Molecular characterization of the 2022 Sudan virus disease outbreak in Uganda.

IMPORTANCE: Ebola disease (EBOD) is a public health threat with a high case fatality rate. Most EBOD outbreaks have occurred in remote locations, but the 2013-2016 Western Africa outbreak demonstrated how devastating EBOD can be when it reaches an urban population. Here, the 2022 Sudan virus disease (SVD) outbreak in Mubende District, Uganda, is summarized, and the genetic relatedness of the new variant is evaluated. The Mubende variant exhibited 96% amino acid similarity with historic SUDV sequences from the 1970s and a high degree of conservation throughout the outbreak, which was important for ongoing diagnostics and highly promising for future therapy development. Genetic differences between viruses identified during the Mubende SVD outbreak were linked with epidemiological data to better interpret viral spread and contact tracing chains. This methodology should be used to better integrate discrete epidemiological and sequence data for future viral outbreaks.

Assessment of the pathogen genomics landscape highlights disparities and challenges for effective AMR Surveillance and outbreak response in the East African community.

The East African Community (EAC) grapples with many challenges in tackling infectious disease threats and antimicrobial resistance (AMR), underscoring the importance of regional and robust pathogen genomics capacities. However, a significant disparity exists among EAC Partner States in harnessing bacterial pathogen sequencing and data analysis capabilities for effective AMR surveillance and outbreak response. This study assesses the current landscape and challenges associated with pathogen next-generation sequencing (NGS) within EAC, explicitly focusing on World Health Organization (WHO) AMR-priority pathogens. The assessment adopts a comprehensive approach, integrating a questionnaire-based survey amongst National Public Health Laboratories (NPHLs) with an analysis of publicly available metadata on bacterial pathogens isolated in the EAC countries. In addition to the heavy reliance on third-party organizations for bacterial NGS, the findings reveal a significant disparity among EAC member States in leveraging bacterial pathogen sequencing and data analysis. Approximately 97% (n = 4,462) of publicly available high-quality bacterial genome assemblies of samples collected in the EAC were processed and analyzed by external organizations, mainly in Europe and North America. Tanzania led in-country sequencing efforts, followed by Kenya and Uganda. The other EAC countries had no publicly available samples or had all their samples sequenced and analyzed outside the region. Insufficient local NGS sequencing facilities, limited bioinformatics expertise, lack of adequate computing resources, and inadequate data-sharing mechanisms are among the most pressing challenges that hinder the EAC's NPHLs from effectively leveraging pathogen genomics data. These insights emphasized the need to strengthen microbial pathogen sequencing and data analysis capabilities within the EAC to empower these laboratories to conduct pathogen sequencing and data analysis independently. Substantial investments in equipment, technology, and capacity-building initiatives are crucial for supporting regional preparedness against infectious disease outbreaks and mitigating the impact of AMR burden. In addition, collaborative efforts should be developed to narrow the gap, remedy regional imbalances, and harmonize NGS data standards. Supporting regional collaboration, strengthening in-country genomics capabilities, and investing in long-term training programs will ultimately improve pathogen data generation and foster a robust NGS-driven AMR surveillance and outbreak response in the EAC, thereby supporting global health initiatives.

The East African Community (EAC) mobile laboratory networks in Kenya, Burundi, Tanzania, Rwanda, Uganda, and South Sudan-from project implementation to outbreak response against Dengue, Ebola, COVID-19, and epidemic-prone diseases.

BACKGROUND: East Africa is home to 170 million people and prone to frequent outbreaks of viral haemorrhagic fevers and various bacterial diseases. A major challenge is that epidemics mostly happen in remote areas, where infrastructure for Biosecurity Level (BSL) 3/4 laboratory capacity is not available. As samples have to be transported from the outbreak area to the National Public Health Laboratories (NPHL) in the capitals or even flown to international reference centres, diagnosis is significantly delayed and epidemics emerge. MAIN TEXT: The East African Community (EAC), an intergovernmental body of Burundi, Rwanda, Tanzania, Kenya, Uganda, and South Sudan, received 10 million € funding from the German Development Bank (KfW) to establish BSL3/4 capacity in the region. Between 2017 and 2020, the EAC in collaboration with the Bernhard-Nocht-Institute for Tropical Medicine (Germany) and the Partner Countries' Ministries of Health and their respective NPHLs, established a regional network of nine mobile BSL3/4 laboratories. These rapidly deployable laboratories allowed the region to reduce sample turn-around-time (from days to an average of 8h) at the centre of the outbreak and rapidly respond to epidemics. In the present article, the approach for implementing such a regional project is outlined and five major aspects (including recommendations) are described: (i) the overall project coordination activities through the EAC Secretariat and the Partner States, (ii) procurement of equipment, (iii) the established laboratory setup and diagnostic panels, (iv) regional training activities and capacity building of various stakeholders and (v) completed and ongoing field missions. The latter includes an EAC/WHO field simulation exercise that was conducted on the border between Tanzania and Kenya in June 2019, the support in molecular diagnosis during the Tanzanian Dengue outbreak in 2019, the participation in the Ugandan National Ebola response activities in Kisoro district along the Uganda/DRC border in Oct/Nov 2019 and the deployments of the laboratories to assist in SARS-CoV-2 diagnostics throughout the region since early 2020. CONCLUSIONS: The established EAC mobile laboratory network allows accurate and timely diagnosis of BSL3/4 pathogens in all East African countries, important for individual patient management and to effectively contain the spread of epidemic-prone diseases.

