Use of GenoType® MTBDRplus assay to assess drug resistance and mutation patterns of multidrug-resistant tuberculosis isolates in northern India.

Purpose: The emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. The diagnosis of MDR-TB is of paramount importance in establishing appropriate clinical management and infection control measures. The aim of this study was to evaluate drug resistance and mutational patterns in clinical isolates MDR-TB by GenoType® MTBDRplus assay. Material and Methods: A total of 350 non-repeated sputum specimens were collected from highly suspected drug-resistant pulmonary tuberculosis (PTB) cases; which were processed by microscopy, culture, differentiation and first line drug susceptibility testing (DST) using BacT/ALERT 3D system. Results: Among a total of 125 mycobacterium tuberculosis complex (MTBC) strains, readable results were obtained from 120 (96%) strains by GenoType® MTBDRplus assay. Only 45 MDR-TB isolates were analysed for the performance, frequency and mutational patterns by GenoType® MTBDRplus assay. The sensitivity of the GenoType® MDRTBplus assay for detecting individual resistance to rifampicin (RIF), isoniazid (INH) and multidrug resistance was found to be 95.8%, 96.3% and 97.7%, respectively. Mutation in codon S531L of the rpoB gene and codon S315T1 of katG genes were dominated in MDR-TB strains, respectively (P < 0.05). Conclusions: The GenoType® MTBDRplus assay is highly sensitive with short turnaround times and a rapid test for the detection of the most common mutations conferring resistance in MDR-TB strains that can readily be included in a routine laboratory workflow.

Changing patterns and trends of multidrug-resistant tuberculosis at referral centre in Northern India: A 4-year experience.

Purpose: India has a high burden of drug-resistant tuberculosis (TB), although there is little data on multidrug-resistant tuberculosis (MDR-TB). Although MDR-TB has existed for long time in India, very few diagnostic laboratories are well-equipped to test drug sensitivity. The objectives of this study were to determine the prevalence of MDR-TB, first-line drug resistance patterns and its changing trends in northern India in the 4 years. Materials and Methods: This was a prospective study from July 2007 to December 2010. Microscopy, culture by Bactec460 and p-nitro-α-acetylamino-β-hydroxypropiophenone (NAP) test was performed to isolate and identify Mycobacterium tuberculosis (M. tb) complex (MTBC). Drug sensitivity testing (DST) was performed by 1% proportional method (Bactec460) for four drugs: Rifampicin, isoniazid, ethambutol and streptomycin. Various clinical and demographical profiles were evaluated to analyse risk factors for development of drug resistance. Results: We found the overall prevalence rate of MDR-TB to be 38.8%, increasing from 36.4% in 2007 to 40.8% in 2010. we found that the prevalence of MDR-TB in new and previously treated cases was 29.1% and 43.3% ( P < 0.05; CI 95%). The increasing trend of MDR-TB was more likely in pulmonary TB when compared with extra-pulmonary TB ( P < 0.05; CI 95%). Conclusions: we found a high prevalence (38.8%) of MDR-TB both in new cases (29.1%) and previously treated cases (43.3%).This study strongly highlights the need to make strategies for testing, surveillance, monitoring and management of such drug-resistant cases.

Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis.

Purpose: Extrapulmonary tuberculosis (EPTB) is emerging problem in developing and developed countries. The diagnosis of EPTB in its different clinical presentations remains a true challenge. IS6110-based polymerase chain reaction (PCR) is used for rapid identification and positivity rate of the Mycobacterium tuberculosis complex in clinical isolates of different sites of EPTB. The present study was carried out to study the prevalence of M. tuberculosis complex in clinical isolates of EPTB at tertiary care centres in Lucknow. Materials and Methods: Seven hundred fifty-six specimens were collected from the suspected cases of EPTB which were processed for Mycobacteria by Ziehl Neelson (ZN) staining and BACTEC culture. All the specimens were also processed for IS6110-based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of the M. tuberculosis complex. Results: Of these 756 specimens, 71(9.3%) were positive for acid fast bacilli (AFB) by ZN staining, 227(30.1%) were positive for mycobacteria by BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 165 (20.7%) isolates. We found a significant difference in sensitivities of different tests (P<0.05). Conclusions: This study reveals the positivity of M. tuberculosis complex in clinical isolates of EPTB case in tertiary care hospitals in Northern India. 72.7% of M. tuberculosis complex was confirmed by IS6110-PCR in culture isolates from different sites of EPTB. The high prevalence of the M. tuberculosis complex was seen in lymph node aspirate and synovial fluid. However, utility of PCR may play a potentially significant role in strengthening the diagnosis of EPTB especially targeting IS6110.

