Abhishek Mewara

Cephalosporin-resistant Shigella flexneri over 9 years (2001–09) in India

OBJECTIVES To determine the pattern and antimicrobial resistance genes of cephalosporin resistance in Shigella flexneri and Shigella dysenteriae over 9 years. METHODS Isolates of Shigella (S. flexneri, n = 119 and S. dysenteriae, n = 24) were tested for resistance to ceftriaxone and cefepime by disc diffusion, for MIC by Etest and for extended-spectrum β-lactamase (ESBL) and AmpC production. The presence of antimicrobial resistance genes was investigated by PCR using specific primers for bla(TEM), bla(OXA-1), bla(CTX-M-15), bla(SHV) and bla(CMY-2) for all the isolates. RESULTS Twenty (16.8%) S. flexneri isolates were resistant/intermediately susceptible to ceftriaxone/cefepime, while all S. dysenteriae were susceptible. In S. flexneri isolates, the MIC(50) values of ceftriaxone and cefepime were found to be 0.032 and 0.125 mg/L, respectively, while their MIC(90) values were 12 and 8 mg/L, respectively. The MIC(50) and MIC(90) for S. dysenteriae were below 1 mg/L for ceftriaxone; however, for cefepime the MIC(90) was found to be 4 mg/L. Of the 20 resistant/intermediately susceptible S. flexneri isolates, 9 were positive for ESBL production and 4 for AmpC production by phenotypic tests. All 20 isolates were found to be positive for bla(TEM), 10 for bla(CTX-M-15), 8 for bla(OXA) and 7 for bla(CMY-2); none was positive for bla(SHV). CONCLUSIONS We report a high level of cephalosporin resistance with high MICs and ESBL- and AmpC-mediated antibiotic resistance in Shigella from north India.

Prevalence of multidrug resistance in Mycobacterium tuberculosis isolates from HIV seropositive and seronegative patients with pulmonary tuberculosis in north India

BackgroundMultidrug resistant (MDR) and extensively-drug resistant (XDR) tuberculosis (TB) are a serious threat to the national TB control programs of developing countries, and the situation is further worsened by the human immunodeficiency virus (HIV) pandemic. The literature regarding MDR/XDR-TB is, however, scanty from most parts of India. We carried out this study to assess the prevalence of MDR/XDR-TB in new and previously treated cases of pulmonary TB and in HIV seropositive and seronegative patients.MethodsSputum and blood specimens were obtained from 2100 patients suspected of pulmonary tuberculosis and subjected to sputum microscopy and culture for TB, and HIV serology at our tertiary care centre in north India. The culture positive Mycobacterium tuberculosis isolates were subjected to drug susceptibility testing (DST) for first line anti-tuberculosis drugs, and the MDR isolates were further subjected to second line DST. Various parameters of the patients’ were analyzed viz. clinical presentation, radiology, previous treatment history, demographic and socioeconomic data and microbiology results.ResultsOf the 2100 patients, sputum specimens of 256 were smear positive for acid-fast bacilli (AFB), 271 (12.9%) grew Mycobacterium spp., and M. tuberculosis was isolated in 219 (10.42%). Of the 219 patients infected with M. tuberculosis, 20.1% (44/219) were found to be seropositive for HIV. Overall, MDR-TB was observed in 17.4% (39/219) isolates. There were 121 newly diagnosed and 98 previously treated patients, of which MDR-TB was found to be associated with 9.9% (12/121) and 27.6% (27/98) cases respectively. There was significantly higher association of MDR-TB (12/44, 27.3%) with HIV seropositive patients as compared to HIV seronegative patients (27/175, 15.4%) after controlling previous treatment status, age, and sex (odd’s ratio, 2.3 [95% CI, 1.000-5.350]; p-value, 0.05). No XDR-TB was found among the MDR-TB isolates.ConclusionThe present study demonstrated a high prevalence of drug resistance amongst pulmonary TB isolates of M. tuberculosis from north India as compared to the WHO estimates for India in 2010, though this could possibly be attributed to the clustering of more serious or referred cases at our tertiary care centre. The prevalence of MDR-TB in HIV seropositive patients was significantly higher than seronegative individuals. The study emphasizes the need to monitor the trends of drug resistance in TB in various populations in order to timely implement appropriate interventions to curb the menace of MDR-TB.

Development and evaluation of multiplex PCR in rapid diagnosis of abdominal tuberculosis

The clinical features of abdominal tuberculosis (TB) are non-specific and establishing a diagnosis remains a challenge. A delay in diagnosis is likely to increase the morbidity in these patients. We developed a multiplex polymerase chain reaction (PCR) using 16SrRNA, IS6110, and devR, and evaluated it in comparison with other conventional tests in clinical suspects of abdominal TB. A total of 183 patients with clinical suspicion of abdominal TB (96 patients with intestinal TB and 87 with peritoneal TB) were enrolled for the study. Endoscopic or intraoperative biopsies were collected from patients suspected of intestinal TB and ascitic fluid was collected from patients with a suspicion of peritoneal TB. Of the intestinal tuberculosis group, there were 40 confirmed cases and 56 controls, while of the peritoneal tuberculosis group there were 37 confirmed cases and 50 controls. Multiplex PCR showed a high sensitivity and specificity in both the intestinal TB and peritoneal TB groups. When combined with histopathology, multiplex PCR could detect 97.5% of all the cases in the intestinal tuberculosis group, while in combination adenosine deaminase levels (ADA) in cases of peritoneal tuberculosis it increased the specificity of diagnosis of peritoneal tuberculosis to 95%. In combination with histopathology in suspected intestinal TB cases, and ADA testing in suspected peritoneal TB cases, it can be used as a highly sensitive, specific, and rapid diagnostic tool with the ability to supplement the limitations of other diagnostic modalities.