A cholera outbreak caused by drinking contaminated river water, Bulambuli District, Eastern Uganda, March 2016.

BACKGROUND: A cholera outbreak started on 29 February in Bwikhonge Sub-county, Bulambuli District in Eastern Uganda. Local public health authorities implemented initial control measures. However, in late March, cases sharply increased in Bwikhonge Sub-county. We investigated the outbreak to determine its scope and mode of transmission, and to inform control measures. METHODS: We defined a suspected case as sudden onset of watery diarrhea from 1 March 2016 onwards in a resident of Bulambuli District. A confirmed case was a suspected case with positive stool culture for V. cholerae. We conducted descriptive epidemiologic analysis of the cases to inform the hypothesis on mode of transmission. To test the hypothesis, we conducted a case-control study involving 100 suspected case-patients and 100 asymptomatic controls, individually-matched by residence village and age. We collected seven water samples for laboratory testing. RESULTS: We identified 108 suspected cases (attack rate: 1.3%, 108/8404), including 7 confirmed cases. The case-control study revealed that 78% (78/100) of case-patients compared with 51% (51/100) of control-persons usually collected drinking water from the nearby Cheptui River (ORMH = 7.8, 95% CI = 2.7-22); conversely, 35% (35/100) of case-patients compared with 54% (54/100) of control-persons usually collected drinking water from borehole pumps (ORMH = 0.31, 95% CI = 0.13-0.65). The index case in Bwikhonge Sub-county had onset on 29 February but the outbreak had been on-going in the neighbouring sub-counties in the previous 3 months. V. cholera was isolated in 2 of the 7 river water samples collected from different locations. CONCLUSIONS: We concluded that this cholera outbreak was caused by drinking contaminated water from Cheptui River. We recommended boiling and/or treating drinking water, improved sanitation, distribution of chlorine tablets to the affected villages, and as a long-term solution, construction of more borehole pumps. After implementing preventive measures, the number of cases declined and completely stopped after 6th April.

Baseline Inflammatory Biomarkers Identify Subgroups of HIV-Infected African Children With Differing Responses to Antiretroviral Therapy.

BACKGROUND: Identifying determinants of morbidity and mortality may help target future interventions for human immunodeficiency virus (HIV)-infected children. METHODS: CD4(+) T-cell count, HIV viral load, and levels of biomarkers (C-reactive protein, tumor necrosis factor α [TNF-α], interleukin 6 [IL-6], and soluble CD14) and interleukin 7 were measured at antiretroviral therapy (ART) initiation in the ARROW trial (case-cohort design). Cases were individuals who died, had new or recurrent World Health Organization clinical stage 4 events, or had poor immunological response to ART. RESULTS: There were 115 cases (54 died, 45 had World Health Organization clinical stage 4 events, and 49 had poor immunological response) and 485 controls. Before ART initiation, the median ages of cases and controls were 8.2 years (interquartile range [IQR], 4.4-11.4 years) and 5.8 years (IQR, 2.3-9.3 years), respectively, and the median percentages of lymphocytes expressing CD4 were 4% (IQR, 1%-9%) and 13% (IQR, 8%-18%), respectively. In multivariable logistic regression, cases had lower age-associated CD4(+) T-cell count ratio (calculated as the ratio of the subject's CD4(+) T-cell count to the count expected in healthy individuals of the same age; P < .0001) and higher IL-6 level (P = .002) than controls. Clustering biomarkers and age-associated CD4(+) and CD8(+) T-cell count ratios identified 4 groups of children. Group 1 had the highest frequency of cases (41% cases; 16% died) and profound immunosuppression; group 2 had similar mortality (23% cases; 15% died), but children were younger, with less profound immunosuppression and high levels of inflammatory biomarkers and malnutrition; group 3 comprised young children with moderate immunosuppression, high TNF-α levels, and high age-associated CD8(+) T-cell count ratios but lower frequencies of events (12% cases; 7% died); and group 4 comprised older children with low inflammatory biomarker levels, lower HIV viral loads, and good clinical outcomes (11% cases; 5% died). CONCLUSIONS: While immunosuppression is the major determinant of poor outcomes during ART, baseline inflammation is an additional important factor, identifying a subgroup of young children with similar mortality. Antiinflammatory interventions may help improve outcomes.

