Submicroscopic carriage of Plasmodium falciparum and Plasmodium vivax in a low endemic area in Ethiopia where no parasitaemia was detected by microscopy or rapid diagnostic test.

BACKGROUND: Motivated by the success in malaria control that was documented over the last decade Ethiopia is aiming at malaria elimination by 2020 in selected districts. It is currently unknown if asymptomatic, submicroscopic malaria parasite carriage may form a hurdle to achieve elimination. The elimination effort may further be complicated by possible glucose-6 phosphate dehydrogenase (G6PD) deficiency which would hinder the use of 8-aminoquinolines in the elimination efforts. METHOD: In February 2014 a community-based cross-sectional survey was conducted in Malo, southwest Ethiopia. Finger-prick blood samples (n = 555) were tested for presence of Plasmodium falciparum and Plasmodium vivax with microscopy, rapid diagnostic test (RDT), and nested polymerase chain reaction (nPCR). Multiplicity of P. falciparum infections was determined based on genotyping the polymorphic merozoite surface protein-2 (MSP-2) gene. Individuals were also genotyped for mutations in the gene that produces G6PD. RESULTS: All study participants were malaria infection negative by microscopy and RDT. Nested PCR revealed P. falciparum mono-infection in 5.2% (29/555), P. vivax mono-infection in 4.3% (24/555) and mixed infection in 0.2% (1/555) of individuals. All parasitemic individuals were afebrile (axillary temperature <37.5°C). None of the study participants carried mutations for the G6PD African A-(202GA) and Mediterranean (563CT) variants. All infections, except one, were single-clone infection by MSP-2 genotyping. CONCLUSION: The detection of a substantial number of subpatent malaria infections in an apparently asymptomatic population without evidence for malaria transmission by conventional diagnostics raises questions about the path to malaria elimination. It is currently unknown how important these infections are for sustaining malaria transmission in the study sites. The absence of G6PD deficiency indicates that 8-aminoquinolines may be safely deployed to accelerate elimination initiatives

Past eight-year malaria data in Gedeo zone, southern Ethiopia: trend, reporting-quality, spatiotemporal distribution, and association with socio-demographic and meteorological variables.

BACKGROUND: Informed decision making is underlined by all tiers in the health system. Poor data record system coupled with under- (over)-reporting of malaria cases affects the country's malaria elimination activities. Thus, malaria data at health facilities and health offices are important particularly to monitor and evaluate the elimination progresses. This study was intended to assess overall reported malaria cases, reporting quality, spatiotemporal trends and factors associated in Gedeo zone, South Ethiopia. METHODS: Past 8 years retrospective data stored in 17 health centers and 5 district health offices in Gedeo Zone, South Ethiopia were extracted. Malaria cases data at each health center with sociodemographic information, between January 2012 and December 2019, were included. Meteorological data were obtained from the national meteorology agency of Ethiopia. The data were analyzed using Stata 13. RESULTS: A total of 485,414 suspected cases were examined for malaria during the previous 8 years at health centers. Of these suspects, 57,228 (11.79%) were confirmed malaria cases with an overall decline during the 8-year period. We noted that 3758 suspected cases and 467 confirmed malaria cases were not captured at the health offices. Based on the health centers records, the proportions of Plasmodium falciparum (49.74%) and P. vivax (47.59%) infection were nearly equivalent (p = 0.795). The former was higher at low altitudes while the latter was higher at higher altitudes. The over 15 years of age group accounted for 11.47% of confirmed malaria cases (p < 0.001). There was high spatiotemporal variation: the highest case record was during Belg (12.52%) and in Dilla town (18,150, 13.17%, p < 0.001) which is located at low altitude. Monthly rainfall and minimum temperature exhibited strong associations with confirmed malaria cases. CONCLUSION: A notable overall decline in malaria cases was observed during the eight-year period. Both P. falciparum and P. vivax were found at equivalent endemicity level; hence control measures should continue targeting both species. The noticed under reporting, the high malaria burden in urban settings, low altitudes and Belg season need spatiotemporal consideration by the elimination program

Gametocyte Sex Ratio: The Key to Understanding Plasmodium falciparum Transmission?

