A Standardized Approach for Collection of Objective Data to Support Outcome Determination for Late-Phase Tuberculosis Clinical Trials.

Rationale: We developed a standardized method, possible poor treatment response (PPTR), to help ascertain efficacy endpoints in Study S31/A5349 (NCT02410772), an open-label trial comparing two 4-month rifapentine-based regimens with a standard 6-month regimen for the treatment of pulmonary tuberculosis (TB). Objectives: We describe the use of the PPTR process and evaluate whether the goals of minimizing bias in efficacy endpoint assessment and attainment of relevant data to determine outcomes for all participants were achieved. Methods: A PPTR event was defined as the occurrence of one or more prespecified triggers. Each PPTR required initiation of a standardized evaluation process that included obtaining multiple sputum samples for microbiology. Measurements and Main Results: Among 2,343 participants with culture-confirmed drug-susceptible TB, 454 individuals (19.4%) had a total of 534 individual PPTR events, of which 76.6% were microbiological (positive smear or culture at or after 17 wk). At least one PPTR event was experienced by 92.4% (133 of 144) of participants with TB-related unfavorable outcome and between 13.8% and 14.7% of participants with favorable and not-assessable outcomes. A total of 75% of participants with TB-related unfavorable outcomes had microbiological confirmation of failure to achieve a disease-free cure. Conclusions: Standardized methodologies, such as our PPTR approach, could facilitate unbiased efficacy outcome determinations, improve discrimination between outcomes that are related and unrelated to regimen efficacy, and enhance the ability to conduct pooled analyses of contemporary trials.

Characterization of Differentially Detectable Mycobacterium tuberculosis in the Sputum of Subjects with Drug-Sensitive or Drug-Resistant Tuberculosis before and after Two Months of Therapy.

Standard methods for enumerating Mycobacterium tuberculosis in patient sputum can miss large populations of viable M. tuberculosis cells that are unable to grow either on solid medium or in liquid medium unless the medium has been extensively diluted. Because these bacteria can be detected in liquid medium after limiting dilution, they have been termed differentially culturable or differentially detectable M. tuberculosis (DD-Mtb). Treatment with isoniazid (H), rifampin (R), pyrazinamide (Z), and ethambutol (E) (HRZE) for 1 to 2 weeks has been shown to increase the representation of DD-Mtb in the sputum of drug-sensitive (DS) tuberculosis (TB) patients. However, little is known about DD-Mtb after longer periods of treatment with HRZE or in patients with drug-resistant (DR) TB who receive second-line therapies. Here, we measured the proportion of DD-Mtb cells in the sputum of 47 subjects, 29 with DS TB and 18 with DR TB, before initiation of treatment and at 2 weeks and 2 months thereafter. Prior to treatment, DD-Mtb cells represented the majority of M. tuberculosis cells in the sputum of 21% of subjects with DS TB, and this proportion rose to 65% after 2 weeks of treatment with first-line drugs. In subjects with DR TB, DD-Mtb cells were found in the sputum of 29% of subjects prior to treatment initiation, and this proportion remained steady at 31% after 2 weeks of treatment with second-line drugs. By 2 months, DD-Mtb cells were detected in the sputum of only 2/15 (13.3%) subjects with DS TB and in 0/15 of subjects with DR TB. One of the DS subjects whose sputum was positive for DD-Mtb at month 2 later experienced treatment failure.

Improved Outcomes With High-dose Isoniazid in Multidrug-resistant Tuberculosis Treatment in Haiti.

We report outcomes for a cohort of patients with multidrug-resistant tuberculosis who received high-dose isoniazid in Haiti. Patients who received high-dose isoniazid had a faster time to culture conversion and higher odds of successful outcome, despite high-level isoniazid resistance. This suggests high-dose isoniazid may have effectiveness even with phenotypic resistance.

High-throughput mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping for Mycobacterium tuberculosis epidemiological studies.

The emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner.

Tuberculosis in the aftermath of the 2010 earthquake in Haiti.

