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 smearnegative 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.Pan African Medical Journal 2016; 2

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>We assessed the performance of a commercial line probe assay (Genotype MTBDR<it>plus</it>) 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

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

<p>Abstract</p> <p>Background</p> <p>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 <it>M. tuberculosis</it>. 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^®MTBDR<it>plus </it>(Hain Life Sciences, Nehren, Germany).</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>Eighteen direct DST reports were analysed: NRA – 4, MODS- 6, Genotype MTBDR^®– 3 and Genotype^®MTBDR<it>plus </it>– 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^®MTBDR<it>plus</it>, 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^®MTBDR<it>plus</it>, 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.</p> <p>Conclusion</p> <p>Direct testing of rifampicin and isoniazid resistance in <it>M. tuberculosis </it>was found to be highly sensitive and specific, and allows prompt detection of MDR TB.</p

Studies on new tuberculosis vaccine candidates in animal models

Background: Tuberculosis (TB) is a major health problem in many countries, especially in low income countries. Globally, TB causes more than 2 million human deaths annually and 1/5 of all adult deaths in developing countries. WHO estimates that one third of the world's population i.e. 1.9 billion people, are infected with M. tuberculosis (Mtb). The most cost effective way to combat infectious diseases is the preventive vaccination. Most of the world's population is vaccinated with the only available TB vaccine, the Bacillus Calmette-Guérin (BCG) vaccine that was developed a century ago. Even though the BCG vaccine protects the young child against disseminated TB disease it has none or little protective effect against adult pulmonary TB (PTB). PTB is the major disease manifestation of TB in adults and it causes death at the most productive age, further adding to poverty in already impoverished countries. Hence, the development of a new more efficient TB vaccine(s) is the highest priority in TB research. Aims: The aim of this thesis was to evaluate protective efficacy of new TB vaccine candidates with the long term goal to improve the efficacy of primary BCG vaccination against adult PTB by boosting with new vaccine(s). Results: New vaccine candidates and adjuvant/delivery systems for mucosal applications were developed and tested in animal models. Lipoarabinomannan (LAM) was purified from a virulent Mtb strain and used to prepare novel oligosaccharide-protein conjugate vaccines. To avoid the immuno-suppressive effects of the intact LAM molecule while taking advantage of its carbohydrate antigen epitopes, LAM was de-lipidated and split into oligosaccharides by partial chemical degradation. The arabinomannan oligosaccharides (AM) were used to prepare AM-protein conjugate (AM-Prot) vaccines and to generate a large number of monoclonal antibodies (MoAbs). Adjuvants can augment the immune response to many vaccine antigens and for some sub-cellular vaccines e.g. Diphteria and Tetanus vaccine, they are essential. There is a vast number of experimental adjuvants but most of them are intrinsically toxic and only few can be considered for use in man. Aluminum salts are so far the only adjuvants approved for large scale human use. In this thesis a new adjuvant L3 was investigated. L3 is non-toxic and approved by the Swedish FDA for human phase I/II trials. The different AM-Prot vaccines and a vaccine based on heat killed whole-cell BCG (H-kBCG) were formulated with L3 and studied for their ability to protect against virulent Mtb challenge in the mouse and guinea pig models - both as primary- and boost-vaccines. Both types of vaccines, when given nasally, evoked specific and robust cellular and humoral immune responses. The vaccines also conferred significant protection, at least equivalent to that induced by the BCG vaccine. Presently the general consensus is that cell mediated immunity (CMI) is decisive for protection against Mtb infections. This, however, has led investigators to neglect the role of the humoral immunity. Importantly, high antibody titers in particular towards LAM are seen in most TB patients but the role of these antibodies is unknown. Therefore, in this thesis the role of antibodies in Mtb infection was re-evaluated in passive protection experiments using an AM-specific MoAb. Mice were infected intravenously with virulent Mtb and the MoAb was added intravenously either prior to or together with the bacteria. The MoAb protected against the infection in terms of a dosedependent reduction in bacterial load in spleens and lungs, reduced weight loss and, importantly, enhanced long-term survival. Conclusions: 1) AM-Prot vaccines can, when formulated with the L3 adjuvant and given nasally, provide as good protection as live BCG, ii) The protection is also conferred by conjugates containing Mtb-irrelevant carrier protein (i.e. tetanus toxoid) showing that antigenic epitopes of AM alone are protective. Therefore we propose that AM epitopes should be considered as part of a future new multi-sub-component vaccine, iii) The AM-Prot vaccine affords protection both when used as a primary vaccine and as a boost vaccine, iv) A H-kBCG vaccine did not protect when administered alone but was protective when given in the L3 adjuvant. The pre-clinical studies of this vaccine candidate are concluded and the vaccine is considered ready for phase 1/11 trials in man, v) None of the vaccines ameliorated the course of TB when used as a therapeutic regimen. Hence, caution is advised in the use of therapeutic vaccines, and in the design of phase 1-111 trials great care must be taken not to include individuals with active TB disease, vi) Certain antiAM antibodies may confer protection, most likely aiding to the early containment of infection and thus giving time for a protective cellular immune response to develop

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 BACTECTM MGITTM 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

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