Acceptability and Feasibility of the Plasma Separation Card for an Integrated Model of Care for HBV and HCV Screening Among People Attending HIV Clinics in Cameroon and Uganda.

BACKGROUND: Sub-Saharan African countries have a high burden of viral hepatitis and poor access to screening and care. The aim of this study was to evaluate the feasibility and acceptability of using the plasma separation card (PSC) for viral hepatitis B and C screening among people living with HIV (PLHIV) in Cameroon and Uganda. METHODS: This is a cross-sectional study carried out between 05/2021 and 03/2023 including 192 PLHIV in Cameroon (n = 104) and Uganda (n = 88). Basic sociodemographic variables and whole blood samples were collected. Adequate filling with blood of PSCs was used to determine feasibility together with participant responses to questions on acceptability. A logistic regression model was carried out to assess the relationship between PSC acceptability and factors of interest. RESULTS: 70% of participants reported PSC as an acceptable viral hepatitis screening tool, and it was significantly more accepted in Uganda than Cameroon (100% vs. 43.2%, p < 0.001). Similarly, 75% of PSCs had at least one spot sample filled and were viable for analysis, 99% were correctly filled in Uganda and 53.4% in Cameroon. Reported ease of method performance (aOR: 24.77 95% CI 2.97-206.42, p = 0.003) and reduced collection time (aOR: 3.73 95% CI 1.26-11.04, p = 0.017) were associated with greater odds of PSC acceptance. HBsAg + and anti-HCV + prevalence were 11.1% and 1.0%, respectively. CONCLUSIONS: In spite of country differences, overall, the PSC was reported as a feasible and acceptable viral hepatitis testing method. Acceptability and feasibility of the method must be explored in heterogeneous target communities and qualitative research to better understand country-specific barriers and facilitators should be carried out.

External quality assessment to support the WHO ProSPeRo study for the evaluation of two dual HIV/syphilis point-of-care tests in seven countries.

BACKGROUND: Sexually transmitted infections (STIs) such as syphilis and HIV remain to be a significant public health issue worldwide. Dual rapid point-of-care tests (POCTs) have shown promise for detecting antibodies to HIV and syphilis but have not been fully evaluated in the field. Our study supported the WHO ProSPeRo study on Sexually Transmitted Infection Point-of-Care Testing (STI POCT) by providing external quality assessment (EQA) for HIV and syphilis testing in reference laboratories and their associated clinical sites in seven countries. METHODS: HIV/syphilis serum liquid and dried tube specimen (DTS) panels were prepared by CDC. Liquid panels were distributed to the reference laboratories for three rounds of testing using commercially and locally available laboratory-based serological tests. DTS panels were sent to the clinical testing sites for 8 rounds of POC testing using the Abbott SD BIOLINE HIV/Syphilis Duo test (hereafter referred to as SD BIOLINE) and the Chembio Dual Path Platform (DPP) HIV-Syphilis assay. EQA panels were tested at CDC using the Rapid Plasma Reagin (RPR) test and the Treponema pallidum Particle Agglutination assay (TP-PA) for syphilis antibodies. Genetic Systems HIV-1/HIV-2 Plus O EIA, Geenius HIV Supplemental Assay and the Oraquick Advance HIV test were used to detect HIV antibodies in the EQA panels. Results from the reference laboratories and POCT sites were compared to those obtained at the CDC and a percentage agreement was calculated. RESULTS: Qualitative RPR and TP-PA performed at the reference laboratories demonstrated 95.4-100% agreement with CDC results while quantitative RPR and TP-PA tests demonstrated 87.7% and 89.2% agreement, respectively. A 93.8% concordance rate was observed for qualitative HIV testing in laboratories. EQA testing at clinical sites using dual tests showed 98.7% and 99.1% agreement for detection of HIV antibodies and eight out of 10 sites had > 95.8% agreement for syphilis testing. However, two clinical sites showed only 65.0-66.7% agreement for SD BIOLINE and 84.0-86.7% for DPP, respectively, for syphilis testing. CONCLUSIONS: Overall, laboratories demonstrated high EQA performance in this study. Both HIV/syphilis POCTs gave expected results in the clinic-based evaluations using DTS. However, testing errors were identified in a few testing sites suggesting the necessity for continuous training and monitoring the quality of POC testing.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

Limited threat of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletion to the utility of HRP2-based malaria RDTs in Northern Uganda.

