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Tuberculosis diagnosis might be delayed or missed in children with severe pneumonia because this diagnosis is usually only considered in cases of prolonged symptoms or antibiotic failure. Systematic tuberculosis detection at hospital admission could increase case detection and reduce mortality.We did a stepped-wedge cluster-randomised trial in 16 hospitals from six countries (Cambodia, Cameroon, Côte d'Ivoire, Mozambique, Uganda, and Zambia) with high incidence of tuberculosis. Children younger than 5 years with WHO-defined severe pneumonia received either the standard of care (control group) or standard of care plus Xpert MTB/RIF Ultra (Xpert Ultra; Cepheid, Sunnyvale, CA, USA) on nasopharyngeal aspirate and stool samples (intervention group). Clusters (hospitals) were progressively switched from control to intervention at 5-week intervals, using a computer-generated random sequence, stratified on incidence rate of tuberculosis at country level, and masked to teams until 5 weeks before switch. We assessed the effect of the intervention on primary (12-week all-cause mortality) and secondary (including tuberculosis diagnosis) outcomes, using generalised linear mixed models. The primary analysis was by intention to treat. We described outcomes in children with severe acute malnutrition in a post hoc analysis. This study is registered with ClinicalTrials.gov (NCT03831906) and the Pan African Clinical Trial Registry (PACTR202101615120643).From March 21, 2019, to March 30, 2021, we enrolled 1401 children in the control group and 1169 children in the intervention group. In the intervention group, 1140 (97·5%) children had nasopharyngeal aspirates and 942 (80·6%) had their stool collected; 24 (2·1%) had positive Xpert Ultra. At 12 weeks, 110 (7·9%) children in the control group and 91 (7·8%) children in the intervention group had died (adjusted odds ratio [OR] 0·986, 95% CI 0·597-1·630, p=0·957), and 74 (5·3%) children in the control group and 88 (7·5%) children in the intervention group had tuberculosis diagnosed (adjusted OR 1·238, 95% CI 0·696-2·202, p=0·467). In children with severe acute malnutrition, 57 (23·8%) of 240 children in the control group and 53 (17·8%) of 297 children in the intervention group died, and 36 (15·0%) of 240 children in the control group and 56 (18·9%) of 297 children in the intervention group were diagnosed with tuberculosis. The main adverse events associated with nasopharyngeal aspirates were samples with blood in 312 (27·3%) of 1147 children with nasopharyngeal aspirates attempted, dyspnoea or SpOSystematic molecular tuberculosis detection at hospital admission did not reduce mortality in children with severe pneumonia. High treatment and microbiological confirmation rates support more systematic use of Xpert Ultra in this group, notably in children with severe acute malnutrition.Unitaid and L'Initiative.For the French translation of the abstract see Supplementary Materials section.

BACKGROUND AND OBJECTIVES Children experience high tuberculosis (TB)-related mortality but causes of death among those with presumptive TB are poorly documented. We describe the mortality, likely causes of death, and associated risk factors among vulnerable children admitted with presumptive TB in rural Uganda. METHODS We conducted a prospective study of vulnerable children, defined as RESULTS Of the 219 children included, 157 (71.7%) were CONCLUSIONS Vulnerable children hospitalized with presumptive TB experienced high mortality. A better understanding of the likely causes of death in this group is important to guide empirical management.

We regret that this article is behind a paywall., Background: High-dose rifampicin is considered to shorten anti-TB treatment duration but its effect on antiretroviral metabolism is unknown. Objectives: To assess the effect of doubling the rifampicin dose (to 20 mg/kg/day, R20) on efavirenz pharmacokinetics (PK) in HIV/TB coinfected patients. Methods: Open-label Phase 2 drug-drug interaction randomized trial. Pulmonary TB, ART-naive adults were randomized to R20 and either efavirenz 600 mg (EFV600) or 800 mg (EFV800), or rifampicin 10 mg/kg/day (R10) and EFV600 with a 1:1:1 ratio. Patients were first started on TB treatment and 2-4 weeks later started on ART. They were switched to R10 and EFV600 after 8 weeks. Full PK sampling was done 4 weeks (on rifampicin) and 24 weeks (off rifampicin) after ART initiation. Transaminases, plasma HIV-1 RNA and sputum cultures were monitored. The efavirenz geometric mean ratio (GMR) of AUC at 4 and 24 weeks after ART initiation within the same patient was calculated in each arm and its 90% CI was compared with a preset range (0.70-1.43). Results: Of 98 enrolled patients (32 in the R20EFV600 arm, 33 in the R20EFV800 arm and 33 in the R10EFV600 arm), 87 had full PK sampling. For the R20EFV600, R20EFV800 and R10EFV600 arms, GMRs of efavirenz AUC were 0.87 (90% CI: 0.75-1.00), 1.12 (90% CI: 0.96-1.30) and 0.96 (90% CI: 0.84-1.10). Twelve weeks after ART initiation, 78.6%, 77.4% and 72.4% of patients had HIV-1 RNA below 100 copies/mL and 85.7%, 86.7% and 80.0% had Week 8 culture conversion, respectively. Two patients per arm experienced a severe increase in transaminases. Conclusions: Doubling the rifampicin dose had a small effect on efavirenz concentrations and was well tolerated.

Background Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries. Methods Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda. Results The sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10− 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2). Conclusions The HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.

We enrolled 250 febrile children in western Uganda to compare the results of malaria rapid diagnostic tests (RDTs) when using capillary vs venous blood. Participants were tested with 4 different RDT types. Polymerase chain reaction testing was performed as the reference standard. Sensitivity and specificity were broadly similar across RDT types and sampling method. Agreement between sample type was high, ranging from 0.95 to 0.99. When following the manufacturer’s recommended interpretation, only 5 tests would have resulted in a different clinical diagnosis. These results demonstrate that malaria RDTs perform similarly when using capillary or venous blood in febrile children with Plasmodium falciparum malaria.