Shigellosis: Epidemiology in India

Shigellosis is one of the major causes of diarrhoea in India. The accurate estimates of morbidity and mortality due to shigellosis are lacking, though it is endemic in the country and has been reported to cause many outbreaks. The limited information available indicates Shigella to be an important food-borne pathogen in India. S. flexneri is the most common species, S. sonnei and non-agglutinable shigellae seem to be steadily surfacing, while S. dysenteriae has temporarily disappeared from the northern and eastern regions. Antibiotic-resistant strains of different Shigella species and serotypes have emerged all over the world. Especially important is the global emergence of multidrug resistant shigellae, notably the increasing resistance to third generation cephalosporins and fluoroquinolones, and also azithromycin. This calls for a continuous and strong surveillance of antibiotic resistance across the country for periodic updation of the local antibiograms. The prevention of shigellosis is desirable as it will substantially reduce the morbidity associated with diarrhoea in the country. Public health measures like provision of safe water and adequate sanitation are of immense importance to reduce the burden of shigellosis, however, the provision of resources to develop such an infrastructure in India is a complex issue and will take time to resolve. Thus, the scientific thrust should be focused towards development of a safe and affordable multivalent vaccine. This review is focused upon the epidemiology, disease burden and the therapeutic challenges of shigellosis in Indian perspective.

Evaluation of in-house loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of M. tuberculosis in pulmonary specimens.

Loop‐mediated isothermal amplification (LAMP) assay has come forward as a rapid, cost‐effective molecular technique for diagnosis of tuberculosis (TB) in developing countries. This study evaluated Mycobacterium tuberculosis–specific in‐house LAMP assay targeting 16s rRNA and compared it with other conventional tests and nucleic acid amplification assay (IS6110 PCR).

Outbreak of scrub typhus in North India: a re-emerging epidemic

Scrub typhus is re-emerging in India. We describe an outbreak of 45 cases from our tertiary care center in north India. This outbreak included city dwellers who had no history of travel to hilly areas. The classical feature of scrub typhus, the eschar, was also noted rarely in these patients. The changing epidemiology of scrub typhus should be kept in mind while attending patients with acute febrile illness.

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.

Rising trends of syphilis in a tertiary care center in North India

Background and Objectives: Syphilis is a classical sexually transmitted disease (STD), caused by Treponema pallidum subsp. pallidum. In this retrospective study, we analyzed trends of syphilis prevalence in patient groups attending our tertiary care center. Materials and Methods: The data was obtained by reviewing laboratory records of the STD laboratory from January 1, 2006 to December 31, 2011. Cases positive by both Venereal Disease Research Laboratory (VDRL) and Treponema pallidum particle agglutination (TPPA) tests were analyzed for seroprevalence of syphilis in different groups, and to analyze the rising or falling trends, if any. Results: A total of 28,920 serum samples were received in the 6-year study period for VDRL testing, of which 972 (3.4%) were found to be reactive. Of these, 1722 sera were also submitted for TPPA testing, 374 (21.7%) of which were positive. A total of 375 samples were submitted for both tests, indicating biological false positivity of 0.27%. A rising trend, though not statistically significant, was observed in pregnant women, drug users and patients from wards/out-patient departments, while a statistically significant rise in prevalence of syphilis was found in HIV-positive individuals. A falling trend (not statistically significant) was observed in STD clinic attendees. Conclusion: An increasing trend of syphilis was observed during the study period when all groups were analyzed together, especially in HIV-seropositive individuals, which calls for continued and sustained efforts for case detection, treatment, and preventive measures to contain the disease.

Combination of adenosine-deaminase and nucleic acid amplification assays for diagnosing tuberculous pleural effusion.

We read with interest the paper by Keng et al., who evaluated pleural adenosine-deaminase (ADA), ADA2, interferon-gamma (IFN-g), and interferon-gamma release assays (IGRA) by enzyme-linked immuno-spot (T-SPOT.TB) assay as diagnostic tools for tuberculous pleural effusion (TPE) and concluded that pleural ADA, ADA2, and IFN-g assays, but not T-SPOT.TB assay, have good diagnostic performances for TB pleurisy. The recent policy statement of the World Health Organization (WHO) has also concluded that serological tests provide inconsistent and imprecise estimates of sensitivity and specificity and should not be used for the diagnosis of pulmonary and extra-pulmonary TB. However, nucleic acid amplification tests (NAAT) have been endorsed by WHO and may play important role in rapid detection of TB. NAATs are now used for the direct detection of Mycobacterium tuberculosis complex in respiratory specimens, mainly in developed countries, because of their high sensitivity and specificity, especially in smear-positive respiratory specimens. In this context, we evaluated ADA, along with NAATs such as Loop-mediated isothermal amplification (LAMP) assay based on the amplification of IS6110 gene of M. tuberculosis complex (MTC) and IS6110 PCR, and conventional techniques (microscopy and culture) for the diagnosis of TPE in 106 patients with pleural effusion. The study was approved by the Institute Ethics Committee and written informed consent was obtained from all patients. The patients were categorized in following groups: Group A (n Z 31) included confirmed TPE patients which were smear or culture positive for M. tuberculosis from pleural fluid and/or with histology showing a caseating granuloma; Group B (n Z 61) included probable TPE patients with signs and symptoms of TB (cough, fever, chest pain, and pleural exudate) or histology showing a chronic inflammation without caseating granuloma, with response to treatment, or pleural effusion patients with a sputum culture positive for M. tuberculosis; and Group C (n Z 14) included pleural effusion patients with an