BACKGROUND: Rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein-2 (PfHRP2) are exclusively deployed in Uganda, but deletion of the pfhrp2/3 target gene threatens their usefulness as malaria diagnosis and surveillance tools. METHODS: A cross-sectional survey was conducted at 40 sites across four regions of Uganda in Acholi, Lango, W. Nile and Karamoja from March 2021 to June 2023. Symptomatic malaria suspected patients were recruited and screened with both HRP2 and pan lactate dehydrogenase (pLDH) detecting RDTs. Dried blood spots (DBS) were collected from all patients and a random subset were used for genomic analysis to confirm parasite species and pfhrp2 and pfhrp3 gene status. Plasmodium species was determined using a conventional multiplex PCR while pfhrp2 and pfhrp3 gene deletions were determined using a real-time multiplex qPCR. Expression of the HRP2 protein antigen in a subset of samples was further assessed using a ELISA. RESULTS: Out of 2435 symptomatic patients tested for malaria, 1504 (61.8%) were positive on pLDH RDT. Overall, qPCR confirmed single pfhrp2 gene deletion in 1 out of 416 (0.2%) randomly selected samples that were confirmed of P. falciparum mono-infections. CONCLUSION: These findings show limited threat of pfhrp2/3 gene deletions in the survey areas suggesting that HRP2 RDTs are still useful diagnostic tools for surveillance and diagnosis of P. falciparum malaria infections in symptomatic patients in this setting. Periodic genomic surveillance is warranted to monitor the frequency and trend of gene deletions and its effect on RDTs.

Atypical B cells consist of subsets with distinct functional profiles.

Atypical B cells are a population of activated B cells that are commonly enriched in individuals with chronic immune activation but are also part of a normal immune response to infection or vaccination. To better define the role of atypical B cells in the human adaptive immune response, we performed single-cell sequencing of transcriptomes, cell surface markers, and B cell receptors in individuals with chronic exposure to the malaria parasite Plasmodium falciparum, a condition known to lead to accumulation of circulating atypical B cells. We identified three previously uncharacterized populations of atypical B cells with distinct transcriptional and functional profiles and observed marked differences among these three subsets in their ability to produce immunoglobulin G upon T-cell-dependent activation. Our findings help explain the conflicting observations in prior studies regarding the function of atypical B cells and highlight their different roles in the adaptive immune response in chronic inflammatory conditions.

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.

Uptake and effectiveness of a mobile application for real-time reporting and quality assurance of decentralized SARS-CoV-2 testing in Uganda.

Background: Effective management of the COVID-19 pandemic required rapid expansion of diagnosis. The introduction of antigen tests presented an opportunity to decentralize testing, but raised challenges with ensuring accurate and timely reporting of testing data, which is essential to guide the response. Digital solutions can help address this challenge and provide more efficient means of monitoring and quality assurance. Methods: Uganda's existing laboratory investigation form was digitized in the form of an Android-based application, eLIF, which was developed by the Central Public Health Laboratory and implemented in 11 high-volume facilities between December 2021 and May 2022. The app enabled healthcare workers to report testing data via mobile phone or tablet. Uptake of the tool was monitored through a dashboard that enabled real-time visibility into data being transmitted from sites, as well as qualitative insights from site visits and online questionnaires. Results and discussion: A total of 15,351 tests were conducted at the 11 health facilities during the study period. Of these, 65% were reported through eLIF, while 12% were reported through preexisting Excel-based tools. However, 23% of tests were only captured in paper registers and not transmitted to the national database, illustrating the need for increased uptake of digital tools to ensure real-time data reporting. While data captured through eLIF were transmitted to the national database within 0-3 days (min, max), data transmitted through Excel were transmitted in within 0-37 days (min, max), and data for paper-based reporting took up to 3 months. The majority of healthcare workers interviewed in an endpoint questionnaire responded that eLIF improved timeliness of patient management, and reduced reporting time. However, some functions of the app were not successfully implemented, such as providing random selections of samples for external quality assurance and enabling seamless linkage of these data. Challenges arose from broader operational complexities, such as staff workload, frequent task-shifting and unexpected changes to facility workflows, which limited adherence to the envisioned study procedures. Ongoing improvements are needed to adjust to these realities, to strengthen the technology and support to healthcare workers using it, to optimize the impact of this digital intervention.

