Pratima Dixit

Multidrug resistant to extensively drug resistant tuberculosis: What is next?

Drug resistant tuberculosis is a man made problem. While tuberculosis is hundred percent curable, multidrug resistant tuberculosis (MDR-TB) is difficult to treat. Inadequate and incomplete treatment and poor treatment adherence has led to a newer form of drug resistance known as extensively drug resistant tuberculosis (XDR-TB). XDR-TB is defined as tuberculosis caused by Mycobacterium tuberculosis strain, which is resistant to at least rifampicin and isoniazid among the first line anti tubercular drugs (MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second line anti tubercular drugs i.e. amikacin, kanamycin and/or capreomycin. Mismanagement of tuberculosis paves the way to drug resistant tuberculosis. Emergence of XDR-TB is reported world wide. Reported prevalence rates of XDR-TB of total MDR cases are; 6.6% overall worldwide, 6.5% in industrialized countries, 13.6% in Russia and Eastern Europe, 1.5% in Asia, 0.6% in Africa and Middle East and 15.4% in Republic of Korea. Better management and control of tuberculosis specially drug resistant TB by experienced and qualified doctors, access to standard microbiology laboratory, co-morbitidy of HIV and tuberculosis, new anti-TB drug regimens, better diagnostic tests, international standards for second line drugs (SLD)-susceptibility testing, invention of newer antitubercular molecules and vaccines and knowing the real magnitude of XDR-TB are some of the important issues to be addressed for effective prevention and management of XDR-TB.

Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis.

BACKGROUND Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available. AIM This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates. METHODS A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein-Jensen medium was used as reference test. RESULTS Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8-14 days. CONCLUSION Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings.

Prevalence of gyrA and B gene mutations in fluoroquinolone-resistant and -sensitive clinical isolates of Mycobacterium tuberculosis and their relationship with MIC of ofloxacin.

The study was done to know the prevalent mutations of gyrA and gyrB genes, and their significance with drug resistance in clinical isolates of Mycobacterium tuberculosis. A total of 100 ofloxacin- (OFX) resistant and 100 OFX-sensitive isolates of M. tuberculosis were consecutively selected from routine Tuberculosis laboratory. Drug resistance pattern of these isolates was recorded. MIC of OFX was tested in all these isolates by absolute concentration method. Quinolone resistance determining region (QRDR) of gyrA and gyrB genes of 320 and 428 bp, respectively, were amplified and sequenced. Sequencing data were analyzed by BLAST on NCBI with reference strain H37Rv. Of 100 OFX-sensitive isolates, 30 were pansusceptible, 28 were monoresistant, 10 were polyresistant and 32 were multidrug resistant (MDR). Among 100 OFX-resistant isolates, 19 were OFX monoresistant, 16 were polyresistant and 65 were MDR. Mutations in gyrA and gyrB genes were observed in 79% and 5% of OFX-resistant isolates, respectively. Most prevalent mutation was found at codon 94 in QRDR of gyrA gene. Double mutations found in gyrA gene and in both gyrA and gyrB genes signifies higher levels of OFX resistance. In one isolate, a substitution at codon 592 (Pro592Ser) was found as a novel mutation outside the QRDR of gyrB gene. Our findings support previous studies that the OFX resistance to M. tuberculosis is associated with mutations in the QRDR of gyrA gene; however, the level of OFX resistance may not be predicted based on the mutation patterns in the gyrA gene.

Pre-XDR & XDR in MDR and Ofloxacin and Kanamycin resistance in non-MDR Mycobacterium tuberculosis isolates

Resistance to second line anti tubercular drugs is a cause of serious concern. The present study reports the prevalence of Ofloxacin (OFX) and Kanamycin (KM) resistance in Mycobacterium tuberculosis isolates from cases of pulmonary tuberculosis, who received anti tubercular treatment for >4 weeks in past. Of total 438 enrolled patients, 361 were culture positive for M. tuberculosis, of which, 95 (26.3%) were OFX resistant & 49 (13.5%) were KM resistant. Total 130 isolates were Multidrug resistant, of which, 55 (42.3%) were resistant to either OFX or KM (Pre-XDR) & 11 (8.5%) were resistant to both KM & OFX (XDR). Resistance to quinolones & aminoglycosides should be routinely assessed in areas endemic for tuberculosis.