PROBLEM: In 2010, Haiti sustained a devastating earthquake that crippled the health-care infrastructure in the capital city, Port-au-Prince, and left 1.5 million people homeless. Subsequently, there was an increase in reported tuberculosis in the affected population. APPROACH: We conducted active tuberculosis case finding in a camp for internally displaced persons and a nearby slum. Community health workers screened for tuberculosis at the household level. People with persistent cough were referred to a physician. The National Tuberculosis Program continued its national tuberculosis reporting system. LOCAL SETTING: Even before the earthquake, Haiti had the highest tuberculosis incidence in the Americas. About half of the tuberculosis cases occur in the Port-au-Prince region. RELEVANT CHANGES: The number of reported tuberculosis cases in Haiti has increased after the earthquake, but data are too limited to determine if this is due to an increase in tuberculosis burden or to improved case detection. Compared to previous national estimates (230 per 100,000 population), undiagnosed tuberculosis was threefold higher in a camp for internally displaced persons (693 per 100,000) and fivefold higher in an urban slum (1165 per 100,000). With funding from the World Health Organization (WHO), active case finding is now being done systematically in slums and camps. LESSONS LEARNT: Household-level screening for prolonged cough was effective in identifying patients with active tuberculosis in this study. Without accurate data, early detection of rising tuberculosis rates is challenging; data collection should be incorporated into pragmatic disease response programmes.

Efficacy of nitazoxanide against clinical isolates of Mycobacterium tuberculosis.

Nitazoxanide (NTZ) has bactericidal activity against the H37Rv laboratory strain of Mycobacterium tuberculosis with a MIC of 16 µg/ml. However, its efficacy against clinical isolates of M. tuberculosis has not been determined. We found that NTZ's MIC against 50 clinical isolates ranged from 12 to 28 µg/ml with a median of 16 µg/ml and was unaffected by resistance to first- or second-line antituberculosis drugs or a diversity of spoligotypes.

Use of Luminex MagPlex magnetic microspheres for high-throughput spoligotyping of Mycobacterium tuberculosis isolates in Port-au-Prince, Haiti.

Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct "spacers" separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.

Multidrug-resistant tuberculosis in Port-au-Prince, Haiti.

OBJECTIVE: To determine the prevalence of multidrug-resistant tuberculosis (MDR-TB) among patients with new smear-positive pulmonary TB in Port-au-Prince, Haiti. METHODS: Sputum samples were cultured from 1 006 patients newly diagnosed with TB in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced. All isolates with rpoB mutations were sent to the New York State reference laboratory for conventional drug susceptibility testing (DST). All isolates were also tested with the GenoType MTBDRplus line-probe assay. RESULTS: Mycobacterium tuberculosis was isolated from 906 patients. Twenty-six (2.9%) of the isolates had missense mutations or deletions in rpoB and were resistant to rifampin by DST. All 26 were also resistant to isoniazid and classified as MDR-TB. Forty-six control isolates without rpoB mutations were found to be rifampin sensitive by DST. The GenoType MTBDRplus line-probe assay correctly identified 26 MDR-TB strains. It misclassified one pansusceptible isolate as rifampin resistant. CONCLUSIONS: This study shows an MDR-TB prevalence of 2.9% in newly diagnosed TB patients in Haiti and suggests that rpoB sequencing and hybridization assays are good screening tools for early detection of MDR-TB.

An optimized method for purifying, detecting and quantifying Mycobacterium tuberculosis RNA from sputum for monitoring treatment response in TB patients.

