Krishnamoorthy Gopinath

Non-tuberculous mycobacteria in TB-endemic countries: are we neglecting the danger?

There are more than 120 members of the genus Mycobacterium, which are diverse in pathogenicity, in vivo adaptation, virulence, response to drugs, and growth characteristics. Mycobacteria other than M. tuberculosis complex and M. leprosy are known as Non-Tuberculous Mycobacteria (NTM) and are known by various acronyms. They attracted abrupt attention only after the AIDS epidemic, but most of the reports were published from TB non-endemic countries [1] and only rarely from TB-endemic countries. This is probably because the chances of missing NTM species are higher in TB-endemic countries, which are poorly equipped and overburdened with other diseases (Box 1). The information regarding their true incidence and prevalence in these countries is scarce [2]. In the absence of such authentic information, the current dogma has been that the NTM are of the least consequence. However, we do not agree with this myth and wish to present our viewpoint on this important aspect and emphasize the need for a fresh look at this neglected aspect. Box 1. Possible Factors for Under-reporting of NTM from TB-Endemic Countries NTM infections are not reportable in any country. Awareness is lacking among treating physicians and microbiologists. Laboratory infrastructure is lacking for culture and identification of non-tuberculous mycobacteria. High burden of TB and HIV attracts the bulk of the attention of the health care system; governmental fiscal inputs toward the costs of these neglected infections continue to be neglected. Standardized or accepted criteria to define NTM respiratory disease are lacking. Prevalence of NTM Infections before and after the AIDS Epidemic We searched methodological search terms and phrases such as “non-tuberculous mycobacteria and AIDS” in Medline records and found that 3,020 articles were published between 1981 and 2009. Using the same phrase, only 59 articles were published between 1900 and 1981, indicating a clear upsurge of NTM disease in the post-AIDS era. However, most of these publications were from TB non-endemic countries [1]–[5], but not much significance could be adhered to these isolations [2]. The disseminated NTM infection is typically seen when the CD4+ T lymphocyte number falls below 50 µl. For this reason, it is argued that in TB-HIV co-endemic countries, AIDS patients usually die of tuberculosis or other infections before their CD4+ count falls low enough for NTM to cause a disease (Box 2). Nevertheless, we feel that besides this argument, in a majority of the patients, the diagnosis of NTM disease gets missed in these countries. Box 2. Facts about Non-tuberculous Mycobacterial Disease AIDS patients are significantly more vulnerable to NTM infections due to severe T cell immunodeficiency. Solid organ transplant patients, even though immunocompromised, are not at as high a risk as their HIV-positive counterparts. Although some genetic and anatomical factors predispose to NTM, no proven associations have been proven among geographical, occupational, or ethnic factors and NTM infections. Anatomical abnormalities and other co-morbidities such as chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis (CF), pneumoconiosis, past history of TB, pulmonary alveolar proteinosis, and esophageal motility disorders are well-established predisposing conditions. Disseminated NTM infections have been associated with specific genetic syndromes such as mutations in interferon (IFN)-γ, interleukin (IL)-12 synthesis, and in response pathways and the nuclear factor-κB essential modulator (NEMO). Conventional methods are not sufficiently sensitive to estimate prevalence and incidence of NTM infections. Monoplex TB-specific PCR needs to be replaced by multiplex PCR systems on relevant clinical samples along with blood and urine samples in tertiary-care settings. Multiplex PCR primers have been designed to amplify genus-specific regions, M. tuberculosis complex specific, M. avium complex specific, M fortuitum complex specific, and species-specific gene targets, that can be performed in a single tube. Geographical Distribution of NTM Some workers also consider that the low detection rate of NTM is due to diversity in the environmental and climatic conditions in the HIV-TB-endemic countries, but this argument is not supported by the literature [3]–[5]. In most of the surveys, the rate of human NTM infections is estimated by non-specific antibody assays or skin tests [6]. Hence, these findings may not be a reliable source of information. The International Union against Tuberculosis and Lung Diseases (IUATLD) reviewed data from 14 countries and found that the M. avium complex (MAC) was the most frequently isolated species in all these countries, which included China, India, and Korea. While M. fortuitum was the most frequently encountered species in Belgium (2.1%), the Czech Republic (17.5%), Denmark (5.3%), Finland (6.7%), France (6.5%), Germany (12.2%), Italy (2.5%), Portugal (16.5%), Spain (10.8%), Switzerland (17.5%), Turkey (33.9%), and the United Kingdom (6.0%), undoubtedly, environment is the main reservoir of NTM. There is no evidence of human-to-human or animal-to-human transmission [6]. Most infections are acquired either from the water (treated or untreated) or soil. MAC and M. fortuitum are frequently isolated from the drinking water distribution systems and swimming pools in both developing and developed countries.

