Dr
Willy Ssengooba
Current Organisation
Makerere University
Current Job Title
Scientific Director of the Mycobacteriology (BSL-3) Facility
Biography
Publications
Not all patients produce sputum, yet most available TB tests use sputum. We investigated the utility of a novel RNA-based quantitative test, the tuberculosis molecular bacterial load assay (TB-MBLA), for the detection and quantification of Mycobacterium tuberculosis in stool. Stools from 100 adult individuals were treated with OMNIgene-sputum reagent and tested using Xpert MTB/RIF ultra (Xpert ultra), auramine O smear microscopy (smear), mycobacterial growth indicator tube (MGIT), and Lowenstein-Jensen (LJ) cultures. The remaining portions were frozen at -20°C and later tested by TB-MBLA. MGIT sputum culture was used as a TB confirmatory test and reference for stool tests. Sixty-one of 100 participants were already confirmed TB positive by MGIT sputum culture, 20 (33%) of whom were HIV coinfected. TB-MBLA detected M. tuberculosis in 57/100 stool samples, including 49 already confirmed for TB. The mean bacterial load measured by stool TB-MBLA was 5.67 ± 1.7 log
10
estimated CFU (eCFU) per mL in HIV-coinfected participants, which was higher than the 4.83 ± 1.59 log
10
eCFU per mL among the HIV-negative participants (
P
= 0.04). The sensitivities (95% confidence intervals [CI]) of stool assays were 80% (68 to 89) and 90% (79 to 98) for TB-MBLA and Xpert ultra, which were both higher than the 44% (32 to 58), 64% (51 to 76), and 62% (45 to 77) for smear, MGIT, and Lowenstein-Jensen (LJ) stool cultures, respectively. The specificity (95% CI) of stool assays was highest for smear, at 97% (87 to 100), followed by Xpert ultra at 91% (76 to 98), TB-MBLA at 79% (63 to 90), LJ at 80% (64 to 91), and MGIT at 62% (45 to 77). Twenty-six percent of MGIT and 21% of LJ stool cultures were indeterminate due to contamination. Detection and quantification of viable M. tuberculosis bacilli in stool raises its utility as an alternative to sputum as a sample type for TB diagnosis.
IMPORTANCE
This paper highlights the value of stool as a sample type for diagnosis of tuberculosis. While other studies have used DNA-based assays like the Xpert MTB/RIF and culture to detect Mycobacterium tuberculosis in stool, this is the first study that has applied TB-MBLA, an RNA-based assay, to quantify TB bacteria in stool. The high microbial density and diversity in stool compromises the specificity and sensitivity of culture-based tests due to overgrowth of non-M. tuberculosis flora. Consequently, TB-MBLA becomes the most sensitive and specific test for the detection and quantification of viable TB bacteria in stool. Most crucially, this study raises the possibility of a nonsputum alternative sample type for diagnosis of TB among people who have difficulty in producing sputum.
Introduction.
Drug resistant tuberculosis remains a worldwide problem that requires prompt diagnosis.
Hypothesis/Gap statement.
The WHO recommended direct, rapid Xpert MTB/RIF is prohibitively costly, therefore, there is a need to validate a rapid, affordable DST for use in low- and middle-income settings.
Aim.
The technical performance and time to results of a simple, direct microscopy-based slide DST (SDST) assay for diagnosis of rifampicin-resistant TB was evaluated in Uganda.
Methodology.
Sputum samples from 122 smear-positive re-treatment TB patients presenting to the TB treatment centre at Uganda's National Referral Hospital, Mulago, Kampala, Uganda were examined. The sputum samples were tested by the direct SDST which was compared to the indirect Lowenstein Jensen Proportion Method (LJDST) method as the gold standard. The time to results was defined as the time from DST setting to results interpretation. The results were further analysed for sensitivity and specificity as well as agreement between LJDST and SDST for rifampicin resistance determination.
Results.