Clinical Profile and Prevalence of Rotavirus Infection in Children Presented with Acute Diarrhea at Tertiary Care Referral Hospital at Northern Part of India.

A prospective analysis of 90 clinically diagnosed cases with acute diarrhea over a period of one year was carried out to determine the prevalence of rotavirus infection in children between 2 months to 2 years of age. Enzyme Linked Immunosorbent Assay (ELISA) and Polyacrylamide Gel Electrophoresis (PAGE) were used for detection of rotavirus from stool sample. Fourteen (15.6%) of them were found to be positive for group A rotavirus, 9 (23%) cases were between 6 months to 1 year of age. Rotavirus excretion was highest (50%) when all three symptoms (diarrhea, vomiting and fever) occurred in the same child. A planned study for surveillance of rotavirus serotypes is required from this area.

Bacterial isolates from mechanically ventilated patients with nosocomial pneumonia within intensive care unit of a tertiary care center.

Nosocomial pneumonia is a common complication in mechanically ventilated patients. A study was carried out to determine the incidence, common bacterial etiologic agents and their antimicrobial susceptibility, and outcome of such pneumonia in an Intensive Care Unit (ICU) of a tertiary care center. In Surgical ICU (SICU) 176 patients required mechanical ventilation for more than 72 hours. A total of 39 (22.1%) of these patients developed nosocomial bacterial pneumonia as determined by microbiological assays. Endotracheal aspirate cultures detected a single bacterial isolate in 22 (56.4%) patients while two and three organisms were isolated from 10 (25.6%) and 7 (17.9%) patients respectively. Fifty three (84.1%) of a total of 63 isolates were Gram negative bacilli. The most frequently encountered pathogens were Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter species among the Gram negative bacilli and Staphylococcus aureus among the Gram positives. Resistance of bacterial isolates varied from 24 to 90% against commonly used antibiotics. Amikacin had the best profile, with 14% to 55% resistance against various isolates. Twenty three (59%) of 39 patients with pneumonia expired in the ICU. P. aeruginosa (25.6%) and K. pneunmoniae (17.9%) were the predominant isolates in these patients. Nosocomial pneumonia with high mortality is a frequent occurrence in mechanically ventilated patients in our ICU setting. Gram negative organisms with high levels of antimicrobial resistance are the most common isolates. Regular surveillance and monitoring of changes in antibiotic susceptibility of bacterial pathogens and appropriate therapeutic measures are likely to reduce the mortality in these patients.

A comparative study of clinico-radiological spectrum of tuberculosis among HIV seropositive and HIV seronegative patients.

BACKGROUND: A study to determine the prevalence of human immunodeficiency virus (HIV) infection among tuberculosis patients and to compare the clinico-radiological spectrum of tuberculosis among HIV seropositive and seronegative patients was carried out in the Department of TB and Chest Diseases, CSM Medical University, Lucknow (Uttar Pradesh), India. METHODS: A total of 1105 radiologically and/or bacteriologically confirmed patients of tuberculosis were screened for HIV infection during the years 1995 to 1997 and from 2000-2001. RESULTS: Out of a total 1105 patients screened, 31(2.8%) were found to be HIV seropositive. Tuberculin positivity was less among HIV seropositive patients as compared to HIV seronegative patients (22.6% vs 76.4%; p < 0.001). There was no statistically significant difference in sputum smear positivity for acid-fast bacilli (AFB) among HIV seropositive and seronegative patients. Among HIV seropositive patients, mid and lower zone involvement, exudative lesions and mediastinal lymphadenopathy was more common as compared to the seronegative patients. CONCLUSION: HIV seropositivity rates among tuberculosis patients was 2.8 percent. The presentation of tuberculosis was more often atypical among these patients.

Cryptococcal infection in patients with clinically diagnosed meningitis in a tertiary care center.