A mosquito needs to ingest at least one male and one female gametocyte to become infected with malaria. The sex of Plasmodium falciparum gametocytes can be determined microscopically but recent transcriptomics studies paved the way for the development of molecular methods that allow sex-ratio assessments at much lower gametocyte densities. These sex-specific gametocyte diagnostics were recently used to examine gametocyte dynamics in controlled and natural infections as well as the impact of different antimalarial drugs. It is currently unclear to what extent sex-specific gametocyte diagnostics obviate the need for mosquito feeding assays to formally assess transmission potential. Here, we review recent and historic assessments of gametocyte sex ratio in relation to host and parasite characteristics, treatment, and transmission potential

Molecular Markers for Sensitive Detection of Plasmodium falciparum Asexual Stage Parasites and their Application in a Malaria Clinical Trial.

Plasmodium falciparum parasite life stages respond differently to antimalarial drugs. Sensitive stage-specific molecular assays may help to examine parasite dynamics at microscopically detectable and submicroscopic parasite densities in epidemiological and clinical studies. In this study, we compared the performance of skeleton-binding protein 1 (SBP1), ring-infected erythrocyte surface antigen, Hyp8, ring-exported protein 1 (REX1), and PHISTb mRNA for detecting ring-stage trophozoite-specific transcripts using quantitative reverse transcriptase polymerase chain reaction. Markers were tested on tightly synchronized in vitro parasites and clinical trial samples alongside established markers of parasite density (18S DNA and rRNA) and gametocyte density (Pfs25 mRNA). SBP1 was the most sensitive marker but showed low-level expression in mature gametocytes. Novel markers REX1 and PHISTb showed lower sensitivity but higher specificity for ring-stage trophozoites. Using in vivo clinical trial samples from gametocyte-negative patients, we observed evidence of persisting trophozoite transcripts for at least 14 days postinitiation of treatment. It is currently not clear if these transcripts represent viable parasites that may have implications for clinical treatment outcome or transmission potential

The shape of the iceberg: quantification of submicroscopic Plasmodium falciparum and Plasmodium vivax parasitaemia and gametocytaemia in five low endemic settings in Ethiopia.

BACKGROUND: The widespread presence of low-density asymptomatic infections with concurrent gametocytes may be a stumbling block for malaria elimination. This study investigated the asymptomatic reservoir of Plasmodium falciparum and Plasmodium vivax infections in schoolchildren from five settings in northwest Ethiopia. METHODS: Two cross-sectional surveys were conducted in June and November 2015, enrolling 551 students from five schools and 294 students from three schools, respectively. Finger prick whole blood and plasma samples were collected. The prevalence and density of P. falciparum and P. vivax parasitaemia and gametocytaemia were determined by 18S rRNA quantitative PCR (qPCR) and pfs25 and pvs25 reverse transcriptase qPCR. Antibodies against blood stage antigens apical membrane antigen-1 (AMA-1) and merozoite surface protein-1 (MSP-119) were measured for both species. RESULTS: Whilst only 6 infections were detected by microscopy in 881 slides (0.7%), 107 of 845 blood samples (12.7%) were parasite positive by (DNA-based) qPCR. qPCR parasite prevalence between sites and surveys ranged from 3.8 to 19.0% for P. falciparum and 0.0 to 9.0% for P. vivax. The median density of P. falciparum infections (n = 85) was 24.4 parasites/µL (IQR 18.0-34.0) and the median density of P. vivax infections (n = 28) was 16.4 parasites/µL (IQR 8.8-55.1). Gametocyte densities by (mRNA-based) qRT-PCR were strongly associated with total parasite densities for both P. falciparum (correlation coefficient = 0.83, p = 0.010) and P. vivax (correlation coefficient = 0.58, p = 0.010). Antibody titers against P. falciparum AMA-1 and MSP-119 were higher in individuals who were P. falciparum parasite positive in both surveys (p < 0.001 for both comparisons). DISCUSSION: This study adds to the available evidence on the wide-scale presence of submicroscopic parasitaemia by quantifying submicroscopic parasite densities and concurrent gametocyte densities. There was considerable heterogeneity in the occurrence of P. falciparum and P. vivax infections and serological markers of parasite exposure between the examined low endemic settings in Ethiopia

Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR.

