Megha Sharma

Biomedical waste management: Incineration vs. environmental safety

Public concerns about incinerator emissions, as well as the creation of federal regulations for medical waste incinerators, are causing many health care facilities to rethink their choices in medical waste treatment. As stated by Health Care Without Harm, non-incineration treatment technologies are a growing and developing field. Most medical waste is incinerated, a practice that is short-lived because of environmental considerations. The burning of solid and regulated medical waste generated by health care creates many problems. Medical waste incinerators emit toxic air pollutants and toxic ash residues that are the major source of dioxins in the environment. International Agency for Research on Cancer, an arm of WHO, acknowledged dioxins cancer causing potential and classified it as human carcinogen. Development of waste management policies, careful waste segregation and training programs, as well as attention to materials purchased, are essential in minimizing the environmental and health impacts of any technology.

IDENTIFICATION OF LYSINE POSITIVE NON-FERMENTING GRAM NEGATIVE BACILLI (STENOTROPHOMONAS MALTOPHILIA AND BURKHOLDERIA CEPACIA COMPLEX)

BACKGROUND The Burkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are closely related groups of non-fermenting gram-negative bacilli (NFGNBs) having a similar spectrum of infections ranging from superficial to deep-seated and disseminated infections. Identification of these lysine decarboxylase-positive NFGNBs lags behind in most Indian laboratories. A simplified identification scheme was devised for these two pathogens that allowed us to isolate them with an increasing frequency at our tertiary care institute. MATERIALS AND METHODS A simple five-tube conventional biochemical identification of these bacteria has been standardized. In the beginning, some of the isolates were confirmed from the International B. cepacia Working group, Belgium. Molecular identification and typing using recA polymerase chain reaction-restriction fragment length polymorphism was also standardized for BCC. For short-term preservation of BCC, an innovative method of preserving the bacteria in Robertsons cooked medium tubes kept in a domestic refrigerator was developed. RESULTS Thirty-nine isolates of BCC isolates were obtained from various specimens (30 from blood cultures) and 22 S. maltophilia (13 blood cultures and 9 respiratory isolates) were isolated during the year 2007 alone. CONCLUSIONS BCC and S. maltophilia can be identified with relative ease using a small battery of biochemical reactions. Use of simplified methods will allow greater recognition of their pathogenic potential and correct antimicrobials should be advised in other clinical laboratories and hospitals.

Multiplex polymerase chain reaction using insertion sequence 6110 (IS6110) and mycobacterial protein fraction from BCG of Rm 0.64 in electrophoresis target genes for diagnosis of tuberculous lymphadenitis.

PURPOSE Tubercular lymphadenitis (TBLA) is a common manifestations of extrapulmonary tuberculosis (EPTB) accounting for 30-40% of cases. Prompt diagnosis and timely initiation of anti-tubercular therapy (ATT) is the key for successful clinical outcome. This study was carried out to evaluate multiplex polymerase chain reaction (MPCR) using MPB64 and IS6110, and compare with the conventional methods for rapid diagnosis of TBLA. MATERIALS AND METHODS In our study, lymph node fine-needle aspirates of patients were evaluated for TBLA. They were classified as Group I: TBLA group, divided into (a) Confirmed TBLA cases (n0 = 80): Culture/smear-positive or cytological examination showing presence of epithelioid cell granuloma with or without multinucleate giant cell and caseation necrosis with presence of AFB, and (b) suspected TBLA cases ( n = 30): Culture/smear-negative and cytological examination showing presence of epithelioid cell granuloma and response to ATT and Group II (Control) (n = 25): Patients of lymphadenopathy confirmed to be caused by other diseases such as sarcoidosis, lymphoma, etc., All samples were subjected to conventional tests and MPCR. For MPCR we used Mycobacterium tuberculosis-specific deoxyribonucleic acid sequences specific for the MPB64 and IS6110 region. RESULTS In the confirmed TBLA group, Ziehl-Neelsen (ZN) smear, cytology, culture, and MPCR positivity was 30%, 70%, 26.3%, and 91.3% respectively. In the suspected TBLA group, smear and culture were negative, and sensitivity of cytology and MPCR was 73.3% and 86.6%, respectively. In the control group all tests were found to be negative, thus giving a specificity of 100% to all the tests in the study. CONCLUSION In conclusion, techniques like MPCR with high sensitivity and specificity can play an important role in rapid diagnosis of TBLA.

