Molecular characterization of Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Khartoum, Sudan.

Background: The aim of this study was to characterize the drug resistance profile, and the specific lineages of Mycobacterium tuberculosis (MTB) strains isolated from patients with pulmonary TB in the state of Khartoum in Sudan. Methods: Consecutive sputum samples and clinical data were collected from 406 smear-positive TB patients with pulmonary TB in 2007-2009. The samples were cultured, and drug susceptibility testing (DST) was performed using the proportion method (PM) on solid Löwenstein-Jensen medium, and species were identified using biochemical methods at the National Reference Laboratory (NRL) in Khartoum. Extracted deoxyribonucleic acid from a total of 120, 60 suspected multidrug-resistant isolates (MDR), and 60 non-MDR isolates were subsequently sent to the WHO supranational reference laboratory (SRL) in Stockholm at the Public Health Agency of Sweden, for confirmation of the drug resistance profile, examinations by line probe assay (LPA), and molecular epidemiology analysis with Spoligotyping. Results: LPA results correlated 100% for non-MDR and 62% for the suspected MDR strains when compared to the DST results obtained by PM at the NRL. Two strains were initially using the PM identified as MDR-TB but later shown by Hain GenoType Mycobacterium CM/AS to belong Mycobacterium avium complex (Mycobacterium intracelluare). These two strains were excluded from the study material for further analysis. The remaining 58 MDR strains were analyzed using LPA, and 36 strains were confirmed as MDR, 10 as rifampicin monoresistant, and eight as isoniazid-monoresistant. Spoligotyping for all the 118 MTB isolates revealed a total of 115 patterns in which four patterns represented major clusters with a total of 108 (91%) of the strains. The CAS1_Delhi/family was the predominant type and detected in 62 isolates (52%), of which 26 were MDR and 36 were susceptible. It was followed by H3/family with 19 (16%) strains, and 11 Latin American Mediterranean3/family, 16 T2/T1, and two strains each of the Beijing and S lineage. Conclusion: Comparison of DST results obtained using PM and LPA showed 100% agreement for the non-MDR strains but only 62% for the MDR strains. Taking in consideration the time, risk of contamination and the cost of labour to identify MDR TB, the LPA have clear advantages in early detection of MDRTB than the PM. Additionally in this study material Spoligotyping revealed the CAS1 Delhi as the most predominant family. We could not see no major difference in lineages between MDR and non-MDR strains.

Cost-effectiveness analysis of microscopic observation drug susceptibility test versus Xpert MTB/Rif test for diagnosis of pulmonary tuberculosis in HIV patients in Uganda.

BACKGROUND: Microscopic Observation Drug Susceptibility (MODS) and Xpert MTB/Rif (Xpert) are highly sensitive tests for diagnosis of pulmonary tuberculosis (PTB). This study evaluated the cost effectiveness of utilizing MODS versus Xpert for diagnosis of active pulmonary TB in HIV infected patients in Uganda. METHODS: A decision analysis model comparing MODS versus Xpert for TB diagnosis was used. Costs were estimated by measuring and valuing relevant resources required to perform the MODS and Xpert tests. Diagnostic accuracy data of the tests were obtained from systematic reviews involving HIV infected patients. We calculated base values for unit costs and varied several assumptions to obtain the range estimates. Cost effectiveness was expressed as costs per TB patient diagnosed for each of the two diagnostic strategies. Base case analysis was performed using the base estimates for unit cost and diagnostic accuracy of the tests. Sensitivity analysis was performed using a range of value estimates for resources, prevalence, number of tests and diagnostic accuracy. RESULTS: The unit cost of MODS was US$ 6.53 versus US$ 12.41 of Xpert. Consumables accounted for 59 % (US$ 3.84 of 6.53) of the unit cost for MODS and 84 % (US$10.37 of 12.41) of the unit cost for Xpert. The cost effectiveness ratio of the algorithm using MODS was US$ 34 per TB patient diagnosed compared to US$ 71 of the algorithm using Xpert. The algorithm using MODS was more cost-effective compared to the algorithm using Xpert for a wide range of different values of accuracy, cost and TB prevalence. The cost (threshold value), where the algorithm using Xpert was optimal over the algorithm using MODS was US$ 5.92. CONCLUSIONS: MODS versus Xpert was more cost-effective for the diagnosis of PTB among HIV patients in our setting. Efforts to scale-up MODS therefore need to be explored. However, since other non-economic factors may still favour the use of Xpert, the current cost of the Xpert cartridge still needs to be reduced further by more than half, in order to make it economically competitive with MODS.