Diagnostics that more accurately detect and quantify viable Mycobacterium tuberculosis (Mtb) in the sputum of patients undergoing therapy are needed. Current culture- and molecular-based tests have shown limited efficacy for monitoring treatment response in TB patients, either due to the presence of viable sub-populations of Mtb which fail to grow under standard culture conditions (termed differentially detectable/culturable Mtb, DD Mtb) or the prolonged half-life of Mtb DNA in sputum. Here, we report an optimized RNA-based method for detecting and quantifying viable Mtb from patient sputum during the course of therapy. We first empirically derived a novel RNA extraction protocol from sputum that improves recovery of Mtb RNA while almost completely eliminating contamination from Mtb DNA and host nucleic acids. Next, we identified five Mtb 16S rRNA primer sets with varying limits of detection that were capable of distinguishing between live versus dead H37Rv Mtb. This combined protocol was then tested on sputa from a longitudinal cohort of patients receiving therapy for drug sensitive (DS) or drug resistant (DR) TB with first-line or second-line regimens, respectively. Results were compared with that of culture, including CFU, BACTEC MGIT, and a limiting dilution assay capable of detecting DD Mtb. The five 16S rRNA primer sets positively identified nearly all (range 94-100%) culture positive sputa, and a portion (19-37%) of culture negative sputa. In comparison, ten highly expressed Mtb mRNAs showed positivity in 72-86% of culture positive sputa, and in 0-13% of culture negative sputa. Two of the five 16S rRNA primer sets were able to positively identify 100% of culture positive sputa, and when tested on culture negative sputa from the DS cohort at 2 months post-initiation of therapy, identified 40% of samples as positive; a percentage that is in line with expected treatment failure rates when first-line therapy is discontinued early. These two primer sets also detected 16S rRNA in 13-20% of sputa at 6 months post-initiation of therapy in the DR cohort. Cycle threshold values for 16S rRNA showed a strong correlation with Mtb numbers as determined by culture (R > 0.87), including as Mtb numbers declined during the course of treatment with first-line and second-line regimens. The optimized molecular assay outlined here may have utility for monitoring treatment response in TB patients.

Transcriptional Biomarkers of Differentially Detectable Mycobacterium tuberculosis in Patient Sputum.

Certain populations of Mycobacterium tuberculosis go undetected by standard diagnostics but can be enumerated using limiting dilution assays. These differentially detectable M. tuberculosis (DD M. tuberculosis) populations may have relevance for persistence due to their drug tolerance. It is unclear how well DD M. tuberculosis from patients is modeled by a recently developed in vitro model in which M. tuberculosis starved in phosphate-buffered saline is incubated with rifampin to produce DD M. tuberculosis (the PBS-RIF model). This study attempted to answer this question. We selected 14 genes that displayed differential expression in the PBS-RIF model and evaluated their expression in patient sputa containing various proportions of DD M. tuberculosis. The expression of 12/14 genes correlated with the relative abundance of DD M. tuberculosis in patient sputa. Culture filtrate (CF), which promotes recovery of DD M. tuberculosis from certain patient sputa, improved these correlations in most cases. The gene whose reduced expression relative to M. tuberculosis 16S rRNA showed the greatest association with the presence and relative abundance of DD M. tuberculosis in patient sputa, icl1, was recently shown to play a functional role in restraining DD M. tuberculosis formation in the PBS-RIF model. Expression of icl1, combined with two additional DD M. tuberculosis-related genes, showed strong performance for predicting the presence or absence of DD M. tuberculosis in patient sputa (receiver operating characteristic [ROC] area under the curve [AUC] = 0.88). Thus, the in vitro DD M. tuberculosis model developed by Saito et al. (K. Saito, T. Warrier, S. Somersan-Karakaya, L. Kaminski, et al., Proc Natl Acad Sci U S A 114:E4832-E4840, 2017, https://doi.org/10.1073/pnas.1705385114) bears a resemblance to DD M. tuberculosis found in tuberculosis (TB) patients, and DD M. tuberculosis transcriptional profiles may be useful for monitoring DD M. tuberculosis populations in patient sputum. IMPORTANCE Differentially detectable M. tuberculosis (DD M. tuberculosis), which is detectable by limiting dilution assays but not by CFU, is present and enriched for in TB patient sputum after initiation of first-line therapy. These cryptic cells may play a role in disease persistence due to their phenotypic tolerance to anti-TB drugs. A recently developed in vitro model of DD M. tuberculosis (the PBS-RIF model) has expanded our understanding of these cells, though how well it translates to DD M. tuberculosis in patients is currently unknown. To answer this question, we selected 14 genes that displayed differential expression in the PBS-RIF model and evaluated their expression in TB patient sputa. We found that 12/14 of these genes showed a similar expression profile in patient sputa that correlated with the relative abundance of DD M. tuberculosis. Further, the expression of three of these genes showed strong performance for predicting the presence or absence of DD M. tuberculosis in patient sputa. The use of DD M. tuberculosis transcriptional profiles may allow for easier monitoring of DD M. tuberculosis populations in patient sputum in comparison to limiting dilution assays.

