Bhagwan Maharjan

Ambulatory-Based Standardized Therapy for Multi-Drug Resistant Tuberculosis: Experience from Nepal, 2005–2006

Objective The aim of this study was to describe treatment outcomes for multi-drug resistant tuberculosis (MDR-TB) outpatients on a standardized regimen in Nepal. Methodology Data on pulmonary MDR-TB patients enrolled for treatment in the Green Light Committee-approved National Programme between 15 September 2005 and 15 September 2006 were studied. Standardized regimen was used (8Z-Km-Ofx-Eto-Cs/16Z-Ofx-Eto-Cs) for a maximum of 32 months and follow-up was by smear and culture. Drug susceptibility testing (DST) results were not used to modify the treatment regimen. MDR-TB therapy was delivered in outpatient facilities for the whole course of treatment. Multivariable analysis was used to explain bacteriological cure as a function of sex, age, initial body weight, history of previous treatment and the region of report. Principal Findings In the first 12-months, 175 laboratory-confirmed MDR-TB cases (62% males) had outcomes reported. Most cases had failed a Category 2 first-line regimen (87%) or a Category 1 regimen (6%), 2% were previously untreated contacts of MDR-TB cases and 5% were unspecified. Cure was reported among 70% of patients (range 38%–93% by Region), 8% died, 5% failed treatment, and 17% defaulted. Unfavorable outcomes were not correlated to the number of resistant drugs at baseline DST. Cases who died had a lower mean body weight than those surviving (40.3 kg vs 47.2 kg, p<0.05). Default was significantly higher in two regions [Eastern OR = 6.2; 95%CL2.0-18.9; Far West OR = 5.0; 95%CL1.0-24.3]. At logistic regression, cure was inversely associated with body weight <36 kg [Adj.OR = 0.1; 95%CL0.0-0.3; ref. 55–75 kg] and treatment in the Eastern region [Adj.OR = 0.1; 95%CL0.0-0.4; ref. Central region]. Conclusions The implementation of an ambulatory-based treatment programme for MDR-TB based on a fully standardized regimen can yield high cure rates even in resource-limited settings. The determinants of unfavorable outcome should be investigated thoroughly to maximize likelihood of successful treatment.

Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolated in Nepal

ABSTRACT Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.

Characterization of extensively drug-resistant Mycobacterium tuberculosis in Nepal

The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global control of TB. Although molecular characterization of drug resistance-associated mutations in multidrug-resistant isolates in Nepal has been made, mutations in XDR isolates and their genotypes have not been reported previously. In this study, we identified and characterized 13 XDR Mycobacterium tuberculosis isolates from clinical isolates in Nepal. The most prevalent mutations involved in rifampicin, isoniazid, ofloxacin, and kanamycin/capreomycin resistance were Ser531Leu in rpoB gene (92.3%), Ser315Thr in katG gene (92.3%), Asp94Gly in gyrA gene (53.9%) and A1400G in rrs gene (61.5%), respectively. Spoligotyping and multilocus sequence typing revealed that 69% belonged to Beijing family, especially modern types. Further typing with 26-loci variable number of tandem repeats suggested the current spread of XDR M. tuberculosis. Our result highlights the need to reinforce the TB policy in Nepal with regard to control and detection strategies.

Simple Multiplex PCR Assay for Identification of Beijing Family Mycobacterium tuberculosis Isolates with a Lineage-Specific Mutation in Rv0679c

ABSTRACT The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to host cell invasion. Here, we conducted a sequence analysis of the Rv0679c gene using clinical isolates and found that a single nucleotide polymorphism, C to G at position 426, can be observed only in the isolates that are identified as members of the Beijing genotype family. Here, we developed a simple multiplex PCR assay to detect this point mutation and applied it to 619 clinical isolates. The method successfully distinguished Beijing lineage clones from non-Beijing strains with 100% accuracy. This simple, quick, and cost-effective multiplex PCR assay can be used for a survey or for monitoring the prevalence of Beijing genotype M. tuberculosis strains.

First Insights into the Phylogenetic Diversity of Mycobacterium tuberculosis in Nepal

Background Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. Methods and Findings We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. Conclusions We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.