Metagenomic next-generation sequencing to characterize potential etiologies of non-malarial fever in a cohort living in a high malaria burden area of Uganda.

Causes of non-malarial fevers in sub-Saharan Africa remain understudied. We hypothesized that metagenomic next-generation sequencing (mNGS), which allows for broad genomic-level detection of infectious agents in a biological sample, can systematically identify potential causes of non-malarial fevers. The 212 participants in this study were of all ages and were enrolled in a longitudinal malaria cohort in eastern Uganda. Between December 2020 and August 2021, respiratory swabs and plasma samples were collected at 313 study visits where participants presented with fever and were negative for malaria by microscopy. Samples were analyzed using CZ ID, a web-based platform for microbial detection in mNGS data. Overall, viral pathogens were detected at 123 of 313 visits (39%). SARS-CoV-2 was detected at 11 visits, from which full viral genomes were recovered from nine. Other prevalent viruses included Influenza A (14 visits), RSV (12 visits), and three of the four strains of seasonal coronaviruses (6 visits). Notably, 11 influenza cases occurred between May and July 2021, coinciding with when the Delta variant of SARS-CoV-2 was circulating in this population. The primary limitation of this study is that we were unable to estimate the contribution of bacterial microbes to non-malarial fevers, due to the difficulty of distinguishing bacterial microbes that were pathogenic from those that were commensal or contaminants. These results revealed the co-circulation of multiple viral pathogens likely associated with fever in the cohort during this time period. This study illustrates the utility of mNGS in elucidating the multiple potential causes of non-malarial febrile illness. A better understanding of the pathogen landscape in different settings and age groups could aid in informing diagnostics, case management, and public health surveillance systems.

Impact of high human genetic diversity in Africa on immunogenicity and efficacy of RTS,S/AS01 vaccine.

In modern medicine, vaccination is one of the most effective public health strategies to prevent infectious diseases. Indisputably, vaccines have saved millions of lives by reducing the burden of many serious infections such as polio, tuberculosis, measles, pneumonia, and tetanus. Despite the recent recommendation by the World Health Organization (WHO) to roll out RTS,S/AS01, this malaria vaccine still faces major challenges of variability in its efficacy partly due to high genetic variation in humans and malaria parasites. Immune responses to malaria vary between individuals and populations. Human genetic variation in immune system genes is the probable cause for this heterogeneity. In this review, we will focus on human genetic factors that determine variable responses to vaccination and how variation in immune system genes affect the immunogenicity and efficacy of the RTS,S/AS01 vaccine.

Virologic Nonsuppression Among Patients With HIV Newly Diagnosed With Cancer at Uganda Cancer Institute: A Cross-Sectional Study.

PURPOSE: AIDS-related mortality declined markedly since the introduction of antiretroviral therapy (ART); however, cancer mortality in Africa was higher than its incidence in 2020. People living with HIV (PLWHIV) are at an increased risk of malignancy and death from malignancy compared with the general population. In Uganda, AIDS-defining malignancies (ADMs), including cervical cancer, Kaposi sarcoma, and non-Hodgkin lymphoma, are among the commonest malignancies. Virologic nonsuppression has been identified as an important predictor of mortality among PLWHIV diagnosed with cancer. This study aimed to determine the prevalence and to identify factors associated with virologic nonsuppression among PLWHIV newly diagnosed with cancer. METHODS: This was a cross-sectional study that was carried out between December 2018 and April 2019 at the Uganda Cancer Institute. PLWHIV who had been on ART for at least 6 months and were newly diagnosed with cancer were enrolled. RESULTS: A total of 167 participants were enrolled. Cervical cancer was the commonest ADM (n = 45; 50.6%) of all ADMs, while esophageal and breast cancers were the commonest non-ADMs, accounting for 17.5% (n = 14) each of all non-ADMs. The prevalence of virologic nonsuppression was 15%. Having Kaposi sarcoma (odds ratio [OR], 8.15; P = .003), being poorly adherent to ART (OR, 4.1; P = .045), and being on second-line ART (OR, 5.68; P = .011) were associated with virologic nonsuppression. CONCLUSION: The prevalence of virologic nonsuppression is high among patients with HIV newly diagnosed with cancer. These findings emphasize the need for strengthening of adherence strategies, optimizing ART regimens, and prioritization of viral load testing among PLWHIV with newly diagnosed malignancy.