BackgroundYellow fever vaccine stocks have been insufficient to cover exceptional demands for outbreak response. Fractional dosing evidence is limited to the 17DD substrain vaccine. We assessed the immunogenicity and safety of one-fifth fractional dose compared to standard dose of each of the four WHO-prequalified yellow fever vaccines produced from three substrains.MethodsWe conducted a randomized, double-blind, non-inferiority trial in Mbarara, Uganda and Kilifi, Kenya. 960 participants aged 18-59 years without previous yellow fever vaccination or infection were recruited from communities and randomized to receive one of four vaccines and to standard or fractional dosage. Vaccine was administered subcutaneously by unblinded nurse. Other study personnel and participants were blinded to vaccine allocation. Primary immunogenicity outcome, seroconversion, was measured in the per-protocol population; safety outcomes included all vaccinated participants. We defined non-inferiority as no more than 10% decrease in seroconversion in fractional compared to standard dose arms 28 days post-vaccination. Seroconversion was defined as ≥4-fold rise in neutralizing antibody titers measured by 50% plaque reduction neutralization test (PRNT50).ClinicalTrials.gov Identifier: NCT02991495, following participants.FindingsBetween 6th November 2017 and 21st February 2018, 960 participants, total sample goal, were randomized. The primary per-protocol analysis includes 899 participants, with 110 to 117 participants per arm. The absolute difference in seroconversion between fractional and standard doses by vaccine was 1.71% (95%CI: −2.60, 5.28), −0.90% (95%CI: −4.24, 3.13), 1.82% (95%CI: −2.75, 5.39), 0.0% (95%CI: −3.32, 3.29). Fractional doses from all four vaccines met the non-inferiority criterion. There were no safety concerns.InterpretationThese results support fractional dosing of all WHO-prequalified yellow fever vaccines for the general adult population for outbreak response in situations of vaccine shortage.FundingThe study was funded by Médecins Sans Frontières Foundation, Wellcome Trust (grant no. 092654) and the UK Department for International Development. Vaccines were donated in kind.Research in contextEvidence before this studyIn July 2016, following major yellow fever (YF) outbreaks in two countries, WHO published a secretariat information paper including a review of studies assessing the immunogenicity of fractional doses of YF vaccines and recommended consideration of fractional doses to manage a vaccine shortage. Following this, fractional doses of YF vaccine produced by Bio-Manguinhos/Fiocruz (17DD substrain) were given to approximately 7.5 million non-pregnant adults and children ≥ 2 years of age in Kinshasa, Democratic Republic of Congo. The evidence base to support this action was limited to a single vaccine substrain and to a specific context. To broaden and simplify recommendations, WHO called for additional research to be conducted. We designed a trial to assess non-inferiority in seroconversion of fractional (one-fifth dose) versus standard dose for each of the four WHO-prequalified YF vaccines at 28 days post-vaccination in an adult population in Kenya and Uganda. We selected vaccine batches as close as possible to each manufacturer’s minimum release specification.Added value of this studyThis is the first randomized controlled trial assessing all four WHO-prequalified YF vaccines, providing information on the immunogenicity and safety of fractional doses of the different vaccine substrains at 10 days, 28 days and one year post-vaccination. The results show that, at 28 days post-vaccination, most participants had high levels of neutralizing antibodies and that seroconversion rates in the fractional dose arms were non-inferior to standard dose for each of the four vaccines. Seroconversion rates and neutralizing antibodies remained high up to one year post-vaccination for both fractional and standard doses for all vaccines. These results are aligned with previous studies using the 17DD substrain vaccine but extend the evidence to randomized comparisons of all four vaccines and to a sub-Saharan Africa context. To our knowledge, this is the first trial assessing immunogenicity of fractional doses at 10 days post-vaccination.Implications of all the available evidenceOur study supports the use of one-fifth fractional doses of all four WHO-prequalified yellow fever vaccines for the general adult population and fills a critical knowledge gap to support WHO policy on the use of fractional dosing of yellow fever vaccine for outbreak response. The immunogenicity and safety of fractional dosing in children and specific populations, such as those living with HIV, is yet to be determined. Long-term studies are warranted to confirm the duration of protection.

BACKGROUND A timely differential diagnostic is essential to identify the etiology of central nervous system (CNS) infections in children, in order to facilitate targeted treatment, manage patients, and improve clinical outcome. OBJECTIVE The Pediatric Infection-Point-of-Care (PI-POC) trial is investigating novel methods to improve and strengthen the differential diagnostics of suspected childhood CNS infections in low-income health systems such as those in Southwestern Uganda. This will be achieved by evaluating (1) a novel DNA-based diagnostic assay for CNS infections, (2) a commercially available multiplex PCR-based meningitis/encephalitis (ME) panel for clinical use in a facility-limited laboratory setting, (3) proteomics profiling of blood from children with severe CNS infection as compared to outpatient controls with fever yet not severely ill, and (4) Myxovirus resistance protein A (MxA) as a biomarker in blood for viral CNS infection. Further changes in the etiology of childhood CNS infections after the introduction of the pneumococcal conjugate vaccine against Streptococcus pneumoniae will be investigated. In addition, the carriage and invasive rate of Neisseria meningitidis will be recorded and serotyped, and the expression of its major virulence factor (polysaccharide capsule) will be investigated. METHODS The PI-POC trial is a prospective observational study of children including newborns up to 12 years of age with clinical features of CNS infection, and age-/sex-matched outpatient controls with fever yet not severely ill. Participants are recruited at 2 Pediatric clinics in Mbarara, Uganda. Cerebrospinal fluid (for cases only), blood, and nasopharyngeal (NP) swabs (for both cases and controls) sampled at both clinics are analyzed at the Epicentre Research Laboratory through gold-standard methods for CNS infection diagnosis (microscopy, biochemistry, and culture) and a commercially available ME panel for multiplex PCR analyses of the cerebrospinal fluid. An additional blood sample from cases is collected on day 3 after admission. After initial clinical analyses in Mbarara, samples will be transported to Stockholm, Sweden for (1) validation analyses of a novel nucleic acid–based POC test, (2) biomarker research, and (3) serotyping and molecular characterization of S. pneumoniae and N. meningitidis. RESULTS A pilot study was performed from January to April 2019. The PI-POC trial enrollment of patients begun in April 2019 and will continue until September 2020, to include up to 300 cases and controls. Preliminary results from the PI-POC study are expected by the end of 2020. CONCLUSIONS The findings from the PI-POC study can potentially facilitate rapid etiological diagnosis of CNS infections in low-resource settings and allow for novel methods for determination of the severity of CNS infection in such environment. CLINICALTRIAL ClinicalTrials.gov NCT03900091; https://clinicaltrials.gov/ct2/show/NCT03900091 INTERNATIONAL REGISTERED REPORT DERR1-10.2196/21430