Diagnostic potential of multi‐targeted LAMP (loop‐mediated isothermal amplification) for osteoarticular tuberculosis

Delay in diagnosing osteoarticular tuberculosis (OATB) contributes significantly to morbidity by causing disfiguration and neurological sequelae. The delay caused by conventional culture and the expertise and expense involved in other nucleic acid based tests, make LAMP (loop‐mediated isothermal amplification) assay a favorable middle path. We evaluated LAMP assay using IS6110 and MPB64 for rapid diagnosis of OATB by comparing with IS6110 PCR and culture. LAMP assay was performed on 140 synovial fluid and pus samples (10 culture‐positive proven cases, 80 culture‐negative probable cases, and 50 negative controls) using three set of primer pairs each for IS6110 and MPB64. LAMP assay, using two‐target approach, had an overall sensitivity and specificity of 90% and 100% in detecting OATB. Sensitivity of IS6110 PCR, IS6110 LAMP, and MPB64 LAMP was 80%, 100%, and 100%, respectively, for confirmed cases and 72.5%, 81.75%, and 86.25%, respectively, for probable cases. Six additional cases were picked using two‐target approach. LAMP assay utilizing IS6110 and MPB64 is a cost‐effective technique for an early and reliable diagnosis of OATB.

Loop-mediated isothermal amplification (LAMP) assay for speedy diagnosis of tubercular lymphadenitis: The multi-targeted 60-minute approach

INTRODUCTION Tuberculous lymphadenitis (TBLA), the most common presentation of tuberculosis, poses a significant diagnostic challenge in the developing countries. Timely, accurate and cost-effective diagnosis can decrease the high morbidity associated with TBLA especially in resource-poor high-endemic regions. The loop-mediated isothermal amplification assay (LAMP), using two targets, was evaluated for the diagnosis of TBLA. MATERIAL AND METHODS LAMP assay using 3 sets of primers (each for IS6110 and MPB64) was performed on 170 fine needle aspiration samples (85 confirmed, 35 suspected, 50 control cases of TBLA). Results were compared against IS6110 PCR, cytology, culture and smear. RESULTS The overall sensitivity and specificity of LAMP assay, using multi-targeted approach, was 90% and 100% respectively in diagnosing TBLA. The sensitivity of multi-targeted LAMP, only MPB64 LAMP, only IS6110 LAMP and IS6110 PCR was 91.7%, 89.4%, 84.7% and 75.2%, respectively among confirmed cases and 85.7%, 77.1%, 68.5% and 60%, respectively among suspected cases of TBLA. Additional 12/120 (10%) cases were detected using multi-targeted method. DISCUSSION The multi-targeted LAMP, with its speedy and reliable results, is a potential diagnostic test for TBLA in low-resource countries.

Whole Genome Sequencing of Mycobacterium tuberculosis Isolates From Extrapulmonary Sites.

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide. Extrapulmonary tuberculosis (EPTB) constitutes around 15-20% of TB cases in immunocompetent individuals. Extrapulmonary sites that are affected by TB include bones, lymph nodes, meningitis, pleura, and genitourinary tract. Whole genome sequencing has emerged as a powerful tool to map genetic diversity among Mycobacterium tuberculosis (MTB) isolates and identify the genomic signatures associated with drug resistance, pathogenesis, and disease transmission. Several pulmonary isolates of MTB have been sequenced over the years. However, availability of whole genome sequences of MTB isolates from extrapulmonary sites is limited. Some studies suggest that genetic variations in MTB might contribute to disease presentation in extrapulmonary sites. This can be addressed if whole genome sequence data from large number of extrapulmonary isolates becomes available. In this study, we have performed whole genome sequencing of five MTB clinical isolates derived from EPTB sites using next-generation sequencing platform. We identified 1434 nonsynonymous single nucleotide variations (SNVs), 143 insertions and 105 deletions. This includes 279 SNVs that were not reported before in publicly available datasets. We found several mutations that are known to confer resistance to drugs. All the five isolates belonged to East-African-Indian lineage (lineage 3). We identified 9 putative prophage DNA integrations and 14 predicted clustered regularly interspaced short palindromic repeats (CRISPR) in MTB genome. Our analysis indicates that more work is needed to map the genetic diversity of MTB. Whole genome sequencing in conjunction with comprehensive drug susceptibility testing can reveal clinically relevant mutations associated with drug resistance.

Evaluation of multiplex polymerase chain reaction utilising multiple targets in Mycobacterium tuberculosis direct test negative but culture positive cases: a potential method for enhancing the diagnosis of tuberculosis.