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims:  Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management.

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.

Urine as an adjunct specimen for the diagnosis of active pulmonary tuberculosis

BACKGROUND The diagnosis of pulmonary tuberculosis (PTB) is conventionally established by examination of three Ziehl-Neelsen stained smears; however, negative results do not preclude active TB. Since tubercle bacilli or their nucleic acids are also expected to be excreted through the kidneys, we assessed spot urine as a supplementary specimen for diagnosing PTB. METHODS A total of 164 respiratory specimens (147 sputum, 15 bronchoalveolar lavage, and two gastric lavage) from 81 suspected PTB cases were prospectively collected and processed. A total of 112 non-TB controls were also included in the study. For three consecutive days, morning urine specimens were collected from all patients and controls, and were processed for culture by BACTEC MGIT 960 (mycobacteria growth indicator tube) and Lowenstein-Jensen methods and for PCR by amplifying a 441-bp fragment of the hsp65 gene (Mycobacterium genus-specific) and a 786-bp fragment of the cfp32 gene (TB complex-specific). RESULTS Of the 81 patients suspected of having PTB, 46 (56.8%) were sputum culture-positive. Of these, 12 (26.1%) were also urine culture-positive for Mycobacterium tuberculosis. Of the 35 sputum culture-negative cases, three (8.6%) were urine culture-positive. The TB complex specific PCR (cfp32) was positive in 52.2% (24/46) of the bacteriologically-confirmed and 28.6% (10/35) of the bacteriologically-negative PTB patients. In none of the control subjects were urine culture or PCR found to be positive for M. tuberculosis. CONCLUSIONS Specific PCR and culture examination of spot urine samples from suspected PTB patients significantly improved the detection rate of PTB and should be encouraged in resource-limited settings and where multiple pulmonary specimens are not feasible.

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.

Prevalence of mycobacteremia in Indian HIV-infected patients detected by the MB/BacT automated culture system

The use of automated blood cultures system, such as MB/BacT, has provided a novel opportunity for laboratories to diagnose mycobacteremia in HIV-infected patients. However, no such study has been carried out in India so far. This prospective study was conducted on 52 HIV-positive patients with suspected tuberculosis who were referred to our tertiary care hospital in New Delhi. In these patients, the prevalence of mycobacteremia was evaluated using the MB/BacT automated culture system (bioMérieux, France). Twenty-seven HIV-negative but suspected tuberculosis patients were also included for comparison. Mycobacteria could be isolated from sputa or fecal samples of 20 HIV-positive patients (38.4%), and in nine (45%) of these 20 cases, mycobacteria could also be isolated simultaneously from their blood specimens. In the remaining 32 patients, all relevant non-hematological clinical samples remained negative for mycobacteria, but the pathogen could be detected from the blood samples of seven (21.87%) of these 32 patients. Therefore, only 25 (48%) clinically suspected patients remained negative in both Löwenstein-Jensen (L-J) and MB/BacT culture methods, and 12 of these responded to anti-tubercular treatment, while in the rest either non-tubercular diagnosis was established or they were lost to follow-up. The study revealed that low

Mycobacterium avium subspecies Paratuberculosis and crohn's regional ileitis: How strong is association?

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Controlling multidrug-resistant tuberculosis in India

counts and poor or no reactivity to purified protein derivative (PPD) were the best clinical predictors for the occurrence of mycobacteremia in HIV-positive patients. Of the 16 isolates from blood, 13 were diagnosed as Mycobacterium tuberculosis and one each were identified as M. avium, M. kansasii, and a mixed infection of M. tuberculosis and M. avium complex. The prevalence rate of mycobacteremia was significantly low (11.1%) in HIV-negative patients. In conclusion, this study showed that blood culture could be an important adjunct investigation for confirming the clinical diagnosis of tuberculosis in HIV-positive patients.