Multidrug-resistant Pseudomonas aeruginosa in healthcare facilities in Port-au-Prince, Haiti.

OBJECTIVES: Pseudomonas aeruginosa is a leading cause of opportunistic infections worldwide, particularly in healthcare settings, and frequently demonstrates resistance to commonly prescribed antimicrobials. Carbapenem resistance is prevalent worldwide, however there are currently limited data available from Haiti. The aim of this study was to characterise and document this phenotype in Port-au-Prince, Haiti, to further inform the need for appropriate infection control, empirical treatment guidelines and laboratory screening measures, both in Haiti and globally. METHODS: A total of 50 P. aeruginosa isolates were characterised by multilocus sequence typing (MLST) and antimicrobial susceptibility testing, of which 8 isolates were also subjected to whole-genome sequencing (WGS) to identify potential genetic correlations of phenotypic resistance. RESULTS: By MLST, 23 sequence types (STs) were identified, including 13 new STs. Nineteen isolates belonged to a single, previously characterised ST (ST654), all of which demonstrated a multidrug-resistant phenotype, including resistance to meropenem, imipenem and ceftazidime; two isolates were also resistant to colistin. WGS revealed the presence of genes encoding several previously characterised resistance determinants in ST654; notably ACC(6')-Ib3-cr and GES-7. Metallo-ß-lactamase genes (blaVIM-5) were also detected in three isolates. CONCLUSION: These findings confirm that drug-resistant clones of P. aeruginosa are present in Haiti, supporting the need for appropriate screening and control measures and confirming that drug-resistant micro-organisms pose a global threat. Further investigations are required to guide appropriate antimicrobial prescribing in this region.

Diagnosis of Tuberculosis Using Gastric Aspirates in Pediatric Patients in Haiti.

BACKGROUND: We aimed to determine whether the Xpert MTB/RIF (Xpert) assay is a useful adjunct to culture for the rapid diagnosis of tuberculosis (TB) using gastric lavage aspirates (GLAs) in children aged < 5 years. METHODS: We reviewed the yield from diagnostic modalities in children suspected of having TB followed at an infectious disease research and treatment center in Port-au-Prince, Haiti, from 2011 to 2016. RESULTS: In 187 children clinically diagnosed with TB, a microbiologic diagnosis could be established in 40 (21%). Cultures, Xpert, and smears were positive in 30 (19%), 28 (17%), and 3 (1.6%) children, respectively. Ten cases that would not have been diagnosed by culture alone were found by the use of the Xpert assay. Collecting 2 GLA samples optimized microbiologic yield. CONCLUSIONS: In GLAs, Xpert increased the yield of microbiologically documented cases by 33%. Additionally, the rapidity of diagnosis potentially makes Xpert a valuable adjunct in initiating treatment for TB in children. Smear microscopy has low sensitivity in GLA and did not add to the documented cases. Our findings also highlight the low rate of microbiologic confirmation of clinically diagnosed TB.

Drug-resistant TB prevalence study in 5 health institutions in Haiti.

