Manimuthu Mani Sankar

High rate of extensively drug-resistant tuberculosis in Indian AIDS patients

Fifty-four full-blown AIDS patients suspected of having HIV–tuberculosis co-infection were investigated for the prevalence of extensively drug-resistant (XDR) Mycobacterium tuberculosis. Out of the 54 patients, M. tuberculosis was isolated from 24 (44.4%). Twelve (50%) isolates of these had resistance to first-line drugs, whereas four (33.33%) were also resistant to second-line drugs. All four patients, in whom XDR M. tuberculosis was isolated, died within 2.6 months of diagnosis.

Molecular characterization of Mycobacterium tuberculosis isolates from North Indian patients with extrapulmonary tuberculosis

Genotypic studies are important to understand the molecular epidemiology and transmission routes of Mycobacterium tuberculosis. In the first and largest study from India, spoligotyping and 24 loci mycobacterial interspersed repetitive units (MIRU) were performed to find genetic profiles of 125 M. tuberculosis strains isolated from patients with extrapulmonary tuberculosis (EPTB) and their drug susceptibility test was performed using BACTEC-MGIT 960. Spoligotyping results were compared with the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). The spoligotyping results showed that 110 (88%) displayed known patterns while 15 (12%) isolates had no matching database. Predominant spoligotypes belonged to CAS family (57.27%). The largest clade comprised of 38 isolates belonging to the CAS1_DEL lineage. Though there was no significant association between specific mycobacterial lineage and extrapulmonary site, a significantly high (p < 0.001) number of Beijing type isolates (28.6%) were isolated from bone and joint samples as compared to cerebrospinal fluid (5%). There was a significant association between Beijing family isolates and multi-drug-resistance, while all MANU genotypes were pan-drug sensitive. The CAS family lineage was most prevalent genotype in the EPTB cases in our population.

In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay

BackgroundNon-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance.ObjectiveTo find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM.MethodsA total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM).ResultsOf the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). M. mucogenicum was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 – 100%.ConclusionTetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.

Genetic diversity and drug susceptibility profile of Mycobacterium tuberculosis isolated from different regions of India.

OBJECTIVES Molecular genotyping profiles of Mycobacterium tuberculosis (MTB) provide a valuable insight into the evolution and transmission of the bacilli. Due to the lack of comprehensive national level data from India on this subject, we performed this study to determine the recent trends and distribution of various MTB lineages circulating in India. METHODS A total of 628 MTB isolates were obtained from North, West, South, Central and Eastern India. Spoligotyping and drug susceptibility testing was performed by using manufacturers instructions. RESULTS Spoligotyping detected 102 distinct spoligo-patterns. A total of 536 (85.3%) isolates were distributed into 85 SITs which matched the pre-existing database, whereas 17 SITs were newly created for 34 (5.4%) isolates. Overall, CAS family genotype was predominant, comprising 222 (35.4%) isolates, followed by EAI in 152 (24.2%), Beijing in 108 (17.2%), Manu in 41 (6.5%), T in 30 (4.8%), H in 6 (0.9%), X in 3 (0.5%) and one (0.2%) each in Ural and AFRI. Drug susceptibility testing identified 134 (21.3%) isolates as multi drug resistant (MDR). CONCLUSIONS The CAS lineage had a pan India presence but EAI lineage was confined to southern parts of India. Beijing genotype of MTB was significantly associated (p-value <0.0001) with MDR.

Incidence and Prevalence of Tuberculosis among Household Contacts of Pulmonary Tuberculosis Patients in a Peri-Urban Population of South Delhi, India

