Diagnostic performance of an ultra-sensitive RDT and a conventional RDT in malaria mass testing, treatment and tracking interventions in southern Ghana.

BACKGROUND: Application of numerous malaria control interventions has led to reduction in clinical malaria cases and deaths but also the realisation that asymptomatic parasite carriers play a key role in sustaining transmission. This study assessed the effectiveness of using the Ultra-sensitive NxTek eliminate RDT (uRDT) and conventional SD Bioline HRP2 RDT (cRDT) in diagnosing asymptomatic parasitaemia while measuring the impact of mass testing, treatment and tracking (MTTT) on the prevalence of asymptomatic malaria over a 1-year period in Ghana. METHODS: A total of 4000 targeted participants from two towns, Obom and Kofi Kwei, with their surrounding villages, were tested for asymptomatic malaria four times over the study period using uRDT (intervention) and the cRDT (control) respectively. Participants carrying malaria parasites were followed by home visit and phone calls for compliance to treatment, and filter paper blood blots collected from participants were used to determine true parasite carriage by PET-PCR. A mathematical model of the study site was developed and used to test the impact of test sensitivity and mass migration on the effect of MTTT. RESULTS: The start and end point sensitivities of the cRDT were 48.8% and 41.7% and those for the uRDT were 52.9% and 59.9% respectively. After a year of MTTTs, asymptomatic parasite prevalence, as determined by PCR, did not differ statistically in the control site (40.6% to 40.1%, P = 0.730) but decreased at the intervention site (55.9% to 46.4%, P < 0.0001). Parasite prevalence by RDT, however, indicated statistical reduction in the control site (25.3% to 22.3%, P = 0.017) and no change in the intervention site (35.1% to 36.0%, P = 0.614). The model predicted a mild effect of both diagnostic sensitivity and human movement in diminishing the impact of MTTT in the study sites. CONCLUSIONS: Asymptomatic parasite prevalence at the molecular level reduced significantly in the site where the uRDT was used but not where the cRDT was used. Overall, the uRDT exhibited higher sensitivity relative to the cRDT. Highly sensitive molecular techniques such as PET-PCR should be included in parasite prevalence estimation during MTTT exercises.

Correction: Genotypic glucose-6-phosphate dehydrogenase (G6PD) deficiency protects against Plasmodium falciparum infection in individuals living in Ghana.

[This corrects the article DOI: 10.1371/journal.pone.0257562.].

Nationwide Surveillance of Pfhrp2 Exon 2 Diversity in Plasmodium falciparum Circulating in Symptomatic Malaria Patients Living in Ghana.

Reports of increasing false-negative HRP2-based rapid diagnostic test results across Africa require constant monitoring of factors associated with these false-negative outcomes, as failure of this diagnostic tool will have severe consequences on malaria treatment and control programs. This study characterized the extent of genetic diversity in the Plasmodium falciparum histidine-rich protein 2 (Pfhrp2) gene in P. falciparum isolates from symptomatic malaria patients across the regions of Ghana. Exon 2 of Pfhrp2 was amplified from gDNA using polymerase chain reaction. All Pfhrp2-negative samples were subjected to Pf18S rRNA and Pfmsp2 gene amplifications. The amplified Pfhrp2 exon 2 fragments from clonal samples were sent for commercial Sanger sequencing. The type and number of PfHRP2 repeats, classified based on repeat types previously reported, were estimated from the sequence data and compared among geographical regions. About 81% (2,333/2,890) of the original microscopy positive DBS were available and used in this study. The Pfhrp2 exon 2 amplification was successful in 98.5% (2,297/2,333) of the tested samples, with band size ranging from 400 bp to 1,050 bp. A total of 13 out of the 24 previously reported repeat types were identified among the samples, with three samples lacking both type 2 and type 7 repeat motifs. This study suggested that the genetic diversity of Pfhrp2 exon 2 identified in P. falciparum circulating in symptomatic malaria patients in Ghana is unlikely to influence the sensitivity and specificity of HRP2 RDT-based diagnosis.

Nationwide molecular surveillance of three Plasmodium species harboured by symptomatic malaria patients living in Ghana.