OBJECTIVES: Tuberculosis (TB) is the leading infectious cause of death in the world. Multi-drug resistant TB (MDR-TB) is a major public health problem as treatment is long, costly, and associated to poor outcomes. Here, we report epidemiological data on the prevalence of drug-resistant TB in Haiti. METHODS: This cross-sectional prevalence study was conducted in five health centers across Haiti. Adult, microbiologically confirmed pulmonary TB patients were included. Molecular genotyping (rpoB gene sequencing and spoligotyping) and phenotypic drug susceptibility testing were used to characterize rifampin-resistant MTB isolates detected by Xpert MTB/RIF. RESULTS: Between April 2016 and February 2018, 2,777 patients were diagnosed with pulmonary TB by Xpert MTB/RIF screening and positive MTB cultures. A total of 74 (2.7%) patients were infected by a drug-resistant (DR-TB) M. tuberculosis strain. Overall HIV prevalence was 14.1%. Patients with HIV infection were at a significantly higher risk for infection with DR-TB strains compared to pan-susceptible strains (28.4% vs. 13.7%, adjusted odds ratio 2.6, 95% confidence interval 1.5-4.4, P = 0.001). Among the detected DR-TB strains, T1 (29.3%), LAM9 (13.3%), and H3 (10.7%) were the most frequent clades. In comparison with previous spoligotypes studies with data collected in 2000-2002 and in 2008-2009 on both sensitive and resistant strains of TB in Haiti, we observed a significant increase in the prevalence of the drug-resistant MTB Spoligo-International-Types (SIT) 137 (X2 clade: 8.1% vs. 0.3% in 2000-02 and 0.9% in 2008-09, p<0.001), 5 (T1 clade: 6.8% vs 1.9 in 2000-02 and 1.7% in 2008-09, P = 0.034) and 455 (T1 clade: 5.4% vs 1.6% and 1.1%, P = 0.029). Newly detected spoligotypes (SIT 6, 7, 373, 909 and 1624) were also recorded. CONCLUSION: This study describes the genotypic and phenotypic characteristics of DR-TB strains circulating in Haiti from April 2016 to February 2018. Newly detected MTB clades harboring multi-drug resistance patterns among the Haitian population as well as the higher risk of MDR-TB infection in HIV-positive people highlights the epidemiological relevance of these surveillance data. The importance of detecting RIF-resistant patients, as proxy for MDR-TB in peripheral sites via molecular techniques, is particularly important to provide adequate patient case management, prevent the transmission of resistant strains in the community and to contribute to the surveillance of resistant strains.

Long-term mortality after tuberculosis treatment among persons living with HIV in Haiti.

INTRODUCTION: Long-term mortality among TB survivors appears to be higher than control populations without TB in many settings. However, data are limited among persons with HIV (PWH). We assessed the association between cured TB and long-term mortality among persons with PWH in Haiti. METHODS: A prospective cohort of PWH from the CIPRA HT-001 trial was followed from study enrolment (August 2005 to July 2008) to study closure (December 2018) to compare mortality between participants with and without TB. The index date for the survival analysis was defined as 240 days after TB diagnosis or randomization date. Time to death was described using Kaplan-Meier curves, and log-rank tests were used to compare time to death between the TB and no-TB cohorts. The association between TB and long-term mortality was estimated with multivariable Cox models. RESULTS: Of the 816 participants in the CIPRA HT-001 trial, 77 were excluded for a history of TB prior to study enrolment and 31 were excluded due to death or attrition prior to the index date, leaving 574 in the no-TB and 134 in the TB cohort. Twenty-four (17.9%) participants in the TB and 48 (8.4%) in the no-TB cohort died during follow-up. Five and 10-year mortality rates were 14.2% and 17.9% respectively, in the TB cohort, and 6.1% and 8.4% in the no-TB cohort. In Kaplan-Meier analysis, participants in the TB cohort had a significantly shorter time to death (log-rank p < 0.001). In multivariable analysis, TB treatment was the only predictor of mortality (HR: 2.78; 95% CI: 1.61, 4.79). Sensitivity analyses, which included only baseline TB cases, an index date of two years after TB diagnosis, and study enrolment and case-control matching yielded results that were consistent with primary analyses. CONCLUSIONS: PWH who are successfully treated for TB have higher long-term mortality than those who are never diagnosed with TB, even after accounting for acute TB-related mortality. A better understanding of the underlying mechanisms associated with TB sequelae is critically needed to guide specific interventions. Until then, more aggressive measures for health promotion and disease prevention are essential to improve long-term survival for PWH after TB treatment.

Adenovirus induction of IRF3 occurs through a binary trigger targeting Jun N-terminal kinase and TBK1 kinase cascades and type I interferon autocrine signaling.