Introduction. The proportion of women with severe maternal morbidity from obstructed labor is between 2 and 12% in resource-limited settings. Maternal vaginal colonization with group B streptococcus (GBS), Escherichia coli, and Enterococcus spp. is associated with maternal and neonatal morbidity. It is unknown if vaginal colonization with these organisms in obstructed labor women is associated with poor outcomes. Objectives. To determine whether vaginal colonization with GBS, E. coli, or Enterococcus is associated with increased morbidity among women with obstructed labor and to determine the risk factors for colonization and antibiotic susceptibility patterns. Methods. We screened all women presenting in labor to Uganda’s Mbarara Regional Referral Hospital maternity ward from April to October 2015 for obstructed labor. Those meeting criteria had vaginal swabs collected prior to Cesarean delivery and surgical antibiotic prophylaxis. Swabs were inoculated onto sterile media for routine bacterial culture and antimicrobial susceptibility testing. Results. Overall, 2,168 women were screened and 276 (13%) women met criteria for obstructed labor. Vaginal swabs were collected from 272 women (99%), and 170 (64%) were colonized with a potential pathogen: 49% with E. coli, 5% with GBS, and 8% with Enterococcus. There was no difference in maternal and fetal clinical outcomes between those colonized and not colonized. The number of hours in labor was a significant independent risk factor for vaginal colonization (aOR 1.02, 95% CI 1.00–1.03, P=0.04). Overall, 38% of GBS was resistant to penicillin; 61% of E. coli was resistant to ampicillin, 4% to gentamicin, and 5% to ceftriaxone and cefepime. All enterococci were ampicillin and vancomycin susceptible. Conclusion. There was no difference in maternal or neonatal morbidity between women with vaginal colonization with E. coli, GBS, and Enterococcus and those who were not colonized. Duration of labor was associated with increased risk of vaginal colonization in women with obstructed labor.

Summary Background WHO recommends Xpert MTB/RIF as initial diagnostic testing for tuberculous meningitis. However, diagnosis remains difficult, with Xpert sensitivity of about 50–70% and culture sensitivity of about 60%. We evaluated the diagnostic performance of the new Xpert MTB/RIF Ultra (Xpert Ultra) for tuberculous meningitis. Methods We prospectively obtained diagnostic cerebrospinal fluid (CSF) specimens during screening for a trial on the treatment of HIV-associated cryptococcal meningitis in Mbarara, Uganda. HIV-infected adults with suspected meningitis (eg, headache, nuchal rigidity, altered mental status) were screened consecutively at Mbarara Regional Referral Hospital. We centrifuged CSF, resuspended the pellet in 2 mL of CSF, and tested 0·5 mL with mycobacteria growth indicator tube culture, 1 mL with Xpert, and cryopreserved 0·5 mL, later tested with Xpert Ultra. We assessed diagnostic performance against uniform clinical case definition or a composite reference standard of any positive CSF tuberculous test. Findings From Feb 27, 2015, to Nov 7, 2016, we prospectively evaluated 129 HIV-infected adults with suspected meningitis for tuberculosis. 23 participants were classified as probable or definite tuberculous meningitis by uniform case definition, excluding Xpert Ultra results. Xpert Ultra sensitivity was 70% (95% CI 47–87; 16 of 23 cases) for probable or definite tuberculous meningitis compared with 43% (23–66; 10/23) for Xpert and 43% (23–66; 10/23) for culture. With composite standard, we detected tuberculous meningitis in 22 (17%) of 129 participants. Xpert Ultra had 95% sensitivity (95% CI 77–99; 21 of 22 cases) for tuberculous meningitis, which was higher than either Xpert (45% [24–68]; 10/22; p=0·0010) or culture (45% [24–68]; 10/22; p=0·0034). Of 21 participants positive by Xpert Ultra, 13 were positive by culture, Xpert, or both, and eight were only positive by Xpert Ultra. Of those eight, three were categorised as probable tuberculous meningitis, three as possible tuberculous meningitis, and two as not tuberculous meningitis. Testing 6 mL or more of CSF was associated with more frequent detection of tuberculosis than with less than 6 mL (26% vs 7%; p=0·014). Interpretation Xpert Ultra detected significantly more tuberculous meningitis than did either Xpert or culture. WHO now recommends the use of Xpert Ultra as the initial diagnostic test for suspected tuberculous meningitis. Funding National Institute of Neurologic Diseases and Stroke, Fogarty International Center, National Institute of Allergy and Infectious Disease, UK Medical Research Council/DfID/Wellcome Trust Global Health Trials, Doris Duke Charitable Foundation.

Setting In early studies, Xpert® MTB/RIF accurately detected culture-proven pulmonary tuberculosis (TB). Recent reports have, however, found a lower than expected specificity in previously treated TB patients. Objective To investigate the diagnostic accuracy of Xpert in presumptive pulmonary TB patients in Southwestern Uganda. Design We obtained demographic and clinical information and collected three sputum samples from each patient for smear microscopy, Xpert and culture. We estimated Xpert sensitivity and specificity against culture, and stratified the analysis by previous treatment and sputum quality status. Results We analyzed results from 860 presumptive TB patients, including 109 (13%) with a previous history of anti-tuberculosis treatment; 205 (24%) were culture-positive. Xpert specificity was lower (91.8%, 95%CI 84.9-96.2) in previously treated than in new TB patients (97.5%, 95%CI 96.1-98.5; P = 0.01). In an adjusted analysis, patients with culture-, Xpert+ results were more likely to have been previously treated for TB (OR 8.3, 95%CI 2.1-32.0; P = 0.002), and to have mucosalivary sputum (OR 4.1, 95%CI 1.1-14.6; P = 0.03), but were less likely to self-report fever (OR 0.23, 95%CI 0.1-0.7; P = 0.008) than patients with concordant positive results. Conclusion Xpert specificity was lower in previously treated patients with suspected TB. The clinical and programmatic impact of culture-, Xpert+ results requires evaluation in future studies.