A retrospective analysis of 326 clinically diagnosed cases with meningitis over a period of five-and-a-half years was carried out to determine the prevalence of cryptococcal infection, its associated risk factors and therapeutic outcome. Fifty-four (16.6%) patients with cryptococcal meningitis were identified by smear examination, culture and/or cryptococcal antigen latex agglutination test. Records of 45 cryptococcal meningitis patients were available; 18 (40%) of them were apparently healthy immunocompetent individuals, 13 (28.9%) had human immunodeficiency virus (HIV) infection, 9 (20%) were renal transplant recipients, 4 (8.9%) were diabetic and 1 (2.2%) had systemic lupus erythematosus. Ten (22.2%) patients died and 11 (24.4%) patients (all HIV-positive) left against medical advice. The present study indicates that cryptococcal infection is associated with high mortality. Presenting symptoms being indistinguishable from other causes of central nervous system infection, all patients with a clinical diagnosis of meningitis, irrespective of their immune status should be investigated for cryptococcal infection.

A pilot study on observations on CD4 & CD8 counts in healthy HIV seronegative individuals.

BACKGROUND & OBJECTIVES: CD4 T lymphocyte count is used to measure the progression of HIV infection and is recommended as part of the standard care of HIV infected person. Information on reference CD4 counts and CD4:CD8 ratio in healthy individuals is lacking in India. Therefore the present study was undertaken to obtain base-line data on CD4 counts and CD4:CD8 ratio of healthy population from north India and to assess the feasibility of using the values as reference in an extended larger study. METHODS: In this pilot study 84 HIV negative healthy volunteers (56 males, 28 females) in the age group of 20-59 yr and who were willing to participate in the study were enrolled after proper counseling. Blood specimens were collected from each subject and processed for anti-HIV antibodies for exclusion of HIV. CD4 and CD8 counts of the samples were performed by fluorescence activated cell sorting (FACS). RESULTS: The mean +/- SD of the absolute numbers of CD4 and CD8 lymphocytes/microliter was 763.6 +/- 226 and 547.5 +/- 190 in males and 797.9 +/- 263 and 567.7 +/- 250 in females. The range of the CD4 and CD8 counts was 365-1328 and 264-991 in males and 415-1257 and 224-1126 in females respectively. The mean +/- SD of the CD4:CD8 ratio was 1.47 +/- 0.42 in males and 1.52 +/- 0.45 in females. INTERPRETATION & CONCLUSION: The results of this study showed that there is wide variability in CD4 count in our population, as is seen in studies reported from other parts of India. A large multicentric study could define the normal range for CD4 and CD8 counts and CD4:CD8 ratio in the Indian population.

Guillain-Barre syndrome and Campylobacter infection.

Campylobacter infection of the gastrointestinal tract has been observed as an antecedent illness in some patients with Guillain-Barre syndrome (GBS); these patients have been reported to have poor prognosis. We investigated 29 patients with GBS, admitted to our hospital from January 1996 to December 1999 for recent Campylobacter enteritis by culture of their stool specimens. Campylobacter upsaliensis and C. jejuni were isolated from stools of one patient each with acute motor axonal neuropathy (AMAN) and acute inflammatory demyelinating polyradiculoneuropathy (AIDP) respectively. The patient with C. upsaliensis infection was a 7 year-old male child who developed features of AMAN, 7 days after onset of diarrhea. He recovered gradually within 24 days with residual deficit in the form of foot drop. This deficit has persisted for last three and half years. The other patient with C. jejuni infection was a 9 year-old boy, who developed AIDP after 9 days of acute diarrhea. This patient recovered completely within 28 days of illness without any deficit. None of the patients had relapse of GBS. The present findings indicate the need of planned systematic studies to explore the role of C. upsaliensis and other campylobacters as agents of antecedent diarrhea in patients of GBS with different clinical presentations and prognosis.

Drug resistant Haemophilus influenzae from respiratory tract infection in a tertiary care hospital in north India.

Haemophilus influenzae is an important respiratory pathogen. Emergence of resistance to various antibiotics is a major problem in patient management. A total of 90 strains of H. influenzae were characterized from specimens obtained from patients of acute respiratory tract infection; 13 (14.4%) belonged to type beta. On biotyping, 90% strains belonged to biotype II. The frequency of resistance to various antibiotics was as follows: cotrimoxazole 33.3% ampicillin 21.1%, cephalexin 7.8%, chloramphenicol 7.8%, ciprofloxacin 2.5% erythromycin and tetracycline 5% each. All the ampicillin-resistant strains produced beta-lactamase as detected by nitrocefin disc method. None of the strains exhibited resistance to cefaclor and third generation cephalosporins. The present study showed emergence of variable resistance to ampicillin, cotrimoxazole and other antibiotics. It is important for the clinical microbiology laboratory to monitor drug resistant strains for instituting appropriate antibiotic therapy of respiratory infections due to H. influenzae.