Outcomes of a clinical diagnostic algorithm for management of ambulatory smear and Xpert MTB/Rif negative HIV infected patients with presumptive pulmonary TB in Uganda: a prospective study.

INTRODUCTION: Diagnostic guidelines for Tuberculosis (TB) in HIV infected patients previously relied on microscopy where the value of initial antibiotic treatment for exclusion of pulmonary TB (PTB) was limited. New guidelines rely on the Xpert MTB Rif test (Xpert). However, the value of the antibiotic treatment remains unclear particularly in individuals who are smear-negative and Xpert-negative-given Xpert has only moderate sensitivity for smear-negative PTB. We assessed an algorithm involving initial treatment with antibiotics prior empiric TB treatment in HIV patients with presumptive PTB who were both smear and Xpert negative. METHODS: We performed a prospective study with six month follow-up to establish patient response to a course of broad spectrum antibiotics prior empiric TB treatment between March 2012 and June 2013. We calculated the proportion of patients who responded to the antibiotic treatment and those who did not. We computed the crude and adjusted odds ratios with their 95% confidence intervals, for response to the antibiotic treatment on various patient characteristics. We report treatment outcomes for patients who received broad spectrum antibiotics only or who were initiated empiric TB treatment. RESULTS: Our cohort comprised 162 smear-negative and Xpert-negative patients, of whom 59% (96 of 162) were female, 81% (131 of 162) were on antiretroviral therapy (ART) for a median of 8.7 months. Overall, 88% (141 of 160) responded to the antibiotic treatment, 8% (12 of 160) got empiric TB treatment and 4% (7 out of 160) were treated for other respiratory disease. The odds of improvement on antibiotics were lower in patients with advanced HIV disease than in patients with early HIV disease. Adjusted odds ratios were significant for HIV clinical stage (AOR; 0.038,) and duration on ART (AOR; 1.038,). CONCLUSION: The majority of HIV patients with presumptive PTB with smear-negative and Xpert negative results improved on the antibiotic treatment and did not require empiric TB treatment. Initial antibiotic treatment appeared more successful in patients with less advanced HIV disease. Findings from our study suggest it is useful to initiate HIV infected patients with presumptive PTB having smear and Xpert negative results on an initial course of antibiotic treatment prior empiric TB treatment.

Meta-analysis to compare the accuracy of GeneXpert, MODS and the WHO 2007 algorithm for diagnosis of smear-negative pulmonary tuberculosis.

BACKGROUND: Smear-negative pulmonary tuberculosis (SN-PTB), which is common in HIV-infected patients, is difficult to diagnose using smear microscopy alone. In 2007, the WHO developed an algorithm to improve the diagnosis and management of smear-negative tuberculosis in HIV prevalent and resource constrained settings. Implementation of the algorithm required individuals with presumptive TB to be initially evaluated using two sputum microscopy examinations followed by clinical diagnosis that may include chest X-ray and antibiotic treatment in smear-negative individuals. Since that time, the WHO has endorsed several new tests for diagnosis of tuberculosis. However, it is unclear how the new tests perform when compared to the WHO 2007 algorithm in diagnosis of SN-PTB. Using meta-analysis study design, we summarized and compared the accuracy of Xpert® MTB/Rif assay (GeneXpert) and Microscopic Observation Drug Susceptibility assay (MODS), with the WHO 2007 algorithm in the diagnosis of SN-PTB. METHODS: A systematic review and meta-analysis of publications on GeneXpert, or MODS, or the WHO 2007 algorithm for diagnosis of SN-PTB, using culture as reference test was performed. Meta-Disc software was used to obtain pooled sensitivity and specificity of the diagnostic methods. Heterogeneity in the accuracy estimates was tested by reviewing the generated forest plots, sROC curves and the Spearman correlation coefficient of the logit of true positive rate versus the logit of false positive rate. RESULTS: Twenty-four publications on all three diagnostic methods were meta-analyzed. The pooled sensitivity and specificity for detection of smear-negative pulmonary tuberculosis were 67% and 98% for GeneXpert, 73% and 91% for MODS, and 61% and 69% for WHO 2007 algorithm, respectively. The sensitivity of GeneXpert reduced from 67% to 54% when sub-group analysis of studies with patient HIV prevalence ≥ 30% was performed. CONCLUSION: The GeneXpert, MODS, and the WHO algorithm have moderate to high accuracy for the diagnosis of SN-PTB. However, the accuracy of the tests is extremely variable. The setting and context under which the tests are conducted in addition to several other factors could explain this variability. There is therefore need to investigate these factors further. The information from these studies would inform the adoption and placement of these new tests.