A novel sputum transport solution eliminates cold chain and supports routine tuberculosis testing in Nepal

This preliminary study evaluated the transport reagent OMNIgene SPUTUM (OMS) in a real-world, resource-limited setting: a zonal hospital and national tuberculosis (TB) reference laboratory, Nepal. The objectives were to: (1) assess the performance of OMS for transporting sputum from peripheral sites without cold chain stabilization; and (2) compare with Nepal’s standard of care (SOC) for Mycobacterium tuberculosis smear and culture diagnostics. Sixty sputa were manually split into a SOC sample (airline-couriered to the laboratory, conventional processing) and an OMS sample (OMS added at collection, no cold chain transport or processing). Smear microscopy and solid culture were performed. Transport was 0–8 days. Forty-one samples (68%) were smear-positive using both methods. Of the OMS cultures, 37 (62%) were positive, 22 (36%) were negative, and one (2%) was contaminated. Corresponding SOC results were 32 (53%), 21 (35%), and seven (12%). OMS “rescued” six (i.e., missed using SOC) compared with one rescue using SOC. Of smear-positives, six SOC samples produced contaminated cultures whereas only one OMS sample was contaminated. OMS reduced culture contamination from 12% to 2%, and improved TB detection by 9%. The results suggest that OMS could perform well as a no cold chain, long-term transport solution for smear and culture testing. The findings provide a basis for larger feasibility studies.

Diagnostic Accuracy of GeneXpert MTB/RIF Assay in Comparison to Conventional Drug Susceptibility Testing Method for the Diagnosis of Multidrug-Resistant Tuberculosis

Xpert MTB/RIF assay is regarded as a great achievement of modern medicine for the rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB). The main purpose of this study was to determine the performance of Xpert MTB/RIF assay compared to conventional drug susceptibility testing (DST) method for the diagnosis of MDR-TB. A comparative cross sectional study was carried out at German-Nepal Tuberculosis Project, Kathmandu, Nepal, from April 2014 to September 2014. A total of 88 culture positive clinical samples (83 pulmonary and 5 extra-pulmonary) received during the study period were analyzed for detection of multidrug-resistant tuberculosis by both GeneXpert MTB/RIF assay and conventional DST method. McNemar chi square test was used to compare the performance of Xpert with that of DST method. A p-value of less than 0.05 was considered as statistically significant. Of total 88 culture positive samples, one was reported as invalid while 2 were found to contain nontuberculous Mycobacteria (NTM). Among remaining 85 Mycobacterium tuberculosis culture positive samples, 69 were found to be MDR-TB positive by both methods. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of GeneXpert MTB/RIF assay were found to be 98.6%, 100%, 100% and 93.8% respectively. Statistically, there was no significant difference between the diagnostic performance of Xpert and conventional DST method for detection of MDR-TB. GeneXpert MTB/RIF assay was found to be highly sensitive, specific and comparable to gold standard conventional DST method for the diagnosis of MDR-TB.

Mycobacterium orygis–Associated Tuberculosis in Free-Ranging Rhinoceros, Nepal, 2015

To the Editor: Mycobacterium orygis, previously described as oryx bacilli, has recently been categorized as a member of M. tuberculosis complex and has been reported to cause tuberculosis (TB) in a variety of animals and in humans. Most reported isolates were of South Asian origin (1). In a previous study (2), we isolated and molecularly characterized M. orygis isolates from wild animals living in a captive facility in Kathmandu, Nepal. The greater one-horned rhinoceros (Rhinoceros unicornis), or Indian rhinoceros, is the largest species of rhinoceros. It is listed in Appendix I of the Convention on International Trade in Endangered Species (https://cites.org/eng/app/appendices.php), designated as vulnerable by the International Union for Conservation of Nature Red List (http://www.iucnredlist.org/search), and designated as a protected species by the Government of Nepal (3). Because of successful conservation efforts, the current wild population of greater one-horned rhinoceros in Nepal and India has increased from 600 in 1975 to 3,555 in mid-2015 (4). As of 2015, the population of these rhinoceros in Nepal was 645, including 605 animals living in Chitwan National Park (CNP) (5). On February 16, 2015, CNP officials observed a sick female rhinoceros in the buffer zone of the western sector of the park near Amaltari. The rhinoceros was dull, depressed, and not feeding. The following day, the animal was found dead in the same area (Technical Appendix Figure 1). Superficial maggot-infested wounds were on both sides of the vulva, indicating that the rhinoceros was not able to naturally remove the maggots and suggesting that the animal was sick for some time. During the necropsy, several granulomatous lesions were observed in the lungs and considered to be compatible with TB infection. The lesions were extensively distributed and well encapsulated and contained caseous necrotic material (Technical Appendix Figure 2). No other pathologic changes were observed in any of the organs examined, leading to the conclusion that the rhinoceros died from TB. A lung tissue sample positive for TB by acid-fast staining was cultured on Lowenstein-Jensen media. We performed spoligotyping and mycobacterial interspersed repetitive units–variable-number tandem-repeat (MIRU-VNTR) procedure on the isolate as previously described (6,7). Spoligotyping analysis, performed as previously described (2), showed that the isolate had a spoligo–international type 587 pattern, indicating it was M. orygis. We also performed multilocus sequence typing on various genes (2), and confirmed that the isolate was M. orygis. We then constructed a dendrogram by comparing the MIRU-VNTR result from rhinoceros isolate with published M. orygis MIRU-VNTR types (Figure) (1,2,8). The rhinoceros M. orygis isolate fell in a unique position in the dendrogram; we identified a difference in only 1 locus (MIRU 424) when we compared the isolate with the largest cluster of reported M. orygis isolates, including those previously reported from Nepal. Figure Phylogeny of Mycobacterium orygis isolates as determine on the basis of mycobacterial interspersed repetitive units–variable-number tandem-repeat (MIRU-VNTR) results of 22 loci. The unweighted pair group method with arithmetic mean dendrogram ... In our earlier study (2), we isolated M. orygis from chital deer (Axis axis) and blue bull (Boselaphus tragocamelus) from a captive wild-animal facility and postulated that the origin of the infection might be from infected animals in CNP, where the deer and blue bull originated. This new finding of a different strain type of M. orygis in a free-ranging rhinoceros in CNP provides evidence for our hypothesis. Other reports of M. orygis in captive wild animals in Nepal (2), cattle and a rhesus monkey in Bangladesh (1), humans in South Asia (1), and an immigrant from India in New Zealand (9) further support this bacterium’s potential widespread distribution in South Asia and attests to the One Health significance of this organism. In a demographic study of rhinoceros in Nepal (10), the animals were found to be living in a narrow area of riverine grassland in CNP. A chronic and devastating disease like TB in this vulnerable and isolated population, which is already threatened from habitat destruction and poaching, is a matter of great conservation concern for the animal’s long-term survivability. Also, CNP is listed by the United Nations Educational, Scientific and Cultural Organization as a World Heritage site because of its rich biodiversity and as an important habitat for endangered animals, including Bengal tigers (Panthera tigris) and Asian elephants (Elephas maximus). Thus, M. orygis–associated TB in rhinoceros in CNP may also indicate a threat to other animals, including some that are endangered. There is a strong possibility of unknown maintenance hosts of M. orygis in and around the national park. Our findings support the need for further investigation to understand the ecology and epidemiology of M. orygis and provide justification for active surveillance of this bacterium in animals in the national park and in livestock and humans in the buffer-zone areas. Furthermore, the increasing evidence for widespread distribution of M. orygis in South Asia provides a new picture of TB and may lead to a new understanding of M. tuberculosis complex. Technical Appendix: Location in Nepal where Mycobacterium orygis–infected rhinoceros was found dead and image of granulomatous tuberculosis lesion. Click here to view.(559K, pdf)