Antibodies to repeat-containing antigens in Plasmodium falciparum are exposure-dependent and short-lived in children in natural malaria infections.

Protection against Plasmodium falciparum, which is primarily antibody-mediated, requires recurrent exposure to develop. The study of both naturally acquired limited immunity and vaccine induced protection against malaria remains critical for ongoing eradication efforts. Towards this goal, we deployed a customized P. falciparum PhIP-seq T7 phage display library containing 238,068 tiled 62-amino acid peptides, covering all known coding regions, including antigenic variants, to systematically profile antibody targets in 198 Ugandan children and adults from high and moderate transmission settings. Repeat elements - short amino acid sequences repeated within a protein - were significantly enriched in antibody targets. While breadth of responses to repeat-containing peptides was twofold higher in children living in the high versus moderate exposure setting, no such differences were observed for peptides without repeats, suggesting that antibody responses to repeat-containing regions may be more exposure dependent and/or less durable in children than responses to regions without repeats. Additionally, short motifs associated with seroreactivity were extensively shared among hundreds of antigens, potentially representing cross-reactive epitopes. PfEMP1 shared motifs with the greatest number of other antigens, partly driven by the diversity of PfEMP1 sequences. These data suggest that the large number of repeat elements and potential cross-reactive epitopes found within antigenic regions of P. falciparum could contribute to the inefficient nature of malaria immunity.

Seroprevalence of Antibodies to SARS-CoV-2 in Rural Households in Eastern Uganda, 2020-2022.

Importance: Estimating the true burden of SARS-CoV-2 infection has been difficult in sub-Saharan Africa owing to asymptomatic infections and inadequate testing capacity. Antibody responses from serologic surveys can provide an estimate of SARS-CoV-2 exposure at the population level. Objective: To estimate SARS-CoV-2 seroprevalence, attack rates, and reinfection in eastern Uganda using serologic surveillance from 2020 to early 2022. Design, Setting, and Participants: This cohort study was conducted in the Tororo and Busia districts of eastern Uganda. Plasma samples from participants in the Program for Resistance, Immunology, Surveillance, and Modeling of Malaria in Uganda Border Cohort were obtained at 4 sampling intervals: October to November 2020, March to April 2021, August to September 2021, and February to March 2022. Each participant contributed up to 4 time points for SARS-CoV-2 serology, with almost half of all participants contributing at all 4 time points, and almost 90% contributing at 3 or 4 time points. Information on SARS-CoV-2 vaccination status was collected from participants, with the earliest reported vaccinations in the cohort occurring in May 2021. Main Outcomes and Measures: The main outcomes of this study were antibody responses to the SARS-CoV-2 spike protein as measured with a bead-based serologic assay. Individual-level outcomes were aggregated to population-level SARS-CoV-2 seroprevalence, attack rates, and boosting rates. Estimates were weighted by the local age distribution according to census data. Results: A total of 1483 samples from 441 participants living in 76 households were tested. Of the 441 participants, 245 (55.6%) were female, and their mean (SD) age was 16.04 (16.04) years. By the end of the Delta wave and before widespread vaccination, adjusted SARS-CoV-2 seroprevalence was 67.7% (95% credible interval [CrI], 62.5%-72.6%) in the study population. During the subsequent Omicron wave, 84.8% (95% CrI, 67.9%-93.7%) of unvaccinated, previously seronegative individuals were infected for the first time, and 50.8% (95% CrI, 40.6%-59.7%) of unvaccinated, already seropositive individuals were likely reinfected, leading to an overall seropositivity of 96.0% (95% CrI, 93.4%-97.9%) in this population. These results suggest a lower probability of reinfection in individuals with higher preexisting antibody levels. There was evidence of household clustering of SARS-CoV-2 seroconversion. No significant associations were found between SARS-CoV-2 seroconversion and gender, household size, or recent Plasmodium falciparum malaria exposure. Conclusions and Relevance: In this cohort study in a rural population in eastern Uganda, there was evidence of very high SARS-CoV-2 infection rates throughout the pandemic inconsistent with national level case data and high reinfection rates during the Omicron wave.

