Epidemiology of anti-tuberculosis drug resistance patterns and trends in tuberculosis referral hospital in Addis Ababa, Ethiopia

ABSTRACT: BACKGROUND: Drug-resistant TB has emerged as a major challenge facing TB prevention and control efforts. In Ethiopia, the extent/trend of drug resistance TB is not well known. The aim of this study was to determine the pattern and trend of resistance to first line anti-TB drugs among culture positive retreatment cases at St.Peter's TB Specialized Hospital. FINDINGS: A hospital based retrospective study was used to assess the pattern of anti-TB drug resistance among previously treated TB patients referred to St.Peter's TB Specialized Hospital from January 2004-December 2008 Gregorian calendar(GC) for better diagnosis and treatment. Among 376 culture positive for M. tuberculosis one hundred and two (27.1%) were susceptible to all of the four first line anti-TB drugs -Isoniazid (INH), Rifampicin (RIF), Ethambutol (ETB) & Streptomycin (STM). While 274 (72.9%) were resistant to at least one drug. Resistance to STM (67.3%) was found to be the most common and the prevalence of MDR-TB was 174 (46.3%). Trend in resistance rate among re-treatment cases from 2004 to 2008 showed a significant increase for any drug as well as for INH, RIF, and MDR resistance (P <0.05 for trend). CONCLUSIONS: There has been an increasing trend in drug resistance in recent years, particularly in retreatment cases. Therefore, establishing advanced diagnostic facilities for early detection of MDR-TB and expanding second line treatment center to treat MDR-TB patients and to prevent its transmission is recommended.     

Comparative genomic analyses of multi-drug resistant Mycobacterium tuberculosis from Nepal and other geographical locations

Nepal exhibits a tuberculosis (TB) incidence rate that is comparable to neighbouring high TB incidence countries. In addition, it records >500 cases of multi-drug resistant (MDR) TB each year. The objective of this study was to perform whole-genome bioinformatic analysis on MDR-TB isolates from Nepal (n = 19) to identify the specific mutations underlying their phenotypic resistance. In addition, we examined the dominant genotype among the Nepal MDR-TB isolates, the East-Asian Beijing sub-lineage, to determine its relatedness to a panel of 1274 genomes of international strains available from public databases. These analyses provided evidence that the XDR-TB isolates in our collection were not derived from importation of primary XDR-TB to Nepal but were more likely the result of acquisition of second-line drug resistance in Nepal. Resistance to fluoroquinolones was detected among a high proportion of the Nepal isolates. This has implications for the management of TB, including appropriate antimicrobial stewardship and susceptibility testing for fluoroquinolones and other second-line TB drugs, to minimise the development of XDR-TB among Nepal TB cases.     

Epidemiology and Clinical Characteristics of Pediatric Drug-Resistant Tuberculosis in Chongqing,China

To gain insight into the epidemiology of childhood drug resistant tuberculosis (DR-TB) in China that has the second largest burden of TB and the largest number of multidrug resistant (MDR) TB cases in the world, we performed the cross-sectional study to investigate drug resistance of four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin and ethambutol) using Mycobacterium tuberculosis isolates from 196 culture-confirmed pediatric TB cases diagnosed in the Children’s Hospital of Chongqing Medical University, China during 2008–2013. Univariate and multivariate logistic regression analyses were performed to assess the associations between patient demographic and clinical characteristics and DR-and MDR-TB, respectively. Twenty-eight percent (56/196) of the study patients exhibited resistance to at least one of the four first-line anti-TB drugs tested. MDR was found in 4.6% (9/196) of the study patients. More than half (5/9, 55.6%) of the MDR cases were from a single county of Chongqing. A significant association was found between being acid-fast bacilli-smear negative and DR-TB (adjusted OR, 2.33; 95% CI, 1.13–4.80) and between having concurrent thoracic-extrathoracic involvement and MDR-TB (adjusted OR, 9.49; 95% CI, 1.05–85.92), respectively. The findings of this study indicate that the rate of DR is high among pediatric TB patients in Chongqing and suggest an urgent need for studies to identify MDR transmission hotspots in Chongqing, thereby contributing to the control DR- and MDR-TB epidemics in China. The study also generates new insight into the pathogenesis of DR and MDR M. tuberculosis strains and highlights the importance of studying childhood TB to the goal of global TB control.     