Rapid Detection of Rifampicin and Isoniazid Resistant Mycobacterium tuberculosis Using Genotype MTBDRplus Assay in Nepal

Rapid line probe assay (LPA) can be a practical and rapid alternative to the slow conventional phenotypic drug susceptibility testing (DST) for detection of drug resistant tuberculosis (TB). The purpose of this study is to determine the diagnostic accuracy of Genotype MTBDRplus, LPA for TB, and compare its performance with conventional DST. A total of 54 culture samples were analyzed for DST using both conventional proportion method and MTBDRplus, where conventional DST identified 43 isolates (79.6%) as drug resistant. Among these 43 drug resistant isolates, 30 isolates (69.7%) were found to be multidrug resistant (MDR). Of all observed mutations using MTBDRplus, codon 531 of rpoB gene and codon 315 of katG gene were found to have highest mutational frequency for RIF resistance (64.7%) and INH resistance (96.8%), respectively. In the present study, MTBDRplus assay was shown to have excellent specificity (100%) for both RIF and INH resistance while sensitivity of the assay was little lower with value of 89.4% for RIF resistance and 91.4% for INH resistance. Therefore, the assay can be a rapid, reliable, and promising molecular test for early detection of MDR-TB in Nepal.

High diversity of multidrug-resistant Mycobacterium tuberculosis Central Asian Strain isolates in Nepal

OBJECTIVES Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) poses a major public health problem in Nepal. Although it has been reported as one of the dominant genotypes of MTB in Nepal, little information on the Central Asian Strain (CAS) family is available, especially isolates related to multidrug resistance (MDR) cases. This study aimed to elucidate the genetic and epidemiological characteristics of MDR CAS isolates in Nepal. METHODS A total of 145 MDR CAS isolates collected in Nepal from 2008 to 2013 were characterized by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis, and drug resistance-associated gene sequencing. RESULTS Spoligotyping analysis showed CAS1_Delhi SIT26 as predominant (60/145, 41.4%). However, by combining spoligotyping and MIRU-VNTR typing, it was possible to successfully discriminate all 145 isolates into 116 different types including 18 clusters with 47 isolates (clustering rate 32.4%). About a half of these clustered isolates shared the same genetic and geographical characteristics with other isolates in each cluster, and some of them shared rare point mutations in rpoB that are thought to be associated with rifampicin resistance. CONCLUSIONS Although the data obtained show little evidence that large outbreaks of MDR-TB caused by the CAS family have occurred in Nepal, they strongly suggest several MDR-MTB transmission cases.