003; 50:197-202 Katoch VM. Newer diagnostic techniques for tuberculosis. Ind J Med Res 2004;120: 418-28 Hazbon MH. Recent advances in molecular methods for early diagnosis of tuberculosis and drug resistant tuberculosis. Biomedica 2004;24 (Suppl):149-62. Blakemore R, Story E, Helb D, Kop JA, Banada P,et al. Evaluation of the Analytical Performance of the Xpert MTB/RIF Assay. J Clin Microbiol 2010; 48(7):2495-501 Pai M, Kalantri S, Dheda K. New tools and emerging technologies in for diagnosis of tuberculosis: Part 1: Latent Tuberculosis. Exp Rev Mol Diagn 2006; 6(3): 412-22

Dr. Shalini Dwivedi and her team won the Belgaum-Mumbai Prize for the best poster in Bacteriology that was presented at the XXXV National Conference of the IAMM held at Varanasi between 23-26 Nov 2011. They presented the findings of their study entiteled “Molecular epidemiology of Neisseria meningitidis serogroup A isolated from invasive meningococcal disease from 2005 to 2011 in Delhi using sequence based MLST and OMP analysis”. The study concluded that P1, 20, 9 being the most prevalent serosubtype of N. meningitidis followed by P1.20,10 and P1.20, 9-10. Interestingly, same clone is circulating in India between the years 2005- 2011. Study and analysis through MLST database shows that serogroup A seems to be a stable group. Other members of the study included Das BK1, Kapil A1, Sood S1, Chaudhry R1, Deb M2, Nair D2, Gupta S3 from Department of Microbiology, 1.All India Institute of Medical Sciences, 2.Vardhman Mahavir Medical College and Safdarjung hospital and 3 National centre for disease control New Delhi

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Crossword puzzle no. 1102 Vikas Manchanda Chacha Nehru Bal Chikitsalaya, Delhi

Answers on page 12

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Jeevanutimes dec 2011  

Newsletter of IAMM - Delhi Chapter

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