Xpert MTB/RIF (Xpert) and culture are the most reliable methods for tuberculosis diagnosis but are still poorly accessible in many low-resource countries. We aimed to assess the effects of OMNIgene Sputum (OM-S) and ethanol in preserving sputum for Xpert and OM-S for mycobacterial growth indicator tube (MGIT) testing over periods of 15 and 8 days, respectively. Sputum samples were collected from newly diagnosed smear-positive patients. For Xpert, pooled samples were split into 5 aliquots: 3 for Xpert on days 0, 7, and 15 without additive and 2 with either OM-S or ethanol at day 15. For MGIT, 2 aliquots were tested without preservative and 2 with OM-S at 0 and 8 days. Totals of 48 and 47 samples were included in the analysis for Xpert and culture. With Xpert, using day 0 as a reference, untreated samples stored for 7 and 15 days showed concordances of 45/46 (97.8%) and 46/48 (95.8%). For samples preserved with OM-S or ethanol for 15 days compared with untreated samples processed at day 0 or after 15 days, OM-S concordances were 46/48 (95.8%) and 47/48 (97.9%), while those of ethanol were 44/48 (91.7%) and 45/48 (93.8%). With MGIT, concordances between untreated and OM-S-treated samples were 21/41 (51.2%) at day 0 and 21/44 (47.7%) at day 8. In conclusion, Xpert equally detected tuberculosis in OM-S-treated and untreated samples up to 15 days but showed slightly lower detection in ethanol-treated samples. Among OM-S-treated samples, MGIT positivity was significantly lower than in untreated samples at both time points.

Improved laboratory diagnosis is critical to reduce the burden of malaria in pregnancy. Peripheral blood smears appear less sensitive than Plasmodium falciparum histidine-rich protein 2–based rapid diagnostic tests (RDTs) for placental malaria infections in studies conducted at delivery. In this study, 81 women in Uganda in the second or third trimester of pregnancy were followed-up until delivery. At each visit, peripheral blood was tested by blood smear, RDT, and nested species-specific polymerase chain reaction (PCR). Sensitivity and specificity of the tests was calculated with PCR, which detected 22 infections of P. falciparum, as the gold standard. The sensitivity and specificity of blood smears were 36.4% (95% confidence interval [CI] = 18.0–59.2%) and 99.6% (95% CI = 97.7–100%), respectively. The corresponding values for RDT were 31.8% (95% CI = 14.7–54.9%) and 100% (95% CI = 98.3–100%). The RDTs could replace blood smears for diagnosis of malaria in pregnancy by virtue of their relative ease of use. Field-based sensitive tests for malaria in pregnancy are urgently needed.

Setting Although it is now widely used for tuberculosis (TB) diagnosis, Xpert(®) MTB/RIF availability remains inadequate in low-resource settings. Moreover, its accuracy in testing stored samples from non-expectorating patients has not been evaluated. Objective To assess the performance of Xpert in frozen samples of induced sputum (IS) and sputum from string test (ST) from non-expectorating individuals with presumed TB. Design This was a laboratory-based study of 377 ST and IS samples collected between March 2010 and March 2013 at a referral hospital in Uganda. Samples were decontaminated, centrifuged and cultured, and the resultant samples were frozen at -20°C before Xpert evaluation. Results TB was detected in ST and IS samples from 19/163 (11.7%) children and 63/201 (29.4%) adults using culture. Xpert sensitivity in frozen sediments from children was 37.5% (95%CI 8.5-75.5) in ST and 41.7% (95%CI 15.2-72.3) in IS samples, with specificities of respectively 100% (95%CI 94.9-100) and 98.6% (95%CI 92.7-100). In adults, sensitivity was respectively 50% (95%CI 31.3-68.7) and 48.5% (95%CI 30.8-66.4) in ST and IS samples, with specificities of respectively 100% (95%CI 95.5-100) and 98.6% (95%CI 92.4-100). Conclusion Given these results, and particularly the high specificity, the use of Xpert on frozen ST and IS sediment samples from both children and adults is promising.

Sputum acid-fast bacilli (AFB) smear microscopy has suboptimal sensitivity but remains the most commonly used laboratory test to diagnose pulmonary tuberculosis (TB). We prospectively evaluated the small membrane filtration (SMF) method that concentrates AFB in a smaller area to facilitate detection to improve the diagnostic performance of microscopy. We enrolled adults with suspicion of pulmonary TB from health facilities in southwestern Uganda. Clinical history, physical examination, and 3 sputum samples were obtained for direct fluorescent AFB smear, SMF, Xpert MTB/RIF, and MGIT culture media. Sensitivity and specificity were estimated for SMF, AFB smear, and Xpert MTB/RIF, using MGIT as the reference standard. The analysis was stratified according to HIV status. From September 2012 to April 2014, 737 participants were included in the HIV-infected stratum (146 [20.5%] were culture positive) and 313 were in the HIV-uninfected stratum (85 [28%] were culture positive). In HIV-infected patients, the sensitivity of a single SMF was 67.4% (95% confidence interval [CI], 59.9% to 74.1%); for AFB, 68.0% (95% CI, 60.6% to 74.6%); and for Xpert MTB/RIF, 91.0% (95% CI, 85.0% to 94.8%). In HIV-uninfected patients, the corresponding sensitivities were 72.5% (95% CI, 62.1% to 80.9%), 80.3% (95% CI, 70.8% to 87.2%), and 93.5% (95% CI, 85.7% to 97.2%). The specificity for all 3 tests in both HIV groups was ≥96%. In this setting, the SMF method did not improve the diagnostic accuracy of sputum AFB. The Xpert MTB/RIF assay performed well in both HIV-infected and -uninfected groups.

