Evaluation of prevalence's of pfdhfr and pfdhps mutations in Angola

<p>Abstract</p> <p>Background</p> <p>Malaria is the major cause of morbidity and mortality in Angola. The most vulnerable groups to <it>Plasmodium falciparum </it>infection are pregnant women and children under five years of age. The use of an intermittent preventive treatment (IPT) with sulphadoxine/pyrimethamine (SP) in pregnant women was introduced in Angola in 2006 by the National Malaria Control Programme, and currently this strategy has been considered to be used for children malaria control. Considering the previous wide use of SP combination in Angola, together to the reported cases of SP treatment failure it is crucial the evaluation of the prevalence of five mutations in <it>pfdhfr </it>and <it>pfdhps </it>genes associated to <it>P</it>. <it>falciparum </it>resistance to SP before the introduction of S/P IPT in children.</p> <p>Methods</p> <p>The study was conducted in five provinces, with different transmission intensities: Huambo, Cabinda, Uíge, Kwanza Norte, and Malanje. The detection of the mutations in <it>pfdhfr </it>and <it>pfdhps </it>genes was carried out in 452 <it>P</it>. <it>falciparum </it>blood samples by PCR RFLP.</p> <p>Results</p> <p>For <it>pfdhfr </it>gene, 90,3% of the samples carried the mutation 51<b>I</b>, with 7.5% of mixed infections; 51% carried wild type allele 59<b>C</b>, with 29.2% mixed infections and; 99.1% of isolates harboured the mutant allele 108<b>N</b>. Concerning, <it>pfdhps </it>gene, 83,1% were mutant type 437<b>G </b>with 11% mixed infections , while 87% of the studied isolates were wild type for codon 540.</p> <p>Discussion</p> <p>This is the first representative epidemiological study of the whole Angola country on the prevalence of the genotypes associated with SP chemoresistance. A high frequency of individual mutations in both genes (51<b>I </b>and 108<b>N </b>in <it>pfdhfr</it>, and 437<b>G </b>in <it>pfdhps</it>) was found, besides a low prevalence of the quintuple mutation.</p> <p>Conclusion</p> <p>The data showed that the implementation IPT using SP in children needs to be reviewed.</p

Performance of antigen concentration thresholds for attributing fever to malaria among outpatients in Angola

The density of malaria parasites is a key determinant of whether an infected individual develops fever. While the pyrogenic threshold for malaria parasite density has been well studied, there are no analogous data on the antigen levels associated with fever during infection. Samples from 797 afebrile and 457 febrile outpatients from two provinces in Angola with known concentrations of histidine-rich protein 2 (HRP2), aldolase, and lactate dehydrogenase (LDH) antigens were analyzed by Bayesian latent class modeling to attribute malarial etiology to the fevers and to estimate the sensitivity and specificity of different antigen thresholds for detection of malaria fevers. Among patients with aldolase or LDH levels detectable with a bead-based assay, the concentrations of these two antigens did not differ between afebrile and febrile patients. In contrast, the concentrations of HRP2 were substantially higher in febrile HRP2-positive patients than in afebrile HRP2-positive patients. When HRP2 concentrations were considered, the malaria-attributable fractions of fever cases were 0.092 in Huambo Province and 0.39 in Uíge Province. Diagnostic tests detecting HRP2 with limits of detection (LODs) in the range of 3,000 to 10,000 pg/µl would provide ideal sensitivity and specificity for determination of malarial etiology among febrile persons

Molecular Markers of Sulfadoxine-Pyrimethamine Resistance in Samples from Children with Uncomplicated Plasmodium falciparum at Three Sites in Angola in 2019

