Ritu Singhal

Microscopy as a diagnostic tool in pulmonary tuberculosis

Tuberculosis continues to cast a huge impact on humanity with its high incidence and mortality, especially in developing countries. For tuberculosis case detection, microscopy continues to be indispensible, given its low cost, rapidity, simplicity of procedure and high specificity. Modifications have attempted to improve the sensitivity of microscopy which include: concentration methods such as centrifugation, N-acetyl cysteine-sodium hydroxide, bleach, ammonium sulfate or chitin. Furthermore, classical Ziehl-Neelsen (ZN) staining has been subjected to varying carbol fuchsin concentrations or replaced by Kinyoun staining, fluorescent microscopy or immune-fluorescence. Currently, light emitting diode fluorescence is recognizably the most plausible method as an alternative to ZN staining.

Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. Results: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

First and second line drug resistance among treatment naïve pulmonary tuberculosis patients in a district under Revised National Tuberculosis Control Programme (RNTCP) in New Delhi.

There is limited information of level of drug resistance to first-line and second line anti-tuberculosis agents in treatment naïve pulmonary tuberculosis (PTB) patients from the Indian region. Therefore, the present prospective study was conducted to determine the antimicrobial susceptibility to first-line and second line anti-TB drug resistance in such patients. Sputum samples from consecutive treatment naïve PTB cases registered in Lala Ram Sarup (LRS) district, under RNTCP containing 12 Directly Observed Treatment Centre’s (DOTS), were enrolled using cluster sampling technology. A total of 453 samples were received from July 2011 to June 2012. All samples were cultured on solid medium followed by drug susceptibility to first and second line anti-tubercular drugs as per RNTCP guidelines. Primary multi-drug resistance (MDR) was found to be 18/453; (4.0%). Extensively drug resistance (XDR) was found in one strain (0.2%), which was found to be resistant to other antibiotics. Data of drug resistant tuberculosis among treatment naïve TB patients are lacking in India. The presence of XDR-TB and high MDR-TB in small population studied, calls for conducting systematic multi-centric surveillance across the country.

Sequence Analysis of Fluoroquinolone Resistance-Associated Genes gyrA and gyrB in Clinical Mycobacterium tuberculosis Isolates from Patients Suspected of Having Multidrug-Resistant Tuberculosis in New Delhi, India

ABSTRACT Fluoroquinolones (FQs) are broad-spectrum antibiotics recommended for the treatment of multidrug-resistant tuberculosis (MDR-TB) patients. FQ resistance, caused by mutations in the gyrA and gyrB genes of Mycobacterium tuberculosis, is increasingly reported worldwide; however, information on mutations occurring in strains from the Indian subcontinent is scarce. Hence, in this study, we aimed to characterize mutations in the gyrA and gyrB genes of acid-fast bacillus (AFB) smear-positive sediments or of M. tuberculosis isolates from AFB smear-negative samples from patients in India suspected of having MDR-TB. A total of 152 samples from patients suspected of having MDR-TB were included in the study. One hundred forty-six strains detected in these samples were characterized by sequencing of the gyrA and gyrB genes. The extracted DNA was subjected to successive amplifications using a nested PCR protocol, followed by sequencing. A total of 27 mutations were observed in the gyrA genes of 25 strains, while no mutations were observed in the gyrB genes. The most common mutations occurred at amino acid position 94 (13/27 [48.1%]); of these, the D94G mutation was the most prevalent. The gyrA mutations were significantly associated with patients with rifampin (RIF)-resistant TB. Heterozygosity was seen in 4/27 (14.8%) mutations, suggesting the occurrence of mixed populations with different antimicrobial susceptibilities. A high rate of FQ-resistant mutations (17.1%) was obtained among the isolates of TB patients suspected of having MDR-TB. These observations emphasize the need for accurate and rapid molecular tests for the detection of FQ-resistant mutations at the time of MDR-TB diagnosis.

Optimization of the preconcentration of selenium IV on palladium nanoparticles (PdNPs), using multivariate analysis for the inorganic speciation of selenium in environmental water samples

A simple method for the characterization and quantification of ultratrace quantities of Se(IV) in environmental water samples by preconcentration of selenium(IV) on PdNPs was optimized by employing the multivariate strategy. 12-run Plackett–Burman factorial design experiments were carried out to identify and the central 23 + star orthogonal composite design was used to optimize the relevant experimental factors. Sodium borohydride (NaBH4) was used to reduce Se(IV) to Se(0) and also to generate PdNPs. The PdNPs were collected and dissolved in a minimum amount of nitric acid and selenium was quantified by ETAAS. Se(VI) in the sample was reduced to Se(IV) and the concentration of Se(VI) was the difference between the concentrations of Se(IV) and total Se(VI) + Se(IV). The standard addition studies of Se(IV) and Se(VI) at the low, mid and high concentration levels of the calibration curve indicated recoveries between 97 and 104%. The detection limit and the preconcentration factor (PF) for the estimation of selenium employing this methodology were 0.025 μg L−1 and 100 respectively. The adsorption capacity of PdNPs in the present study for Se(0) was found to be 28 mg g−1. The proposed procedure was validated by applying it for the determination of the content of total Se in Certified Reference Material BND 701-02 (NPL, India) and applied for the determination of sub ppm to ppm levels of Se(IV) and Se(VI) in ground water samples from the Nawanshahar–Hoshiarpur region, Punjab, India.

Performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis

BACKGROUND Fluorescence microscopy (FM) over the years has shown the potential for increasing the performance of microscopy. The present study was aimed to access the performance of the LED microscope for the detection of acid fast bacilli in a tuberculosis (TB) endemic country. METHODS The study was conducted at a National Reference Laboratory (NRL) in New Delhi, India. Sputum samples were collected from suspected TB patients. Each sample was processed with Auramine O and ZN methods. Auramine O stained smears were evaluated using two different excitatory light sources (MVP and LED); and ZN stained smears were examined under light microscope. The mean time required to read the smears with different modalities was recorded. Bacterial cultures provided the reference standard. RESULTS A total of 200 patients were included in this study. Sensitivity and specificity for the LED assessment, MVP assessment and light microscopy were 83.1% and 82.4%, 78.5% and 87.5% and 81.6% and 83.5%, respectively. Mean reading time was approximately three times faster than ZN microscopy. The mean time to read a negative smear was 2min with fluorescence microscopy and 5min with light microscopy with time savings of 60%. CONCLUSION Although the use of LED-FM only marginally increased sensitivity, the considerable time saving ability combined with very good acceptance and ease of use makes it a reliable alternative to other conventional methods available.

Drug resistance detection and mutation patterns of multidrug resistant tuberculosis strains from children in Delhi

A total of 312 sputum samples from pediatric patients presumptive of multidrug resistant tuberculosis were tested for the detection of drug resistance using the GenoTypeMTBDRplus assay. A total of 193 (61.8%) patients were smear positive and 119 (38.1%) were smear negative by Ziehl–Neelsen staining. Line probe assay (LPA) was performed for 208 samples/cultures (193 smear positive samples and 15 cultures from smear negative samples). Valid results were obtained from 198 tests. Of these, 125/198 (63.1%) were sensitive to both rifampicin (RIF) and isoniazid (INH). 73/198 (36.9%) were resistant to at least INH/RIF, out of which 49 (24.7%) were resistant to both INH and RIF (multidrug resistant). Children with tuberculosis are often infected by someone close to them, so strengthening of contact tracing in the program may help in early diagnosis to identify additional cases within the household. There is a need to evaluate newer diagnostic assays which have a high sensitivity in the case of smear negative samples, additional samples other than sputum among young children not able to expectorate, and also to fill the gap between estimated and reported cases under the program.

Phylogenetic associations with drug-resistant Mycobacterium tuberculosis isolates in a paediatric population

SETTING Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India. OBJECTIVES As paediatric tuberculosis (TB) is a surrogate marker for actively transmitted disease in a community, we investigated drug resistance patterns of 97 Mycobacterium tuberculosis complex strains isolated from children and explored their phylogenetic associations. DESIGN A total of 111 paediatric patients who attended the out-patient department during the study period 2009-2011 and whose sputum samples were sent to the Microbiology Department for liquid culture and drug susceptibility testing (DST) were included in this study. DST and spoligotyping were performed on cultures positive for M. tuberculosis complex. RESULTS DST against four first-line drugs showed that 31 of 97 (32%) strains were pan-susceptible, while 66/97 (68%) were resistant to at least one drug, including 55/97 (56.7%) that were resistant to at least isoniazid and rifampicin (i.e., multidrug-resistant). The majority of the isolates (n = 81/90, 90%) belonged to the principal genetic group 1 strains, the most predominant spoligotyping clusters being spoligotyping international type (SIT)1/Beijing (n = 28), SIT26/CAS1-Delhi (n = 27) and SIT53/T1 (n = 6). CONCLUSION The involvement of Beijing and CAS1-Delhi clades in paediatric TB patients suggests that these two lineages play a major role in ongoing active transmission.

Phenol-ammonium sulfate microscopy method for diagnosis of pulmonary tuberculosis

BACKGROUND Collection and processing of sputum samples for the detection of acid fast bacilli (AFB) is hazardous for health-workers in developing countries with limited facilities. The phenol ammonium sulfate (PhAS) method involves smear microscopy and Ziehl-Neelson (ZN) staining of precipitates/ floccules formed in sputum samples when PhAS is added. The present study has been designed to assess the performance and safety of this method. MATERIALS AND METHODS The study was conducted from January 2011 to March 2011 at the Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, New Delhi. A total of 1038 sputum samples were subjected to ZN staining before and after treatment with PhAS. The smear microscopy results of the PhAS treated and untreated samples were compared. In addition, 200 representative samples were inoculated after processing by petroffs method directly for culture and after treatment with PhAS. RESULT The sensitivity, specificity, positive predictive value and negative predictive value of the PhAS solution treated ZN smear microscopy method were found to be 98.8%, 88.5%, 98.0% and 92.7% respectively in comparison with direct smear microscopy. The overall correlation between the two methods was found to be 97.3%. None of the PhAS treated samples grew Mycobacterium tuberculosis on culture. CONCLUSION Sputum microscopy with PhAS solution is a safe, reliable and inexpensive alternative for direct microscopy. This method can be conveniently applied for usage in microscopy centers with limited bio-safety facilities.

Multidrug-resistant tuberculosis among different types of suspected cases: Study from New Delhi

There are limited data of multidrug-resistant tuberculosis (MDR-TB) diagnosed in various patient categories by implementing Programmatic Management of Drug Resistant TB (PMDT) using line probe assay (LPA) from our country. Samples from presumptive MDR-TB from five districts of New Delhi were subjected to LPA from 1st October 2011 to 31st December 2014. The MDR-TB diagnosed in 4th & 5th month follow-up positives were significantly higher than other categories of the patients. Only 3/232 (2.2%) RIF resistants were diagnosed among smear negative re-treatment cases. The data suggest interim cost-benefit analysis of the program especially among smear negatives retreatment cases.