A total of 117 smear positive sputum samples with valid results for both tests were compared. The median time to results for SDST was 14 days with an interquartile range (IQR) of 10-14 days compared to 60 days with IQR of 60-75 days for LJDST. The number for rifampicin resistance by the gold standard LJDST was 26. The SDST had a sensitivity of 96 % (95 %; CI 81-99 %) and a specificity of 97.8 % (95 %; CI 93-100 %). The Positive Predictive and Negative Predictive values for SDST were 92.3 % (95 %; CI 76.8-99 %) and 98.9 % (95 %; CI 94-100 %), respectively. The
kappa
agreement between SDST and LJDST was 92.3 %.
Conclusion.
The SDST was found to be a rapid and accurate direct test for the detection of rifampicin resistance among retreatment TB cases in low-income settings.
Introduction
Despite the limited evidence for its effectiveness, thermal screening at points of entry has increasingly become a standard protocol in numerous parts of the globe in response to the COVID-19 pandemic. We sought to determine the effectiveness of thermal screening as a key step in diagnosing COVID-19 in a resource-limited setting.
Materials and methods
This was a retrospective cross-sectional study based on a review of body temperature and Xpert Xpress SARS CoV-2 test results records for truck drivers entering Uganda through Mutukula between 15th May and 30th July 2020. All records missing information for body temperature, age, gender, and Xpert Xpress SARS CoV-2 status were excluded from the data set. A data set of 7,181 entries was used to compare thermal screening and Xpert Xpress SARS CoV-2 assay test results using the diagnostic statistical test in STATAv15 software. The prevalence of COVID-19 amongst the truck drivers based on Xpert Xpress SARS CoV-2 assay results was determined. The sensitivity, specificity, positive predictive value, negative predictive value, positive and negative Likelihood ratios were obtained using Xpert Xpress SARS CoV-2 assay as the gold standard.
Results
Based on our gold standard test, the proportion of persons that tested positive for COVID-19 was 6.7% (95% CI: 6.1-7.3). Of the 7,181 persons that were thermally screened, 6,844 (95.3%) were male. The sample median age was 38 years (interquartile range, IQR: 31-45 years). The median body temperature was 36.5°C (IQR: 36.3-36.7) and only n (1.2%) had a body temperature above 37.5°C. The sensitivity and specificity of thermal screening were 9.9% (95% CI: 7.4-13.0) and 99.5% (95% CI: 99.3-99.6) respectively. The positive and negative predictive values were 57.8 (95% CI: 46.5-68.6) and 93.9 (95% CI: 93.3-94.4) respectively. The positive and negative Likelihood Ratios (LRs) were 19 (95% CI: 12.4-29.1) and 0.9 (95% CI: 0.88-0.93) respectively.
Conclusion
In this study population, the use of Thermal screening alone is ineffective in the detection of potential COVID-19 cases at point of entry. We recommend a combination of screening tests or additional testing using highly sensitive molecular diagnostics such as Polymerase Chain Reaction.
INTRODUCTION:Susceptibility testing for pyrazinamide (PZA), a cornerstone anti-TB drug is not commonly done in Uganda because it is expensive and characterized with technical difficulties thus resistance to this drug is less studied. Resistance is commonly associated with mutations in the pncA gene and its promoter region. However, these mutations vary geographically and those conferring phenotypic resistance are unknown in Uganda. This study determined the prevalence of PZA resistance and its association with pncA mutations. MATERIALS AND METHODS:Using a cross-sectional design, archived isolates collected during the Uganda national drug resistance survey between 2008-2011 were sub-cultured. PZA resistance was tested by BACTEC Mycobacterial Growth Indicator Tube (MGIT) 960 system. Sequence reads were downloaded from the NCBI Library and bioinformatics pipelines were used to screen for PZA resistance-conferring mutations. RESULTS:The prevalence of phenotypic PZA resistance was found to be 21%. The sensitivity and specificity of pncA sequencing were 24% (95% CI, 9.36-45.13%) and 100% (73.54% - 100.0%) respectively. We identified four mutations associated with PZA phenotypic resistance in Uganda; K96R, T142R, R154G and V180F. CONCLUSION:There is a high prevalence of phenotypic PZA resistance among TB patients in Uganda. The low sensitivity of pncA gene sequencing confirms the already documented discordances suggesting other mechanisms of PZA resistance in Mycobacterium tuberculosis.
