Prevalence of yaws and syphilis in the Ashanti region of Ghana and occurrence of H. ducreyi, herpes simplex virus 1 and herpes simplex virus 2 in skin lesions associated with treponematoses.

Yaws affects children in tropical regions, while syphilis primarily affects sexually active adults worldwide. Despite various campaigns towards the eradication of yaws and elimination of syphilis, these two diseases are still present in Ghana. The aetiological agents of both diseases, two Treponema pallidum subspecies, are genetically similar. This study aimed to assess the prevalence of these treponematoses and the occurrence of pathogens causing similar skin lesions in the Ashanti region of Ghana. A point-of-care test was used to determine the seroprevalence of the treponematoses. Both yaws and syphilis were identified in the Ashanti region of Ghana. Multiplex PCR was used to identify treponemes and other pathogens that cause similar skin lesions. The results indicated that the seroprevalences of T. pallidum in individuals with yaws-like and syphilis-like lesions were 17.2% and 10.8%, respectively. Multiplex PCR results showed that 9.1%, 1.8% and 0.9% of yaws-like lesions were positive for Haemophilus ducreyi, herpes simplex virus-1 (HSV-1) and T. pallidum respectively. Among syphilis-like lesions, 28.3% were positive for herpes simplex virus -2 (HSV-2) by PCR. To our knowledge, this is the first time HSV-I and HSV-2 have been reported from yaws-like and syphilis-like lesions, respectively, in Ghana. The presence of other organisms apart from T. pallidum in yaws-like and syphilis-like lesions could impede the total healing of these lesions and the full recovery of patients. This may complicate efforts to achieve yaws eradication by 2030 and the elimination of syphilis and warrants updated empirical treatment guidelines for skin ulcer diseases.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Hepatitis B Virus Prevalence and Transmission in the Households of Pregnant Women in Kinshasa, Democratic Republic of Congo.

Background: The World Health Organization Africa region has high regional hepatitis B virus (HBV) prevalence, and evidence suggests more frequent horizontal HBV transmission than other regions. Context-specific epidemiological studies are needed to inform additional HBV prevention measures. Methods: In the cross-sectional Horizontal and Vertical Transmission of Hepatitis B (HOVER-HBV) study, we introduced HBV surface antigen (HBsAg) screening alongside existing HIV screening as part of routine antenatal care in high-volume maternity clinics in Kinshasa, Democratic Republic of Congo. We recruited households of pregnant women ("index mothers") who were HBsAg-positive and HBsAg-negative, defining households as index-positive and index-negative, respectively. Household members underwent HBsAg testing and an epidemiological survey. We evaluated HBsAg prevalence and potential transmission correlates. Results: We enrolled 1006 participants from 200 households (100 index-positive, 100 index-negative) across Kinshasa. HBsAg-positivity prevalence was more than twice as high in index-positive households (5.0% [95% confidence interval {CI}, 2.8%-7.1%]) as in index-negative households (1.9% [95% CI, .6%-3.2%]). HBsAg-positivity prevalence was 3.3 (95% CI, .9-11.8) times as high among direct offspring in index-positive versus index-negative households. Factors associated with HBsAg positivity included older age, marriage, and having multiple recent partners or any new sexual partners among index mothers; and older age, lower household wealth, sharing nail clippers, and using street salons among offspring in index-positive households. Conclusions: Vertical and horizontal HBV transmission within households is ongoing in Kinshasa. Factors associated with infection reveal opportunities for HBV prevention efforts, including perinatal prevention, protection during sexual contact, and sanitation of shared personal items.

Evaluation of Malaria Rapid Diagnostic Test Performance and pfhrp2 Deletion in Tanzania School Surveys, 2017.