A study on pre-XDR & XDR tuberculosis & their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India

Background & objectives: Pre-extensively drug resistant (pre-XDR) and extensively drug resistant tuberculosis (XDR-TB) have been areas of growing concern, and are posing threat to global efforts of TB control. The present study was planned to study the presence of pre-XDR and XDR Mycobacterium tuberculosis and their genotypes in clinical isolates obtained from previously treated cases of pulmonary TB. Methods: A total of 219 isolates obtained from previously treated cases of pulmonary TB were subjected to first-line (streptomycin, isoniazid, rifampicin and ethambutol) and second-line (ofloxacin, kanamycin, capreomycin and amikacin) drug susceptibility testing on solid Lowenstein-Jensen medium by proportion method. Genotyping was done for pre-XDR and XDR-TB isolates using 12 loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR). Results: Multi-drug resistance was observed in 39.7 per cent (87/219) isolates. Pre-XDR and XDR M. tuberculosis isolates amongst 87 multi-drug resistant (MDR) TB isolates were 43 (49.4%) and 10 (11.4%), respectively. Two most dominant genotypes among pre-XDR and XDR M. tuberculosis isolates were Beijing and Delhi/CAS types. Interpretation & conclusions: Resistance to second-line anti-tubercular drugs should be routinely assessed in areas endemic for TB. Similar genotype patterns were seen in pre-XDR and XDR-TB isolates. Beijing and Delhi/CAS were predominant genotypes.

Performance of newer and conventional diagnostic methods in detection of drug sensitive and resistant tuberculous meningitis

Abstract Objective To compare the performance of traditional and newer diagnostic methods for diagnosis of tuberculous meningitis (TBM) along with detection of drug resistant TBM. Methods Single cerebrospinal fluids sample from 281 suspected TBM patients was processed during August 2011 to July 2012 for acid fast bacilli (AFB) examination by Ziehl-Neelsen and auramine staining methods, AFB culture on Lowenstein-Jensen culture media, by microscope observation of drug susceptibility testing assay, and nucleic acid amplification tests by in-house conventional PCR and in-house real time PCR targeting IS6110 insertion sequence. All the isolates were subjected to drug susceptibility testing for isoniazid, rifampicin, ethambutol and streptomycin by 1% proportion method. Results The percentage positivity by AFB smear examination and culture on Lowenstein-Jensen media was 21% (59/281), 11.4% (32/281) respectively. The detection rate by conventional PCR was 33.1% (93/281). The real time PCR showed positivity rate of 44.8% (126/281). The extra detection by real time PCR was 11.7%. Only one isolate was multiple drug resistant and 22 (68.8%) were pan-susceptible. Remaining eight isolates showed either mono/poly drug resistant to first line antitubercular drugs. Conclusions Real time PCR is a more sensitive and rapid method if it is appropriately adopted in clinical practice for diagnosis of TBM. Prevalence of multiple drug resistance Mycobacterium tuberculosis causing TBM is low.

A novel gyrB gene mutation in fluoroquinolone resistant clinical isolates of Mycobacterium tuberculosis

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis can be conferred by mutations in gyrA or gyrB gene. Recent studies suggest that amino acid substitutions in gyrB gene may also play a crucial role in resistance, but genetic studies of these mutations in M. tuberculosis are lacking.

Clinical and radiological prognostic indicators in childhood tuberculous meningitis

Tuberculous meningitis (TBM), the severest form of tuberculosis, continues to affect children in India. The disease has a high rate of mortality and disabling consequences. Scarce literature exists on prognostic indicators in children and none on radiological prognostic features. Our aim is to delineate clinical, laboratory and radiological prognostic indicators in childhood TBM. Children with TBM diagnosed according to pre-decided criteria and admitted to a teaching hospital in Northern India were enrolled for study. Demographic, clinical, laboratory and radiologic features at admission were charted. Patients were followed up to 3 months from discharge or until death. Features at admission were compared between those with good and bad outcome by univariate and logistic regression analysis. A total of 53 of 68 enrolled children were followed for 3 months. Significant factors on univariate analysis were age, headache, Glasgow coma score (GCS), muscle tone, extensor plantar response, decerebrate posturing, clinical stage of disease and infarcts on computerized tomography scan head. On logistic regression, only age ≤ 3 yr, GCS < 10 and increased muscle tone were significantly associated with bad outcome. Younger age, lower GCS and increased muscle tone at initial diagnosis are significant independent predictors of bad outcome in TBM in children.