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BackgroundSerious invasive infections in newborns are a major cause of death. Lack of data on etiological causes hampers progress towards reduction of mortality. This study aimed to identify pathogens responsible for such infections in young infants in sub-Saharan Africa and to describe their antibiotics resistance profile.MethodsBetween September 2016 and April 2018 we implemented an observational study in two rural sites in Burkina Faso and Tanzania enrolling young infants aged 0-59 days old with serious invasive infection. Blood samples underwent blood culture and molecular biology.ResultsIn total 634 infants with clinical diagnosis of serious invasive infection were enrolled and 4.2% of the infants had a positive blood culture. The most frequent pathogens identified by blood culture were Klebsiella pneumonia and Staphylococcus aureus, followed by Escherichia coli. Gram-negative isolates were only partially susceptible to first line WHO recommended treatment for neonatal sepsis at community level. A total of 18.6% of the infants were PCR positive for at least one pathogen and Escherichia coli and Staphylococcus aureus were the most common bacteria detected. Among infants enrolled, 60/634 (9.5%) died. Positive blood culture but not positive PCR was associated with risk of death. For most deaths, no pathogen was identified either by blood culture or molecular testing, and hence a causal agent remained unclear. Mortality was associated with low body temperature, tachycardia, respiratory symptoms, convulsions, history of difficult feeding, movement only when stimulated or reduced level of consciousness, diarrhea and/or vomiting.ConclusionWhile Klebsiella pneumonia and Staphylococcus aureus, as well as Escherichia coli were pathogens most frequently identified in infants with clinical suspicion of serious invasive infections, most cases remain without definite diagnosis, making more accurate diagnostic tools urgently needed. Antibiotics resistance to first line antibiotics is an increasing challenge even in rural Africa.

BackgroundStunting among children in low-resource settings is associated with enteric pathogen carriage and micronutrient deficiencies. Our goal was to test whether administration of scheduled antimicrobials and daily nicotinamide improved linear growth in a region with a high prevalence of stunting and enteric pathogen carriage.Methods and findingsWe performed a randomized, 2 × 2 factorial, double-blind, placebo-controlled trial in the area around Haydom, Tanzania. Mother-child dyads were enrolled by age 14 days and followed with monthly home visits and every 3-month anthropometry assessments through 18 months. Those randomized to the antimicrobial arm received 2 medications (versus corresponding placebos): azithromycin (single dose of 20 mg/kg) at months 6, 9, 12, and 15 and nitazoxanide (3-day course of 100 mg twice daily) at months 12 and 15. Those randomized to nicotinamide arm received daily nicotinamide to the mother (250 mg pills months 0 to 6) and to the child (100 mg sachets months 6 to 18). Primary outcome was length-for-age z-score (LAZ) at 18 months in the modified intention-to-treat group. Between September 5, 2017 and August 31, 2018, 1,188 children were randomized, of whom 1,084 (n = 277 placebo/placebo, 273 antimicrobial/placebo, 274 placebo/nicotinamide, and 260 antimicrobial/nicotinamide) were included in the modified intention-to-treat analysis. The study was suspended for a 3-month period by the Tanzanian National Institute for Medical Research (NIMR) because of concerns related to the timing of laboratory testing and the total number of serious adverse events (SAEs); this resulted in some participants receiving their final study assessment late. There was a high prevalence of stunting overall (533/1,084, 49.2%). Mean 18-month LAZ did not differ between groups for either intervention (mean LAZ with 95% confidence interval [CI]: antimicrobial: -2.05 CI -2.13, -1.96, placebo: -2.05 CI -2.14, -1.97; mean difference: 0.01 CI -0.13, 0.11, p = 0.91; nicotinamide: -2.06 CI -2.13, -1.95, placebo: -2.04 CI -2.14, -1.98, mean difference 0.03 CI -0.15, 0.09, p = 0.66). There was no difference in LAZ for either intervention after adjusting for possible confounders (baseline LAZ, age in days at 18-month measurement, ward, hospital birth, birth month, years of maternal education, socioeconomic status (SES) quartile category, sex, whether the mother was a member of the Datoga tribe, and mother's height). Adverse events (AEs) and SAEs were overall similar between treatment groups for both the nicotinamide and antimicrobial interventions. Key limitations include the absence of laboratory measures of pathogen carriage and nicotinamide metabolism to provide context for the negative findings.ConclusionsIn this study, we observed that neither scheduled administration of azithromycin and nitazoxanide nor daily provision of nicotinamide was associated with improved growth in this resource-poor setting with a high force of enteric infections. Further research remains critical to identify interventions toward improved early childhood growth in challenging conditions.Trial registrationClinicalTrials.gov NCT03268902.