Human brucellosis, a chronic disease contracted through contact with animals and consuption of unpasteurized dairy products is underreported in limited-resource countries. This cross-sectional study aimed to determine the prevalence and risk factors of brucellosis among febrile patients attending a community hospital in South western Uganda. A questionnaire that captured socio-demographic, occupational and clinical data was administered. Blood samples were tested for Brucella antibodies using Rose Bengal Plate Test (RBPT) and blood culture with standard aerobic BACTEC bottle was done. Of 235 patients enrolled, prevalence of brucellosis (RBPT or culture confirmed) was 14.9% (95% CI 10.6–20.1) with a culture confrmation in 4.3% of the participants. The factors independently associated with brucellosis were consumption of raw milk (aOR 406.15, 95% CI 47.67–3461.69); history of brucellosis in the family (aOR 9.19, 95% CI 1.98–42.54); and selling hides and skins (aOR 162.56, 95% CI 2.86–9256.31). Hepatomegaly (p

The Xpert MTB/RIF assay is a major advance for diagnosis of tuberculosis (TB) in high-burden countries but is limited in children by their difficulty to produce sputum. We investigated TB in sputum and stool from children with the aim of improving paediatric TB diagnosis. A prospective cohort of children with presumptive TB, provided two sputum or induced sputum at enrolment in a regional referral hospital in Uganda. Stool was collected from those started on TB treatment. All specimen were tested for Xpert MTB/RIF, mycobacteria growth indicator tube (MGIT), Lowenstein Jensen cultures and microscopy (except stool). We compared TB detection between age categories and assessed the performance of Xpert MTB/RIF in sputum and stool. Of the 392 children enrolled, 357 (91.1%) produced at least one sputum sample. Sputum culture yield was 13/357 (3.6%): 3/109 (2.6%), 3/89 (3.2%), 3/101 (2.6%) and 4/44 (8.2%) among children of 2, 2-5, ≥ 5-10 and 10 years, respectively (p = 0.599). Xpert MTB/RIF yield was 14/350 (4.0%): 3/104 (2.9%), 4/92 (4.3%), 3/88 (2.9%) and 4/50 (.0%), respectively (p = 0.283). Sensitivity and specificity of Xpert MTB/RIF in sputum against sputum culture were 90.9% (95% CI 58.7-99.8) and 99.1% (99.1-99.8). In stool, it was 55.6% (21.2-86.3) and 98.2% (98.2-100) against Xpert MTB/RIF and culture in sputum. Only a minority of children had microbiologically confirmed TB with a higher proportion in children above 10 years. Although sensitivity of Xpert MTB/RIF in stool was low, with good optimization, it might be a good alternative to sputum in children.

Background Multi-drug resistant (MDR) Enterobacteriaceae are on the increase worldwide and their spread has become a global challenge. Escalating the challenge is the possibility that many of these are Carbapenemase-producing Enterobacteriaceae (CPE). This further complicates patient management. The magnitude of MDR-CPE in many developed settings has been reported, however, there is paucity of data from resource limited settings. We evaluated the epidemiology of MDR-CPE of clinical origin in South Western Uganda. Methods From September 2013 to June 2014, all Enterobacteriaceae isolated from diverse specimens obtained from patients attending Mbarara Regional Referral Hospital, South-western Uganda, were screened for MDR in a laboratory-based cross sectional study. Isolates found to be MDR were screened for carbapenem susceptibility/resistance phenotypically by Kirby Bauer disc diffusion method following CLSI guidelines and genetically using the multiplex real-time Polymerase Chain Reaction (RT-PCR). Results Of the 658 strains isolated, 183 (27.8%) were MDR and 68 (37.15%) of those MDR exhibited at least one form of carbapenem resistance with 23 (12.57%) and 56 (30.60%) isolates expressing phenotypic and genetic resistance, respectively. Eleven MDR-CPE (6.01%) isolates exhibited both phenotypic and genotypic resistance to carbapenems. Only blaVIM and blaOXA-48 genes were detected among the genetically resistant isolates. Conclusion The high prevalence of MDR-CPE calls for aggressive infection control and prevention strategies, including reinforcement of hand hygiene, using contact precautions and early detection of CPE through use of targeted surveillance and molecular techniques in resource limited settings.

Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for PCR testing to confirm Plasmodium infections or for epidemiological studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of Plasmodium species using real-time PCR. We extracted DNA from archived blood smears and corresponding red blood cell pellets collected from asymptomatic children in southwestern Uganda in 2010. We then performed real-time PCR followed by high-resolution melting (HRM) to identify Plasmodium species, and we compared our results to those of microscopy. We analyzed a total of 367 blood smears and corresponding red blood cell pellets, including 185 smears (50.4%) that were positive by microscopy. Compared to microscopy, PCR-HRM analysis of smear DNA had a sensitivity of 93.0% (95% confidence interval [CI], 88.2 to 96.2%) and a specificity of 96.7% (95% CI, 93.0 to 98.8%), and PCR-HRM analysis of pellet DNA had a sensitivity of 100.0% (95% CI, 98.0 to 100.0%) and a specificity of 94.0% (95% CI, 89.4 to 96.9%). Identification of positive PCR-HRM results to the species level revealed Plasmodium falciparum (92.0%), Plasmodium ovale (5.6%), and Plasmodium malariae (2.4%). PCR-HRM analysis of DNA extracts from Giemsa-stained thick blood smears or corresponding blood pellets had high sensitivity and specificity for malaria diagnosis, compared to microscopy. Therefore, blood smears can provide an adequate source of DNA for confirmation of Plasmodium species infections and can be used for retrospective genetic studies.