Effects of anti-malarial prophylaxes on maternal transfer of Immunoglobulin-G (IgG) and association to immunity against Plasmodium falciparum infections among children in a Ugandan birth cohort.

BACKGROUND: The in-utero transfer of malaria specific IgG to the fetus in Plasmodium falciparum infected pregnant women potentially plays a role in provision of immune protection against malaria in the first birth year. However, the effect of Intermittent Prophylactic Treatment in Pregnancy (IPTp) and placental malaria on the extent of in-utero antibody transfer in malaria endemic regions like Uganda remain unknown. The aim of this study was thus to establish the effect of IPTp on in-utero transfer of malaria specific IgG to the fetus and the associated immune protection against malaria in the first birth year of children born to mothers who had P. falciparum infection during pregnancy in Uganda. METHODS: We screened a total of 637 cord blood samples from a double blinded randomized clinical trial on Sulfadoxine-Pyrimethamine (SP) and Dihydroartemisinin-Piperaquine (DP) IPTp in a Ugandan birth cohort; study conducted from Busia, Eastern Uganda. Luminex assay was used to measure the cord levels of IgG sub-types (IgG1, IgG2, IgG3 and IgG4) against 15 different P. falciparum specific antigens, with tetanus toxoid (t.t) as a control antigen. Man-Whitney U test (non-parametric) in STATA (ver15) was used in statistical analysis of the samples. In addition, Multivariate cox regression analysis was used to determine the effect of maternal transfer of IgG on the incidence of malaria in the first birth year of children under study. RESULTS: Mothers on SP expressed higher levels of cord IgG4 against erythrocyte binding antigens (EBA140, EBA175 and EBA181) (p<0.05). Placental malaria did not affect cord levels of IgG sub-types against selected P. falciparum specific antigens (p>0.05). Children who expressed higher levels (75th percentile) of total IgG against the six key P. falciparum antigens (Pf SEA, Rh4.2, AMA1, GLURP, Etramp5Ag1 and EBA 175) had higher risk of malaria in the first birth year; AHRs: 1.092, 95% CI: 1.02-1.17 (Rh4.2); 1.32, 95% CI: 1.00-1.74 (PfSEA); 1.21, 95%CI: 0.97-1.52 (Etramp5Ag1); 1.25, 95%CI: 0.98-1.60 (AMA1); 1.83, 95%CI: 1.15-2.93 (GLURP) (GLURP), and 1.35,; 95%CI: 1.03-1.78 (EBA175). Children born to mothers categorized as poorest had the highest risk of malaria infections in the first birth year (AHR: 1.79, 95% CI: 1.31-2.4). Children born to mothers who had malaria infections during gestation had higher risk of getting malaria in the first birth year (AHR 1.30; 95%CI: 0.97-1.7). CONCLUSION: Malaria prophylaxis in pregnant mothers using either DP or SP does not affect expression of antibodies against P. falciparum specific antigens in the cord blood. Poverty and malaria infections during pregnancy are key risk factors of malaria infections in the first birth year of growth of children. Antibodies against P. falciparum specific antigens does not protect against parasitemia and malaria infections in the first birth year of children born in malaria endemic areas.

HIV Viral Load Scale-up Among Children and Adolescents: Trends in Viral Load Suppression, Sample Type and Processing in 7 PEPFAR Countries, 2015-2018.