Regional Evaluation of Two SARS-CoV-2 Antigen Rapid Diagnostic Tests in East Africa.

The clinical performance of two rapid antigen tests for the diagnosis of Severe Acute Respiratory Coronavirus (SARS-CoV-2) were regionally evaluated in East African populations. Swabs were collected from 1,432 individuals from five Partner States of the East African Community (Tanzania, Uganda, Burundi, Rwanda and South Sudan). The two rapid antigen tests (Bionote NowCheck COVID-19 Ag and SD Biosensor STANDARD Q COVID-19 Ag) were evaluated against the detection of SARS-CoV-2 RNA by the Reverse Transcription PCR (RT-PCR) gold standard. Of the concordant results with both RT-PCR and rapid antigen test data (862 for Bionote and 852 for SD Biosensor), overall clinical sensitivity was 60% and 50% for the Bionote NowCheck and the SD Biosensor STANDARD Q, respectively. Stratification by viral load, including samples with RT-PCR cycle thresholds (Ct) of <25, improved sensitivity to 90% for both rapid diagnostic tests (RDTs). Overall specificity was good at 99% for both antigen tests. Taken together, the clinical performance of both Ag-RDTs in real world settings within the East African target population was lower than has been reported elsewhere and below the acceptable levels for sensitivity of >80%, as defined by the WHO. Therefore, the rapid antigen test alone should not be used for diagnosis but could be used as part of an algorithm to identify potentially infectious individuals with high viral load. IMPORTANCE Accurate diagnostic tests are essential to both support the management and containment of outbreaks, as well as inform appropriate patient care. In the case of the SARS-CoV-2 pandemic, antigen Rapid Diagnostic Tests (Ag-RDTs) played a major role in this function, enabling widespread testing by untrained individuals, both at home and within health facilities. In East Africa, a number of SARS-CoV-2 Ag-RDTs are available; however, there remains little information on their true test performance within the region, in the hands of the health workers routinely carrying out SARS-CoV-2 diagnostics. This study contributes test performance data for two commonly used SARS-CoV-2 Ag-RDTs in East Africa, which will help inform the use of these RDTs within the region.

The East African Community mobile laboratory network prepares for monkeypox outbreaks.

In response to the largest recorded monkeypox virus outbreak outside of endemic Central and Western Africa, the East African Community (EAC), in cooperation with the Bernhard-Nocht- Institute for Tropical Medicine, coordinated an emergency monkeypox diagnostic training for the East African Region. As of June 2022, the Democratic Republic of Congo reported a steady increase of suspected monkeypox cases, increasing the risk of spill-over into the remaining six EAC Partner States. Within the existing EAC Mobile Laboratories project, laboratory experts of the National Public Health Laboratories of the remaining six EAC Partner States (Burundi, Rwanda, Tanzania, Kenya, Uganda, and South Sudan) participated in the workshop and were trained in the reception of suspect samples, DNA extraction and diagnosis using real-time polymerase chain reaction (RT-PCR). The EAC region is now equipped with the tools to prepare and rapidly respond to any emerging monkeypox outbreak.

The impact of viraemia on inflammatory biomarkers and CD4+ cell subpopulations in HIV-infected children in sub-Saharan Africa.