Infections of the central nervous system (CNS) are severe conditions, leading to neurological sequelae or death. Knowledge of the causative agents is essential to develop guidelines for case management in resource-limited settings. Between August 2009 and October 2012, we conducted a prospective descriptive study of the aetiology of suspected CNS infections in children two months to 12 years old, with fever and at least one sign of CNS involvement in Mbarara Hospital, Uganda. Children were clinically evaluated on admission and discharge, and followed-up for 6 months for neurological sequelae. Pathogens were identified from cerebrospinal fluid (CSF) and blood using microbiological and molecular methods. We enrolled 459 children. Plasmodium falciparum (36.2%) and bacteria in CSF (13.3%) or blood (3.3%) were the most detected pathogens. Viruses were found in 27 (5.9%) children. No pathogen was isolated in 207 (45.1%) children. Patterns varied by age and HIV status. Eighty-three (18.1%) children died during hospitalisation, and 23 (5.0%) during follow-up. Forty-one (13.5%) children had neurological sequelae at the last visit. While malaria remains the main aetiology in children with suspected CNS infections, no pathogen was isolated in many children. The high mortality and high rate of neurological sequelae highlight the need for efficient diagnosis.

Northern Uganda hosts a large population of refugees from South Sudan, and malaria is one of the major health problems in the area. In 2015, intermittent preventive treatment for malaria (IPTc) was implemented in two refugee camps among children aged 6 months to 14 years. Three distributions of dihydroartemisinin–piperaquine (DP) were conducted at 8-week intervals. The first dose was directly administered at IPTc distribution sites and the second and third doses were given to caregivers to administer at home. A multi-faceted evaluation was implemented, including coverage surveys, malaria prevalence surveys, reinforced surveillance, and pharmacovigilance. Programme coverage exceeded 90% during all three distributions with a total of 40,611 participants. Compared to same period during the previous year (only available data), the incidence of malaria in the target populations was reduced (IRR 0.73, 95% CI 0.69–0.77 among children under 5 years old; IRR 0.70, 95% CI 0.67–0.72 among children aged 5–14 years). Among those not targeted for intervention, the incidence between the 2 years increased (IRR 1.49, 95% CI 1.42–1.56). Cross-sectional surveys showed a prevalence of parasitaemia (microscopy or PCR) of 12.9–16.4% (95% CI 12.6–19.3) during the intervention, with the highest prevalence among children aged 5–14 years, but with a large increase 8 weeks after the final distribution. A total of 57 adverse events were reported during the intervention period, including one severe adverse event (death from varicella). Adverse events were of mild to moderate severity, and were mainly dermatologic and gastrointestinal. This is the first documentation of an IPTc programme in a refugee camp. The positive impact of DP on the incidence of malaria, together with its favourable safety profile, should lead to further use of IPTc in similar settings. Expanding coverage groups and decreasing intervals between distributions might provide more benefit, but would need to be balanced with the operational implications of a broader, more frequent distribution schedule.

Background: Information on Streptococcus pneumoniae nasopharyngeal (NP) carriage before the pneumococcal conjugate vaccine (PCV) introduction is essential to monitor impact. The 10-valent PCV (PCV10) was officially introduced throughout Ugandan national childhood immunization programs in 2013 and rolled-out countrywide during 2014. We aimed to measure the age-specific Streptococcus pneumoniae carriage and serotype distribution across all population age groups in the pre-PCV10 era in South Western Uganda. Methods: We conducted a two-stage cluster, age-stratified, cross-sectional community-based study in Sheema North sub-district between January and March 2014. One NP swab was collected and analyzed for each participant in accordance with World Health Organization guidelines. Results: NP carriage of any pneumococcal serotype was higher among children < 2years old (77%; n=387) than among participants aged ≥15years (8.5%; n=325) (chi 2 p < 0.001).Of the 623 positive cultures, we identified 49 serotypes among 610 (97.9%) isolates; thirteen (2.1%) isolates were non-typeable. Among < 2. years old, serotypes 6A, 6B, 14, 15B, 19F and 23F accounted for half of all carriers. Carriage prevalence with PCV10 serotypes was 29.4% among individuals aged < 2. years (n = 387), 23.4% in children aged 2-4. years (n = 217), 11.4% in 5-14. years (n = 417), and 0.4% among individuals ≥15. years of age (n = 325). The proportion of carried pneumococci serotypes contained in PCV10 was 38.1% (n = 291), 32.8% (n = 154), 29.4% (n = 156), and 4.4% (n = 22) among carriers aged < 2. years, 2-4. years, 5-14. years and ≥15. years, respectively. Discussion: In Sheema district, the proportion of PCV10 serotypes was low ( < 40%), across all age groups, especially among individuals aged 15. years or older ( < 5%). PCV10 introduction is likely to impact transmission among children and to older individuals, but less likely to substantially modify pneumococcal NP ecology among individuals aged 15. years or older.

Background: To confirm presence of Mycobacterium tuberculosis complex, some tuberculosis culture laboratories still rely on para-nitrobenzoic acid (PNB), a traditional technique that requires sub-culturing of clinical isolates and two to three weeks to give results. Rapid identification tests have improved turnaround times for mycobacterial culture results. Considering the challenges of the PNB method, we assessed the performance of the SD Bioline TB Ag MPT64 assay by using PNB as gold standard to detect M. tuberculosis complex from acid-fast bacilli (AFB) positive cultures. Objectives: The aim of this study was to determine the sensitivity, specificity and turnaround time of the SD MPT64 assay for identification of M. tuberculosis complex, in a setting with high prevalence of tuberculosis and HIV. Methods: A convenience sample of 690 patients, with tuberculosis symptoms, was enrolled at Epicentre Mbarara Research Centre between April 2010 and June 2011. The samples were decontaminated using NALC-NaOH and re-suspended sediments inoculated in Mycobacterium Growth Indicator Tubes (MGIT) media, then incubated at 37 °C for a maximum of eight weeks. A random sample of 50 known negative cultures and 50 non-tuberculous mycobacteria isolates were tested for specificity, while sensitivity was based on AFB positivity. The time required from positive culture to reporting of results was also assessed with PNB used as the gold standard. Results: Of the 138 cultures that were AFB-positive, the sensitivity of the SD MPT64 assay was 100.0% [95% CI: 97.3 – 100] and specificity was 100.0% (95% CI, 96.4 – 100). The median time from a specimen receipt to confirmation of strain was 10 days [IQR: 8–12] with SD MPT64 and 24 days [IQR: 22–26] with PNB. Conclusion: The SD MPT64 assay is comparable to PNB for identification of M. tuberculosis complex and reduces the time to detection.