Funding Information: The study was funded by the U.S. President’s Malaria Initiative and the Advanced Molecular Detection program at CDC. Funding Information: We thank all study staff and participants, Venceslau Mambi Pelenda, José Bumba da Cunha, Oliveira Kiatoko, Felismina Caquece, Luzala Elisabeth Armando Garcia, Djos Kialanda, Garcia Pembele, Domingos Jandondo, and Belmira José Bondo. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention or the U.S. Agency for International Development. The study was funded by the U.S. President’s Malaria Initiative and the Advanced Molecular Detection program at CDC. P.R.D., C.M.F., B.N.A., S.L., R.H., J.F.M.M., F.F., J.F.M., and M.M.P. designed and participated in the specimen collection. P.R.D., A.L.M.C., S.R.R., and J.K. performed the laboratory analysis. P.R.D., A.L.M.C., J.K., S.R.R., J.-H.M.O., and E.T. analyzed the sequencing data. S.R.R. and M.M.P. wrote the manuscript. All authors read and approved the final version of the manuscript. Publisher Copyright: Copyright © 2023 Rosillo et al.Sulfadoxine-pyrimethamine (SP) is used for prevention of malaria in pregnant women in Angola. We sequenced the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes, implicated in SP resistance, in samples collected during a 2019 study of artemisinin-based combination therapy efficacy in Benguela, Lunda Sul, and Zaire provinces. A total of 90 day 0 and day of failure samples were individually sequenced, while 508 day 0 samples from participants without recurrent parasitemia were pooled after DNA extraction into 61 pools. The N51I, C59R, and S108N pfdhfr mutations and A437G pfdhps mutations were present at high proportions in all provinces (weighted allele frequencies, 62% to 100%). The K540E pfdhps mutation was present at lower proportions (10% to 14%). The A581G pfdhps mutation was only observed in Zaire, at a 4.6% estimated prevalence. The I431V and A613S mutations were also only observed in Zaire, at a prevalence of 2.8% to 2.9%. The most common (27% to 66%) reconstructed haplotype in all three provinces was the canonical quadruple pfdhfr pfdhps mutant. The canonical quintuple mutant was absent in Lunda Sul and Benguela and present in 7.9% of samples in Zaire. A single canonical sextuple (2.6%) mutant was observed in Zaire Province. Proportions of the pfdhps K540E and A581G mutations were well below the World Health Organization thresholds for meaningful SP resistance (prevalence of 95% for K540E and 10% for A581G). Samples from therapeutic efficacy studies represent a convenient source of samples for monitoring SP resistance markers.publishersversionpublishe

Prevalence of molecular markers of artemisinin and lumefantrine resistance among patients with uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2015

Abstract Background Artemisinin-based combination therapy is the first-line anti-malarial treatment for uncomplicated Plasmodium falciparum infection in Angola. To date, the prevalence of polymorphisms in the pfk13 gene, associated with artemisinin resistance, and pfmdr1, associated with lumefantrine resistance, have not been systematically studied in Angola. Methods DNA was isolated from pretreatment and late treatment failure dried blood spots collected during the 2015 round of therapeutic efficacy studies in Benguela, Lunda Sul, and Zaire Provinces in Angola. The pfk13 propeller domain and pfmdr1 gene were sequenced and analysed for polymorphisms. Pfmdr1 copy number variation was assessed using a real-time PCR method. The association between pfmdr1 and pfk13 mutations and treatment failure was investigated. Results The majority of pretreatment (99%, 466/469) and all late treatment failure (100%, 50/50) samples were wild type for pfk13. Three of the pretreatment samples (1%) carried the A578S mutation commonly observed in Africa and not associated with artemisinin resistance. All 543 pretreatment and day of late treatment failure samples successfully analysed for pfmdr1 copy number variation carried one copy of pfmdr1. The NYD haplotype was the predominant pfmdr1 haplotype, present in 63% (308/491) of pretreatment samples, followed by NFD, which was present in 32% (157/491) of pretreatment samples. The pfmdr1 N86 allele was overrepresented in day of late treatment failure samples from participants receiving artemether–lumefantrine (p value 0.03). Conclusions The pretreatment parasites in patients participating in therapeutic efficacy studies in 2015 in Angola’s three sentinel sites showed genetic evidence of susceptibility to artemisinins, consistent with clinical outcome data showing greater than 99% day 3 clearance rates. The lack of increased pfmdr1 copy number is consistent with previous reports from sub-Saharan Africa. Although pfmdr1 NYD and NFD haplotypes were overrepresented in artemether–lumefantrine late treatment failure samples, their role as markers of resistance was unclear given that these haplotypes were also present in the majority of successfully treated patients in the artemether–lumefantrine treatment arms

Malaria surveys using rapid diagnostic tests and validation of results using post hoc quantification of Plasmodium falciparum histidine-rich protein 2

Abstract Background Rapid diagnostic test (RDT) positivity is supplanting microscopy as the standard measure of malaria burden at the population level. However, there is currently no standard for externally validating RDT results from field surveys. Methods Individuals’ blood concentration of the Plasmodium falciparum histidine rich protein 2 (HRP2) protein were compared to results of HRP2-detecting RDTs in participants from field surveys in Angola, Mozambique, Haiti, and Senegal. A logistic regression model was used to estimate the HRP2 concentrations corresponding to the 50 and 90% level of detection (LOD) specific for each survey. Results There was a sigmoidal dose–response relationship between HRP2 concentration and RDT positivity for all surveys. Variation was noted in estimates for field RDT sensitivity, with the 50% LOD ranging between 0.076 and 6.1 ng/mL and the 90% LOD ranging between 1.1 and 53 ng/mL. Surveys conducted in two different provinces of Angola using the same brand of RDT and same study methodology showed a threefold difference in LOD. Conclusions Measures of malaria prevalence estimated using population RDT positivity should be interpreted in the context of potentially large variation in RDT LODs between, and even within, surveys. Surveys based on RDT positivity would benefit from external validation of field RDT results by comparing RDT positivity and antigen concentration

Evaluating malaria case management at public health facilities in two provinces in Angola

Abstract Background Malaria accounts for the largest portion of healthcare demand in Angola. A pillar of malaria control in Angola is the appropriate management of malaria illness, including testing of suspect cases with rapid diagnostic tests (RDTs) and treatment of confirmed cases with artemisinin-based combination therapy (ACT). Periodic systematic evaluations of malaria case management are recommended to measure health facility readiness and adherence to national case management guidelines. Methods Cross-sectional health facility surveys were performed in low-transmission Huambo and high-transmission Uíge Provinces in early 2016. In each province, 45 health facilities were randomly selected from among all public health facilities stratified by level of care. Survey teams performed inventories of malaria commodities and conducted exit interviews and re-examinations, including RDT testing, of a random selection of all patients completing outpatient consultations. Key health facility readiness and case management indicators were calculated adjusting for the cluster sampling design and utilization. Results Availability of RDTs or microscopy on the day of the survey was 71% (54–83) in Huambo and 85% (67–94) in Uíge. At least one unit dose pack of one formulation of an ACT (usually artemether–lumefantrine) was available in 83% (66–92) of health facilities in Huambo and 79% (61–90) of health facilities in Uíge. Testing rates of suspect malaria cases in Huambo were 30% (23–38) versus 69% (53–81) in Uíge. Overall, 28% (13–49) of patients with uncomplicated malaria, as determined during the re-examination, were appropriately treated with an ACT with the correct dose in Huambo, compared to 60% (42–75) in Uíge. Incorrect case management of suspect malaria cases was associated with lack of healthcare worker training in Huambo and ACT stock-outs in Uíge. Conclusions The results reveal important differences between provinces. Despite similar availability of testing and ACT, testing and treatment rates were lower in Huambo compared to Uíge. A majority of true malaria cases seeking care in health facilities in Huambo were not appropriately treated with anti-malarials, highlighting the importance of continued training and supervision of healthcare workers in malaria case management, particularly in areas with decreased malaria transmission

Bead-based immunoassay allows sub-picogram detection of histidine-rich protein 2 from <i>Plasmodium falciparum</i> and estimates reliability of malaria rapid diagnostic tests

<div><p>Detection of histidine-rich protein 2 (HRP2) from the malaria parasite <i>Plasmodium falciparum</i> provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different <i>P</i>. <i>falciparum</i> transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate <i>P</i>. <i>falciparum</i> transmission intensity and measure the impact of interventions.</p></div