Background Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of mortality and morbidity across all age groups throughout the world, especially in developing countries. Methodology/Principal Findings In this study, we have included 432 open index cases with their 1608 household contacts in a prospective cohort study conducted from May 2007 to March 2009. The follow-up period was 2 years. All Index cases were diagnosed on the basis of suggestive signs and symptoms and sputum being AFB positive. Among the 432 index patients, 250 (57.9%) were males and 182 (42.1%) females; with mean age of 34±14.4 yr and 26±11.1 yr, respectively. Out of 1608 household contacts, 866 (53.9%) were males and 742 (46.1%) females; with mean age of 26.5±15.8 and 26.5±16.0 yr, respectively. Of the total 432 households, 304 (70.4%) had ≤4 members and 128 (29.6%) had ≥5 members. The median size of the family was four. Of the 1608 contacts, 1206 were able to provide sputum samples, of whom 83 (6.9%) were found MTB culture positive. Household contacts belonging to adult age group were predominantly (74, 89.2%) infected as compared to the children (9, 10.8%). On screening the contact relationship status with index patients, 52 (62.7%) were first-degree relatives, 18 (34.6%) second-degree relatives and 12 (14.5%) spouses who got infected from their respective index patients. Co-prevalent and incident tuberculosis was found in 52 (4.3%) and 31 (2.6%) contacts, respectively. In incident cases, the diagnosis could be made between 4 to 24 months of follow-up, after their baseline evaluation. Conclusion Active household contact investigation is a powerful tool to detect and treat tuberculosis at early stages and the only method to control TB in high-TB-burden countries.

Diagnostic usefulness of Xpert MTB/RIF assay for detection of tuberculous meningitis using cerebrospinal fluid

OBJECTIVE Tuberculous meningitis (TBM) is the most severe form of extra-pulmonary tuberculosis (TB) due to association of diseases with high rates of mortality and morbidity. Diagnosis continues to be a clinical challenge as microbiological confirmation is rare and time consuming resulting in delayed treatment. Xpert MTB/RIF assay is a rapid and simple test, which has been endorsed by World Health Organization as an initial diagnostic test for the diagnosis of TBM. However, evidence still lacks for its performance on cerebrospinal fluid (CSF) for the diagnosis of TBM especially from India. METHODS A total of 267 CSF samples from patients with high clinico-radiological suspicion of TBM were included in this study. Ziehl-Neelsen (ZN) staining, BACTEC Mycobacterial Growth Indicator Tube (MGIT-960) culture system, and Xpert MTB/RIF assay (using cartridge version G4) were tested on all samples. RESULTS Of total 267 samples, all were negative for smear AFB and 52 (19.5%) were culture positive by MGIT-960 culture system. However, out of 52 (19.5%) cultures detected positive by MGIT-960, 5 (9.6%) were detected as resistant to rifampicin. Xpert MTB/RIF assay was positive in 38 (14.2%) samples and negative in 223 (83.5%) samples. Cartridge error was detected in 6 (2.2%) samples, which could not be repeated due to insufficient sample volume. The sensitivity and specificity of Xpert MTB/RIF assay in comparison to MGIT-960 was 55.1% (95%, CI: 40.2-69.3) and 94.8% (95%, CI: 90.9-97.4) respectively. Overall, Xpert MTB/RIF assay detected 38 (14.2%) as positive for MTB of which 4 (10.5%), 31 (81.6%) and 3 (7.9%) were found to be rifampicin resistant, sensitive and indeterminate respectively. CONCLUSION Xpert MTB/RIF assay showed lower sensitivity as compared to MGIT 960 culture for the diagnosis of TBM from CSF samples.

High prevalence of non-tuberculous mycobacterial disease among non-HIV infected individuals in a TB endemic country--experience from a tertiary center in Delhi, India.