As part of malaria nationwide monitoring and evaluation initiatives, there is an increasing trend of incorporating malaria rapid diagnostic tests (mRDTs) in surveys conducted within primary schools to detect malaria parasites. However, mRDTs based on the detection of histidine-rich protein 2 (HRP2) are known to yield false-positive results due to persistent antigenemia, and false-negative results may result from low parasitemia or Plasmodium falciparum hrp2/3 gene deletion. We evaluated diagnostic performance of an HRP2 and pan-parasite lactate dehydrogenase (HRP2/pLDH) mRDT against polymerase chain reaction (PCR) for detection of P. falciparum among 17,051 primary school-age children from eight regions of Tanzania in 2017. According to PCR, the prevalence of P. falciparum was 19.2% (95% CI: 18.6-19.8). Using PCR as reference, the sensitivity and specificity of mRDT was 76.2% (95% CI: 74.7-77.7) and 93.9% (95% CI: 93.5-94.3), respectively. Test agreement was lowest in low transmission areas, where true-positive mRDTs were outnumbered by false-negatives due to low parasitemia. Discordant samples (mRDT-negative but PCR-positive) were screened for pfhrp2/3 deletion by real-time PCR. Among those with a parasite density sufficient for analysis, pfhrp2 deletion was confirmed in 60 samples, whereas pfhrp3 deletion was confirmed in two samples; one sample had both pfhrp2 and pfhrp3 deletions. The majority of samples with gene deletions were detected in the high-transmission Kagera region. Compared with mRDTs, PCR and other molecular methods offer increased sensitivity and are not affected by pfhrp2/3 deletions, making them a useful supplement to mRDTs in schools and other epidemiological surveys.

Genomic insights into Plasmodium vivax population structure and diversity in central Africa.

BACKGROUND: Though Plasmodium vivax is the second most common malaria species to infect humans, it has not traditionally been considered a major human health concern in central Africa given the high prevalence of the human Duffy-negative phenotype that is believed to prevent infection. Increasing reports of asymptomatic and symptomatic infections in Duffy-negative individuals throughout Africa raise the possibility that P. vivax is evolving to evade host resistance, but there are few parasite samples with genomic data available from this part of the world. METHODS: Whole genome sequencing of one new P. vivax isolate from the Democratic Republic of the Congo (DRC) was performed and used in population genomics analyses to assess how this central African isolate fits into the global context of this species. RESULTS: Plasmodium vivax from DRC is similar to other African populations and is not closely related to the non-human primate parasite P. vivax-like. Evidence is found for a duplication of the gene PvDBP and a single copy of PvDBP2. CONCLUSION: These results suggest an endemic P. vivax population is present in central Africa. Intentional sampling of P. vivax across Africa would further contextualize this sample within African P. vivax diversity and shed light on the mechanisms of infection in Duffy negative individuals. These results are limited by the uncertainty of how representative this single sample is of the larger population of P. vivax in central Africa.

Global risk of selection and spread of Plasmodium falciparum histidine-rich protein 2 and 3 gene deletions.

In the thirteen years since the first report of pfhrp2-deleted parasites in 2010, the World Health Organization (WHO) has found that 40 of 47 countries surveyed worldwide have reported pfhrp2/3 gene deletions. Due to a high prevalence of pfhrp2/3 deletions causing false-negative HRP2 RDTs, in the last five years, Eritrea, Djibouti and Ethiopia have switched or started switching to using alternative RDTs, that target pan-specific-pLDH or P. falciparum specific-pLDH alone of in combination with HRP2. However, manufacturing of alternative RDTs has not been brought to scale and there are no WHO prequalified combination tests that use Pf-pLDH instead of HRP2 for P. falciparum detection. For these reasons, the continued spread of pfhrp2/3 deletions represents a growing public health crisis that threatens efforts to control and eliminate P. falciparum malaria. National malaria control programmes, their implementing partners and test developers desperately seek pfhrp2/3 deletion data that can inform their immediate and future resource allocation. In response, we use a mathematical modelling approach to evaluate the global risk posed by pfhrp2/3 deletions and explore scenarios for how deletions will continue to spread in Africa. We incorporate current best estimates of the prevalence of pfhrp2/3 deletions and conduct a literature review to estimate model parameters known to impact the selection of pfhrp2/3 deletions for each malaria endemic country. We identify 20 countries worldwide to prioritise for surveillance and future deployment of alternative RDT, based on quickly selecting for pfhrp2/3 deletions once established. In scenarios designed to explore the continued spread of deletions in Africa, we identify 10 high threat countries that are most at risk of deletions both spreading to and subsequently being rapidly selected for. If HRP2-based RDTs continue to be relied on for malaria case management, we predict that the major route for pfhrp2 deletions to spread is south out from the current hotspot in the Horn of Africa, moving through East Africa over the next 20 years. We explore the variation in modelled timelines through an extensive parameter sensitivity analysis and despite wide uncertainties, we identify three countries that have not yet switched RDTs (Senegal, Zambia and Kenya) that are robustly identified as high risk for pfhrp2/3 deletions. These results provide a refined and updated prediction model for the emergence of pfhrp2/3 deletions in an effort to help guide pfhrp2/3 policy and prioritise future surveillance efforts and innovation.

