Correction: Limitations of rapid diagnostic tests in malaria surveys in areas with varied transmission intensity in Uganda 2017-2019: Implications for selection and use of HRP2 RDTs.

[This corrects the article DOI: 10.1371/journal.pone.0244457.].

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.

Evaluation of malaria outbreak detection methods, Uganda, 2022.

BACKGROUND: Malaria outbreaks are detected by applying the World Health Organization (WHO)-recommended thresholds (the less sensitive 75th percentile or mean + 2 standard deviations [2SD] for medium-to high-transmission areas, and the more sensitive cumulative sum [C-SUM] method for low and very low-transmission areas). During 2022, > 50% of districts in Uganda were in an epidemic mode according to the 75th percentile method used, resulting in a need to restrict national response to districts with the highest rates of complicated malaria. The three threshold approaches were evaluated to compare their outbreak-signaling outputs and help identify prioritization approaches and method appropriateness across Uganda. METHODS: The three methods were applied as well as adjusted approaches (85th percentile and C-SUM + 2SD) for all weeks in 2022 for 16 districts with good reporting rates ( ≥ 80%). Districts were selected from regions originally categorized as very low, low, medium, and high transmission; district thresholds were calculated based on 2017-2021 data and re-categorized them for this analysis. RESULTS: Using district-level data to categorize transmission levels resulted in re-categorization of 8/16 districts from their original transmission level categories. In all districts, more outbreak weeks were detected by the 75th percentile than the mean + 2SD method (p < 0.001). For all 9 very low or low-transmission districts, the number of outbreak weeks detected by C-SUM were similar to those detected by the 75th percentile. On adjustment of the 75th percentile method to the 85th percentile, there was no significant difference in the number of outbreak weeks detected for medium and low transmission districts. The number of outbreak weeks detected by C-SUM + 2SD was similar to those detected by the mean + 2SD method for all districts across all transmission intensities. CONCLUSION: District data may be more appropriate than regional data to categorize malaria transmission and choose epidemic threshold approaches. The 75th percentile method, meant for medium- to high-transmission areas, was as sensitive as C-SUM for low- and very low-transmission areas. For medium and high-transmission areas, more outbreak weeks were detected with the 75th percentile than the mean + 2SD method. Using the 75th percentile method for outbreak detection in all areas and the mean + 2SD for prioritization of medium- and high-transmission areas in response may be helpful.

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.

Genetic diversity and genetic relatedness in Plasmodium falciparum parasite population in individuals with uncomplicated malaria based on microsatellite typing in Eastern and Western regions of Uganda, 2019-2020.

BACKGROUND: Genetic diversity and parasite relatedness are essential parameters for assessing impact of interventions and understanding transmission dynamics of malaria parasites, however data on its status in Plasmodium falciparum populations in Uganda is limited. Microsatellite markers and DNA sequencing were used to determine diversity and molecular characterization of P. falciparum parasite populations in Uganda. METHODS: A total of 147 P. falciparum genomic DNA samples collected from cross-sectional surveys in symptomatic individuals of 2-10 years were characterized by genotyping of seven highly polymorphic neutral microsatellite markers (n = 85) and genetic sequencing of the Histidine Rich Protein 2 (pfhrp2) gene (n = 62). ArcGIS was used to map the geographical distribution of isolates while statistical testing was done using Student's t-test or Wilcoxon's rank-sum test and Fisher's exact test as appropriate at P ≤ 0.05. RESULTS: Overall, 75.5% (95% CI 61.1-85.8) and 24.5% (95% CI14.2-38.9) of parasites examined were of multiclonal (mixed genotype) and single clone infections, respectively. Multiclonal infections occurred more frequently in the Eastern region 73.7% (95% CI 48.8-89.1), P < 0.05. Overall, multiplicity of infection (MOI) was 1.9 (95% CI 1.7-2.1), P = 0.01 that was similar between age groups (1.8 vs 1.9), P = 0.60 and regions (1.9 vs 1.8), P = 0.43 for the < 5 and ≥ 5 years and Eastern and Western regions, respectively. Genomic sequencing of the pfhrp2 exon2 revealed a high level of genetic diversity reflected in 96.8% (60/62) unique sequence types. Repeat type AHHAAAHHATD and HRP2 sequence Type C were more frequent in RDT-/PCR + samples (1.9% vs 1.5%) and (13% vs 8%), P < 0.05 respectively. Genetic relatedness analysis revealed small clusters of gene deleted parasites in Uganda, but no clustering with Eritrean parasites. CONCLUSION: High level of genetic diversity of P. falciparum parasites reflected in the frequency of multiclonal infections, multiplicity of infection and variability of the pfhrp2 gene observed in this study is consistent with the high malaria transmission intensity in these settings. Parasite genetic analysis suggested spontaneous emergence and clonal expansion of pfhrp2 deleted parasites that require close monitoring to inform national malaria diagnosis and case management policies.

Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017-2019.

BACKGROUND: Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda. METHODS: Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR. RESULTS: Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6-13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4-5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7-20.0%) compared to the western region 3.1% (95% CI 0.8-7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02-23.55), p = 0.003. CONCLUSIONS: This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.

Systematic review of the status of pfhrp2 and pfhrp3 gene deletion, approaches and methods used for its estimation and reporting in Plasmodium falciparum populations in Africa: review of published studies 2010-2019.

BACKGROUND: Malaria rapid diagnostic tests based on histidine-rich protein-2 have played a vital role in improving malaria case management and surveillance particularly in Africa, where Plasmodium falciparum is predominant. However, their usefulness has been threatened by the emergence of gene deletion on P. falciparum histidine rich protein 2 (pfhrp2) and P. falciparum histidine rich protein 3 (pfhrp3). Use of standard and recommended methods is key for accurate investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion. METHODS: A systematic review was conducted to assess the status, methods and approaches that have been used for investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion in Africa. An online search was done using PubMed and MEDLINE Google Scholar for all articles published in English on pfhrp2/3 gene deletion in Africa. Relevant articles that met the inclusion criteria were summarized and assessed based on the protocol recommended by the World Health Organization for confirmation and reporting of pfhrp2/3 gene deletion. RESULTS: The search identified a total of 18 articles out of which 14 (77.7%) fulfilled the criteria for inclusion and were retained for review. The articles were distributed across 12 countries where the pfhrp2 and pfhrp3 gene deletion studies were conducted and reported. The level of pfhrp2/3 gene deletion across selected studies in Africa ranged from the highest 62% to the lowest 0.4%. There was wide variation in methods and approaches including study designs, size and sampling and whether both pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion were investigated, with a wide variation in laboratory methods. CONCLUSION: Based on the review, there is evidence of the presence of pfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. Countries that are considering plans to investigate, confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.

The contribution of malaria control interventions on spatio-temporal changes of parasitaemia risk in Uganda during 2009-2014.

BACKGROUND: In Uganda, malaria vector control interventions and case management with Artemisinin Combination Therapies (ACTs) have been scaled up over the last few years as a result of increased funding. Data on parasitaemia prevalence among children less than 5 years old and coverage of interventions was collected during the first two Malaria Indicator Surveys (MIS) conducted in 2009 and 2014, respectively. In this study, we quantify the effects of control interventions on parasitaemia risk changes between the two MIS in a spatio-temporal analysis. METHODS: Bayesian geostatistical and temporal models were fitted on the MIS data of 2009 and 2014. The models took into account geographical misalignment in the locations of the two surveys and adjusted for climatic changes and socio-economic differentials. Parasitaemia risk was predicted over a 2 × 2 km2 grid and the number of infected children less than 5 years old was estimated. Geostatistical variable selection was applied to identify the most important ITN coverage indicators. A spatially varying coefficient model was used to estimate intervention effects at sub-national level. RESULTS: The coverage of Insecticide Treated Nets (ITNs) and ACTs more than doubled at country and sub-national levels during the period 2009-2014. The coverage of Indoor Residual Spraying (IRS) remained static at all levels. ITNs, IRS, and ACTs were associated with a reduction in parasitaemia odds of 19% (95% BCI: 18-29%), 78% (95% BCI: 67-84%), and 34% (95% BCI: 28-66%), respectively. Intervention effects varied with region. Higher socio-economic status and living in urban areas were associated with parasitaemia odds reduction of 46% (95% BCI: 0.51-0.57) and 57% (95% BCI: 0.40-0.53), respectively. The probability of parasitaemia risk decline in the country was 85% and varied from 70% in the North-East region to 100% in Kampala region. The estimated number of children infected with malaria declined from 2,480,373 in 2009 to 825,636 in 2014. CONCLUSIONS: Interventions have had a strong effect on the decline of parasitaemia risk in Uganda during 2009-2014, albeit with varying magnitude in the regions. This success should be sustained by optimizing ITN coverage to achieve universal coverage.

Factors associated with malaria parasitaemia among children under 5 years in Uganda: a secondary data analysis of the 2014 Malaria Indicator Survey dataset.