Non-tuberculous mycobacteria (NTM) considered mostly as colonizers or ignored as environmental contaminants in the past, are now increasingly recognized as important pulmonary pathogens in both immunocompromised and immunocompetent population.1 Specific risk factors identified are HIV infection, cystic fibrosis, underlying chronic lung disease, previous tuberculosis (TB), and work in the mining industry.1 These are also reported to cause surgical-site infections, post-injection abscesses, osteomyelitis, catheter-related blood-stream infections, and central nervous system infections.2 Non-tuberculous mycobacteria rates of infection and disease has significantly increased in recent years and rates vary widely depending on population and geographic location.1 Most reports are from developed countries that have low rates of TB. However, in countries with high burden of TB, including India, NTM pulmonary disease often goes unrecognized and is misdiagnosed as pulmonary TB because clinical presentation of NTM and Mycobacterium tuberculosis (MTB) diseases are indistinguishable from each other. Prevalence of NTM is unknown in India as NTM disease is not a reportable condition and there is lack of awareness among clinicians coupled with lack of laboratory capacity to diagnose these infections.3 Among few reports available, NTM isolation rates are reported to range from 0.5 to 8.6% in India.4 A recent study from central India reported prevalence of NTM increased from 1.0% in 2005 to 3.5% in 2008 and 88.6% of the NTM isolated were clinically relevant.4 Identification of NTM is of clinical relevance as most of the NTM are notably resistant or only partially susceptible to the standard anti-tubercular drugs and the treatment strategies and the duration of these infections differ from MTB. We conducted retrospective review of isolation rates of Mycobacterium tuberculosis complex (MTC) and NTM, the species of NTM characterized and their clinical significance during January 2011–June 2012 at our tertiary care hospital (700 bedded) in Delhi, India. The study was approved by the ethical committee of our institute. A total of 436 clinical specimens were processed for mycobacteria culture, 237 from pulmonary (sputum, bronchoalveolar lavage, bronchial wash, and endotracheal aspirates) and 199 from extrapulmonary sites (urine, pus, peritoneal fluid, lymph node aspirate, synovial fluid, endometrial biopsy, CSF). Specimens were digested and decontaminated by the standard N-acetyl-l-cysteine–NaOH method and inoculated into BACTEC mycobacteria growth indicator tube (MGIT) 960 vials (BD Diagnostics, Sparks, MD, USA) and Lowenstein–Jensen (LJ) medium slants (Hi Media laboratories, Mumbai, India). Smear microscopic examination was performed using Zeihl Neelsen (ZN) and fluorochrome stains. All positive MGIT vials were subjected to identification of MTC, by p-nitrobenzoic acid (PNBA) assay on MGIT 960 as described elsewhere5 and SD TB MPT64 antigen rapid assay by immunochromatographic method (Standard Diagnostics, Seoul, South Korea). p-Nitrobenzoic acid resistant and/or MPB 64 antigen negative isolates (suspected to be NTM) were subjected to a multiplex PCR using primers for mycobacterium genus (targeting hsp65), MTC (targeting ESAT6), and Mycobacterium avium complex (targeting MAC) specific genes.3 Confirmed NTM isolates were further speciated by pigment production, growth on MacConkey agar, rate of growth, nitrate reduction test, tellurite reduction, urease production, tween-80 hydrolysis, arylsulfatase, heat-stable catalase, iron uptake, and tolerance to 5% NaCl.6 Drug-susceptibility testing was performed for MTC for PSIRE (pyrazinamide, streptomycin, isoniazid, rifampicin, and ethambutol) by BACTEC MGIT 960 (1% proportional method). Of 436 specimens, 109 (25.6%) were positive for acid-fast bacilli by smear microscopy (ZN and/or fluorescent) and 131 (30.7%) specimens were culture positive. Of 131 positive cultures, 118 (90.1%) isolates were identified as MTC (71.2% from pulmonary and 28.8% from extrapulmonary samples) and 13 (9.9%) isolates as NTM species. The NTM were isolated from pulmonary specimens in nine (69.2%) patients, and four (30.8%) were isolated from non-pulmonary specimens, one each from urine, ascitic fluid, bone, and blood specimens. The most common NTM species characterized was Mycobacterium kansasii (4) followed by Mycobacterium chelonae (3), Mycobacterium xenopi (2), Mycobacterium scrofulaceum (1), M. avium (1), Mycobacterium asiaticum (1), and Mycobacterium fortuitum (1). Clinical details and speciation of the NTMs isolated are shown in Table 1. Fluorescent staining was more sensitive as it detected 12 additional culture positive cases that were negative by ZN stain (of these, four were NTM). As NTM