Poor specificity may negatively impact rapid diagnostic test (RDT)-based diagnostic strategies for malaria. We performed real-time PCR on a subset of subjects who had undergone diagnostic testing with a multiple-antigen (histidine-rich protein 2 and pan -lactate dehydrogenase pLDH [HRP2/pLDH]) RDT and microscopy. We determined the sensitivity and specificity of the RDT in comparison to results of PCR for the detection of Plasmodium falciparum malaria. We developed and evaluated a two-step algorithm utilizing the multiple-antigen RDT to screen patients, followed by confirmatory microscopy for those individuals with HRP2-positive (HRP2 + )/pLDH-negative (pLDH − ) results. In total, dried blood spots (DBS) were collected from 276 individuals. There were 124 (44.9%) individuals with an HRP2 + /pLDH + result, 94 (34.1%) with an HRP2 + /pLDH − result, and 58 (21%) with a negative RDT result. The sensitivity and specificity of the RDT compared to results with real-time PCR were 99.4% (95% confidence interval [CI], 95.9 to 100.0%) and 46.7% (95% CI, 37.7 to 55.9%), respectively. Of the 94 HRP2 + /pLDH − results, only 32 (34.0%) and 35 (37.2%) were positive by microscopy and PCR, respectively. The sensitivity and specificity of the two-step algorithm compared to results with real-time PCR were 95.5% (95% CI, 90.5 to 98.0%) and 91.0% (95% CI, 84.1 to 95.2), respectively. HRP2 antigen bands demonstrated poor specificity for the diagnosis of malaria compared to that of real-time PCR in a high-transmission setting. The most likely explanation for this finding is the persistence of HRP2 antigenemia following treatment of an acute infection. The two-step diagnostic algorithm utilizing microscopy as a confirmatory test for indeterminate HRP2 + /pLDH − results showed significantly improved specificity with little loss of sensitivity in a high-transmission setting.

Background The performance of different malaria rapid diagnostic tests (RDT) may be influenced by transmission intensity and by the length of time each test requires to become negative after treatment and patient’s recovery. Methods Results of three RDTs (two HRP2 and one pLDH antigen-based tests) were compared to blood smear microscopy (the gold standard method) in children under 5 years of age living in a high versus low malaria intensity setting in southwestern Uganda. In each setting, 212 children, who tested positive by at least one RDT and by microscopy, were treated with artemether-lumefantrine. RDTs and microscopy were then repeated at fixed intervals to estimate each test’s time to negativity after treatment and patient recovery. Results In the two settings, sensitivities ranged from 98.4 to 99.2 % for the HRP2 tests and 94.7 to 96.1 % for the pLDH test. Specificities were 98.9 and 98.8 % for the HRP2 tests and 99.7 % for the pLDH test in the low-transmission setting and 79.7, 80.7 and 93.9 %, respectively, in the high-transmission setting. Median time to become negative was 35–42 or more days for the HRP2 tests and 2 days for the pLDH test. Conclusions High transmission contexts and a long time to become negative resulted in considerably reduced specificities for the HRP2 tests. Choice of RDT for low- versus high-transmission settings should balance risks and benefits of over-treatment versus missing malaria cases. Trial registration: Registry number at ClinicalTrial.gov: NCT01325974 Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1529-6) contains supplementary material, which is available to authorized users.

Plasmodium vivax is considered to be rare in the predominantly Duffy negative populations of Sub-Saharan Africa, as this red blood cell surface antigen is essential for invasion by the parasite. However, despite only very few reports of molecularly confirmed P. vivax from tropical Africa, serological evidence indicated that 13% of the persons sampled in Congo had been exposed to P. vivax. We identified P. vivax by microscopy in 8 smears from Ugandan pregnant women who had been enrolled in a longitudinal study of malaria in pregnancy. A nested polymerase chain reaction (PCR) protocol was used to detect and identify the Plasmodium parasites present. PCR analysis confirmed the presence of P. vivax for three of the women and analysis of all available samples from these women revealed clinically silent chronic low-grade vivax infections for two of them. The parasites in one woman carried pyrimethamine resistance-associated double non-synonymous mutations in the P. vivax dihydrofolate reductase gene. The three women found infected with P. vivax were Duffy positive as were nine of 68 women randomly selected from the cohort. The data presented from these three case reports is consistent with stable transmission of malaria in a predominantly Duffy negative African population. Given the substantial morbidity associated with vivax infection in non-African endemic areas, it will be important to investigate whether the distribution and prevalence of P. vivax have been underestimated in Sub-Saharan Africa. This is particularly important in the context of the drive to eliminate malaria and its morbidity.

BACKGROUND: Malaria in pregnancy is associated with maternal and fetal morbidity and mortality. In 2006, WHO recommended use of artemisinin-based combination treatments during the second or third trimesters, but data on efficacy and safety in Africa were scarce. We aimed to assess whether artemether-lumefantrine was at least as efficacious as oral quinine for the treatment of uncomplicated falciparum malaria during the second and third trimesters of pregnancy in Mbarara, Uganda. METHODS: We did an open-label, randomised, non-inferiority trial between October, 2006, and May, 2009, at the antenatal clinics of the Mbarara University of Science and Technology Hospital in Uganda. Pregnant women were randomly assigned (1:1) by computer generated sequence to receive either quinine hydrochloride or artemether-lumefantrine, and were followed up weekly until delivery. Our primary endpoint was cure rate at day 42, confirmed by PCR. The non-inferiority margin was a difference in cure rate of 5%. Analysis of efficacy was for all randomised patients without study deviations that could have affected the efficacy outcome. This study was registered with ClinicalTrials.gov, number NCT00495508. FINDINGS: 304 women were randomly assigned, 152 to each treatment group. By day 42, 16 patients were lost to follow-up and 25 were excluded from the analysis. At day 42, 137 (99.3%) of 138 patients taking artemether-lumefantrine and 122 (97.6%) of 125 taking quinine were cured-difference 1.7% (lower limit of 95% CI -0.9). There were 290 adverse events in the quinine group and 141 in the artemether-lumefantrine group. INTERPRETATION: Artemisinin derivatives are not inferior to oral quinine for the treatment of uncomplicated malaria in pregnancy and might be preferable on the basis of safety and efficacy. FUNDING: Médecins Sans Frontières and the European Commission.