BACKGROUND: In the midst of success with malaria reduction in Uganda, there are areas that still have high prevalence of malaria parasitaemia. This project aimed at investigating factors associated with this prevalence and its relationship with anaemia. METHODS: This is a secondary data analysis of the 2014 Malaria Indicator Survey dataset of children under 5 years. All had a blood sample taken by finger or heel prick for determination of malaria parasitaemia and estimation of haemoglobin level for anaemia status. The main outcome was the presence of malaria parasitaemia by microscopy and independent variables included: age, gender, residence (urban vs rural), use of a long-lasting, insecticidal-treated net, indoor residual spraying (IRS) of household in the past 6 months, mother's highest education level, mother heard malaria prevention message in the past 6 months, and household wealth status. RESULTS: The analysis included 4930 children and of these, 938 (19.04%: 95% CI 16.63-21.71) tested positive for malaria parasites. Malaria parasite prevalence significantly increased from 11.08 (95% CI 9.12-13.40) among children with no anaemia to 50.99% (95% CI 39.13-62.74) with severe anaemia (Chi-square p-value = 0.001). Additionally, prevalence significantly rose from the youngest age group (under 6 months) by 1.62 times (95% CI 1.04-2.52, p = 0.033) among the age group of 7-12 months and to four times (95% CI 2.57-6.45, p = 0.001) among those who were between 49 and 59 months. The following were associated with reduced parasitaemia: IRS use (AOR 0.23 [0.08-0.61], p = 0.004), educated mothers (primary AOR 0.75 [0.59-0.96], p = 0.023 to tertiary AOR 0.11 [0.02-0.53], 0.006), mother heard malaria message (AOR 0.78 [0.62-0.99], p = 0.037), and wealthier households (richest AOR 0.17 [0.08-0.36], p = 0.001). CONCLUSIONS: Increasing malaria parasite prevalence among children under 5 years is still related to increasing age and severity of anaemia even in the context of decreasing malaria prevalence. Designing interventions that include the use of IRS and behaviour change communication tailored to include older children, especially in areas with high malaria prevalence, could be of added value. All this should be done in an environment that improves the socio-economic status and equity of such populations.

Geostatistical modelling of malaria indicator survey data to assess the effects of interventions on the geographical distribution of malaria prevalence in children less than 5 years in Uganda.

BACKGROUND: Malaria burden in Uganda has declined disproportionately among regions despite overall high intervention coverage across all regions. The Uganda Malaria Indicator Survey (MIS) 2014-15 was the second nationally representative survey conducted to provide estimates of malaria prevalence among children less than 5 years, and to track the progress of control interventions in the country. In this present study, 2014-15 MIS data were analysed to assess intervention effects on malaria prevalence in Uganda among children less than 5 years, assess intervention effects at regional level, and estimate geographical distribution of malaria prevalence in the country. METHODS: Bayesian geostatistical models with spatially varying coefficients were used to determine the effect of interventions on malaria prevalence at national and regional levels. Spike-and-slab variable selection was used to identify the most important predictors and forms. Bayesian kriging was used to predict malaria prevalence at unsampled locations. RESULTS: Indoor Residual Spraying (IRS) and Insecticide Treated Nets (ITN) ownership had a significant but varying protective effect on malaria prevalence. However, no effect was observed for Artemisinin Combination-based Therapies (ACTs). Environmental factors, namely, land cover, rainfall, day and night land surface temperature, and area type were significantly associated with malaria prevalence. Malaria prevalence was higher in rural areas, increased with the child's age, and decreased with higher household socioeconomic status and higher level of mother's education. The highest prevalence of malaria in children less than 5 years was predicted for regions of East Central, North East and West Nile, whereas the lowest was predicted in Kampala and South Western regions, and in the mountainous areas in Mid-Western and Mid-Eastern regions. CONCLUSIONS: IRS and ITN ownership are important interventions against malaria prevalence in children less than 5 years in Uganda. The varying effects of the interventions calls for selective implementation of control tools suitable to regional ecological settings. To further reduce malaria burden and sustain malaria control in Uganda, current tools should be supplemented by health system strengthening, and socio-economic development.

How Do Patients and Health Workers Interact around Malaria Rapid Diagnostic Testing, and How Are the Tests Experienced by Patients in Practice? A Qualitative Study in Western Uganda.