Abstract Background Tuberculosis (TB) is the leading cause of death from an infectious disease and the roll-out of rapid molecular diagnostics for rifampin resistance has resulted in a steady rise in the number of patients with multidrug-resistant (MDR)-TB referred for treatment. Pyrazinamide is used in susceptible TB treatment for 6 months when used in combination with rifampin, isoniazid and ethambutol and is an important companion drug in novel MDR-TB trials. This study was undertaken to determine the prevalence of pyrazinamide resistance by either phenotypic or pncA testing among patients admitted to a referral hospital in Tanzania for drug-susceptible and MDR-TB treatment. Methods Surveillance sputa were sent among subjects beginning TB therapy at the national MDR-TB referral hospital during a 6 month period in 2013–2014. Mycobacterial cultures of pretreatment sputa were performed at the Kilimanjaro Clinical Research Institute (KCRI) in the BACTEC mycobacterial growth indicator tubes (MGIT) 960 system. Speciation of M. tuberculosis complex was confirmed by MTBc assay. Isolates were sub-cultured on to Lowenstein-Jensen (LJ) slants. Phenotypic resistance to pyrazinamide was performed in the MGIT system while a real-time PCR with High Resolution Melt (HRM) technique was used to determine mutation in the pncA gene from the same pure subculture. Sputa were then collected monthly to determine the time to culture negativity. Final treatment outcome was determined. Results Ninety-one M. tuberculosis isolates from individual patients were available for analysis of which 30 (32.9%) had MDR-TB, the mean (±SD) age was 33 ± 10 years, and the majority 23 (76.7%) were males. Of the 30 MDR-TB patients, 15(50%) had isolates with pyrazinamide resistance by conventional MGIT testing. This proportion expectedly exceeded the number with pyrazinamide resistance in the 61 patients without MDR-TB, 13 (21.3%) (p = 0.008). Six (20%) of MDR-TB patients had a poor outcome including treatment failure. Among patients with treatment failure, 5 (83%) had pyrazinamide resistance compared to only 10 (41.6%) with treatment success (p = 0.08). Two patients died, and both had isolates with pyrazinamide resistance. No other pretreatment characteristic was associated with treatment outcome. Conclusion Pyrazinamide susceptibility appears to be important in clinical outcomes for MDR-TB patients, and susceptibility testing appears to be a critical adjunct to TB care. The high proportion of PZA resistance in non-MDR TB cases calls for further local investigation.

Growth impairment is a major public health issue for children in Tanzania. The question remains as to whether dietary mycotoxins play a role in compromising children's growth. We examined children's exposures to dietary aflatoxin and fumonisin and potential impacts on growth in 114 children under 36 months of age in Haydom, Tanzania. Plasma samples collected from the children at 24 months of age (N = 60) were analyzed for aflatoxin B1-lysine (AFB1-lys) adducts, and urine samples collected between 24 and 36 months of age (N = 94) were analyzed for urinary fumonisin B1 (UFB1). Anthropometric, socioeconomic, and nutritional parameters were measured and growth parameter z-scores were calculated for each child. Seventy-two percent of the children had detectable levels of AFB1-lys, with a mean level of 5.1 (95% CI: 3.5, 6.6) pg/mg albumin; and 80% had detectable levels of UFB1, with a mean of 1.3 (95% CI: 0.8, 1.8) ng/ml. This cohort had a 75% stunting rate [height-for-age z-scores (HAZ)