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB,katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.     

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.     

非一线抗结核药物耐药机制及耐药性诊断研究进展

结核病(Tuberculosis, TB)至今仍是世界三大传染疾病之一。2014年,TB导致的死亡人数已经超过HIV。二线抗TB药物是临床治疗耐多药TB(Multidrug-resistant TB, MDR-TB)的主要药物,然而某些MDR-TB患者由于未及时诊断、治疗方案不合理、所处区域医疗条件差等原因,逐渐发展成为广泛耐药TB(Extensively drug-resistant TB, XDR-TB),使治疗更加困难,其死亡率甚至与肺癌接近。目前结核分枝杆菌(Mycobacterium tuberculosis)的耐药性机制研究已经转向非一线药物,如二线、三线和一些新研发的抗TB药物,揭示这些非一线药物的耐药机制对于耐药TB的治疗和新型抗TB药物的研发具有重要意义。本文对目前临床上使用的主要非一线药物的耐药机制研究进行了综述,并对目前常用的TB耐药性诊断方法的优缺点进行了归纳比较。     

Anti-Tuberculosis Drug Resistance among New and Previously Treated Sputum Smear-Positive Tuberculosis Patients in Uganda: Results of the First National Survey

Background

Multidrug resistant and extensively drug resistant tuberculosis (TB) have become major threats to control of tuberculosis globally. The rates of anti-TB drug resistance in Uganda are not known. We conducted a national drug resistance survey to investigate the levels and patterns of resistance to first and second line anti-TB drugs among new and previously treated sputum smear-positive TB cases.

Methods

Sputum samples were collected from a nationally representative sample of new and previously treated sputum smear-positive TB patients registered at TB diagnostic centers during December 2009 to February 2011 using a weighted cluster sampling method. Culture and drug susceptibility testing was performed at the national TB reference laboratory.

Results

A total of 1537 patients (1397 new and 140 previously treated) were enrolled in the survey from 44 health facilities. HIV test result and complete drug susceptibility testing (DST) results were available for 1524 (96.8%) and 1325 (85.9%) patients, respectively. Of the 1209 isolates from new cases, resistance to any anti-TB drug was 10.3%, 5% were resistant to isoniazid, 1.9% to rifampicin, and 1.4% were multi drug resistant. Among the 116 isolates from previously treated cases, the prevalence of resistance was 25.9%, 23.3%, 12.1% and 12.1% respectively. Of the 1524 patients who had HIV testing 469 (30.7%) tested positive. There was no association between anti-TB drug resistance (including MDR) and HIV infection.

Conclusion

The prevalence of anti-TB drug resistance among new patients in Uganda is low relative to WHO estimates. The higher levels of MDR-TB (12.1%) and resistance to any drug (25.3%) among previously treated patients raises concerns about the quality of directly observed therapy (DOT) and adherence to treatment. This calls for strengthening existing TB control measures, especially DOT, routine DST among the previously treated TB patients or periodic drug resistance surveys, to prevent and monitor development and transmission of drug resistant TB.     

Performance Comparison of Three Rapid Tests for the Diagnosis of Drug-Resistant Tuberculosis

Background

The aim of this study was to compare the performance of several recently developed assays for the detection of multi- and extensively drug-resistant tuberculosis (M/XDR-TB) in a large, multinational field trial.

Methods

Samples from 1,128 M/XDR-TB suspects were examined by Line Probe Assay (LPA), Pyrosequencing (PSQ), and Microscopic Observation of Drug Susceptibility (MODS) and compared to the BACTEC MGIT960 reference standard to detect M/XDR-TB directly from patient sputum samples collected at TB clinics in India, Moldova, and South Africa.