Pathogen recognition is a critical function of immune sentinel cells. Naïve macrophages or dendritic cells (DCs) undergo pathogen-directed activation and maturation, and as mature antigen-presenting cells (APCs), they contribute essential functions to both innate and adaptive immunity. Using recombinant adenovirus (rAdV) as a model for murine APC activation by DNA viruses, we demonstrate a critical role for stress kinase activation in cell intrinsic and extrinsic antiviral signaling cascades. We propose two viral triggers, viral capsid and viral DNA, are required for APC activation. Endosomal escape and presentation of cytosolic rAdV DNA induces phosphorylation of TANK-binding kinase 1 (TBK1) at serine 172 but does not induce IkappaB kinase epsilon activity as determined by in vitro kinase assays. However, induction of TBK1 alone is not sufficient for interferon regulatory factor 3 (IRF3) phosphorylation. We show that capsid-dependent activation of Jun N-terminal kinase (JNK) stress kinase is a necessary step, licensing TBK1 phosphorylation of IRF3 at Ser 396. A second later phase of JNK activity is required to coordinate phosphorylation of JNK-dependent transcription factors (c-Jun/ATF2) with activated IRF3 in the induction of primary IRF3-responsive transcripts. Finally, we demonstrate that maximal JNK/TBK1/IRF3 stimulation by rAdV depends on an intact type I interferon (IFN) signaling cascade. By requiring multiple viral triggers and type I IFN autocrine regulation, APCs have an inherent fail-safe mechanism against inappropriate activation and maturation.

Genotypic drug resistance using whole-genome sequencing of Mycobacterium tuberculosis clinical isolates from North-western Tanzania.

BACKGROUND: Drug resistant Tuberculosis (TB) is considered a global public health threat. Whole-genome sequencing (WGS) is a new technology for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles and genotypes for epidemiologic surveillance. Therefore, we used WGS to determine genotypic drug resistance profiles and genetic diversity of drug resistant Mycobacterium tuberculosis isolates from Mwanza, North-western Tanzania. METHODS: A cross-sectional study was conducted at the Bugando Medical Center (BMC) from September 2014 to June 2015. Consecutively, smear-positive newly diagnosed TB patients aged ≥18 years were enrolled. Sputum samples were cultured on Löwenstein-Jensen (LJ) slants. Mycobacterial genomic DNA was extracted for WGS to determine drug resistant mutations for first and second line drugs as well as the spoligotypes. RESULTS: A total of 78 newly diagnosed patients with pulmonary TB with a median age of 37 [IQR: 30-46] years were enrolled. Of these, 57.8% (45/74) were males and 34.6% (27/78) were HIV positive. Mycobacterium tuberculosis genomic DNA for WGS was obtained from isolates in 74 (94.9%) patients. Of the 74 isolates, six (8.1%) isolates harbored mutations for resistance to at least one drug. The resistance to the drugs was isoniazid 3/74 (4.1%), rifampicin mono-resistant 2/74 (2.7%), ethambutol 2/74 (2.7%) and streptomycin 1/74 (1.4%). None was isoniazid mono-resistant. Of the 74 only one (1.4%) patient had MDR-TB. The resistance to ethionamide, the second line drug, was detected in one patient (1.4%). None was resistant to pyrazinamide, fluoroquinolones, kanamycin, amikacin, or capreomycin. The mutations detected were mabA-inhA promoter region C(-15)T and katG Ser513Thr for isoniazid; rpoB His526Leu and rpoB Ser531Leu for rifampicin; embB Met306Val and embB Met306Ile for ethambutol; rpsL Lys43Arg for streptomycin; and mabA-inhA promoter region C(-15)T for ethionamide. The spoligotypes of the drug resistant Mycobacterium tuberculosis were distinct to all six isolates and belonged to T1, T2, T3-ETH, CAS1-DELHI, EAI5 and LAM11-ZWE lineages. CONCLUSION: The genetic drug resistance profile of Mycobacterium tuberculosis isolates from North-western Tanzania comprises of the common previously reported mutations. The prevalence of resistance to first and second line drugs including MDR-TB is low. Six drug resistant strains exhibited different spoligotypes, suggesting limited transmission of drug resistant strains in the region.

Differentially Detectable Mycobacterium tuberculosis Cells in Sputum from Treatment-Naive Subjects in Haiti and Their Proportionate Increase after Initiation of Treatment.