Background: Environmental enteropathy (EE), the adverse impact of frequent and numerous enteric infections on the gut resulting in a state of persistent immune activation and altered permeability, has been proposed as a key determinant of growth failure in children in low- and middle-income populations. A theory-driven systems model to critically evaluate pathways through which enteropathogens, gut permeability, and intestinal and systemic inflammation affect child growth was conducted within the framework of the Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) birth cohort study that included children from eight countries. Methods: Non-diarrheal stool samples (N = 22,846) from 1253 children from multiple sites were evaluated for a panel of 40 enteropathogens and fecal concentrations of myeloperoxidase, alpha-1-antitrypsin, and neopterin. Among these same children, urinary lactulose:mannitol (L:M) (N = 6363) and plasma alpha-1-acid glycoprotein (AGP) (N = 2797) were also measured. The temporal sampling design was used to create a directed acyclic graph of proposed mechanistic pathways between enteropathogen detection in non-diarrheal stools, biomarkers of intestinal permeability and inflammation, systemic inflammation and change in length- and weight- for age in children 0–2 years of age. Findings: Children in these populations had frequent enteric infections and high levels of both intestinal and systemic inflammation. Higher burdens of enteropathogens, especially those categorized as being enteroinvasive or causing mucosal disruption, were associated with elevated biomarker concentrations of gut and systemic inflammation and, via these associations, indirectly associated with both reduced linear and ponderal growth. Evidence for the association with reduced linear growth was stronger for systemic inflammation than for gut inflammation; the opposite was true of reduced ponderal growth. Although Giardia was associated with reduced growth, the association was not mediated by any of the biomarkers evaluated. Interpretation: The large quantity of empirical evidence contributing to this analysis supports the conceptual model of EE. The effects of EE on growth faltering in young children were small, but multiple mechanistic pathways underlying the attribution of growth failure to asymptomatic enteric infections had statistical support in the analysis. The strongest evidence for EE was the association between enteropathogens and linear growth mediated through systemic inflammation. Funding: Bill & Melinda Gates Foundation.

Critical illness from tuberculosis (TB) bloodstream infection results in a high case fatality rate for people living with human immunodeficiency virus (HIV). Critical illness can lead to altered pharmacokinetics and suboptimal drug exposures. We enrolled adults living with HIV and hospitalized with sepsis, with and without meningitis, in Mbarara, Uganda that were starting first-line anti-TB therapy. Serum was collected two weeks after enrollment at 1-, 2-, 4-, and 6-h post-dose and drug concentrations quantified by validated LC-MS/MS methods. Non-compartmental analyses were used to determine total drug exposure, and population pharmacokinetic modeling and simulations were performed to determine optimal dosages. Eighty-one participants were enrolled. Forty-nine completed pharmacokinetic testing: 18 (22%) died prior to testing, 13 (16%) were lost to follow-up and one had incomplete testing. Isoniazid had the lowest serum attainment, with only 4.1% achieving a target exposure over 24 h (AUC0–24) of 52 mg·h/L despite appropriate weight-based dosing. Simulations to reach target AUC0–24 found necessary doses of rifampin of 1800 mg, pyrazinamide of 2500–3000 mg, and for isoniazid 900 mg or higher. Given the high case fatality ratio of TB-related critical illness in this population, an early higher dose anti-TB therapy should be trialed.

Antimicrobial resistance (AMR) is an emerging public health problem and methods for surveillance are needed. We designed 85 sequence-specific PCR reactions to detect 79 genes or mutations associated with resistance across 10 major antimicrobial classes, with a focus on E. coli. The 85 qPCR assays demonstrated >99.9% concordance with sequencing. We evaluated the correlation between genotypic resistance markers and phenotypic susceptibility results on 239 E. coli isolates. Both sensitivity and specificity exceeded 90% for ampicillin, ceftriaxone, cefepime, imipenem, ciprofloxacin, azithromycin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, tetracycline, and chloramphenicol phenotypic susceptibility results. We then evaluated the assays on direct stool specimens and observed a sensitivity of 97% ± 5 but, as expected, a lower specificity of 75% ± 31 versus the genotype of the E. coli cultured from stool. Finally, the assays were incorporated into a convenient TaqMan Array Card (TAC) format. These assays may be useful for tracking AMR in E. coli isolates or directly in stool for targeted testing of the fecal antibiotic resistome.