Hepatitis B virus prevalence and transmission in the households of pregnant women in Kinshasa, Democratic Republic of Congo.

Background: Despite routine infant vaccination and blood donor screening, the Democratic Republic of Congo (DRC) has high hepatitis B virus (HBV) prevalence compared to the United States and Europe. Through the cross-sectional Horizontal and Vertical Transmission of Hepatitis B (HOVER-HBV) study, we characterized household prevalence in DRC's capital, Kinshasa, to inform additional prevention efforts. Methods: We introduced HBV surface antigen (HBsAg) screening alongside existing HIV screening as part of routine antenatal care (ANC) in high-volume maternity clinics in Kinshasa. We recruited households of pregnant women who were HBsAg-positive and HBsAg-negative, defining households as "exposed" and "unexposed," respectively. Household members underwent HBsAg testing and an epidemiological survey. We evaluated HBsAg prevalence and potential transmission correlates. Results: We enrolled 1,006 participants from 200 households (100 exposed, 100 unexposed) across Kinshasa. HBsAg prevalence was more than twice as high in exposed households (5.0%; 95% CI: 2.8%-7.1%) as in unexposed households (1.9%; 0.6%-3.2%). Exposed direct offspring had 3.3 (0.9, 11.8) times the prevalence of unexposed direct offspring. Factors associated with HBsAg-positivity included older age, marriage, and having multiple recent partners or any new sexual partners among index mothers; and older age, lower household wealth, sharing nail clippers, and using street salons among exposed offspring. Conclusions: Vertical and horizontal HBV transmission within households is ongoing in Kinshasa. Factors associated with infection reveal opportunities for HBV prevention efforts, including perinatal prevention, protection during sexual contact, and sanitation of shared personal items.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI 2.7-18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

A novel duplex qualitative real-time PCR assay for the detection and differentiation of Plasmodium ovale curtisi and Plasmodium ovale wallikeri malaria.

Background: P. ovale spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species, P. ovale curtisi (Poc) and P. ovale wallikeri (Pow). These species are thought to differ in clinical symptomatology and latency, but existing diagnostic assays have limited ability to detect and distinguish them. In this study, we developed a new duplex assay for the detection and differentiation of Poc and Pow that can be used to improve our understanding of these parasites. Methods: Repetitive sequence motifs were identified in available Poc and Pow genomes and used for assay development and validation. We evaluated the analytical sensitivity and specificity of the best-performing assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), then validated its performance using 55 P. ovale spp. samples and 40 non-ovale Plasmodium samples from the DRC. Poc and Pow prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated. Results: The best-performing Poc and Pow targets had 9 and 8 copies within the reference genomes, respectively. Our duplex assay had 100% specificity and 95% confidence lower limits of detection of 4.2 and 41.2 parasite genome equivalents/µl for Poc and Pow, respectively. Species was determined in 80% of all P. ovale spp.-positive field samples and 100% of those with >10 parasites/µl. Most P. ovale spp. field samples from the DRC were found to be Poc infections. Conclusions: We identified promising multi-copy targets for molecular detection and differentiation of Poc and Pow and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density Pow infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of P. ovale spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC.

Epidemiology of Plasmodium malariae and Plasmodium ovale spp. in Kinshasa Province, Democratic Republic of Congo.

Reports suggest non-falciparum species are an underappreciated cause of malaria in sub-Saharan Africa but their epidemiology is ill-defined, particularly in highly malaria-endemic regions. We estimated incidence and prevalence of PCR-confirmed non-falciparum and Plasmodium falciparum malaria infections within a longitudinal study conducted in Kinshasa, Democratic Republic of Congo (DRC) between 2015-2017. Children and adults were sampled at biannual household surveys and routine clinic visits. Among 9,089 samples from 1,565 participants, incidences of P. malariae, P. ovale spp., and P. falciparum infections by 1-year were 7.8% (95% CI: 6.4%-9.1%), 4.8% (95% CI: 3.7%-5.9%) and 57.5% (95% CI: 54.4%-60.5%), respectively. Non-falciparum prevalences were higher in school-age children, rural and peri-urban sites, and P. falciparum co-infections. P. falciparum remains the primary driver of malaria in the DRC, though non-falciparum species also pose an infection risk. As P. falciparum interventions gain traction in high-burden settings, continued surveillance and improved understanding of non-falciparum infections are warranted.