are ubiquitous in nature, NTM pulmonary infections consistent with the American thoracic society (ATS) and British thoracic society (BTS) guidelines (defined in Table 1) were considered clinically relevant.7,8 Of nine patients with pulmonary NTM isolated, seven (77.9%) had definite NTM disease and two (15.4%) had probable NTM disease; among four patients with extrapulmonary specimens, two (50%) had definite NTM disease, one (25%) had probable NTM disease, and one (25%) had possible disease or colonization (Table 1). Majority of patients were males (69.2%) and 46.1% (6/13) were older in age (≧60 years). The main underlying risk factors found to be associated with NTM infections were pre-existing pulmonary disease (54%), chronic obstructive pulmonary disease (COPD; 23.1%), past and/or present history of TB (30.8%, two pulmonary TB and one Pott’s spine cases were diagnosed by both smear for AFB and culture positivity for MTB and a case of osteomyelitis was smear positive for AFB), chronic smoking (15.4%), diabetes (23.1%), steroids (7.6%), and malignancy (7.6%). No predisposing condition was detected in three patients (23.1%). All 13 patients with NTM disease were HIV seronegative (ARCHITECT® HIV Ag/Ab Combo Assay, Abbott diagnostics). Table 1 Clinical and microbiological features of non-tuberculous mycobacteria (NTM) isolated Between PNBA and MPB64 antigen assay results, 100% concordance was observed. All mycobacteria grew both in MGIT and LJ medium; however mean isolation time was much shorter with MGIT than with LJ (11 vs 28 days). Furthermore, mean recovery time of MTC from pulmonary specimens was lesser than that of non-pulmonary specimens (10 vs 16 days) by MGIT culture. Among NTMs, slow-growing mycobacteria grew in mean time of 12.9 days. Rapidly growing mycobacteria (RGM) bloodstream infection due to M. chelonae in one of the patient could be detected by MGIT in 2.2 days. Of the MTC, 67% were sensitive to all tested drugs, 14.7% were multidrug-resistant (MDR), and 17.6% were mono-resistant. Treatment details of the patients were not retrieved. While clinical diagnosis of NTM pulmonary disease is challenging, reliance only on positive smear microscopy for the initiation of treatment for suspected TB in most TB endemic countries and not on Mycobacteria culture, species identification, and drug-resistance testing results, is unfortunate as acid-fast bacilli visualized in smears may not necessarily be MTB. Moreover, NTM co-infections with MTB disease are not infrequent, though rarely diagnosed.10 As NTM strains exhibits high drug-resistance to first-line anti-tubercular drugs, many of NTM infections would be considered TB treatment failures, and subsequently treated for multidrug-resistant TB (MDR-TB) disease. The same was highlighted in a recent report from China, where NTM accounted for 30% of suspected MDR-TB cases and 4% of retreatment TB cases.11 In another study from New Delhi, India, detected NTM were detected by multiplex PCR in 17.6% of the suspected MDR-pulmonary TB cases and in 12.4% of the suspected extrapulmonary TB cases.3 These findings highlight the necessity of laboratory speciation of mycobacteria and initiation of treatment for mycobacterial infections based on in vitro susceptibility testing. In the present study, 12 of the 13 NTM infections (92.3%) were provisionally diagnosed as pulmonary or extrapulmonary TB based on clinical presentation and smear microscopy findings. Of these, three patients (23.1%) having past history of TB were considered for retreatment (MDR-TB suspected) and one patient was found to be co-infected with MTB (pulmonary NTM and Pott’s spine). None of the patient in this series was HIV seropositive. In a recent 3 years study from India, 42% of AFB cultures were positive and approximately 2% of these were NTM. Pulmonary infections represented 81% of all NTM cases and the major predisposing condition was underlying lung disease (54%) including 40% patients with a past or present history of pulmonary TB while only 2% patients were known HIV-positive.12 High prevalence of NTM infections observed among non-HIV seropositive individuals in a TB endemic country in this report, underscores the need for increased awareness of these emerging human pathogens and importance of mycobacteria speciation to reduce morbidity and mortality resulting from these diseases. Use of liquid culture medium and molecular methods in clinical laboratories can significantly reduce turn-around time particularly for diagnosis of infections due to slow-growing NTM and MDR-TB. However, with improved facilities for recovery of NTMs that may result in increased frequency of isolation of these organisms, NTM disease requires clinical correlation and differentiation from colonization.