Summary: Background: Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. Methods: We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0–2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics. Findings: We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6–71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8–38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6–39·5) was more common than bacterial (25·0%, 23·4–28·4) and parasitic diarrhoea (3·5%, 3·0–5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8–29·9), sapovirus (22·8, 18·9–27·5), rotavirus (20·7, 18·8–23·0), adenovirus 40/41 (19·0, 16·8–23·0), enterotoxigenic Escherichia coli (18·8, 16·5–23·8), norovirus (15·4, 13·5–20·1), astrovirus (15·0, 12·0–19·5), Campylobacter jejuni or C coli (12·1, 8·5–17·2), Cryptosporidium (5·8, 4·3–8·3), and typical enteropathogenic E coli (5·4, 2·8–9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7–54·1], specificity 84·0% [83·0–84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1–17·3], specificity 96·5% [96·0–97·0]). Interpretation: Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings. Funding: Bill & Melinda Gates Foundation.

Summary: Background: Enteropathogen infections in early childhood not only cause diarrhoea but contribute to poor growth. We used molecular diagnostics to assess whether particular enteropathogens were associated with linear growth across seven low-resource settings. Methods: We used quantitative PCR to detect 29 enteropathogens in diarrhoeal and non-diarrhoeal stools collected from children in the first 2 years of life obtained during the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) multisite cohort study. Length was measured monthly. We estimated associations between aetiology-specific diarrhoea and subclinical enteropathogen infection and quantity and attained length in 3 month intervals, at age 2 and 5 years, and used a longitudinal model to account for temporality and time-dependent confounding. Findings: Among 1469 children who completed 2 year follow-up, 35 622 stool samples were tested and yielded valid results. Diarrhoeal episodes attributed to bacteria and parasites, but not viruses, were associated with small decreases in length after 3 months and at age 2 years. Substantial decrements in length at 2 years were associated with subclinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction −0·14, 95% CI −0·27 to −0·01), enteroaggregative Escherichia coli (−0·21, −0·37 to −0·05), Campylobacter (−0·17, −0·32 to −0·01), and Giardia (−0·17, −0·30 to −0·05). Norovirus, Cryptosporidium, typical enteropathogenic E coli, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigella and E bieneusi were associated with the largest decreases in LAZ per log increase in quantity per g of stool (−0·13 LAZ, 95% CI −0·22 to −0·03 for Shigella; −0·14, −0·26 to −0·02 for E bieneusi). Based on these models, interventions that successfully decrease exposure to Shigella, enteroaggregative E coli, Campylobacter, and Giardia could increase mean length of children by 0·12–0·37 LAZ (0·4–1·2 cm) at the MAL-ED sites. Interpretation: Subclinical infection and quantity of pathogens, particularly Shigella, enteroaggregative E coli, Campylobacter, and Giardia, had a substantial negative association with linear growth, which was sustained during the first 2 years of life, and in some cases, to 5 years. Successfully reducing exposure to certain pathogens might reduce global stunting. Funding: Bill & Melinda Gates Foundation.

[This corrects the article DOI: 10.1371/journal.pone.0176522.].

Amplicon-based Next Generation Sequencing (NGS) is an emerging method for Mycobacterium tuberculosis drug susceptibility testing (DST) but has not been well described. We examined 158 clinical multidrug-resistant M. tuberculosis isolates via NGS of 11 resistance-associated gene regions covering 3519 nucleotides. Across these gene regions, complete resistance or heteroresistance (defined as 1%-99% mutation) was present in at least one isolate in 6.3% of loci. The number of isolates with heteroresistance was highest for gyrA codon 94, rpoB codons 526 and 531, and embB codons 306, 372 and 406 (range 11-26% of isolates exhibited heteroresistance). 57% of MDR strains had heteroresistance of one or more recognized resistance-associated mutation. Heteroresistant loci generally exhibited high or low degrees of mutation (>90% or

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.