Epidemiology of Plasmodium malariae and Plasmodium ovale spp. in a highly malaria-endemic country: a longitudinal cohort study in Kinshasa Province, Democratic Republic of Congo.

Background: Increasing reports suggest that non-falciparum species are an underappreciated cause of malaria in sub-Saharan Africa, but their epidemiology is not well-defined. This is particularly true in regions of high P. falciparum endemicity such as the Democratic Republic of Congo (DRC), where 12% of the world's malaria cases and 13% of deaths occur. Methods and Findings: The cumulative incidence and prevalence of P. malariae and P. ovale spp. infection detected by real-time PCR were estimated among children and adults within a longitudinal study conducted in seven rural, peri-urban, and urban sites from 2015-2017 in Kinshasa Province, DRC. Participants were sampled at biannual household survey visits (asymptomatic) and during routine health facility visits (symptomatic). Participant-level characteristics associated with non-falciparum infections were estimated for single- and mixed-species infections. Among 9,089 samples collected from 1,565 participants over a 3-year period, the incidence of P. malariae and P. ovale spp. infection was 11% (95% CI: 9%-12%) and 7% (95% CI: 5%-8%) by one year, respectively, compared to a 67% (95% CI: 64%-70%) one-year cumulative incidence of P. falciparum infection. Incidence continued to rise in the second year of follow-up, reaching 26% and 15% in school-age children (5-14yo) for P. malariae and P. ovale spp., respectively. Prevalence of P. malariae, P. ovale spp., and P. falciparum infections during household visits were 3% (95% CI: 3%-4%), 1% (95% CI: 1%-2%), and 35% (95% CI: 33%-36%), respectively. Non-falciparum malaria was more prevalent in rural and peri-urban vs. urban sites, in school-age children, and among those with P. falciparum co-infection. A crude association was detected between P. malariae and any anemia in the symptomatic clinic population, although this association did not hold when stratified by anemia severity. No crude associations were detected between non-falciparum infection and fever prevalence. Conclusions: P. falciparum remains the primary driver of malaria morbidity and mortality in the DRC. However, non-falciparum species also pose an infection risk across sites of varying urbanicity and malaria endemicity within Kinshasa, DRC, particularly among children under 15 years of age. As P. falciparum interventions gain traction in high-burden settings like the DRC, continued surveillance and improved understanding of non-falciparum infections are warranted.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

P. malariae is found worldwide and causes chronic parasitism in its human hosts. We developed a P. malariae (Pm) diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies /µL (~1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was 35 minutes. Combined with simplified DNA extraction methods, the assay has potential for future field-deployable point-of-care use to detect a parasite species that remains largely undiagnosed.

Early syphilis in Guangzhou, China: presentation, molecular detection of Treponema pallidum , and genomic sequences in clinical specimens and isolates obtained by rabbit infectivity testing.

Background: The global resurgence of syphilis requires novel prevention strategies. Whole genome sequencing (WGS) of Treponema pallidum ( TPA ) using different specimen types is essential for vaccine development. Methods: Patients with primary (PS) and secondary (SS) syphilis were recruited in Guangzhou, China. We collected ulcer exudates and blood from PS participants, and skin biopsies and blood from SS participants for TPA polA polymerase chain reaction (PCR); ulcer exudates and blood were also used to isolate TPA strains by rabbit infectivity testing (RIT). TPA WGS was performed on 52 ulcer exudates and biopsy specimens and 25 matched rabbit isolates. Results: We enrolled 18 PS and 51 SS participants from December 2019 to March 2022. Among PS participants, TPA DNA was detected in 16 (89%) ulcer exudates and three (17%) blood specimens. Among SS participants, TPA DNA was detected in 50 (98%) skin biopsies and 27 (53%) blood specimens. TP A was isolated from 48 rabbits, with a 71% (12/17) success rate from ulcer exudates and 69% (36/52) from SS bloods. Twenty-three matched SS14 clade genomes were virtually identical, while two Nichols clade pairs had discordant tprK sequences. Forty-two of 52 unique TPA genomes clustered in an SS14 East Asia subgroup, while ten fell into two East Asian Nichols subgroups. Conclusions: Our TPA detection rate was high from PS ulcer exudates and SS skin biopsies and over 50% from SS whole blood, with RIT isolation in over two-thirds of samples. Our results support the use of WGS from rabbit isolates to inform vaccine development. Summary: We performed Treponema pallidum molecular detection and genome sequencing from multiple specimens collected from early syphilis patients and isolates obtained by rabbit inoculation. Our results support the use of whole genome sequencing from rabbit isolates to inform syphilis vaccine development.