BACKGROUND: Successful scale-up in the use of malaria rapid diagnostic tests (RDTs) requires that patients accept testing and treatment based on RDT results and that healthcare providers treat according to test results. Patient-provider communication is a key component of quality care, and leads to improved patient satisfaction, higher adherence to treatment and better health outcomes. Voiced or perceived patient expectations are also known to influence treatment decision-making among healthcare providers. While there has been a growth in literature on provider practices around rapid testing for malaria, there has been little analysis of inter-personal communication around the testing process. We investigated how healthcare providers and patients interact and engage throughout the diagnostic and treatment process, and how the testing service is experienced by patients in practice. METHODS: This research was conducted alongside a larger study which explored determinants of provider treatment decision-making following negative RDT results in a rural district (Kibaale) in mid-western Uganda, ten months after RDT introduction. Fifty-five patients presenting with fever were observed during routine outpatient visits at 12 low-level public health facilities. Observation captured communication practices relating to test purpose, results, diagnosis and treatment. All observed patients or caregivers were immediately followed up with in-depth interview. Analysis followed the 'framework' approach. A summative approach was also used to analyse observation data. RESULTS: Providers failed to consistently communicate the reasons for carrying out the test, and particularly to RDT-negative patients, a diagnostic outcome or the meaning of test results, also leading to confusion over what the test can detect. Patients appeared to value testing, but were frustrated by the lack of communication on outcomes. RDT-negative patients were dissatisfied by the absence of information on an alternative diagnosis and expressed uncertainty around adequacy of proposed treatment. CONCLUSIONS: Poor provider communication practices around the testing process, as well as limited inter-personal exchange between providers and patients, impacted on patients' perceptions of their proposed treatment. Patients have a right to health information and may be more likely to accept and adhere to treatment when they understand their diagnosis and treatment rationale in relation to their perceived health needs and visit expectations.

Why do health workers give anti-malarials to patients with negative rapid test results? A qualitative study at rural health facilities in western Uganda.

BACKGROUND: The large-scale introduction of malaria rapid diagnostic tests (RDTs) promises to improve management of fever patients and the rational use of valuable anti-malarials. However, evidence on the impact of RDT introduction on the overprescription of anti-malarials has been mixed. This study explored determinants of provider decision-making to prescribe anti-malarials following a negative RDT result. METHODS: A qualitative study was conducted in a rural district in mid-western Uganda in 2011, ten months after RDT introduction. Prescriptions for all patients with negative RDT results were first audited from outpatient registers for a two month period at all facilities using RDTs (n = 30). Facilities were then ranked according to overall prescribing performance, defined as the proportion of patients with a negative RDT result prescribed any anti-malarial. Positive and negative deviant facilities were sampled for qualitative investigation; positive deviants (n = 5) were defined ex post facto as <0.75% and negative deviants (n = 7) as >5%. All prescribing clinicians were targeted for qualitative observation and in-depth interview; 55 fever cases were observed and 22 providers interviewed. Thematic analysis followed the 'framework' approach. RESULTS: 8344 RDT-negative patients were recorded at the 30 facilities (prescription audit); 339 (4.06%) were prescribed an anti-malarial. Of the 55 observed patients, 38 tested negative; one of these was prescribed an anti-malarial. Treatment decision-making was influenced by providers' clinical beliefs, capacity constraints, and perception of patient demands. Although providers generally trusted the accuracy of RDTs, anti-malarial prescription was driven by perceptions of treatment failure or undetectable malaria in patients who had already taken artemisinin-based combination therapy prior to facility arrival. Patient assessment and other diagnostic practices were minimal and providers demonstrated limited ability to identify alternative causes of fever. Provider perceptions of patient expectations sometimes appeared to influence treatment decisions. CONCLUSIONS: The study found high provider adherence to RDT results, but that providers believed in certain clinical exceptions and felt they lacked alternative options. Guidance on how the RDT works and testing following partial treatment, better methods for assisting providers in diagnostic decision-making, and a context-appropriate provider behaviour change intervention package are needed.

Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda.

BACKGROUND: Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex for field deployment. A new commercial molecular assay based on loop-mediated isothermal amplification (LAMP) was assessed for field use. METHODS: Malaria LAMP (Eiken Chemical, Japan) was evaluated for samples from 272 outpatients at a rural Ugandan clinic and compared with expert microscopy, nested PCR, and quantitative PCR (qPCR). Two technicians performed the assay after 3 days of training, using 2 alternative blood sample-preparation methods and visual interpretation of results by fluorescence assay. RESULTS: Compared with 3-well nested PCR, the sensitivity of both LAMP and single-well nested PCR was 90%; the microscopy sensitivity was 51%. For samples with a Plasmodium falciparum qPCR titer of ≥ 2 parasites/µL, LAMP sensitivity was 97.8% (95% confidence interval, 93.7%-99.5%). Most false-negative LAMP results involved samples with parasitemia levels detectable by 3-well nested PCR but very low or undetectable by qPCR. CONCLUSIONS: Malaria LAMP in a remote Ugandan clinic achieved sensitivity similar to that of single-well nested PCR in a United Kingdom reference laboratory. LAMP dramatically lowers the detection threshold achievable in malaria-endemic settings, providing a new tool for diagnosis, surveillance, and screening in elimination strategies.