BACKGROUND:We aimed to evaluate the antibody in lymphocyte supernatant (ALS) assay as a biomarker to diagnose tuberculosis among adults from Tanzania with and without HIV. METHODS:Adults admitted with suspicion for tuberculosis had sputa obtained for GeneXpert MTB/RIF, acid-fast bacilli smear and mycobacterial culture; blood was obtained prior to treatment initiation and after 4 weeks. Adults hospitalized with non-infectious conditions served as controls. Peripheral blood mononuclear cells were cultured unstimulated for 72 hours. Anti-mycobacterial antibodies were measured from culture supernatants by ELISA, using BCG vaccine as the coating antigen. Median ALS responses were compared between cases and controls at baseline and between cases over time. RESULTS:Of 97 TB cases, 85 were microbiologically confirmed and 12 were clinically diagnosed. Median ALS responses from TB cases (0.366 OD from confirmed cases and 0.285 from clinical cases) were higher compared to controls (0.085, p

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P

Background: Most studies of the causes of diarrhoea in low-income and middle-income countries have looked at severe disease in people presenting for care, and there are few estimates of pathogen-specific diarrhoea burdens in the community. Methods: We undertook a birth cohort study with not only intensive community surveillance for diarrhoea but also routine collection of non-diarrhoeal stools from eight sites in South America, Africa, and Asia. We enrolled children within 17 days of birth, and diarrhoeal episodes (defined as maternal report of three or more loose stools in 24 h, or one loose stool with visible blood) were identified through twice-weekly home visits by fieldworkers over a follow-up period of 24 months. Non-diarrhoeal stool specimens were also collected for surveillance for months 1–12, 15, 18, 21, and 24. Stools were analysed for a broad range of enteropathogens using culture, enzyme immunoassay, and PCR. We used the adjusted attributable fraction (AF) to estimate pathogen-specific burdens of diarrhoea. Findings: Between November 26, 2009, and February 25, 2014, we tested 7318 diarrhoeal and 24 310 non-diarrhoeal stools collected from 2145 children aged 0–24 months. Pathogen detection was common in non-diarrhoeal stools but was higher with diarrhoea. Norovirus GII (AF 5·2%, 95% CI 3·0–7·1), rotavirus (4·8%, 4·5–5·0), Campylobacter spp (3·5%, 0·4–6·3), astrovirus (2·7%, 2·2–3·1), and Cryptosporidium spp (2·0%, 1·3–2·6) exhibited the highest attributable burdens of diarrhoea in the first year of life. The major pathogens associated with diarrhoea in the second year of life were Campylobacter spp (7·9%, 3·1–12·1), norovirus GII (5·4%, 2·1–7·8), rotavirus (4·9%, 4·4–5·2), astrovirus (4·2%, 3·5–4·7), and Shigella spp (4·0%, 3·6–4·3). Rotavirus had the highest AF for sites without rotavirus vaccination and the fifth highest AF for sites with the vaccination. There was substantial variation in pathogens according to geography, diarrhoea severity, and season. Bloody diarrhoea was primarily associated with Campylobacter spp and Shigella spp, fever and vomiting with rotavirus, and vomiting with norovirus GII. Interpretation: There was substantial heterogeneity in pathogen-specific burdens of diarrhoea, with important determinants including age, geography, season, rotavirus vaccine usage, and symptoms. These findings suggest that although single-pathogen strategies have an important role in the reduction of the burden of severe diarrhoeal disease, the effect of such interventions on total diarrhoeal incidence at the community level might be limited. Funding: Bill & Melinda Gates Foundation.

ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. IMPORTANCE Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.