Background
Two thirds of children with tuberculosis have nonsevere disease, which may be treatable with a shorter regimen than the current 6-month regimen.
Methods
We conducted an open-label, treatment-shortening, noninferiority trial involving children with nonsevere, symptomatic, presumably drug-susceptible, smear-negative tuberculosis in Uganda, Zambia, South Africa, and India. Children younger than 16 years of age were randomly assigned to 4 months (16 weeks) or 6 months (24 weeks) of standard first-line antituberculosis treatment with pediatric fixed-dose combinations as recommended by the World Health Organization. The primary efficacy outcome was unfavorable status (composite of treatment failure [extension, change, or restart of treatment or tuberculosis recurrence], loss to follow-up during treatment, or death) by 72 weeks, with the exclusion of participants who did not complete 4 months of treatment (modified intention-to-treat population). A noninferiority margin of 6 percentage points was used. The primary safety outcome was an adverse event of grade 3 or higher during treatment and up to 30 days after treatment.
Results
From July 2016 through July 2018, a total of 1204 children underwent randomization (602 in each group). The median age of the participants was 3.5 years (range, 2 months to 15 years), 52% were male, 11% had human immunodeficiency virus infection, and 14% had bacteriologically confirmed tuberculosis. Retention by 72 weeks was 95%, and adherence to the assigned treatment was 94%. A total of 16 participants (3%) in the 4-month group had a primary-outcome event, as compared with 18 (3%) in the 6-month group (adjusted difference, -0.4 percentage points; 95% confidence interval, -2.2 to 1.5). The noninferiority of 4 months of treatment was consistent across the intention-to-treat, per-protocol, and key secondary analyses, including when the analysis was restricted to the 958 participants (80%) independently adjudicated to have tuberculosis at baseline. A total of 95 participants (8%) had an adverse event of grade 3 or higher, including 15 adverse drug reactions (11 hepatic events, all but 2 of which occurred within the first 8 weeks, when the treatments were the same in the two groups).
Conclusions
Four months of antituberculosis treatment was noninferior to 6 months of treatment in children with drug-susceptible, nonsevere, smear-negative tuberculosis. (Funded by the U.K. Medical Research Council and others; SHINE ISRCTN number, ISRCTN63579542.).
Background
Second-line drug resistance (SLD) among tuberculosis (TB) patients is a serious emerging challenge towards global control of the disease. We characterized SLD-resistance conferring-mutations among TB patients with rifampicin and/or isoniazid (RIF and/or INH) drug-resistance tested at the Uganda National TB Reference Laboratory (NTRL) between June 2017 and December 2019.
Methods
This was a descriptive cross-sectional secondary data analysis of 20,508 M. tuberculosis isolates of new and previously treated patients' resistant to RIF and/or INH. DNA strips with valid results to characterise the SLD resistance using the commercial Line Probe Assay Genotype MTBDRsl Version 2.0 Assay (Hain Life Science, Nehren, Germany) were reviewed. Data were analysed with STATAv15 using cross-tabulation for frequency and proportions of known resistance-conferring mutations to injectable agents (IA) and fluoroquinolones (FQ).