Acute central nervous system (CNS) infections in children in sub-Saharan Africa are often fatal. Potential contributors include late presentation, limited diagnostic capacity and inadequate treatment. A more nuanced understanding of treatment practices with a goal of optimizing such practices is critical to prevent avoidable case fatality. We describe empiric antimicrobial treatment, antibiotic resistance and treatment adequacy in a prospective cohort of 459 children aged two months to 12 years hospitalised for suspected acute CNS infections in Mbarara, Uganda, from 2009 to 2012. Among these 459 children, 155 had a laboratory-confirmed diagnosis of malaria (case-fatality rate [CFR] 14%), 58 had bacterial infections (CFR 24%) and 6 children had mixed malaria and bacterial infections (CFR 17%). Overall case fatality was 18.1% (n = 83). Of 219 children with laboratory-confirmed malaria and/or bacterial infections, 182 (83.1%) received an adequate antimalarial and/or antibiotic on the day of admission and 211 (96.3%) within 48 hours of admission. The proportion of those receiving adequate treatment was similar among survivors and non-survivors. All bacterial isolates were sensitive to ceftriaxone except one Escherichia coli isolate with extended-spectrum beta-lactamase (ESBL). The observed high mortality was not a result of inadequate initial antimicrobial treatment at the hospital. The epidemiology of CNS infection in this setting justifies empirical use of a third-generation cephalosporin, however antibiotic resistance should be monitored closely.

Treatment with inhaled nitric oxide as an adjuvant therapy for pediatric patients with cerebral malaria for 48 hours did not result in a significant difference in plasma Angiopoietin-1 levels when compared with placebo in a phase II open-label clinical trial., Background. Children with cerebral malaria (CM) have high rates of mortality and neurologic sequelae. Nitric oxide (NO) metabolite levels in plasma and urine are reduced in CM. Methods. This randomized trial assessed the efficacy of inhaled NO versus nitrogen (N2) as an adjunctive treatment for CM patients receiving intravenous artesunate. We hypothesized that patients treated with NO would have a greater increase of the malaria biomarker, plasma angiopoietin-1 (Ang-1) after 48 hours of treatment. Results. Ninety-two children with CM were randomized to receive either inhaled 80 part per million NO or N2 for 48 or more hours. Plasma Ang-1 levels increased in both treatment groups, but there was no difference between the groups at 48 hours (P = not significant [NS]). Plasma Ang-2 and cytokine levels (tumor necrosis factor-α, interferon-γ, interleukin [IL]-1β, IL-6, IL-10, and monocyte chemoattractant protein-1) decreased between inclusion and 48 hours in both treatment groups, but there was no difference between the groups (P = NS). Nitric oxide metabolite levels—blood methemoglobin and plasma nitrate—increased in patients treated with NO (both P < .05). Seven patients in the N2 group and 4 patients in the NO group died. Five patients in the N2 group and 6 in the NO group had neurological sequelae at hospital discharge. Conclusions. Breathing NO as an adjunctive treatment for CM for a minimum of 48 hours was safe, increased blood methemoglobin and plasma nitrate levels, but did not result in a greater increase of plasma Ang-1 levels at 48 hours.

Introduction Puerperal sepsis causes 10% of maternal deaths in Africa, but prospective studies on incidence, microbiology and antimicrobial resistance are lacking. Methods We performed a prospective cohort study of 4,231 Ugandan women presenting to a regional referral hospital for delivery or postpartum care, measured vital signs after delivery, performed structured physical exam, symptom questionnaire, and microbiologic evaluation of febrile and hypothermic women. Malaria rapid diagnostic testing, blood and urine cultures were performed aseptically and processed at Epicentre Mbarara Research Centre. Antimicrobial susceptibility and breakpoints were determined using disk diffusion per EUCAST standards. Hospital diagnoses, treatments and outcomes were abstracted from patient charts. Results Mean age was 25 years, 12% were HIV-infected, and 50% had cesarean deliveries. Approximately 5% (205/4176) with ≥1 temperature measurement recorded developed postpartum fever or hypothermia; blood and urine samples were collected from 174 (85%), and 17 others were evaluated clinically. Eighty-four (48%) had at least one confirmed source of infection: 39% (76/193) clinical postpartum endometritis, 14% (25/174) urinary tract infection (UTI), 3% (5/174) bloodstream infection. Another 3% (5/174) had malaria. Overall, 30/174 (17%) had positive blood or urine cultures, and Acinetobacter species were the most common bacteria isolated. Of 25 Gram-negatives isolated, 20 (80%) were multidrug-resistant and cefepime non-susceptible. Conclusions For women in rural Uganda with postpartum fever, we found a high rate of antibiotic resistance among cultured urinary and bloodstream infections, including cephalosporin-resistant Acinetobacter species. Increasing availability of microbiology testing to inform appropriate antibiotic use, development of antimicrobial stewardship programs, and strengthening infection control practices should be high priorities.

We compared Mycobacterium tuberculosis sputum culture recovery and contamination rates between Lowenstein-Jensen medium (LJ) containing the following decontaminants and LJ alone: (i) PANTA ( n = 299), (ii) Selectatab-MB ( n = 299), and (iii) penicillin G ( n = 234). The contamination rate for LJ alone was approximately 31%, versus 5.0% for PANTA-containing, 2% for Selectatab-containing, and 9% for penicillin-containing media ( P < 0.001). M. tuberculosis isolation rates were 9.8%, 17%, 18%, and 12% for standard LJ, PANTA, Selectatab, and penicillin cultures, respectively.