Nanopore sequencing for malaria molecular surveillance: opportunities and challenges.

Nanopore-based sequencing platforms offer the potential for affordable malaria molecular surveillance (MMS) in resource-limited settings to track and ultimately counteract emerging threats, such as drug resistance and diagnostic escape. Here, we discuss opportunities and challenges to implementing MMS using nanopore sequencing, highlighting priority areas for technical development and innovation.

Clinical and genomic diversity of Treponema pallidum subsp. pallidum: A global, multi-center study of early syphilis to inform vaccine research.

Background: The continuing increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We conducted a multi-center, observational study to explore Treponema pallidum subsp. pallidum ( TPA ) molecular epidemiology essential for vaccine research by analyzing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. Methods: We enrolled patients with primary (PS), secondary (SS) or early latent (ELS) syphilis from clinics in China, Colombia, Malawi and the United States between November 2019 - May 2022. Inclusion criteria included age ≥18 years, and syphilis confirmation by direct detection methods and/or serological testing. TPA detection and WGS were conducted on lesion swabs, skin biopsies/scrapings, whole blood, and/or rabbit-passaged isolates. We compared our WGS data to publicly available genomes, and analysed TPA populations to identify mutations associated with lineage and geography. Findings: We screened 2,820 patients and enrolled 233 participants - 77 (33%) with PS, 154 (66%) with SS, and two (1%) with ELS. Median age of participants was 28; 66% were cis -gender male, of which 43% reported identifying as "gay", "bisexual", or "other sexuality". Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants demonstrated a predominance of SS14-lineage strains with geographic clustering. Phylogenomic analysis confirmed that Nichols-lineage strains are more genetically diverse than SS14-lineage strains and cluster into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models demonstrated population-specific substitutions, some in outer membrane proteins (OMPs) of interest. Interpretation: Our study involving participants from four countries substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains will be vital for vaccine development and improved understanding of syphilis pathogenesis on a population level. Funding: National Institutes of Health, Bill and Melinda Gates Foundation.

Plasmodium falciparum resistant to artemisinin and diagnostics have emerged in Ethiopia.

Diagnosis and treatment of Plasmodium falciparum infections are required for effective malaria control and are pre-requisites for malaria elimination efforts; hence we need to monitor emergence, evolution and spread of drug- and diagnostics-resistant parasites. We deep sequenced key drug-resistance mutations and 1,832 SNPs in the parasite genomes of 609 malaria cases collected during a diagnostic-resistance surveillance study in Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused by P. falciparum carrying the candidate artemisinin partial-resistance kelch13 (K13) 622I mutation, which was less common in diagnostic-resistant parasites mediated by histidine-rich proteins 2 and 3 (pfhrp2/3) deletions than in wild-type parasites (P = 0.03). Identity-by-descent analyses showed that K13 622I parasites were significantly more related to each other than to wild type (P < 0.001), consistent with recent expansion and spread of this mutation. Pfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Of concern, 8.2% of K13 622I parasites also carried the pfhrp2/3 deletions. Close monitoring of the spread of combined drug- and diagnostic-resistant parasites is needed.

Impact of malaria diagnostic choice on monitoring of Plasmodium falciparum prevalence estimates in the Democratic Republic of the Congo and relevance to control programs in high-burden countries.

Malaria programs rely upon a variety of diagnostic assays, including rapid diagnostic tests (RDTs), microscopy, polymerase chain reaction (PCR), and bead-based immunoassays (BBA), to monitor malaria prevalence and support control and elimination efforts. Data comparing these assays are limited, especially from high-burden countries like the Democratic Republic of the Congo (DRC). Using cross-sectional and routine data, we compared diagnostic performance and Plasmodium falciparum prevalence estimates across health areas of varying transmission intensity to illustrate the relevance of assay performance to malaria control programs. Data and samples were collected between March-June 2018 during a cross-sectional household survey across three health areas with low, moderate, and high transmission intensities within Kinshasa Province, DRC. Samples from 1,431 participants were evaluated using RDT, microscopy, PCR, and BBA. P. falciparum parasite prevalence varied between diagnostic methods across all health areas, with the highest prevalence estimates observed in Bu (57.4-72.4% across assays), followed by Kimpoko (32.6-53.2%), and Voix du Peuple (3.1-8.4%). Using latent class analysis to compare these diagnostic methods against an "alloyed gold standard," the most sensitive diagnostic method was BBA in Bu (high prevalence) and Voix du Peuple (low prevalence), while PCR diagnosis was most sensitive in Kimpoko (moderate prevalence). RDTs were consistently the most specific diagnostic method in all health areas. Among 9.0 million people residing in Kinshasa Province in 2018, the estimated P. falciparum prevalence by microscopy, PCR, and BBA were nearly double that of RDT. Comparison of malaria RDT, microscopy, PCR, and BBA results confirmed differences in sensitivity and specificity that varied by endemicity, with PCR and BBA performing best for detecting any P. falciparum infection. Prevalence estimates varied widely depending on assay type for parasite detection. Inherent differences in assay performance should be carefully considered when using community survey and surveillance data to guide policy decisions.