Results
Among the eligible participants, 12,993/20,508 (63.4%) were male and median (IQR) age 32 (24-43). A total of 576/20,508 (2.8%) of the M. tuberculosis isolates from participants had resistance to RIF and/or INH. These included; 102/576 (17.7%) single drug-resistant and 474/576 (82.3%) multidrug-resistant (MDR) strains. Only 102 patients had test results for FQ of whom 70/102 (68.6%) and 01/102 (0.98%) had resistance-conferring mutations in the gyrA locus and gyrB locus respectively. Among patients with FQ resistance, gyrAD94G 42.6% (30.0-55.9) and gyrA A90V 41.1% (28.6-54.3) mutations were most observed. Only one mutation, E540D was detected in the gyrB locus. A total of 26 patients had resistance-conferring mutations to IA in whom, 20/26 77.0% (56.4-91.0) had A1401G mutation in the rrs gene locus.
Conclusions
Our study reveals a high proportion of mutations known to confer high-level fluoroquinolone drug-resistance among patients with rifampicin and/or isoniazid drug resistance. Utilizing routinely generated laboratory data from existing molecular diagnostic methods may aid real-time surveillance of emerging tuberculosis drug-resistance in resource-limited settings.
Tuberculous meningitis (TBM) is the most lethal manifestation of tuberculosis and requires rapid diagnosis and initiation of treatment to prevent death and serious neurological disability.….
BACKGROUND:Determining mycobacterial burden is important in assessing severity of disease, evaluating infectiousness and predicting patient treatment outcomes. Mycobacterial burden assessed by smear microscopy grade and time to culture positivity is clearly interpretable by most physicians. GeneXpert (Xpert) has been recommended by WHO as a first line tuberculosis (TB) diagnostic test as an alternative to smear microscopy. Xpert gives cycle threshold (Ct) values as a potential measure for mycobacterial burden. For physicians to clearly interpret Ct values as measures of mycobacterial burden, this study compared the Xpert quantification capabilities with those of smear microscopy and culture. The study also determined a linear relationship between Xpert Ct values and MGIT culture time to positivity (MGIT-TTP) and associated factors. A cut off Ct value which best predicts smear positivity was also determined using the Receiver Operator Curve analysis method. RESULTS:Excluding missing results and rifampicin resistant TB cases, a moderately strong correlation of 0.55 between Xpert Ct value and smear grade was obtained. A weak correlation of 0.37 was obtained between Xpert Ct values and MGIT time to positivity while that between Xpert Ct values and LJ culture was 0.34. The Xpert Ct values were found to increase by 2.57 for every unit increase in days to positive and HIV status was significantly associated with this relationship. A cut off Ct value of 23.62 was found to best predict smear positivity regardless of HIV status. CONCLUSION:Our study findings show that GeneXpert Ct values are comparable to smear microscopy as a measure of M. tuberculosis burden and can be used to replace smear microscopy. However, given the low correlation between Xpert Ct value and culture positivity, Xpert Ct values cannot replace culture as a measure of M. tuberculosis burden among TB patients.
Tuberculosis (TB) is the leading cause of death in humans by a single infectious agent worldwide with approximately two billion humans latently infected with the bacterium Mycobacterium tuberculosis. Currently, the accepted method for controlling the disease is Tuberculosis Directly Observed Treatment Shortcourse (TB-DOTS). This program is not preventative and individuals may transmit disease before diagnosis, thus better understanding of disease transmission is essential. Using whole-genome sequencing and single nucleotide polymorphism analysis, we analyzed genomes of 145 M. tuberculosis clinical isolates from active TB cases from the Rubaga Division of Kampala, Uganda. We established that these isolates grouped into M. tuberculosis complex (MTBC) lineages 1, 2, 3, and 4, with the most isolates grouping into lineage 4. Possible transmission pairs containing ≤12 SNPs were identified in lineages 1, 3, and 4 with the prevailing transmission in lineages 3 and 4. Furthermore, investigating DNA codon changes as a result of specific SNPs in prominent virulence genes including plcA and plcB could indicate potentially important modifications in protein function. Incorporating this analysis with corresponding epidemiological data may provide a blueprint for the integration of public health interventions to decrease TB transmission in a region.