Identification of enteric pathogens in HIV-positive patients with diarrhoea in northern India.

Enteric pathogens associated with chronic diarrhoea in HIV-positive patients were studied. The study was conducted during January 1995-December 1998. Stool specimens from all diarrhoea patients (n = 26) were examined microscopically for ova and parasites using wet preparations and stained smears. Stool samples from diarrhoea patients were also cultured on appropriate media to isolate enteric bacterial pathogens. Of the 59 patients, 26 (44%) had prolonged diarrhoea for more than 4 weeks. Enteric pathogens were detected in 19 (73%) of the 26 patients: 17 patients harboured a single pathogen, and 2 patients had mixed pathogens. The detection rate of emerging parasites, including Isospora, Cryptosporidium, Blastocystis hominis, and Strongyloides stercoralis as a single agent, was significantly higher than conventional pathogens (50% vs 19.2%; p < 0.05). Only one patient harboured both conventional and emerging pathogens (Entamoeba histolytica and Cryptosporidium). Isospora belli was detected in 8 (31%) of the 26 diarrhoea patients: in 7 (27%) patients as a single agent and in one patient with S. stercoralis. Cryptosporidium was identified in 3 (11%) diarrhoea patients: in 2 (8%) patients as a single agent and in one patient with E. histolytica, followed by B. hominis in 2 (8%) patients. E. histolytica was most commonly isolated (3/26; 11.5%), followed by Giardia lamblia, enteropathogenic Escherichia coli, and Campylobacter jejuni (one patient each). Parasitic pathogens were frequently associated with HIV-positive patients with diarrhoea in northern India. I. belli was the most frequent parasite isolated, followed by Cryptosporidium. Stools of all HIV-positive patients with diarrhoea should thoroughly be investigated to identify aetiologic agents for proper management.

Chlamydia trachomatis infection among pregnant women: prevalence and prenatal importance.

BACKGROUND: Chlamydia trachomatis infection in pregnant women is suspected to result in low birth-weight and premature infants. We conducted studies to ascertain the prevalence of this infection among pregnant women in our setting and whether its presence is a risk factor for low birth-weight or prematurity. METHODS: In the first study, 94 pregnant women between 26 and 30 weeks of gestation were screened for infection with Chlamydia trachomatis. The second investigated a cohort of 172 pregnant women presenting in spontaneous labour. The infection status was related to perinatal outcome in terms of birth-weight and gestation. In both the studies, Chlamydia trachomatis infection was diagnosed using the Chlamydiazyme test performed on endocervical swabs. RESULTS: The prevalence of Chlamydia trachomatis infection in mid-pregnancy and at labour was 17% (16/94) and 18.6% (32/172), respectively. Women with infection were relatively older than those without it [mean (SD) age: 26.6 (4.5) years v. 24.8 (3.6) years, p = 0.01]. The mean (SD) birth-weight [2869 (611) g v. 2814 (496) g], gestation [38.5 (2.6) weeks v. 38.3 (2.0) weeks], and incidence of low birth-weight [18.7% v. 20.7%] as well as prematurity [9.4% v. 10.7%] were similar among neonates born to women with or without infection. Neonates born to infected mothers experienced purulent conjunctivitis more frequently than those born to non-infected mothers [12.5% v. 2.8%, p = 0.04]. CONCLUSION: Chlamydia trachomatis is a relatively common infection in pregnant women. However, it was not associated with either low birth-weight or prematurity.

Adverse foetal outcome in specific IgM positive Chlamydia trachomatis infection in pregnancy.

A study was undertaken to determine the adverse foetal outcome in antichlamydial IgM positive asymptomatic pregnant females. An indirect immunoperoxidase assay was done to detect IgM in 78 apparently normal asymptomatic pregnant women during the third trimester and follow up was done till delivery to study the effect of chlamydial infection on foetal outcome. A total of 28 (35.9%) women were positive for antichlamydial IgM while only 3.33 per cent asymptomatic non-pregnant normal women (controls) were positive. 28.7 per cent IgM positive mothers delivered low birth weight (LBW) babies (P less than 0.05), 9.7 per cent had premature labour (PTL) and 4.7 per cent had intrauterine death (IUD). The findings are significant as none of the IgM negative mothers had PTL and IUD and only 2.6 per cent had LBW babies.