BACKGROUND: Clinical presentations of malaria in Ghana are primarily caused by infections containing microscopic densities of Plasmodium falciparum, with a minor contribution from Plasmodium malariae and Plasmodium ovale. However, infections containing submicroscopic parasite densities can result in clinical disease. In this study, we used PCR to determine the prevalence of three human malaria parasite species harboured by suspected malaria patients attending healthcare facilities across the country. METHODS: Archived dried blood spots on filter paper that had been prepared from whole blood collected from 5260 patients with suspected malaria attending healthcare facilities across the country in 2018 were used as experimental material. Plasmodium species-specific PCR was performed on DNA extracted from the dried blood spots. Demographic data and microscopy data for the subset of samples tested were available from the original study on these specimens. RESULTS: The overall frequency of P. falciparum, P. malariae and P. ovale detected by PCR was 74.9, 1.4 and 0.9%, respectively. Of the suspected symptomatic P. falciparum malaria cases, 33.5% contained submicroscopic densities of parasites. For all regions, molecular diagnosis of P. falciparum, P. malariae and P. ovale was significantly higher than diagnosis using microscopy: up to 98.7% (75/76) of P. malariae and 97.8% (45/46) of P. ovale infections detected by PCR were missed by microscopy. CONCLUSION: Plasmodium malariae and P. ovale contributed to clinical malaria infections, with children aged between 5 and 15 years harbouring a higher frequency of P. falciparum and P. ovale, whilst P. malariae was more predominant in individuals aged between 10 and 20 years. More sensitive point-of-care tools are needed to detect the presence of low-density (submicroscopic) Plasmodium infections, which may be responsible for symptomatic infections.

Genotypic glucose-6-phosphate dehydrogenase (G6PD) deficiency protects against Plasmodium falciparum infection in individuals living in Ghana.

INTRODUCTION: The global effort to eradicate malaria requires a drastic measure to terminate relapse from hypnozoites as well as transmission via gametocytes in malaria-endemic areas. Primaquine has been recommended for the treatment of P. falciparum gametocytes and P. vivax hypnozoites, however, its implementation is challenged by the high prevalence of G6PD deficient (G6PDd) genotypes in malaria endemic countries. The objective of this study was to profile G6PDd genotypic variants and correlate them with malaria prevalence in Ghana. METHODS: A cross-sectional survey of G6PDd genotypic variants was conducted amongst suspected malaria patients attending health care facilities across the entire country. Malaria was diagnosed using microscopy whilst G6PD deficiency was determined using restriction fragment length polymorphisms at position 376 and 202 of the G6PD gene. The results were analysed using GraphPad prism. RESULTS: A total of 6108 subjects were enrolled in the study with females representing 65.59% of the population. The overall prevalence of malaria was 36.31%, with malaria prevalence among G6PDd genotypic variants were 0.07% for A-A- homozygous deficient females, 1.31% and 3.03% for AA- and BA- heterozygous deficient females respectively and 2.03% for A- hemizygous deficient males. The odd ratio (OR) for detecting P. falciparum malaria infection in the A-A- genotypic variant was 0.0784 (95% CI: 0.0265-0.2319, p<0.0001). Also, P. malariae and P. ovale parasites frequently were observed in G6PD B variants relative to G6PD A- variants. CONCLUSION: G6PDd genotypic variants, A-A-, AA- and A- protect against P. falciparum, P. ovale and P. malariae infection in Ghana.

Dynamics of the Composition of Plasmodium Species Contained within Asymptomatic Malaria Infections in the Central Region of Ghana.

BACKGROUND: Monitoring changes in the composition of the Plasmodium species circulating within the population over a period can inform appropriate treatment recommendations. This study monitored variations in the prevalence of four common human Plasmodium species carried by children with asymptomatic malaria infections over a two-year period. METHODS: Two cross-sectional studies were conducted in November 2017 and December 2019. A total of 210 children aged between 4 and 13 years were recruited in 2017, and 164 similarly aged children were recruited in 2019. Approximately 150 µl of finger-pricked blood was used to prepare thick and thin blood smears as well as spot Whatman® #3 filter paper. Genomic DNA was extracted from the dried blood spots and used in PCR to amplify the 18S rRNA gene from four different human Plasmodium parasites. RESULTS: Parasite prevalence by microscopy and the prevalence of P. falciparum detected by PCR was relatively similar at the two time points (Pearson chi-square = 0.405, p=0.525, and Pearson chi-square = 0.452, p=0.501, respectively). However, the prevalence of PCR detectable P. malariae increased by 8.5-fold, whilst P. ovale increased from 0 to 9% in the children sampled in 2019 relative to the children sampled in 2017. The only parasite species identified by microscopy in this study was P. falciparum, and no P. vivax was identified by either microscopy or PCR in the study population during the study period. CONCLUSION: There is the need to implement molecular diagnostic tools for malaria parasite surveillance in Ghana. This will enable the identification and treatment of all circulating malaria parasites including P. malariae and P. ovale, whose population is expanding in parts of Ghana including Simiw.