Background
In resource-limited settings, sputum smear conversion is used to document treatment response. Many People living with HIV (PLHIV) are smear-negative at baseline. The Xpert MTB/RIF test can indirectly measure bacterial load through cycle threshold (ct) values. This study aimed to determine if baseline Xpert MTB/RIF could predict time to culture negativity in PLHIV with newly diagnosed TB.
Methods
A subset of 138 PLHIV from the 'SOUTH' study on outcomes related to TB and antiretroviral drug concentrations were included. Bacterial load was estimated by Mycobacterium Growth Indicator Tubes (MGIT) culture time-to-positivity (TTP) and Lowenstein Jensen (LJ) colony counts. Changes in TTP and colony counts were analyzed with Poisson Generalised Estimating Equations (GEE) and multilevel ordered logistic regression models, respectively, while time to culture negativity analysed with Cox proportional hazard models. ROC curves were used to explore the accuracy of the ct value in predicting culture negativity.
Results
A total of 81 patients (58.7%) were males, median age 34 (IQR 29 ̶ 40) years, median CD4 cell count of 180 (IQR 68 ̶ 345) cells/μL and 77.5% were ART naive. The median baseline ct value was 25.1 (IQR 21.0 ̶ 30.1). A unit Increase in the ct value was associated with a 5% (IRR = 1.05 95% CI 1.04 ̶ 1.06) and 3% (IRR = 1.03 95% CI 1.03 ̶ 1.04) increase in TTP at week 2 and 4 respectively. With LJ culture, a patient's colony grade was reduced by 0.86 times (0R = 0.86 95% CI 0.74 ̶ 0.97) at week 2 and 0.84 times (OR = 0.84 95% CI 0.79 ̶ 0.95 P = 0.002) at week 4 for every unit increase in the baseline ct value. There was a 3% higher likelihood of earlier conversion to negativity for every unit increase in the ct value. A ct cut point ≥28 best predicted culture negativity at week 4 with a sensitivity of 91. 7% & specificity 53.7% while a cut point ≥23 best predicted culture negativity at week 8.
Conclusion
Baseline Xpert MTB/RIF ct values predict sputum conversion in PLHIV on anti-TB treatment. Surrogate biomarkers for sputum conversion in PLHIV are still a research priority.
Background
Chest X-ray (CXR) interpretation remains a central component of the current World Health Organization recommendations as an adjuvant test in diagnosis of smear-negative tuberculosis (TB). With its low specificity, high maintenance and operational costs, utility of CXR in diagnosis of smear-negative TB in high HIV/TB burden settings in the Xpert MTB/RIF era remains unpredictable. We evaluated accuracy and additive value of CXR to Xpert MTB/RIF in the diagnosis of TB among HIV-positive smear-negative presumptive TB patients.
Methods
HIV co-infected presumptive TB patients were recruited from the Infectious Diseases Institute outpatient clinic and in-patient medical wards of Mulago Hospital, Uganda. CXR films were reviewed by two independent radiologists using a standardized evaluation form. CXR interpretation with regard to TB was either positive (consistent with TB) or negative (normal or unlikely TB). Sensitivity, specificity and predictive values of CXR and CXR combined with Xpert MTB/RIF for diagnosis of smear-negative TB in HIV-positive patients were calculated using sputum and/or blood mycobacterial culture as reference standard.
Results
Three hundred sixty-six HIV co-infected smear-negative participants (female, 63.4%; hospitalized, 68.3%) had technically interpretable CXR. Median (IQR) age was 32 (28-39) years and CD4 count 112 (23-308) cells/mm
3
. Overall, 22% (81/366) were positive for Mycobacterium tuberculosis (Mtb) on culture; 187/366 (51.1%) had CXR interpreted as consistent with TB, of which 55 (29.4%) had culture-confirmed TB. Sensitivity and specificity of CXR interpretation in diagnosis of culture-positive TB were 67.9% (95%CI 56.6-77.8) and 53.7% (95%CI 47.7-59.6) respectively, while Xpert MTB/RIF sensitivity and specificity were 65.4% (95%CI 54.0-75.7) and 95.8% (95%CI 92.8-97.8) respectively. Addition of CXR to Xpert MTB/RIF had overall sensitivity and specificity of 87.7% (95%CI 78.5-93.9) and 51.6% (95%CI 45.6-57.5) respectively; 86.2% (95%CI 75.3-93.5) and 48.1% (95%CI 40.7-55.6) among inpatients and 93.8% (95%CI 69.8-99.8) and 58.0% (95%CI 47.7-67.8) among outpatients respectively.