Results

Specificity for all three assays was excellent: 97–100% for isoniazid (INH), rifampin (RIF), moxifloxacin (MOX) and ofloxacin (OFX) and 99–100% for amikacin (AMK), capreomycin (CAP) and kanamycin (KAN) resistance. Sensitivities were lower, but still very good: 94–100% for INH, RIF, MOX and OFX, and 84–90% for AMK and CAP, but only 48–62% for KAN. In terms of agreement, statistically significant differences were only found for detection of RIF (MODS outperformed PSQ) and KAN (MODS outperformed LPA and PSQ) resistance. Mean time-to-result was 1.1 days for LPA and PSQ, 14.3 days for MODS, and 24.7 days for MGIT.

Conclusions

All three rapid assays evaluated provide clinicians with timely detection of resistance to the drugs tested; with molecular results available one day following laboratory receipt of samples. In particular, the very high specificity seen for detection of drug resistance means that clinicians can use the results of these rapid tests to avoid the use of toxic drugs to which the infecting organism is resistant and develop treatment regiments that have a higher likelihood of yielding a successful outcome.     

High Rates of Ofloxacin Resistance in Mycobacterium tuberculosis among Both New and Previously Treated Patients in Tamil Nadu,South India

Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB) control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP) and previously treated (PT) pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs) in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH), rifampicin (RMP), ofloxacin (OFX) and kanamycin (KAN). Human Immunodeficiency Virus (HIV) status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB) was highest in the treatment failure (35%) group, followed by relapse (13%) and treatment after default (10%) groups. Extensively drug resistant TB (XDRTB) was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.     

Efflux Pump Gene Expression in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.     

Drug resistance-conferring mutations in Mycobacterium tuberculosis from Madang, Papua New Guinea

ABSTRACT: BACKGROUND: Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. RESULTS: Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. CONCLUSIONS: We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.     

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.     

MTB新疆临床分离株MDR-TB和XDR-TB耐药情况初步调查

目的:分析新疆喀什地区结核分枝杆菌(MTB)临床分离株对4种一线和7种二线抗结核药物的耐药情况,初步探讨本地区耐多药结核病(MDR-TB)和广泛耐药结核病(XDR-TB)的流行情况。方法:收集2008.11.1~2009.10.31日期间新疆喀什地区结核病患者痰液标本,进行分离培养及菌种鉴定,并应用比例法对所分离得到的菌株进行耐药性检测。结果:102株分枝杆菌中,88株(86.3%)属于结核分枝杆菌复合群,7株(6.9%)属于牛型分枝杆菌,7株(6.9%)属于非结核分枝杆菌。对一种以上的抗结核药物具有耐药性的有20株,总耐药率为22.7%(20/88),耐多药率为6.8%(6/88),pre-XDR为33.3%(2/6),无XDR-TB病例。结论:新疆喀什地区结核病患者耐药率较高,在结核病治疗工作中应给予重视,尤其应加强对Pre-XDR的重视,以免发展成XDR-TB。     

Low Resistance to First and Second Line Anti-Tuberculosis Drugs among Treatment Naive Pulmonary Tuberculosis Patients in Southwestern Uganda

BackgroundThere are limited data on region-specific drug susceptibility of tuberculosis (TB) in Uganda. We performed resistance testing on specimens collected from treatment-naive patients with pulmonary TB in Southwestern Uganda for first and second line anti-TB drugs. We sought to provide data to guide regional recommendations for empiric TB therapy.MethodsArchived isolates, obtained from patients at Mbarara Regional Referral Hospital from February 2009 to February 2013, were tested for resistance to isoniazid and rifampicin using the MTBDRplus and Xpert MTB/RIF assays. A subset of randomly selected isolates was tested for second line agents, including fluoroquinolones (FQs), aminoglycosides, cyclic peptides, and ethambutol using the MTBDRsl assay. We performed confirmatory testing for FQ resistance using repeated MTBDRsl, the Mycobacteria growth indicator tube (MGIT) assay, and sequencing of the gyrA and gyrB genes.ResultsWe tested isolates from 190 patients. The cohort had a median age of 33 years (IQR 26-43), 69% (131/190) were male, and the HIV prevalence was 42% (80/190). No isolates (0/190) were rifampicin-resistant and only 1/190 (0.5%) was isoniazid-resistant. Among 92 isolates tested for second-line drug resistance, 71 (77%) had interpretable results, of which none were resistant to aminoglycosides, cyclic peptides or ethambutol. Although 7 (10%) initially tested as resistant to FQs by the MTBDRsl assay, they were confirmed as susceptible by repeat MTBDRsl testing as well as by MGIT and gyrase gene sequencingConclusionWe found no MDR-TB and no resistance to ethambutol, FQs, or injectable anti-TB drugs in treatment naïve patients with pulmonary TB in Southwestern Uganda. Standard treatment guidelines for susceptible TB should be adequate for most patients with TB in this population. Where possible, molecular susceptibility testing methods should be routinely validated by culture methods.     