OBJECTIVE: To determine the impact of virological control on inflammation and cluster of differentiation 4 depletion among HIV-infected children initiating antiretroviral therapy (ART) in sub-Saharan Africa. DESIGN: Longitudinal cohort study. METHODS: In a sub-study of the ARROW trial (ISRCTN24791884), we measured longitudinal HIV viral loads, inflammatory biomarkers (C-reactive protein, tumour necrosis factor alpha, interleukin 6 (IL-6), soluble CD14) and (Uganda only) whole blood immunophenotype by flow cytometry in 311 Zimbabwean and Ugandan children followed for median 3.5 years on first-line ART. We classified each viral load measurement as consistent suppression, blip/post-blip, persistent low-level viral load or rebound. We used multi-level models to estimate rates of increase or decrease in laboratory markers, and Poisson regression to estimate the incidence of clinical events. RESULTS: Overall, 42% children experienced viral blips, but these had no significant impact on immune reconstitution or inflammation. Persistent detectable viraemia occurred in one-third of children and prevented further immune reconstitution, but had little impact on inflammatory biomarkers. Virological rebound to ≥5000 copies/ml was associated with arrested immune reconstitution, rising IL-6 and increased risk of clinical disease progression. CONCLUSIONS: As viral load testing becomes more available in sub-Saharan Africa, repeat testing algorithms will be required to identify those with virological rebound, who need switching to prevent disease progression, whilst preventing unnecessary second-line regimen initiation in the majority of children with detectable viraemia who remain at low risk of disease progression.

Inflammatory biomarkers in HIV-infected children hospitalized for severe malnutrition in Uganda and Zimbabwe.

OBJECTIVES: A proportion of HIV-infected children with advanced disease develop severe malnutrition soon after antiretroviral therapy (ART) initiation. We tested the hypothesis that systemic inflammation underlies the pathogenesis of severe malnutrition in HIV-infected children. DESIGN: Cross-sectional laboratory substudy in 613 HIV-infected children initiating ART in Uganda and Zimbabwe. METHODS: We measured C-reactive protein (CRP), TNFα, IL-6 and soluble CD14 by ELISA in cryopreserved plasma at baseline (pre-ART) and week-4 (children with severe malnutrition only). Independent associations between baseline biomarkers and subsequent hospitalization for severe malnutrition were identified using multivariable fractional polynomial logistic regression. RESULTS: Compared with children without severe malnutrition (n = 574, median age 6.3 years, median baseline weight-for-age Z-score -2.2), children hospitalized for severe malnutrition post-ART (n = 39, median age 2.3 years, median baseline weight-for-age Z-score -4.8) had higher baseline CRP [median 13.5 (interquartile range 5.5, 41.1) versus 4.1 (1.4, 14.4) mg/l; P = 0.003] and IL-6 [median 9.2 (6.7, 15.6) versus 5.9 (4.6, 9.3) pg/ml; P < 0.0001], but similar overall TNFα, soluble CD14 and HIV viral load (all P > 0.06). In a multivariable model, higher pre-ART IL-6, lower TNFα and lower weight-for-age were independently associated with subsequent hospitalization for severe malnutrition. Between weeks 0 and 4, there was a significant rise in CRP, IL-6 and soluble CD14, and fall in TNFα and HIV viral load in children hospitalized for severe malnutrition (all P < 0.02). CONCLUSION: Pre-ART IL-6 and TNFα were more strongly associated with hospitalization for severe malnutrition than CD4 cell count or viral load, highlighting the importance of inflammation at the time of ART initiation in HIV-infected children.

Cotrimoxazole reduces systemic inflammation in HIV infection by altering the gut microbiome and immune activation.

Long-term cotrimoxazole prophylaxis reduces mortality and morbidity in HIV infection, but the mechanisms underlying these clinical benefits are unclear. Here, we investigate the impact of cotrimoxazole on systemic inflammation, an independent driver of HIV mortality. In HIV-positive Ugandan and Zimbabwean children receiving antiretroviral therapy, we show that plasma inflammatory markers were lower after randomization to continue (n = 144) versus stop (n = 149) cotrimoxazole. This was not explained by clinical illness, HIV progression, or nutritional status. Because subclinical enteropathogen carriage and enteropathy can drive systemic inflammation, we explored cotrimoxazole effects on the gut microbiome and intestinal inflammatory biomarkers. Although global microbiome composition was unchanged, viridans group Streptococci and streptococcal mevalonate pathway enzymes were lower among children continuing (n = 36) versus stopping (n = 36) cotrimoxazole. These changes were associated with lower fecal myeloperoxidase. To isolate direct effects of cotrimoxazole on immune activation from antibiotic effects, we established in vitro models of systemic and intestinal inflammation. In vitro cotrimoxazole had modest but consistent inhibitory effects on proinflammatory cytokine production by blood leukocytes from HIV-positive (n = 16) and HIV-negative (n = 8) UK adults and reduced IL-8 production by gut epithelial cell lines. Collectively we demonstrate that cotrimoxazole reduces systemic and intestinal inflammation both indirectly via antibiotic effects on the microbiome and directly by blunting immune and epithelial cell activation. Synergy between these pathways may explain the clinical benefits of cotrimoxazole despite high antimicrobial resistance, providing further rationale for extending coverage among people living with HIV in sub-Saharan Africa.