Conclusion
In this high prevalence TB/HIV setting, CXR interpretation added sensitivity to Xpert MTB/RIF test at the expense of specificity in the diagnosis of culture-positive TB in HIV-positive individuals presenting with TB symptoms and negative smear. CXR interpretation may not add diagnostic value in settings where Xpert MTB/RIF is available as a TB diagnostic tool.
Information on microbiota dynamics in pulmonary tuberculosis (TB) in Africa is scarce. Here, we sequenced sputa from 120 treatment-naïve TB patients in Uganda, and investigated changes in microbiota of 30 patients with treatment-response follow-up samples. Overall, HIV-status and anti-TB treatment were associated with microbial structural and abundance changes. The predominant phyla were Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria and Actinobacteria, accounting for nearly 95% of the sputum microbiota composition; the predominant genera across time were Prevotella, Streptococcus, Veillonella, Haemophilus, Neisseria, Alloprevotella, Porphyromonas, Fusobacterium, Gemella, and Rothia. Treatment-response follow-up at month 2 was characterized by a reduction in abundance of Mycobacterium and Fretibacterium, and an increase in Ruminococcus and Peptococcus; month 5 was characterized by a reduction in Tannerella and Fusobacterium, and an increase in members of the family Neisseriaceae. The microbiota core comprised of 44 genera that were stable during treatment. Hierarchical clustering of this core's abundance distinctly separated baseline (month 0) samples from treatment follow-up samples (months 2/5). We also observed a reduction in microbial diversity with 9.1% (CI 6-14%) of the structural variation attributed to HIV-status and anti-TB treatment. Our findings show discernible microbiota signals associated with treatment with potential to inform anti-TB treatment response monitoring.
Background
The Global Laboratory Initiative (GLI) guidelines recommend repeat for GeneXpertMTB/RIF (XpertMTB/RIF) in patients with a low pretest probability of rifampicin resistance (RR).
Methods
This was a cross-sectional study using results of sputum specimens collected from participants screened for the STREAM 2 trial. We recruited all patients with XpertMTB/RIF RR-TB detected who were referred for RR/multidrug-resistant (MDR) TB treatment initiation at Mulago National Referral Hospital, Kampala, between September 2017 and October 2019. At baseline, smear microscopy, repeat XpertMTB/RIF, Xpert Ultra, and MTBDR
plus
assays were done on sputum specimens. Culture-based drug susceptibility testing (DST) was performed on discordant specimens. We analyzed the prevalence and factors associated with discordance between initial and repeat XpertMTB/RIF RR and false XpertMTB/RIF RR. False XpertMTB/RIF RR was defined as no RR detected by any of Xpert Ultra, LPA, or culture DST (reference comparator).
Results
A total of 126/130 patients had repeat XpertMTB/RIF results, of whom 97 (77.0%) had
M. tuberculosis
detected, 81 (83.5%) had RR detected, and 1 (1.0%) had RR indeterminate. The prevalence of discordant XpertMTB/RIF RR was 15/96 (15.6%), whereas false XpertMTB/RIF RR prevalence was 10/96 (10.4%).Low-bacillary load sputum specimens were more likely to have discordant XpertMTB/RIF RR and false XpertMTB/RIF RR results (adjusted odds ratio [aOR], 0.04; 95% CI, 0.00-0.37;
P
= .01; aOR, 0.02; 95% CI, 0.01-0.35;
P
= .01, respectively).