HIV-positive children and adolescents face gaps in viral load (VL) testing. To understand trends in pediatric/adolescent VL testing, 7 countries collected data from Laboratory Information Management Systems. Results showed increasing proportion of VL tests done through dried blood spot (DBS) and decreased sample rejection rates for DBS compared with plasma, supporting use of DBS VL when skilled phlebotomy is unavailable.

Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria.

Natural killer (NK) cells likely play an important role in immunity to malaria, but the effect of repeated malaria on NK cell responses remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor γ-chain, increased histone methylation, and increased protein expression of LAG-3, KIR, and LILRB1. CD56neg NK cells were highly functional and displayed greater antibody-dependent cellular cytotoxicity than CD56dim NK cells. Higher frequencies of CD56neg NK cells were associated with protection against symptomatic malaria and high parasite densities. After marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to Plasmodium falciparum is required to maintain this modified, adaptive-like NK cell subset.

Asymptomatic malaria infection, associated factors and accuracy of diagnostic tests in a historically high transmission setting in Northern Uganda.

BACKGROUND: Asymptomatic malaria infections are important parasite reservoirs and could sustain transmission in the population, but they are often unreported. A community-based survey was conducted to investigate the prevalence and factors associated with asymptomatic malaria infections in a historically high transmission setting in northern Uganda. METHODS: Using a cross-sectional design, 288 children aged 2-15 years were enrolled and tested for the presence of malaria parasites using rapid diagnostic tests (RDTs) and blood smear microscopy between January to May 2022. Statistical analysis was performed using the exact binomial and Fisher's exact test with p ≤ 0.05 indicating significance. The logistic regression was used to explore factors associated with asymptomatic malaria infections. RESULTS: Overall, the prevalence of asymptomatic infection was 34.7% (95% CI 29.2-40.5) with the highest observed in children 5-10 years 45.9% (95% CI 35.0-57.0). Gweri village accounted for 39.1% (95% CI 27.6-51.6) of malaria infections. Median parasite density was 1500 parasites/µl of blood. Plasmodium falciparum was the dominant species (86%) followed by Plasmodium malariae (5%). Factors associated with asymptomatic malaria infection were sleeping under mosquito net (Adjusted Odds Ratio (aOR) 0.27; 95% CI 0.13-0.56), p = 0.001 and presence of village health teams (VHTs) (aOR 0.02; 95% CI 0.01-0.45), p = 0.001. Sensitivity and specificity were higher for the P. falciparum/pLDH RDTs compared to HRP2-only RDTs, 90% (95% CI 86.5-93.5) and 95.2% (95% CI 92.8-97.7), p = 0.001, respectively. CONCLUSION: Asymptomatic malaria infections were present in the study population and this varied with place and person in the different age groups. Plasmodium falciparum was the dominant parasite species however the presence of P. malariae and Plasmodium ovale was observed, which may have implication for the choice and deployment of diagnostic tools. Individuals who slept under mosquito net or had presence of functional VHTs were less likely to have asymptomatic malaria infection. P.f/pLDH RDTs performed better than the routinely used HRP2 RDTs. In view of these findings, investigation and reporting of asymptomatic malaria reservoirs through community surveys is recommended for accurate disease burden estimate and better targeting of control.

LLIN Evaluation in Uganda Project (LLINEUP2)-Factors associated with coverage and use of long­lasting insecticidal nets following the 2020-21 national mass distribution campaign: a cross-sectional survey of 12 districts.