Diagnostic performance of an ultrasensitive HRP2-based malaria rapid diagnostic test kit used in surveys of afebrile people living in Southern Ghana.

BACKGROUND: The Alere™ Malaria Ag P.f Ultra-sensitive RDT (UsmRDT) kit is an HRP2-based malaria rapid diagnostic test (RDT) with enhanced sensitivity relative to the SD Bioline Malaria Ag P.f RDT (mRDT) kit. However, the diagnostic performance of the UsmRDT kit has not been evaluated in Ghana. METHODS: A total of 740 afebrile participants aged between 3 and 88 years old were recruited from the Central and Greater Accra Regions of Ghana during the off-peak malaria season. Axillary body temperature was measured, and a volume of 1 ml venous blood was drawn from each participant. Prior to separating the blood into plasma and packed cell pellets via centrifugation, the blood was spotted onto one UsmRDT and one mRDT kit and also used to prepare thick and thin blood smears as well as filter paper blood spots. Plasmodium falciparum specific polymerase chain reaction (PCR) was performed on gDNA extracted from 100 µl of the whole blood. RESULTS: The overall positivity rate for microscopy, PCR, UsmRDT and mRDT kit were 20.4%, 40.8%, 31.3% and 30.8%, respectively. Overall, the UsmRDT identified 9.3% (28/302) more PCR positive samples than the mRDT kits. All samples that were negative by the UsmRDT kit were also negative by the mRDT kit. Overall, the sensitivity and specificity of the UsmRDT was 73% (221/302) and 89% (388/436), respectively, while that for the mRDT kit was 58% and 90%, respectively. CONCLUSION: Although the UsmRDT kit was not as sensitive as PCR at detecting asymptomatic P. falciparum carriage, it correctly identified P. falciparum in 9.3% of the study participants that were not captured by the mRDT kit. In malaria endemic settings, the UsmRDT would provide an added advantage by identifying more asymptomatic P. falciparum carriers than the mRDT kit for targeted treatment interventions.

Asymptomatic carriage of Plasmodium falciparum by individuals with variant blood groups and haemoglobin genotypes in southern Ghana.

BACKGROUND: The ABO and the Rhesus blood group systems, as well as various abnormal haemoglobin (Hb) variants (haemoglobinopathies) are known to influence malaria parasite carriage and disease severity in individuals living in malaria endemic areas. This study identified the blood group and Hb variant distribution and Plasmodium falciparum infection status of afebrile individuals living in southern Ghana. METHODS: Afebrile participants were recruited from Obom (358) in the Greater Accra Region and Ewim (100) and Simiw (329) in the Central Region of Ghana. Venous blood (1 ml) was collected into EDTA vacutainer tubes. Three 20 µl drops of blood were used for blood group analysis using the tile method. Another 500 µl aliquot was used for the qualitative sickling test using sodium metabisulphite and haemoglobin electrophoresis. Genomic DNA was extracted from 100 µl of whole blood and used in P. falciparum species-specific PCR. RESULTS: The most abundant blood group and abnormal haemoglobin variant in both sites was blood group O + (47.4%) and HbAS (15.8%). A total of 13 (1.7%) of the participants had full haemoglobinopathies (SS, SC and CC), whilst 196 (25.4%) were carriers (AS and AC). Although there was a significantly higher prevalence of sickling positive participants from the Central Region, genotyping identified a similar prevalence of each of the abnormal haemoglobin genes in both sites. Asymptomatic parasite carriage estimated by PCR was 40.9% in the Central Region and 41.8% in the Greater Accra Region. CONCLUSIONS: Asymptomatic carriage of P. falciparum parasite in the study population was not associated with any particular blood group variant or haemoglobin genotype.