BACKGROUND: The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut. This method is labour-intensive, does not allow processing of large numbers of mosquitoes and can be challenging in terms of objective classification of oocysts. Here, a semi-high-throughput bead-beating ELISA method is proposed for detection of the circumsporozoite protein (CSP) followed by confirmation by quantitative PCR (qPCR). METHODS: Cultured Plasmodium falciparum gametocytes were offered to Anopheles stephensi mosquitoes and examined by microscopy. After bead-beating, mosquito homogenate was examined by CSP-ELISA and 18S qPCR. As negative controls, mosquitoes that were offered a heat-inactivated gametocyte blood meal were used. The CSP-ELISA/qPCR methodology was applied to high and low-intensity infections of cultured P. falciparum gametocytes. A similar methodology optimized for P. vivax was used on mosquitoes that were offered blood from Ethiopian donors who were naturally infected with P. vivax. RESULTS: There was considerable variation in CSP-ELISA signal and qPCR values in mosquitoes with low oocyst intensities. There was a strong agreement mosquito positivity by CSP-ELISA and by qPCR in mosquitoes that fed on cultured P. falciparum material (agreement 96.9%; kappa = 0.97) and naturally infected P. vivax parasite carriers [agreement 92.4% (kappa = 0.83)]. CONCLUSIONS: The proposed bead-beating CSP-ELISA/qPCR methodology considerably increases throughput for the detection of mosquito infection. qPCR remains necessary to confirm infections in mosquitoes with low CSP-ELISA signal. This methodology may prove particularly useful for studies where very low mosquito infection prevalence is expected and study sites where experience with oocyst detection is limited

Comparison of infectivity of Plasmodium vivax to wild-caught and laboratory-adapted (colonized) Anopheles arabiensis mosquitoes in Ethiopia

BACKGROUND: Mosquito-feeding assays that assess transmission of Plasmodium from man-to-mosquito typically use laboratory mosquito colonies. The microbiome and genetic background of local mosquitoes may be different and influence Plasmodium transmission efficiency. In order to interpret transmission studies to the local epidemiology, it is therefore crucial to understand the relationship between infectivity in laboratory-adapted and local mosquitoes. METHODS: We assessed infectivity of Plasmodium vivax-infected patients from Adama, Ethiopia, using laboratory-adapted (colony) and wild-caught (wild) mosquitoes raised from larval collections in paired feeding experiments. Feeding assays used 4-6 day-old female Anopheles arabiensis mosquitoes after starvation for 12 h (colony) and 18 h (wild). Oocyst development was assessed microscopically 7 days post-feeding. Wild mosquitoes were identified morphologically and confirmed by genotyping. Asexual parasites and gametocytes were quantified in donor blood by microscopy. RESULTS: In 36 paired experiments (25 P. vivax infections and 11 co-infections with P. falciparum), feeding efficiency was higher in colony (median: 62.5%; interquartile range, IQR: 47.0-79.0%) compared to wild mosquitoes (median: 27.8%; IQR: 17.0-38.0%; Z = 5.02; P < 0.001). Plasmodium vivax from infectious individuals (51.6%, 16/31) infected a median of 55.0% (IQR: 6.7-85.7%; range: 5.5-96.7%; n = 14) of the colony and 52.7% (IQR: 20.0-80.0%; range: 3.2-95.0%; n = 14) of the wild mosquitoes. A strong association (ρ(16) = 0.819; P < 0.001) was observed between the proportion of infected wild and colony mosquitoes. A positive association was detected between microscopically detected gametocytes and the proportion of infected colony (ρ(31) = 0.452; P = 0.011) and wild (ρ(31) = 0.386; P = 0.032) mosquitoes. CONCLUSIONS: Infectivity assessments with colony and wild mosquitoes yielded similar infection results. This finding supports the use of colony mosquitoes for assessments of the infectious reservoir for malaria in this setting whilst acknowledging the importance of mosquito factors influencing sporogonic development of Plasmodium parasites

The epidemiology and detectability of asymptomatic plasmodium vivax and plasmodium falciparum infections in low, moderate and high transmission settings in Ethiopia