2D-QSAR model development and analysis on variant groups of anti-tuberculosis drugs

A quantitative structure activity relationship study was performed on different groups of anti-tuberculosis drug compound for establishing quantitative relationship between biological activity and their physicochemical /structural properties. In recent years, a large number of herbal drugs are promoted in treatment of tuberculosis especially due to the emergence of MDR (multi drug resistance) and XDR (extensive drug resistance) tuberculosis. Multidrug-resistant TB (MDR-TB) is resistant to front-line drugs (isoniazid and rifampicin, the most powerful anti-TB drugs) and extensively drug-resistant TB (XDR-TB) is resistant to front-line and second-line drugs. The possibility of drug resistance TB increases when patient does not take prescribed drugs for defined time period. Natural products (secondary metabolites) isolated from the variety of sources including terrestrial and marine plants and animals, and microorganisms, have been recognized as having antituberculosis action and have recently been tested preclinically for their growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. A quantitative structure activity relationship (QSAR) studies were performed to explore the antituberculosis compound from the derivatives of natural products . Theoretical results are in accord with the in vitro experimental data with reported growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. Antitubercular activity was predicted through QSAR model, developed by forward feed multiple linear regression method with leave-one-out approach. Relationship correlating measure of QSAR model was 74% (R(2) = 0.74) and predictive accuracy was 72% (RCV(2) = 0.72). QSAR studies indicate that dipole energy and heat of formation correlate well with anti-tubercular activity. These results could offer useful references for understanding mechanisms and directing the molecular design of new lead compounds with improved anti-tubercular activity. The generated QSAR model revealed the importance of structural, thermodynamic and electro topological parameters. The quantitative structure activity relationship provides important structural insight in designing of potent antitubercular agent.     

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosis isolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.     

Management of extensively drug-resistant tuberculosis in Peru: cure is possible

Aim

To describe the incidence of extensive drug-resistant tuberculosis (XDR-TB) reported in the Peruvian National multidrug-resistant tuberculosis (MDR-TB) registry over a period of more than ten years and present the treatment outcomes for a cohort of these patients.

Methods

From the Peruvian MDR-TB registry we extracted all entries that were approved for second-line anti-TB treatment between January 1997 and June of 2007 and that had Drug Susceptibility Test (DST) results indicating resistance to both rifampicin and isoniazid (i.e. MDR-TB) in addition to results for at least one fluoroquinolone and one second-line injectable (amikacin, capreomycin and kanamycin).

Results

Of 1,989 confirmed MDR-TB cases with second-line DSTs, 119(6.0%) XDR-TB cases were detected between January 1997 and June of 2007. Lima and its metropolitan area account for 91% of cases, a distribution statistically similar to that of MDR-TB. A total of 43 XDR-TB cases were included in the cohort analysis, 37 of them received ITR. Of these, 17(46%) were cured, 8(22%) died and 11(30%) either failed or defaulted treatment. Of the 14 XDR-TB patients diagnosed as such before ITR treatment initiation, 10 (71%) were cured and the median conversion time was 2 months.

Conclusion

In the Peruvian context, with long experience in treating MDR-TB and low HIV burden, although the overall cure rate was poor, a large proportion of XDR-TB patients can be cured if DST to second-line drugs is performed early and treatment is delivered according to the WHO Guidelines.     

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.     

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.