Association between domesticated animal ownership and Plasmodium falciparum parasite prevalence in the Democratic Republic of the Congo: a national cross-sectional study.

BACKGROUND: Domesticated animal ownership is an understudied aspect of the human environment that influences mosquito biting behaviour and malaria transmission, and is a key part of national economies and livelihoods in malaria-endemic regions. In this study, we aimed to understand differences in Plasmodium falciparum prevalence by ownership status of common domesticated animals in DR Congo, where 12% of the world's malaria cases occur and anthropophilic Anopheles gambiae vectors predominate. METHODS: In this cross-sectional study, we used survey data from individuals aged 15-59 years in the most recent (2013-14) DR Congo Demographic and Health Survey and previously performed Plasmodium quantitative real-time PCR (qPCR) to estimate P falciparum prevalence differences by household ownership of cattle; chickens; donkeys, horses, or mules; ducks; goats; sheep; and pigs. We used directed acyclic graphs to consider confounding by age, gender, wealth, modern housing, treated bednet use, agricultural land ownership, province, and rural location. FINDINGS: Of 17 701 participants who had qPCR results and covariate data, 8917 (50·4%) of whom owned a domesticated animal, we observed large differences in malaria prevalence across types of animals owned in both crude and adjusted models. Household chicken ownership was associated with 3·9 (95% CI 0·6 to 7·1) more P falciparum infections per 100 people, whereas cattle ownership was associated with 9·6 (-15·8 to -3·5) fewer P falciparum infections per 100 people, even after accounting for bednet use, wealth, and housing structure. INTERPRETATION: Our finding of a protective association conferred by cattle ownership suggests that zooprophylaxis interventions might have a role in DR Congo, possibly by drawing An gambiae feeding away from humans. Studies of animal husbandry practices and associated mosquito behaviours could reveal opportunities for new malaria interventions. FUNDING: The National Institutes of Health and the Bill & Melinda Gates Foundation. TRANSLATIONS: For the French and Lingala translations of the abstract see Supplementary Materials section.

Therapeutic efficacy of pyronaridine-artesunate (Pyramax®) against uncomplicated Plasmodium falciparum infection at Hamusit Health Centre, Northwest Ethiopia.

BACKGROUND: Early case detection and prompt treatment are important malaria control and elimination strategies. However, the emergence and rapid spread of drug-resistant strains present a major challenge. This study reports the first therapeutic efficacy profile of pyronaridine-artesunate against uncomplicated Plasmodium falciparum in Northwest Ethiopia. METHODS: This single-arm prospective study with 42-day follow-up period was conducted from March to May 2021 at Hamusit Health Centre using the World Health Organization (WHO) therapeutic efficacy study protocol. A total of 90 adults ages 18 and older with uncomplicated falciparum malaria consented and were enrolled in the study. A standard single-dose regimen of pyronaridine-artesunate was administered daily for 3 days, and clinical and parasitological outcomes were assessed over 42 days of follow-up. Thick and thin blood films were prepared from capillary blood and examined using light microscopy. Haemoglobin was measured and dried blood spots were collected on day 0 and on the day of failure. RESULTS: Out of 90 patients, 86/90 (95.6%) completed the 42-day follow-up study period. The overall PCR-corrected cure rate (adequate clinical and parasitological response) was very high at 86/87 (98.9%) (95% CI: 92.2-99.8%) with no serious adverse events. The parasite clearance rate was high with fast resolution of clinical symptoms; 86/90 (95.6%) and 100% of the study participants cleared parasitaemia and fever on day 3, respectively. CONCLUSION: Pyronaridine-artesunate was highly efficacious and safe against uncomplicated P. falciparum in this study population.