Recovery of Mycobacterium tuberculosis from Sputum Treated with Cetyl Pyridinium Chloride

Transportation of sputum samples from remote areas to laboratories for culture of mycobacteria may take several days, which can lead to overgrowth of commensal flora and possible loss of viable mycobacteria ([1][1], [3][2], [5][3], [8][4]). To overcome this problem, cetyl pyridinum chloride (CPC) is

Catheter-related bacteremia due to M. chelonae in an immunocompromised patient: An emerging nosocomial pathogen

In the present study, a total of 64 P. falciparum isolates were collected from different malaria endemic regions of India. We observed intraand inter-regional variations in minimum detection limits as well as fragment size of both target genes [Figure 1]. Amplicon size of Pfhrp2 and Pfhrp3 ranged from 577 bp to 1000 bp and 477 bp to 832 bp, respectively. The average minimum detection limit was found to be less in Orissa (62.5 p/μl) and more in Chhattisgarh (295.6 p/μl). These observations led us to conclude that genetic diversity in these diagnostic genes may be a major factor behind the variable sensitivity of RDTs. More studies should be undertaken for the diagnosis of antigens of Indian P. falciparum population so that, the highly polymorphic genes Pfhrp2/Pfhrp3 along with other non-HRP2-based RDTs antigens can be analyzed to improve the malaria rapid diagnostic tests.

Predominance of Beijing lineage among pre-extensively drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis: A tertiary care center experience

Objective/background: Increasing resistance to various first-line and second-line drugs has become a major concern in India. However, it is not known if some genotypes are more associated with second-line drug resistance. Thus, the main aim of this study was to find out the predominant genotype associated with second-line drug resistance. Methods: During the study, a total of 234 multidrug resistant (MDR) strains of Mycobacterium tuberculosis, isolated between 2008 and 2015, were randomly selected and screened for pre-extensively drug-resistant (XDR) and XDR patterns using second-drug susceptibility testing with BACTEC MGIT 960. All the MDR isolates were tested against ofloxacin (2 μg/mL), kanamycin (2.5 μg/mL), amikacin (1 μg/mL), and capreomycin (2.5 μg/mL). Based on the resistance pattern pre-XDR was defined as M. tuberculosis isolates resistant to fluoroquinolone alone. The identified pre-XDR and XDR isolates were further characterized using spoligotyping. The spoligo patterns obtained were compared and analyzed using SITVIT_WEB Unweighted Pair Group Method with Arithmetic Mean, and Minimum Spanning Tree was derived using MIRU-VNTRplus. Results: Among the 234 MDR strains of M. tuberculosis, 85 (36.3%) were detected as pre-XDRs and 15 (6.4%) as XDRs. All the pre-XDR strains were ofloxacin resistant, whereas among the XDR strains, 10 (66.6%) were resistant to ofloxacin, kanamycin, and capreomycin, four (26.6%) were resistant to ofloxacin, kanamycin, and amikacin, and one (6.6%) isolate was resistant to ofloxacin and kanamycin. Upon spoligotyping analysis, the Beijing lineage was found to be the single most dominant lineage among the pre-XDR strains (38.8%) followed by CAS (30.5%), X (7%), T (5.8%), Haarlem (3.5%), EAI (2.3%), and MANU (2.3%). Among the XDR isolates, seven (46.6%) belonged to Beijing, three (20%) belonged to CAS, and one (6.6%) to each of the EAI, T, URAL, and X lineages. Within the Beijing family, ST1 was the most common in both pre-XDR (94%) and XDR isolates. All the isolates belonged to the ST1 sublineage. Conclusion: The Beijing lineage was found to be the single most dominant genotype among the pre-XDR and XDR isolates.