Recent reports indicate that the sputum of 80% or more of treatment-naive subjects with tuberculosis recruited in England or South Africa contained more viable Mycobacterium tuberculosis cells detected by limiting dilution (LD) in liquid culture than detected as CFU. Efforts to generate such differentially detectable (DD) M. tuberculosis populations in vitro have been difficult to reproduce, and the LD assay is prone to artifact. Here, we applied a stringent version of the LD assay to sputum from 33 treatment-naive, HIV-negative Haitian subjects with drug-sensitive tuberculosis (TB) and to a second sputum sample after two weeks of standard treatment with isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) for 13 of these subjects. Twenty-one percent had statistically defined levels of DD M. tuberculosis in their pretreatment sputum at an average proportional excess over CFU of 3-fold. Sixty-nine percent of those who received HRZE had statistically defined levels of DD M. tuberculosis in their sputum, and of these, the mean proportionate excess over CFU was 7.9-fold. Thus, DD M. tuberculosis is detectable in pretreatment sputum from a significant proportion of subjects in the Western Hemisphere, and certain drugs or drug regimens, while reducing CFU, may at the same time increase the proportional representation of DD M. tuberculosis among the surviving bacilli. Monitoring DD M. tuberculosis may improve our ability to predict the efficacy of efforts to shorten treatment.IMPORTANCE Measurement of the reduction in CFU in sputum of patients with TB up to 2 weeks after the initiation of treatment is the gateway test for a new TB treatment. Reports have suggested that CFU assays fail to detect the majority of viable M. tuberculosis cells in sputum samples from the majority of patients when the number of M. tuberculosis is estimated by limiting dilution (LD). In an effort to avoid potential methodologic confounders, we applied a modified version of the LD assay in a study of a geographically distinct population. We confirmed that differentially detectable (DD) M. tuberculosis is often found before treatment, albeit at lower proportionate levels than in earlier reports. Strikingly, the prevalence and proportionate representation of DD M. tuberculosis increased during standard treatment. Sublethal exposure to certain antibiotics may help generate DD M. tuberculosis cells or enrich their representation among the surviving bacteria, and this may contribute to the need for prolonged treatment with those agents in order to achieve durable cures.

Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis screening and treatment of pregnant women in Port-au-Prince, Haiti.

In Haiti, routine screening for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) among pregnant women is not conducted; yet these sexually transmitted infections (STIs) are associated with adverse birth and newborn health outcomes. We aimed to assess the acceptability and feasibility of screening and the prevalence of STIs among pregnant women in Port-au-Prince, Haiti. Pregnant women of at least 18 years of age who attend Haitian Study Group for Kaposi's sarcoma and Opportunistic Infections (GHESKIO) clinics in Port-au-Prince, Haiti provided self-collected vaginal swab specimens. Laboratory testing was done with Xpert® CT/NG and Xpert® TV. The results of this study showed that of the 322 pregnant women who visited GHESKIO for their regular scheduled appointments, 300 (93.2%) consented for CT, NG, and TV testing. Of those, 107 women (35.7%) tested positive for at least one STI. There were 42 (14.7%) cases of CT, 8 (2.8%) NG, and 83 (29.0%) TV infections. Most infections were treated - 122 of 133 (91.7%). In summary, we found that it was highly acceptable and feasible to implement CT, NG, and TV screening among pregnant women in Port-au-Prince, Haiti. We found high prevalence of STIs among pregnant women, which suggest that STI screening in this population may be warranted.

Active Tuberculosis Case Finding in Haiti.

In 2010, Haiti suffered from a devastating earthquake; data on the impact on the tuberculosis (TB) epidemic are limited. From January to June 2013, we conducted active case finding at the household level in a slum in Port-au-Prince. Community health workers identified individuals with cough ≥ 2 weeks, and referred them for evaluation. Contact tracing was conducted for patients with active TB. Of an estimated 7,500 residents screened, 394 (5%) had cough and were tested for TB. One hundred (25%) were diagnosed with active TB; 53 (53%) were smear positive. Ninety of these TB index cases provided 317 contacts, and 44 (14%) were diagnosed with active TB; 17 (39%) were smear positive. Overall, 144 TB cases were detected in 6 months (1,920/100,000; national estimate 200/100,000). We found a high burden of undiagnosed TB in Port-au-Prince 3 years after the earthquake. Further assessment of the burden of TB is indicated.

False-positive rifampin resistant results with Xpert MTB/RIF version 4 assay in clinical samples with a low bacterial load.

We report investigation of 22 TB cases with positive Xpert MTB/RIF result for resistance to Rifampin and "Very Low" MTB detection level. Twelve cases were false positive without rpoB mutations, 2 were false-positives with a silent mutation in rpoB codon T508, and only 10 were true positives.