Multidrug-resistant tuberculosis (MDR-TB) treatment in Bangladesh is empiric or based on qualitative drug-susceptibility testing (DST) by comparative growth in culture media with and without a single drug concentration.Adult patients were enrolled throughout Bangladesh during the period of 2011-2013 at MDR-TB treatment initiation. Quantitative DST by minimum inhibitory concentration (MIC) testing for 12 first and second-line anti-TB drugs was compared to pretreatment clinical characteristics and treatment outcomes. MIC values at or one dilution lower than the resistance breakpoint used for qualitative DST were categorized as borderline susceptible, and MIC values one or two dilutions greater as borderline resistant.Seventy-four patients were enrolled with a mean age of 35 ± 15 years, and 51 (69%) were men. Of the rifampin isolates with MIC >1.0 μg/ml, 12 (19%) were fully susceptible or borderline susceptible to rifabutin (MIC ≤ 0.5 μg/ml). Amikacin was fully susceptible in 73 isolates (99%), but kanamycin in only 54 (75%) (p

Individual pharmacokinetic variability may be common in patients treated for multidrug-resistant tuberculosis (MDR-TB) but data are sparse from resource-limited settings and across the early treatment interval.Plasma drug activity, as measured by the TB Drug Activity (TDA) assay at 2 and 4 weeks of treatment with a standardized MDR-TB regimen was performed in patients with pulmonary MDR-TB from Tanzania. TDA values were correlated with measures of early treatment outcome including every two week collection of sputum for time-to-positivity (TTP) in liquid culture from the MGIT 960 automated system. Patients were evaluated at 24 weeks and those surviving without delayed sputum culture conversion (>8 weeks), culture reversion after previously negative, or weight loss were defined as having a favorable outcome.Twenty-five patients were enrolled with a mean age of 37 ±12 years. All were culture positive from the pretreatment sputum sample with a mean TTP in MGIT of 257 ±134 hours, and the median time to culture conversion on treatment was 6 weeks. Twenty patients (80%) had an increase in TDA, with the overall mean TDA at 2 weeks of 2.1 ±0.7 compared to 2.4 ±0.8 at 4 weeks (p = 0.005). At 2 weeks 13 subjects (52%) had a TDA value > 2-log killing against their own M. tuberculosis isolate compared to 17 subjects (68%) at 4 weeks (McNemar's exact test p = 0.29). An interim treatment outcome was able to be determined in 23 patients (92%), of whom 7 had a poor outcome (30%). An increase in TDA from week 2 to week 4 was associated with favorable outcome, [unadjusted OR = 20.0, 95% CI: 1.61-247.98, exact p = 0.017 and adjusted OR = 19.33, 95% CI: 1.55-241.5, exact p = 0.023].The majority of patients with MDR-TB in Tanzania had an increase in plasma drug activity from week 2 to week 4 of treatment as measured by the TDA assay. Understanding the etiology and full impact of this dynamic may inform therapeutic intervention.

SettingKibong'oto National Tuberculosis Hospital (KNTH), Kilimanjaro, Tanzania.ObjectiveCharacterize the diagnostic process and interim treatment outcomes from patients treated for multidrug-resistant tuberculosis (MDR-TB) in Tanzania.DesignA retrospective cohort study was performed among all patients treated at KNTH for pulmonary MDR-TB between November 2009 and September 2011.ResultsSixty-one culture-positive MDR-TB patients initiated therapy, 60 (98%) with a prior history of TB treatment. Forty-one (67%) were male and 9 (14%) were HIV infected with a mean CD4 count of 424 (±106) cells/µl. The median time from specimen collection to MDR-TB diagnosis and from diagnosis to initiation of MDR-TB treatment was 138 days (IQR 101-159) and 131 days (IQR 32-233), respectively. Following treatment initiation four (7%) patients died (all HIV negative), 3 (5%) defaulted, and the remaining 54 (89%) completed the intensive phase. Most adverse drug reactions were mild to moderate and did not require discontinuation of treatment. Median time to culture conversion was 2 months (IQR 1-3) and did not vary by HIV status. In 28 isolates available for additional second-line drug susceptibility testing, fluoroquinolone, aminoglycoside and para-aminosalicylic acid resistance was rare yet ethionamide resistance was present in 9 (32%).ConclusionThe majority of MDR-TB patients from this cohort had survived a prolonged referral process, had multiple episodes of prior TB treatment, but did not have advanced AIDS and converted to culture negative early while completing an intensive inpatient regimen without serious adverse event. Further study is required to determine the clinical impact of second-line drug susceptibility testing and the feasibility of alternatives to prolonged hospitalization.