Conclusions
Our findings show a high false-positive rifampicin resistance rate in low-TB burden patients, which calls for repeat testing in order to prevent unnecessary prescription of anti-MDR-TB therapy.
Project Title
Evaluation of alternative bacteriological measures of response to therapy during the initial 16-weeks of MDR-TB treatment
EDCTP Project
TMA2018CDF-2351
EDCTP Program
EDCTP2
EDCTP Project Call
Career Development Fellowship (CDF)
Host Organisation
| Department | Institution | Country |
|---|---|---|
| Medical Microbiology | Makerere University | UG |
Project Objectives
During the first 16 weeks of MDR-TB treatment I will: 1) To examine the correlations of FDA smear microscopy, PMA-Xpert Ct value &16s rRNA (MBLA) with MGIT-TTP as a reference standard. 2) To evaluate the ability of FDA smear microscopy, PMA-Xpert Ct value, MBLA in predicting 16-weeks MGIT culture conversion (outcome). 3) To evaluate the influence of patient clinical characteristics, baseline resistance profiles, adherence and HIV-status to the performance of the alternative measures of response
Study Design
Prospective observational study
Project Summary
Background: Effective treatment monitoring is vital not only for proper patient management but also for preventing the unnecessary continuation of failing treatment and emergence of more drug-resistant M. tuberculosis (Mtb). Therefore, the urgent need for better treatment monitoring methods cannot be more emphasized. These methods will protect the currently effective drugs and streamline the development of new drugs for multi-drug resistant tuberculosis (MDR-TB). Treatment monitoring by sputum culture is the gold standard for TB, however, this method remains less accessible due to high operational cost, high skills demand, longer turnaround time and contamination. I, therefore, propose a study to evaluate the effectiveness of alternative methods compared with culture in the mycobacterial growth indicator tube, culture time to positivity (MGIT-TTP) for measuring response to therapy during the initial 16 weeks of MDR-TB treatment. We will evaluate how these alternative methods may predict outcomes to shorter MDR-TB treatment regimen. The methods include; fluorescein-di-acetate (FDA) AFB vital smear microscopy, XpertMTB/RIF assay done using sputum pre-treated with propidium mono-azide (PMA), and Mtb16sr RNA assays (Molecular Bacterial Load Assay; MBLA). This study will inform the possible alternative methods for MDR-TB treatment response monitoring in resource-limited settings allowing a quick decision making. This will help in protecting the best available treatment regimens and novel TB drugs, improve the efficiency of phase II trials/treatment regimens and lead to reduced delayed results-associated costs in the development of novel MDR-TB drugs which will eventually lead to better MDR-TB control. Objectives: 1. To examine the correlations of FDA smear microscopy, PMA-Xpert Ct value &16s rRNA (MBLA) with MGIT-TTP as a reference standard. 2. To evaluate the ability of FDA smear microscopy, PMA-Xpert Ct value, MBLA in predicting 16-weeks MGIT culture conversion (outcome). 3. To evaluate the influence of patient clinical characteristics, baseline resistance profiles, adherence and HIV-status to the performance of the alternative measures of response. Methods: A prospective study of 59 patients’ ≥ 18 years of age with MDR-TB diagnosed according to local standards will be enrolled into the study. Two sputum samples, a spot, and an overnight will be taken at weeks 0, 1, 2, 3, 4, 5, 6, 7, 8, 12 and 16 of MDR-TB treatment. Samples will be pooled and homogenized and divided into three portions; one for; 1) FDA- treated AFB smear microscopy 2) PMA-Xpert/ULTRA assay 3) liquid culture for Mtb-TTP. Portion two for 16s rRNA detection in an MBLA assay. Portion three will be used for TTP determination in MGIT culture. Data will be analyzed for correlation with the alternative methods results with increasing MGIT-TTP. The alternative methods will be analysed to determine how well they predict MDR-TB patient treatment 16 weeks’ culture conversion and the influence of clinical characteristics to their performance.