BACKGROUND: In 2020-2021, long-lasting insecticidal nets (LLINs) were distributed nationwide in Uganda during the COVID-19 pandemic. A cross-sectional survey was conducted in 12 districts to evaluate the impact of the campaign 1-5 months after LLIN distribution. METHODS: During April-May 2021, households were randomly selected from target areas (1-7 villages) surrounding 12 government-run health facilities established as Malaria Reference Centres; at least 50 households were enrolled per cluster. Outcomes included household ownership of LLINs distributed through the universal coverage campaign (UCC) (at least one UCC LLIN), adequate coverage of UCC LLINs (at least one UCC LLIN per 2 residents), and use of LLINs (resident slept under a LLIN the previous night). Multivariate logistic regression models were used to identify household- and individual-level factors associated with outcomes, controlling for clustering around health facilities. RESULTS: In total, 634 households, with 3342 residents and 1631 bed-nets, were included. Most households (93.4%) owned at least 1 UCC LLIN, but only 56.8% were adequately covered by UCC LLINs. In an adjusted analysis, the factor most strongly associated with adequate coverage by UCC LLINs was fewer household residents (1-4 vs 7-14; adjusted odds ratio [aOR] 12.96, 95% CI 4.76-35.26, p < 0.001; 5-6 vs 7-14 residents; aOR 2.99, 95% CI 1.21-7.42, p = 0.018). Of the 3166 residents of households that owned at least one UCC LLIN, only 1684 (53.2%) lived in adequately covered households; 89.9% of these used an LLIN the previous night, compared to 1034 (69.8%) of 1482 residents living in inadequately covered households. In an adjusted analysis, restricted to residents of inadequately covered households, LLIN use was higher in children under-five than those aged 5-15 years (aOR 3.04, 95% CI 2.08-4.46, p < 0.001), and higher in household heads than distantly-related residents (aOR 3.94, 95% CI 2.38-6.51, p < 0.001). CONCLUSIONS: Uganda's 2021-21 campaign was successful, despite the COVID-19 pandemic. In future campaigns, strategies should be adopted to ensure high LLIN coverage, particularly for larger households. A better understanding of the drivers of LLIN use within households is needed to guide future interventions, educational messages, and behaviour change communication strategies; school-aged children and distantly-related residents appear vulnerable and could be targeted.

Reconstructing the SARS-CoV-2 epidemic in eastern Uganda through longitudinal serosurveillance in a malaria cohort.

Importance: Estimating the true burden of SARS-CoV-2 infection has been difficult in sub-Saharan Africa due to asymptomatic infections and inadequate testing capacity. Antibody responses from serologic surveys can provide an estimate of SARS-CoV-2 exposure at the population level. Objective: To estimate SARS-CoV-2 seroprevalence, attack rates, and re-infection in eastern Uganda using serologic surveillance from 2020 to early 2022. Design: Plasma samples from participants in the Program for Resistance, Immunology, Surveillance, and Modeling of Malaria in Uganda (PRISM) Border Cohort were obtained at four sampling intervals: October-November 2020; March-April 2021; August-September 2021; and February-March 2022. Setting: Tororo and Busia districts, Uganda. Participants: 1,483 samples from 441 participants living in 76 households were tested. Each participant contributed up to 4 time points for SARS-CoV-2 serology, with almost half of all participants contributing at all 4 time points, and almost 90% contributing at 3 or 4 time points. Information on SARS-CoV-2 vaccination status was collected from participants, with the earliest reported vaccinations in the cohort occurring in May 2021. Main Outcomes and Measures: The main outcomes of this study were antibody responses to the SARS-CoV-2 spike protein as measured with a bead-based serologic assay. Individual-level outcomes were aggregated to population-level SARS-CoV-2 seroprevalence, attack rates, and boosting rates. Estimates were weighted by the local age distribution based on census data. Results: By the end of the Delta wave and before widespread vaccination, nearly 70% of the study population had experienced SARS-CoV-2 infection. During the subsequent Omicron wave, 85% of unvaccinated, previously seronegative individuals were infected for the first time, and ~50% or more of unvaccinated, already seropositive individuals were likely re-infected, leading to an overall 96% seropositivity in this population. Our results suggest a lower probability of re-infection in individuals with higher pre-existing antibody levels. We found evidence of household clustering of SARS-CoV-2 seroconversion. We found no significant associations between SARS-CoV-2 seroconversion and gender, household size, or recent Plasmodium falciparum malaria exposure. Conclusions and Relevance: Findings from this study are consistent with very high infection rates and re-infection rates for SARS-CoV-2 in a rural population from eastern Uganda throughout the pandemic.

Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray.

The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro transcription/translation (IVTT) systems-a similarly high-throughput protein expression method-are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on the same protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from different expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clear correlation between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.