Evaluation of the Xpert® MTB/Rif test, microscopic observation drug susceptibility test and nitrate reductase assay, for rapid and accurate diagnosis of smear-negative tuberculosis in HIV patients.

Diagnosis of smear-negative tuberculosis (TB), which is frequently seen in HIV-infected patients, is a challenge without conventional culture methods. Since 2007, the WHO (World Health Organization) has endorsed new or improved tests for increased and rapid diagnosis of TB. This study was undertaken in an effort to evaluate the accuracy of two rapid culture methods: the Microscopic Observation Drug Susceptibility assay (MODS) and Nitrate Reductase Assay (NRA), and the molecular based test Xpert® MTB/Rif (Xpert), for diagnosis of smear-negative TB in HIV patients using the mycobacteria growth indicator tube (MGIT) in the BACTEC(TM) MGIT(TM) 960 system as the reference test. 430 smear-negative patients with presumptive TB were enrolled in a cross-sectional study at a tertiary care facility in Uganda. Their sputum was tested on MODS, NRA, Xpert and MGIT. Of the 430 patients, 373 had complete results to compute test accuracy. Mycobacterium tuberculosis (MTB) was detected in 43 patients by MGIT. The sensitivity and specificity were 24.4% and 98.1% for MODS, 41.5% and 92% for NRA, 48.8% and 95.1% for Xpert, respectively. The low sensitivity of the tests implies that additional diagnostics such as chest X-ray and conventional liquid culture methods might still be needed to detect TB in smear-negative HIV patients. The high specificity of the tests is useful to confirm TB in HIV patients with symptoms suggestive of TB.

Rapid screening of MDR-TB using molecular Line Probe Assay is feasible in Uganda.

BACKGROUND: About 500 new smear-positive Multidrug-resistant tuberculosis (MDR-TB) cases are estimated to occur per year in Uganda. In 2008 in Kampala, MDR-TB prevalence was reported as 1.0% and 12.3% in new and previously treated TB cases respectively. Line probe assays (LPAs) have been recently approved for use in low income settings and can be used to screen smear-positive sputum specimens for resistance to rifampicin and isoniazid in 1-2 days. METHODS: We assessed the performance of a commercial line probe assay (Genotype MTBDRplus) for rapid detection of rifampicin and isoniazid resistance directly on smear-positive sputum specimens from 118 previously treated TB patients in a reference laboratory in Kampala, Uganda. Results were compared with MGIT 960 liquid culture and drug susceptibility testing (DST). LPA testing was also performed in parallel in a University laboratory to assess the reproducibility of results. RESULTS: Overall, 95.8% of smear-positive specimens gave interpretable results within 1-2 days using LPA. Sensitivity, specificity, positive and negative predictive values were 100.0%, 96.1%, 83.3% and 100.0% for detection of rifampicin resistance; 80.8%, 100.0%, 100.0% and 93.0% for detection of isoniazid resistance; and 92.3%, 96.2%, 80.0% and 98.7% for detection of multidrug-resistance compared with conventional results. Reproducibility of LPA results was very high with 98.1% concordance of results between the two laboratories. CONCLUSIONS: LPA is an appropriate tool for rapid screening for MDR-TB in Uganda and has the potential to substantially reduce the turnaround time of DST results. Careful attention must be paid to training, supervision and adherence to stringent laboratory protocols to ensure high quality results during routine implementation.

Direct susceptibility testing for multi drug resistant tuberculosis: a meta-analysis.

BACKGROUND: One of the challenges facing the tuberculosis (TB) control programmes in resource-limited settings is lack of rapid techniques for detection of drug resistant TB, particularly multi drug resistant tuberculosis (MDR TB). Results obtained with the conventional indirect susceptibility testing methods come too late to influence a timely decision on patient management. More rapid tests directly applied on sputum samples are needed. This study compared the sensitivity, specificity and time to results of four direct drug susceptibility testing tests with the conventional indirect testing for detection of resistance to rifampicin and isoniazid in M. tuberculosis. The four direct tests included two in-house phenotypic assays - Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS), and two commercially available tests - Genotype MTBDR and Genotype MTBDRplus (Hain Life Sciences, Nehren, Germany). METHODS: A literature review and meta-analysis of study reports was performed. The Meta-Disc software was used to analyse the reports and tests for sensitivity, specificity, and area under the summary receiver operating characteristic (sROC) curves. Heterogeneity in accuracy estimates was tested with the Spearman correlation coefficient and Chi-square. RESULTS: Eighteen direct DST reports were analysed: NRA - 4, MODS- 6, Genotype MTBDR - 3 and Genotype MTBDRplus - 5. The pooled sensitivity and specificity for detection of resistance to rifampicin were 99% and 100% with NRA, 96% and 96% with MODS, 99% and 98% with Genotype MTBDR, and 99% and 99% with the new Genotype MTBDRplus, respectively. For isoniazid it was 94% and 100% for NRA, 92% and 96% for MODS, 71% and 100% for Genotype MTBDR, and 96% and 100% with the Genotype MTBDRplus, respectively. The area under the summary receiver operating characteristic (sROC) curves was in ranges of 0.98 to 1.00 for all the four tests. Molecular tests were completed in 1 - 2 days and also the phenotypic assays were much more rapid than conventional testing. CONCLUSION: Direct testing of rifampicin and isoniazid resistance in M. tuberculosis was found to be highly sensitive and specific, and allows prompt detection of MDR TB.