BACKGROUND: As countries move to malaria elimination, detecting and targeting asymptomatic malaria infections might be needed. Here, the epidemiology and detectability of asymptomatic Plasmodium falciparum and Plasmodium vivax infections were investigated in different transmission settings in Ethiopia. METHOD: A total of 1093 dried blood spot (DBS) samples were collected from afebrile and apparently healthy individuals across ten study sites in Ethiopia from 2016 to 2020. Of these, 862 were from community and 231 from school based cross-sectional surveys. Malaria infection status was determined by microscopy or rapid diagnostics tests (RDT) and 18S rRNA-based nested PCR (nPCR). The annual parasite index (API) was used to classify endemicity as low (API > 0 and < 5), moderate (API ≥ 5 and < 100) and high transmission (API ≥ 100) and detectability of infections was assessed in these settings. RESULTS: In community surveys, the overall prevalence of asymptomatic Plasmodium infections by microscopy/RDT, nPCR and all methods combined was 12.2% (105/860), 21.6% (183/846) and 24.1% (208/862), respectively. The proportion of nPCR positive infections that was detectable by microscopy/RDT was 48.7% (73/150) for P. falciparum and 4.6% (2/44) for P. vivax. Compared to low transmission settings, the likelihood of detecting infections by microscopy/RDT was increased in moderate (Adjusted odds ratio [AOR]: 3.4; 95% confidence interval [95% CI] 1.6-7.2, P = 0.002) and high endemic settings (AOR = 5.1; 95% CI 2.6-9.9, P < 0.001). After adjustment for site and correlation between observations from the same survey, the likelihood of detecting asymptomatic infections by microscopy/RDT (AOR per year increase = 0.95, 95% CI 0.9-1.0, P = 0.013) declined with age. CONCLUSIONS: Conventional diagnostics missed nearly half of the asymptomatic Plasmodium reservoir detected by nPCR. The detectability of infections was particularly low in older age groups and low transmission settings. These findings highlight the need for sensitive diagnostic tools to detect the entire parasite reservoir and potential infection transmitters

Spatial and genetic clustering of Plasmodium falciparum and Plasmodium vivax infections in a low-transmission area of Ethiopia.

The distribution of malaria infections is heterogeneous in space and time, especially in low transmission settings. Understanding this clustering may allow identification and targeting of pockets of transmission. In Adama district, Ethiopia, Plasmodium falciparum and P. vivax malaria patients and controls were examined, together with household members and immediate neighbors. Rapid diagnostic test and quantitative PCR (qPCR) were used for the detection of infections that were genetically characterized by a panel of microsatellite loci for P. falciparum (26) and P. vivax (11), respectively. Individuals living in households of clinical P. falciparum patients were more likely to have qPCR detected P. falciparum infections (22.0%, 9/41) compared to individuals in control households (8.7%, 37/426; odds ratio, 2.9; 95% confidence interval, 1.3-6.4; P = .007). Genetically related P. falciparum, but not P. vivax infections showed strong clustering within households. Genotyping revealed a marked temporal cluster of P. falciparum infections, almost exclusively comprised of clinical cases. These findings uncover previously unappreciated transmission dynamics and support a rational approach to reactive case detection strategies for P. falciparum in Ethiopia

Naturally acquired antibodies to gametocyte antigens are associated with reduced transmission of Plasmodium vivax gametocytes to Anopheles arabiensis mosquitoes

Naturally acquired antibodies may reduce the transmission of Plasmodium gametocytes to mosquitoes. Here, we investigated associations between antibody prevalence and P. vivax infectivity to mosquitoes. A total of 368 microscopy confirmed P. vivax symptomatic patients were passively recruited from health centers in Ethiopia and supplemented with 56 observations from asymptomatic P. vivax parasite carriers. Direct membrane feeding assays (DMFA) were performed to assess mosquito infectivity; for selected feeds these experiments were also performed after replacing autologous plasma with malaria naïve control serum (n=61). The prevalence of antibodies against 6 sexual stage antigens (Pvs47, Pvs48/45, Pvs230, PvsHAP2, Pvs25 and PvCelTOS) and an array of asexual antigens was determined by ELISA and multiplexed bead-based assays. Gametocyte (ρ&lt; 0.42; p = 0.0001) and parasite (ρ = 0.21; p = 0.0001) densities were positively associated with mosquito infection rates. Antibodies against Pvs47, Pvs230 and Pvs25 were associated with 23 and 34% reductions in mosquito infection rates (p&lt;0.0001), respectively. Individuals who showed evidence of transmission blockade in serum-replacement DMFAs (n=8) were significantly more likely to have PvsHAP2 or Pvs47 antibodies. Further studies may demonstrate causality for the observed associations, improve our understanding of the natural transmission of P. vivax and support vaccine development