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Science education and research have the potential to drive profound change in low- and middle-income countries (LMICs) through encouraging innovation, attracting industry, and creating job opportunities. However, in LMICs, research capacity is often limited, and acquisition of funding and access to state-of-the-art technologies is challenging. The Alliance for Global Health and Science (the Alliance) was founded as a partnership between the University of California, Berkeley (USA) and Makerere University (Uganda), with the goal of strengthening Makerere University's capacity for bioscience research. The flagship program of the Alliance partnership is the MU/UCB Biosciences Training Program, an in-country, hands-on workshop model that trains a large number of students from Makerere University in infectious disease and molecular biology research. This approach nucleates training of larger and more diverse groups of students, development of mentoring and bi-directional research partnerships, and support of the local economy. Here, we describe the project, its conception, implementation, challenges, and outcomes of bioscience research workshops. We aim to provide a blueprint for workshop implementation, and create a valuable resource for bioscience research capacity strengthening in LMICs.

Not all patients produce sputum, yet most available TB tests use sputum. We investigated the utility of a novel RNA-based quantitative test, the tuberculosis molecular bacterial load assay (TB-MBLA), for the detection and quantification of Mycobacterium tuberculosis in stool. Stools from 100 adult individuals were treated with OMNIgene-sputum reagent and tested using Xpert MTB/RIF ultra (Xpert ultra), auramine O smear microscopy (smear), mycobacterial growth indicator tube (MGIT), and Lowenstein-Jensen (LJ) cultures. The remaining portions were frozen at -20°C and later tested by TB-MBLA. MGIT sputum culture was used as a TB confirmatory test and reference for stool tests. Sixty-one of 100 participants were already confirmed TB positive by MGIT sputum culture, 20 (33%) of whom were HIV coinfected. TB-MBLA detected M. tuberculosis in 57/100 stool samples, including 49 already confirmed for TB. The mean bacterial load measured by stool TB-MBLA was 5.67 ± 1.7 log10 estimated CFU (eCFU) per mL in HIV-coinfected participants, which was higher than the 4.83 ± 1.59 log10 eCFU per mL among the HIV-negative participants (P = 0.04). The sensitivities (95% confidence intervals [CI]) of stool assays were 80% (68 to 89) and 90% (79 to 98) for TB-MBLA and Xpert ultra, which were both higher than the 44% (32 to 58), 64% (51 to 76), and 62% (45 to 77) for smear, MGIT, and Lowenstein-Jensen (LJ) stool cultures, respectively. The specificity (95% CI) of stool assays was highest for smear, at 97% (87 to 100), followed by Xpert ultra at 91% (76 to 98), TB-MBLA at 79% (63 to 90), LJ at 80% (64 to 91), and MGIT at 62% (45 to 77). Twenty-six percent of MGIT and 21% of LJ stool cultures were indeterminate due to contamination. Detection and quantification of viable M. tuberculosis bacilli in stool raises its utility as an alternative to sputum as a sample type for TB diagnosis. IMPORTANCE This paper highlights the value of stool as a sample type for diagnosis of tuberculosis. While other studies have used DNA-based assays like the Xpert MTB/RIF and culture to detect Mycobacterium tuberculosis in stool, this is the first study that has applied TB-MBLA, an RNA-based assay, to quantify TB bacteria in stool. The high microbial density and diversity in stool compromises the specificity and sensitivity of culture-based tests due to overgrowth of non-M. tuberculosis flora. Consequently, TB-MBLA becomes the most sensitive and specific test for the detection and quantification of viable TB bacteria in stool. Most crucially, this study raises the possibility of a nonsputum alternative sample type for diagnosis of TB among people who have difficulty in producing sputum.

We explored associations between Plasmodium falciparum drug resistance-mediating polymorphisms and clinical presentations in parasitemic children enrolled in a cross-sectional survey in Tororo, Uganda, using a retrospective case-control design. All 243 febrile children (cases) and 243 randomly selected asymptomatic children (controls) were included. In a multivariate analysis adjusting for age, complexity of infection, and parasite density, the prevalence of wild-type genotypes was significantly higher in febrile children compared to asymptomatic children (pfcrt K76T: odds ratio [OR] 4.41 [95% confidence interval {CI}, 1.28-15.1]; pfmdr1 N86Y: OR 4.08 [95% CI, 2.01-8.31], and pfmdr1 D1246Y: OR 4.90 [95% CI, 1.52-15.8]), suggesting greater virulence for wild-type parasites.

Persons living with HIV are known to be at increased risk of developing tuberculosis (TB) disease upon infection with Mycobacterium tuberculosis (Mtb). However, it has remained unclear how HIV co-infection affects subsequent Mtb transmission from these patients. Here, we customized a Bayesian phylodynamic framework to estimate the effects of HIV co-infection on the Mtb transmission dynamics from sequence data. We applied our model to four Mtb genomic datasets collected in sub-Saharan African countries with a generalized HIV epidemic. Our results confirm that HIV co-infection is a strong risk factor for developing active TB. Additionally, we demonstrate that HIV co-infection is associated with a reduced effective reproductive number for TB. Stratifying the population by CD4+ T-cell count yielded similar results, suggesting that, in this context, CD4+ T-cell count is not a better predictor of Mtb transmissibility than HIV infection status alone. Together, our genome-based analyses complement observational household contact studies, and more firmly establish the negative association between HIV co-infection and Mtb transmissibility. Author summary: Many sub-Saharan African countries have seen a considerable rise in TB incidence since the introduction of HIV, suggesting a strong interaction between HIV and TB epidemics. HIV infection is recognized as an important risk factor for developing TB, but the contribution of HIV-infected TB cases to further Mtb transmission is poorly understood. In this study, we analyzed four sets of Mtb genomic sequences collected in different countries, comprising sequences from HIV-negative and HIV-positive TB patients. We applied a phylodynamic model to these sequences, aimed at inferring transmission dynamics within and between different host populations. While our findings support that HIV is a strong risk factor for TB, we show that HIV-positive TB cases generate a significantly lower number of secondary TB cases than HIV-negative cases. This suggests that HIV-positive cases mainly act as sinks in Mtb transmission chains, while HIV-negative cases are a major source of transmission. [ABSTRACT FROM AUTHOR]

Abstract Background Nucleic acid amplification tests (NAATs) have offered hope for rapid diagnosis of tuberculosis (TB). However, their efficiency with smear-negative samples has not been widely studied in low income settings. Here, we evaluated in-house PCR assay for diagnosis of smear-negative TB using Lowenstein-Jensen (LJ) culture as the baseline test. Two hundred and five pulmonary TB (PTB) suspects with smear-negative sputum samples, admitted on a short stay emergency ward at Mulago Hospital in Kampala, Uganda, were enrolled. Two smear-negative sputum samples were obtained from each PTB suspect and processed simultaneously for identification of MTBC using in-house PCR and LJ culture. Results Seventy two PTB suspects (35%, 72/205) were LJ culture positive while 128 (62.4%, 128/205) were PCR-positive. The sensitivity and specificity of in-house PCR for diagnosis of smear-negative PTB were 75% (95% CI 62.6-85.0) and 35.9% (95% CI 27.2-45.3), respectively. The positive and negative predictive values were 39% (95% CI 30.4-48.2) and 72.4% (95% CI 59.1-83.3), respectively, while the positive and negative likelihood ratios were 1.17 (95% CI 0.96-1.42) and 0.70 (95% CI 0.43-1.14), respectively. One hundred and seventeen LJ culture-negative suspects (75 PCR-positive and 42 PCR-negative) were enrolled for follow-up at 2 months. Of the PCR-positive suspects, 45 (60%, 45/75) were still alive, of whom 29 (64.4%, 29/45) returned for the follow-up visit; 15 (20%, 15/75) suspects died while another 15 (20%, 15/75) were lost to follow-up. Of the 42 PCR-negative suspects, 22 (52.4%, 22/42) were still alive, of whom 16 (72.7%, 16/22) returned for follow-up; 11 (26.2%, 11/42) died while nine (21.4%, 9/42) were lost to follow-up. Overall, more PCR-positive suspects were diagnosed with PTB during follow-up visits but the difference was not statistically significant (27.6%, 8/29 vs. 25%, 4/16, p = 0.9239). Furthermore, mortality was higher for the PCR-negative suspects but the difference was also not statistically significant (26.2% vs. 20% p = 0.7094). Conclusion In-house PCR correlates poorly with LJ culture for diagnosis of smear-negative PTB. Therefore, in-house PCR may not be adopted as an alternative to LJ culture.

Background: Drug-Resistant Tuberculosis (DR-TB) is one of the major challenges to TB control. Design and methods: This was a blinded, laboratory-based cross-sectional study using sputum samples or culture isolates. Samples were from patients with rifampicin-resistant—TB and/or with high risk for isoniazid (INH) resistance and/or 2nd line fluoroquinolones (FQ) and injectable agents (IAs). The diagnostic accuracy of the Xpert® MTB/XDR test was compared to MGIT960 and the Hain Genotype® MTBDRplus and MDRsl assays (LPA) as reference DST methods. Factors for laboratory uptake of the Xpert® MTB/XDR test were also evaluated. Results: Of the 100 stored sputum samples included in this study, 65/99 (65.6%) were resistant to INH, 5/100 (5.0%) were resistant to FQ and none were resistant to IAs using MGIT960. The sensitivity and specificity, n (%; 95% Confidence Interval, CI) of Xpert® MTB/XDR test for; INH was 58 (89.2; 79.1–95.5) and 30 (88.2; 72.5–96.6) and for FQ; 4 (80.0; 28.3–99.4) and 95 (100; 96.2–100), respectively. Using LPA as a reference standard, a total of 52/98 (53.1%) were resistant to INH, 3/100 (3.0%) to FQ, and none to IA. The sensitivity and specificity, n (%; 95%CI) of Xpert® MTB/XDR test compared to LPA for; INH was 50 (96.1; 86.7–99.5) and 34 (74.0; 58.8–85.7) for FQ 3 (100; 29.2–100) and 96 (99.0; 94.3–99.9) respectively. The factors for laboratory uptake and roll-out of the Xpert® MTB/XDR test included: no training needed for technicians with, and one day for those without, previous Xpert-ultra experience, recording and reporting needs were not different from those of Xpert-ultra, the error rate was 4/100 (4%), one (1%) indeterminate rate and test turn-around-time were 1hr/45 minutes. Conclusion: There is high sensitivity and specificity of Xpert® MTB/XDR test for isoniazid and fluoroquinolones. There are acceptable Xpert® MTB/XDR test attributes for the test uptake and roll-out. [ABSTRACT FROM AUTHOR]

Chronic Obstructive Pulmonary Disease (COPD) has significantly contributed to global mortality, with three million deaths reported annually. This impact is expected to increase over the next 40 years, with approximately 5 million people predicted to succumb to COPD-related deaths annually. Immune mechanisms driving disease progression have not been fully elucidated. Airway microbiota have been implicated. However, it is still unclear how changes in the airway microbiome drive persistent immune activation and consequent lung damage. Mechanisms mediating microbiome-immune crosstalk in the airways remain unclear. In this review, we examine how dysbiosis mediates airway inflammation in COPD. We give a detailed account of how airway commensal bacteria interact with the mucosal innate and adaptive immune system to regulate immune responses in healthy or diseased airways. Immune-phenotyping airway microbiota could advance COPD immunotherapeutics and identify key open questions that future research must address to further such translation. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis remains a major public health problem worldwide accounting for 1.4 million deaths annually. LL-37 is an effector molecule involved in immunity with both antimicrobial and immunomodulatory properties. The purpose of this study was to compare LL-37 circulatory levels among participants with active and latent tuberculosis and to determine its ability to discriminate between the two infectious states. Methods: A cross-sectional study was performed among 56 active tuberculosis patients, 49 latent tuberculosis individuals, and 43 individuals without tuberculosis infection. The enzyme-linked immunosorbent assay was used to assess LL-37 levels. Data analysis was performed using STATA software and Graph pad Prism version 8. Mann-Whitney U test was used for correlation between variables with two categories and the Kruskal-Wallis test for three or more categories. Results: The study had more female participants than males, with similar median ages across the three groups, 29.5, 25.0, and 23.0 years respectively. Active tuberculosis patients had significantly higher LL-37 levels compared to those with latent tuberculosis and without tuberculosis. The median/interquartile ranges were 318.8 ng/ml (157.9–547.1), 242.2 ng/ml (136.2–579.3), 170.9 ng/ml (129.3–228.3); p = 0.002 respectively. Higher LL-37 was found in the male participant with median/interquartile range, 424.8 ng/ml (226.2–666.8) compared to the females 237.7 ng/ml (129.6–466.6); p = 0.045. LL-37 had better discriminatory potential between active tuberculosis and no tuberculosis (AUC = 0.71, sensitivity 71.4% specificity = 69.8%) than with latent tuberculosis (AUC = 0.55, sensitivity = 71.4%, specificity = 44.9%). There was moderate differentiation between latent tuberculosis and no tuberculosis (AUC = 0.63, sensitivity = 44.9% specificity = 90.7%). Conclusion: Significantly higher LL-37 levels were observed among active tuberculosis patients than those without tuberculosis infection and were, therefore able to discriminate between active tuberculosis and other tuberculosis infectious states, especially with no tuberculosis. Further assessment of this biomarker as a screening tool to exclude tuberculosis is required. [ABSTRACT FROM AUTHOR]

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by [G.sub.ST]. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.

The gab operon (gabDTPC) in Escherichia coli functions in the conversion of [gamma]-aminobutyrate to succinate. One component of gab operon regulation involves the RpoS sigma factor, which mediates activation at high cell density. Transposon mutagenesis was used to identify new genes that regulate gab operon expression in rich media. A Tn5tmp insertion in the hldD (formerly rfaD) gene increased gabT::lacZ expression 12-fold. The hldD gene product, an ADP-L-glycerol-D-mannoheptose-6-epimerase, catalyzes the conversion of ADP-D-glycerol-D-mannoheptose to ADP-L-glycerol-D-mannoheptose, a precursor for the synthesis of inner-core lipopolysaccharide (LPS). Defined mutations in hidE, required for heptose synthesis, and waaF, required for the addition of the second heptose to the inner core, also resulted in high-level gabT::lacZ expression. The hldD, hidE, and waaF mutants exhibited a mucoid colony phenotype due to production of a colanic acid capsule. However, in the hldD::cat background, the high-level expression of gab T::lacZ was independent of the regulatory components for colanic acid synthesis (rcsA, rcsB, and rcsC) and also independent of manC (cpsB), a structural gene for colanic acid synthesis. Activation of gab T::lacZ in the hldD::cat background was dependent on the RpoS sigma factor. The hldD::cat mutation resulted in a sixfold increase in the levels of a translational RpoS-LacZ fusion and had a marginal effect on a transcriptional fusion. This study reveals a stress-induced pathway, mediated by loss of the LPS inner core, that increases RpoS translation and gab operon expression in E. coli.

Background: Tuberculosis remains a global threat and a public health problem that has eluded attempts to eradicate it. Low vitamin D levels have been identified as a risk factor for tuberculosis infection and disease. The human cathelicidin LL-37 has both antimicrobial and immunomodulatory properties and is dependent on vitamin D status. This systematic review attempts to compare vitamin D andLL-37 levels among adult pulmonary tuberculosis patients to non-pulmonary TB individuals between 16–75 years globally and to determine the association between vitamin D and cathelicidin and any contributing factor among the two study groups. Methods/Design: We performed a search, through PubMed, HINARI, Google Scholar, EBSCOhost, and databases. A narrative synthesis through evaluation of vitamin D and LL-37 levels, the association of vitamin D and LL-37, and other variables in individual primary studies were performed. A random-effect model was performed and weighted means were pooled at a 95% confidence interval. This protocol is registered under the International Prospective Register of Systematic Reviews (PROSPERO), registration number CRD42019127232. Results: Of the 2507 articles selected12 studies were eligible for the systematic review and of these only nine were included in the meta-analysis for vitamin D levels and six for LL-37 levels. Eight studies were performed in Asia, three in Europe, and only one study in Africa. The mean age of the participants was 37.3±9.9 yrs. We found low vitamin D and high cathelicidin levels among the tuberculosis patients compared to non-tuberculosis individuals to non-tuberculosis. A significant difference was observed in both vitamin D and LL-37 levels among tuberculosis patients and non-tuberculosis individuals (p = < 0.001). Conclusion: This study demonstrated that active pulmonary tuberculosis disease is associated with hypovitaminosis D and elevated circulatory cathelicidin levels with low local LL-37 expression. This confirms that vitamin D status has a protective role against tuberculosis disease. [ABSTRACT FROM AUTHOR]

Background The Global Laboratory Initiative (GLI) guidelines recommend repeat for GeneXpertMTB/RIF (XpertMTB/RIF) in patients with a low pretest probability of rifampicin resistance (RR). Methods This was a cross-sectional study using results of sputum specimens collected from participants screened for the STREAM 2 trial. We recruited all patients with XpertMTB/RIF RR-TB detected who were referred for RR/multidrug-resistant (MDR) TB treatment initiation at Mulago National Referral Hospital, Kampala, between September 2017 and October 2019. At baseline, smear microscopy, repeat XpertMTB/RIF, Xpert Ultra, and MTBDR plus assays were done on sputum specimens. Culture-based drug susceptibility testing (DST) was performed on discordant specimens. We analyzed the prevalence and factors associated with discordance between initial and repeat XpertMTB/RIF RR and false XpertMTB/RIF RR. False XpertMTB/RIF RR was defined as no RR detected by any of Xpert Ultra, LPA, or culture DST (reference comparator). Results A total of 126/130 patients had repeat XpertMTB/RIF results, of whom 97 (77.0%) had M. tuberculosis detected, 81 (83.5%) had RR detected, and 1 (1.0%) had RR indeterminate. The prevalence of discordant XpertMTB/RIF RR was 15/96 (15.6%), whereas false XpertMTB/RIF RR prevalence was 10/96 (10.4%). Low–bacillary load sputum specimens were more likely to have discordant XpertMTB/RIF RR and false XpertMTB/RIF RR results (adjusted odds ratio [aOR], 0.04; 95% CI, 0.00–0.37; P =.01; aOR, 0.02; 95% CI, 0.01–0.35; P =.01, respectively). Conclusions Our findings show a high false-positive rifampicin resistance rate in low–TB burden patients, which calls for repeat testing in order to prevent unnecessary prescription of anti-MDR-TB therapy. [ABSTRACT FROM AUTHOR]

Abstract Background The occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda. Results Twenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2). Conclusions There is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.

Abstract Background Capilia TB is a simple immunochromatographic assay based on the detection of MPB64 antigen specifically secreted by the Mycobacterium tuberculosis complex (MTC). Capilia TB was evaluated for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 systems in Kampala, Uganda. Since most studies have mainly dealt with respiratory samples, the performance of Capilia TB on blood culture samples was also evaluated. Methods One thousand samples from pulmonary and disseminated tuberculosis (TB) suspects admitted to the JCRC clinic and the TB wards at Old Mulago hospital in Kampala, Uganda, were cultured in automated BACTEC MGIT 960 and BACTEC 9120 blood culture systems. BACTEC-positive samples were screened for purity by sub-culturing on blood agar plates. Two hundred and fifty three (253) samples with Acid fast bacilli (AFB, 174 BACTEC MGIT 960 and 79 BACTEC 9120 blood cultures) were analyzed for presence of MTC using Capilia TB and in-house PCR assays. Results The overall Sensitivity, Specificity, Positive and Negative Predictive values, and Kappa statistic for Capilia TB assay for identification of MTC were 98.4%, 97.6%, 97.7%, 98.4% and 0.96, respectively. Initially, the performance of in-house PCR on BACTEC 9120 blood cultures was poor (Sensitivity, Specificity, PPV, NPV and Kappa statistic of 100%, 29.3%,7%, 100% and 0.04, respectively) but improved upon sub-culturing on solid medium (Middlebrook 7H10) to 100%, 95.6%, 98.2%, 100% and 0.98, respectively. In contrast, the Sensitivity and Specificity of Capilia TB assay was 98.4% and 97.9%, respectively, both with BACTEC blood cultures and Middlebrook 7H10 cultured samples, revealing that Capilia was better than in-house PCR for identification of MTC in blood cultures. Additionally, Capilia TB was cheaper than in-house PCR for individual samples ($2.03 vs. $12.59, respectively), and was easier to perform with a shorter turnaround time (20 min vs. 480 min, respectively). Conclusion Capilia TB assay is faster and cheaper than in-house PCR for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 culture systems in real-time testing of AFB positive cultures.