Drug resistance and genotypic analysis of Mycobacterium tuberculosis strains from Thai tuberculosis patients.

The aim of this study was the molecular characterization of primary drug-resistant Mycobacterium tuberculosis strains in Thailand. We examined a group of M. tuberculosis isolates from newly registered tuberculosis (TB) cases, collected at the largest university hospital, the Siriraj Hospital, in Thailand. Of 76 selected drug-resistant M. tuberculosis strains recovered from previously untreated pulmonary TB patients whose sputum samples were sent to this hospital, 29 (38%) were single-drug resistant, 26(34%) multidrug resistant and two (2.6%) extensively drug resistant. Fifty (66%) strains belonged to Beijing genotype. The study demonstrated a severe problem of drug resistance among recently detected TB patients, and two large clusters of genetically similar strains indicated ongoing transmission of drug-resistant TB.

Recent developments in tuberculosis vaccines.

PURPOSE OF REVIEW: The aim is to review findings related to the use of Bacille Calmette-Guerin (BCG) vaccine, focusing on its limitations and benefits in controlling tuberculosis (TB). Some new TB vaccines, which have entered or are expected to enter clinical trials, are highlighted. RECENT FINDINGS: BCG is currently the only available vaccine against TB, and is widely administered within the World Health Organization Expanded Programme for Immunization. Several trials have shown that the protective efficacy of BCG varies between different populations. Recently, a 60-year follow-up study of American Indians reported the long-term efficacy of BCG to be 52%. The reasons for the low efficacy of the BCG vaccine may be generic differences in the BCG strains, differences in immunological properties of study populations or exposure to environmental factors such as mycobacteria. The low efficacy of the BCG vaccine has encouraged the search for a new vaccine. Among new vaccine candidates are live attenuated Mycobacterium tuberculosis vaccines, recombinant BCG, DNA vaccines, subunit vaccines and fusion proteins with novel adjuvants and delivery systems. SUMMARY: Today, most of the world's population is vaccinated with BCG. It is generally accepted that BCG protects against childhood TB but this immunity wanes with age, resulting in no or insufficient protection against TB. Using modern techniques, several research groups have developed more than 200 new vaccine candidates. Some of these vaccines are now in clinical trials. The clinical evaluation of these new vaccines should be designed to cover a heterogeneous population with great variation in immune responses.

Schistosoma mansoni infection reduces the protective efficacy of BCG vaccination against virulent Mycobacterium tuberculosis.

We hypothesized that the ability of BCG vaccination to protect against Mycobacterium tuberculosis is less in hosts exposed to chronic helminthes infection compared to unexposed individuals. To test this hypothesis we evaluated the efficacy of BCG vaccination in protecting against M. tuberculosis challenge in Schistosoma mansoni pre-infected mice by analyzing their ability to limit the replication of TB bacilli in the lung and liver and the histology of lung sections. The results show that BCG vaccinated mice with prior S. mansoni infection show significantly higher number of colony forming units of TB bacilli as well as significant reduction in air exchange area in the lung compared to controls. In addition, spleen cells from S. mansoni infected mice were found to produce significantly less IFN-gamma and nitric oxide when stimulated in vitro with PPD and several fold higher soluble egg antigen (SEA) and Concanavalin A induced IL-4 and IL-5 secretion. Taken together, our data show that S. mansoni infection reduces the protective efficacy of BCG vaccination against M. tuberculosis possibly by attenuation of protective immune responses to mycobacterial antigens and/or by polarizing the general immune responses to the Th2 profile.

Mycobacterium tuberculosis arabinomannan-protein conjugates protect against tuberculosis.

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.