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Introduction:SARS-CoV-2 is a fatal disease of global public health concern. Measures to reduce its spread critically depend on timely and accurate diagnosis of virus-infected individuals. Biobanks can have a pivotal role in elucidating disease etiology, translation, and advancing public health. In this article, we show how a biobank has been a critical resource in the rapid response to coronavirus disease of 2019 (COVID-19) in Uganda.Materials and Methods:The Integrated Biorepository of H3Africa Uganda established a COVID-19 biobank. Standard Operating Procedures for sample and data collection, sample processing, and storage were developed. An e-questionnairedata tool was used to collect sociodemographic factors. Samples were collected at 7-day intervals from patients, analyzed for key parameters, processed, annotated, characterized, and stored at appropriate temperatures.Results:Stored samples have been used in validation of 17 diagnostic kits, the Cepheid Xpert Xpress SARS-CoV-2 assay, as well as a sample pooling technique for mass screening and polymerase chain reaction assay validation. Kits that passed validation were deployed for mass screening boosting early detection, isolation, and treatment of COVID-19 cases. Also, 10 applications from researchers and biotech companies have been received and approved and 4 grants have been awardedConclusion:The CoV-Bank has proven to be an invaluable resource in the fight against the COVID-19 pandemic in Uganda, as samples have been resources in the validation and development of COVID-19 diagnostic tools, which are important in tracing and isolation of infected cases to confront, delay, and stop the spread of the SARS-CoV-2 virus.

Tuberculosis is a leading cause of global childhood mortality; however, interventions to detect undiagnosed tuberculosis in children are underused. Child contact tracing has been widely recommended but poorly implemented in resource-constrained settings. WHO has proposed a pragmatic screening approach for managing child contacts. We assessed the effectiveness of this screening approach and alternative symptom-based algorithms in identifying secondary tuberculosis in a prospectively followed cohort of Ugandan child contacts.

ABSTRACTScience 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.

ABSTRACTThe Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosiscomplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosismc26030 cells into distilled water and M. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Genedrive to that of the Xpert MTB/RIF assay using M. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/µl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 104CFU/ml and 2.5 × 105CFU/ml for cells spiked into water and sputum, respectively. False-positive rpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus, Mycobacterium gordonae, or Mycobacterium thermoresistibile. In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Genedrive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Genedrive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

ABSTRACTThe specificities and sensitivities of the Bactec mycobacterial growth indicator tube (MGIT) system for the recovery of Mycobacterium tuberculosisfrom pleural fluid are not statistically different than those of the Myco/F lytic liquid culture system. The time to positivity is shorter in the MGIT system (12.7 versus 20.7 days, respectively; P= 0.007). The Myco/F lytic culture system may be an alternative to the MGIT system for diagnosing pleural tuberculosis.

ABSTRACTThe Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection of Mycobacterium tuberculosisin sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions of M. tuberculosisinto sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained from M. tuberculosisculture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in which M. tuberculosiswas detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%; P= 0.03) and for smear-negative specimens (67% versus 58%; P= 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increased M. tuberculosisdetection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.

ABSTRACTSurveillance of the circulating Mycobacterium tuberculosiscomplex (MTC) strains in a given locality is important for understanding tuberculosis (TB) epidemiology. We performed molecular epidemiological studies on sputum smear-positive isolates that were collected for anti-TB drug resistance surveillance to establish the variability of MTC lineages with anti-TB drug resistance and HIV infection. Spoligotyping was performed to determine MTC phylogenetic lineages. We compared patients' MTC lineages with drug susceptibility testing (DST) patterns and HIV serostatus. Out of the 533 isolates, 497 (93.2%) had complete DST, PCR, and spoligotyping results while 484 (90.1%) participants had results for HIV testing. Overall, the frequency of any resistance was 75/497 (15.1%), highest among the LAM (34.4%; 95% confidence interval [CI], 18.5 to 53.2) and lowest among the T2 (11.5%; 95% CI, 7.6 to 16.3) family members. By multivariate analysis, LAM (adjusted odds ratio [ORadj], 5.0; 95% CI, 2.0 to 11.9; P< 0.001) and CAS (ORadj, 2.9; 95% CI, 1.4.0 to 6.3; P= 0.006) families were more likely to show any resistance than was T2. All other MTC lineages combined were more likely to be resistant to any of the anti-TB drugs than were the T2 strains (ORadj, 1.7; 95% CI, 1.0 to 2.9; P= 0.040). There were no significant associations between multidrug resistance and MTC lineages, but numbers of multidrug-resistant TB strains were small. No association was established between MTC lineages and HIV status. In conclusion, the T2 MTC lineage negatively correlates with anti-TB drug resistance, which might partly explain the reported low levels of anti-TB drug resistance in Kampala, Uganda. Patients' HIV status plays no role with respect to the MTC lineage distribution.

ABSTRACTFor Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosisdrug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosisisolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results for M. tuberculosissusceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

ABSTRACTOne of the most effective and widely used antituberculosis (anti-TB) drugs is isoniazid (INH), a prodrug activated via oxidation that forms an adduct with NAD+to inhibit NADH-dependent targets of Mycobacterium tuberculosis, such as enoyl-acyl carrier protein reductase (InhA). The metabolic by-products and potentially toxic intermediates resulting from INH therapy have been identified through a large body of work. However, an INH-NAD adduct or structures related to this adduct have not been identified in specimens from human TB patients or animal models of TB. Analyses by mass spectrometry of urine collected from TB patients in a study conducted by the NIAID-funded Tuberculosis Research Unit identified 4-isonicotinoylnicotinamide (C12H9N3O2) as a novel metabolite of INH therapy. This compound was formed by M. tuberculosisstrains in a KatG-dependent manner but could also be produced by mice treated with INH independent of an M. tuberculosisinfection. Thus, the 4-isonicotinoylnicotinamide observed in human urine samples is likely derived from the degradation of oxidized INH-NAD adducts and provides direct evidence of host INH activation.

Streptococcus pneumoniaeaffects both children and adults worldwide. It colonizes the nasopharynx, and this precedes and serves as a reservoir for invasive disease, for example, pneumonia, bacteremia, and meningitis. It is a leading cause of morbidity and mortality among the elderly and those with underlying medical conditions like immunosuppression and chronic medical illnesses. The increasing number of persons with immunosuppression susceptible to pneumococcal infection has had an impact on the disease burden and the clinical characteristics of the pneumococcal disease in many areas. People with human immunodeficiency virus (HIV) are particularly at risk for persistent infection because of the numerous abnormalities in their immune process like defects in their T cells, B cells, macrophages, and polymorphonuclear cells such as neutrophils. There are limited data on prevalence, outcome, and antimicrobial susceptibility of S. pneumoniaebacteremia among hospitalized patients in Uganda.

Tuberculosis (TB) resistance to rifampicin, the most powerful drug leads to increase in mortality. Globally, half a million new patients develop such resistant TB each year, coupled with both inappropriate diagnosis and treatment initiation.

ABSTRACTThe Etest method for susceptibility testing of Mycobacterium tuberculosiswas compared to the agar proportion method using four first-line agents and two fluoroquinolones. Catergorical agreement between the methods was 100% for rifampin, ethambutol, streptomycin, and ofloxacin and 98% for isoniazid. Results were obtained in 6 to 10 days by Etest. The Etest method is suitable for testing the agents evaluated against M. tuberculosis.