PURPOSE To evaluate multiplex Polymerase Chain Reaction (MPCR) utilising multiple targets (IS6110, Protein b [Pab] and MPB64 genes) in Mycobacterium tuberculosis Direct Test (MTD) negative but culture positive cases and comparison of MPCR with Real-Time polymerase chain reaction (RT-PCR) for diagnosis of tuberculosis. MATERIALS AND METHODS MPCR was carried out on 28 culture positive sputum samples. Out of 28 culture positive samples, 17 were originally reported, as MTD test negative and 11 were MTD test positive, respectively. The results of MPCR were compared with RT-PCR. To check the specificity of the tests, MPCR and RT-PCR were also evaluated with 16 non-tuberculous mycobacterial (NTM) isolates. RESULTS Out of 28 culture positive sputum samples, MPCR was positive in all 28/28 samples, whereas RT-PCR was positive in 27/28 samples and MTD test was originally tested positive in six sputum samples and on repeating MTD testing, five more sputum samples were positive and thus total number of MTD positive were 11/28 sputum samples, respectively. All the tests were negative on evaluation with all the 16 NTMs, thus giving specificity of 100% to all the tests; sensitivity of MPCR, RT-PCR and MTD tests were 100%, 96.42% and 39.28%, respectively, in these specifically selected samples. CONCLUSIONS MPCR may be an important tool in the rapid diagnosis of tuberculosis especially in disease endemic, resource limited countries.

Real-time PCR followed by high-resolution melting curve analysis: A rapid and pragmatic approach for screening of multidrug-resistant extrapulmonary tuberculosis

INTRODUCTION Multidrug resistance (MDR) in extrapulmonary tuberculosis (EPTB) is a diagnostic challenge in an endemic country like India. Timely detection of MDR-TB can contribute to a better patient outcome. OBJECTIVE To perform real-time PCR (qPCR) using rpoB, mpb64 and IS6110 gene on a variety of EPTB samples and to compare the performance of different gene targets. All qPCR positive samples were subjected to high resolution melt-curve analysis (HRM analysis) for rpoB and katG gene to evaluate its potential for MDR screening among different sample types. METHODS Real-time PCR using rpoB, mpb64 and IS6110 genes was carried out on 200 cases of study group and 100 cases of non-TB control group. The study group consisted of 100 culture-confirmed and 100 clinically suspected cases of EPTB. Phenotypic drug susceptibility testing (DST) for culture isolates was performed by the 1% indirect agar proportion method. DNA extracted from all qPCR positive samples was subjected to rpoB and katG HRM analysis for screening of MDR. Sequencing was used to confirm the results of HRM analysis and the results were also compared with phenotypic DST in all culture positive cases. RESULTS The sensitivity of qPCR using rpoB, mpb64 and IS6110 was 86.5%, 86.5% and 76.5%, respectively. All isolates from the control group were negative by all the three targets, giving a specificity of 100%. HRM analysis detected MDR in 22/200 (11%) isolates. 3/200 (1.5%) had mono-rifampicin resistance while 8/200 (4%) had mono-isoniazid resistance. HRM analysis identified an additional 4 MDR cases directly from the samples which were negative by culture. On sequencing, mutations were observed at codon 531 (60%); 533 (16%); 516 (12%) and 526 (12%) of the rpoB gene and at codon 315 (100%) of the katG gene. There was 100% concordance in the results of phenotypic DST, HRM analysis and sequencing. CONCLUSION The HRM analysis can play a promising role in the reliable and rapid screening of EPTB samples for detection of MDR.

Hepatitis E Virus Induced Acute Liver Failure with Scrub Typhus Coinfection in a Pregnant Woman

Coinfections contribute significantly to diagnostic challenges of acute febrile illnesses, especially in endemic areas. The confusion caused by overlapping clinical features impedes timely management. Herein, we report an unusual, previously unreported case of a pregnant woman suffering from a coinfection of scrub typhus and hepatitis E virus. A 25-year-old, 31-week pregnant woman presented with jaundice for 5 days and altered sensorium for 2 days. She had features of both viral acute liver failure (ALF) and tropical infections mimicking ALF, including hyperbilirubinemia, coagulopathy, anemia, thrombocytopenia, intravascular hemolysis, and hepatosplenomegaly. Etiological workup revealed rare coinfection of hepatitis E and scrub typhus. Despite all supportive measures, the patient succumbed to her illness (i.e., absent brainstem reflexes and intracranial bleed secondary to coagulopathy) and had poor fetal outcome, which resulted in stillbirth. ALF in a pregnant woman is a medical and obstetric emergency. It can result from varied etiologies that though differ in their incidence, mode of occurrence, and pregnancy outcome, can clinically masquerade as each other, causing diagnostic dilemma. This unusual case report highlights the significance of keeping all such possibilities in mind while managing a pregnant woman with ALF, especially in a country like India where maternal and perinatal mortality rates, the core indicators of national health, are still among the highest in the world.

Rapid identification of medically important mosquitoes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BackgroundAccurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it.MethodsMosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens.ResultsReproducible MALDI-TOF MS spectra spanning over 2–14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55–60 for Anopheles, 80–100 for Aedes, 30–60 for Culex and 45–50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8).ConclusionsMALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs.