Carriage of sub-microscopic sexual and asexual Plasmodium falciparum stages in the dry season at Navrongo, Ghana

Background: We investigated the prevalence of sub-microscopic Plasmodium falciparum infections and gametocyte carriage in asymptomatic individuals in Navrongo in northern Ghana, an area of seasonal malaria transmission.Design: A cross sectional study of 209 randomly selected participants of all age-groups was conducted in February and March, 2015.Methods: Capillary blood samples collected from these individuals were used for the detection of both asexual and gametocyte stage parasites by microscopy, reverse transcriptase polymerase chain reaction (RT-PCR) and conventional nested PCR methods. The prevalence data as determined by microscopy and molecular methods were comparedusing chi-square tests.Results: Parasitaemia from these asymptomatic infections ranged from 40 to 3,520 parasites/l of blood (geometric mean parasitaemia = 732 parasites/l). The prevalence of asymptomatic P. falciparum carriage was 4.8% (10/209) and 13.9% (29/209) using microscopy and RT-PCR respectively. The overall prevalence of sub-microscopic infectionsin the total number of samples analysed was 9.1% (19/209) and 66% (19/29) of the asymptomatic infections. P. falciparum gametocytemia detected by microscopy was 1% (2/209) and 3.8% (8/209) by PCR.Conclusion: This is the first report of sub-microscopic asexual and gametocytes infections in the dry season in a seasonal malaria transmission area in Ghana. It has established that persistent latent malaria infections occur and that these could supply the source of parasites for the next transmission season. The findings highlight the presence of sub-microscopic infections and therefore the need for active case detection surveillance to eliminate "asymptomatic reservoir" parasites and consequently break the transmission of the disease in Ghana.Funding: Bill and Melinda Gates Foundation grant awarded to Noguchi Memorial Institute for Medical Research Postdoctoral and Postgraduate Training in Infectious Diseases Research (Global Health Grant # OPP52155); National Institutes of Health grant (NIH-NIAID RO1 # 1RO1AI099623) to MDW; European Developing Countries Clinical Trials Partnership (EDCTP)-West African Network of Excellence for Clinical Trials in TB, AIDS and Malaria (WANETAM) (Project code CB.07.41700.007).Keywords: Plasmodium falciparum, asymptomatic infections, sub-microscopic infections, microscopy, reverse transcriptase PCR, Navrong

Copy number variation of plasmepsins 2 and 3 genes in Plasmodium falciparum isolates and implication for dihydroartemisinin-piperaquine resistance in Ghana

Background: In 2008, dihydroartemisinin-piperaquine and artemether-lumefantrine were introduced to supplement artesunate-amodiaquine for the treatment of uncomplicated malaria in Ghana. Drug pressure over the years enhances the development of parasite resistance to drugs. The World Health Organization recommends the detection of copy number variations of plasmepsins 2 (PfPm2) and plasmepsins 3 (PfPm3) genes linked to dihydroartemisinin-piperaquine resistance in treatment efficacy studies.Objective: This study investigated the copy number variations of PfPm2 and PfPm3 genes in the malaria parasite population in Ghana.Methods: Overall, 313 blood samples from children ≤ 9 years presenting with uncomplicated malaria at three sentinel sites used for monitoring antimalarial drug efficacy and resistance in Ghana were used for genetic investigations. The samples were collected in the malaria transmission seasons of 2015 and 2016. Malaria parasite DNA extraction from the blood samples followed by real-time quantitative PCR was used to determine the copy number of the PfPm2 and PfPm3 genes. The gene copy number was calculated by the relative expression formula 2-ΔΔCt for quantification, where ΔΔ is the relative delta-delta, and Ct is the cycle threshold. ΔΔCt was calculated as (Ctβ-tubulin − Ctpfpm2/3) - (Ctβ-tubulin cal − Ctpfpm2/3 cal), where cal is the calibration control of genomic 3D7 DNA with one copy of both the β-tubulin endogenous control and pfpm2 and pfpm3. A change in Ct (ΔCt = Ct PfPm2/3 - Ct Pfβ-tubulin) where is the difference in Ct values for the target gene of interest PfPm2 and PfPm3 and the reference gene Pfβ-tubulin. Statistical significance was defined as p < 0.05.Results: Of the parasites analyzed, 79.2% (n = 228/288) and 80.5% (n = 227/282) had one gene copy for PfPm2 and PfPm3, respectively. For PfPm2, 14.9% (n = 43/288), 3.8% (n = 11/288), and 2.1% (n = 6/288) of the isolates had copy numbers 2, 3 and 4 respectively. For PfPm3, gene copies of 2, 3 and 4 were observed in 16.3% (n = 46/282), 2.1% (n = 6/282), and 1.1% (n = 3/282) of isolates. Analysis of the copy number variation across the three study sites in Cape-Coast, Begoro, and the Navrongo areas showed no significant difference for PfPm2 (p = 0.93) and PfPm3 (p = 0.94) genes.Conclusion: After over a decade of the use of dihydroartemisinin-piperaquine, the mutations associated with resistance to the drug have been observed in Ghanaian P. falciparum isolates. This serves as baseline data for further monitoring of this molecular marker extensively as part of ongoing surveillance of antimalarial drug efficacy studies in Ghana

Therapeutic efficacy of dihydroartemisinin-piperaquine combination for the treatment of uncomplicated malaria in Ghana

In 2020, Dihydroartemisinin-Piperaquine (DHAP) was adopted as a second-line antimalarial for treatment of uncomplicated malaria in Ghana following a review of the country’s antimalarial medicines policy. Available data obtained in 2007 had shown PCR-uncorrected therapeutic efficacy of 93.3% using a 28-day follow-up schedule. In 2020, the standard 42-day follow-up schedule for DHAP was used to estimate efficacy levels among febrile children aged 6 months to 9 years in three malaria sentinel sites representing the three main ecological zones of the country- savannah, forest, and coastal. PCR genotyping distinguished between recrudescence and re-infection using merozoite surface protein 2 (MSP2)-specific primers for FC27 and 3D7 strains. Per protocol analyses showed day 28 efficacy of 100% in all three sentinel sites with day 42 PCR-corrected efficacy ranging between 90.3% (95% CI: 80.1 – 96.4%) in the savannah zone and 100% in the forest and coastal zones, yielding a national average of 97.0% (95% CI: 93.4 – 98.8). No day 3 parasitemia was observed in all three sites. Prevalence of measured fever (axillary temperature ≥ 37.5°C) declined from 50.0 - 98.8% on day 0 to 7.1-11.5% on day 1 whilst parasitemia declined from 100% on day 0 to 1.2 - 2.3% on day 1. Mean haemoglobin levels on days 28 and 42 were significantly higher than pre-treatment levels in all three sites. We conclude that DHAP is highly efficacious in the treatment of uncomplicated malaria in Ghana. This data will serve as baseline for subsequent DHAP efficacy studies in the country

A review of the frequencies of Plasmodium falciparum Kelch 13 artemisinin resistance mutations in Africa.

Artemisinin resistance (AR) emerged in South East Asia 13 years ago and the identification of the resistance conferring molecular marker, Plasmodium falciparum Kelch 13 (Pfk13), 7 years ago has provided an invaluable tool for monitoring AR in malaria endemic countries. Molecular Pfk13 surveillance revealed the resistance foci in the Greater Mekong Subregion, an independent emergence in Guyana, South America, and a low frequency of mutations in Africa. The recent identification of the R561H Pfk13 AR associated mutation in Tanzania, Uganda and in Rwanda, where it has been associated with delayed parasite clearance, should be a concern for the continent. In this review, we provide a summary of Pfk13 resistance associated propeller domain mutation frequencies across Africa from 2012 to 2020, to examine how many other countries have identified these mutations. Only four African countries reported a recent identification of the M476I, P553L, R561H, P574L, C580Y and A675V Pfk13 mutations at low frequencies and with no reports of clinical treatment failure, except for Rwanda. These mutations present a threat to malaria control across the continent, since the greatest burden of malaria remains in Africa. A rise in the frequency of these mutations and their spread would reverse the gains made in the reduction of malaria over the last 20 years, given the lack of new antimalarial treatments in the event artemisinin-based combination therapies fail. The review highlights the frequency of Pfk13 propeller domain mutations across Africa, providing an up-to-date perspective of Pfk13 mutations, and appeals for an urgent and concerted effort to monitoring antimalarial resistance markers in Africa and the efficacy of antimalarials by re-establishing sentinel surveillance systems

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Comparative efficacy of antimalarial drugs including ACTs in the treatment of uncomplicated malaria among children under 5 years in Ghana.

The emergence and spread of Plasmodium falciparum resistance to commonly used antimalarials such as chloroquine and sulphadoxine/pyrimethamine poses major challenges to malaria control in sub-Saharan Africa. We undertook a study on the efficacy of some antimalarial drugs in 2003 with the view of supporting the National Malaria Control Programme in the review of the antimalarial drug treatment policy in Ghana. Children aged 6-59 months with signs/symptoms of uncomplicated malaria including axillary temperature > or =37.5 degrees C; mono infection with P. falciparum; and parent's willingness to give consent, were randomized into four treatment groups and followed up for a maximum of 28 days. The treatment groups were chloroquine (CHQ), sulphadoxine/pyrimethamine (SP), amodiaquine+artesunate (ADQ+ART) combination, and artemether+lumefantrine (Coartem) combination. Clinical evaluation of 168 children studied showed that cumulative pcr-corrected cure rates on day 28 were 100% for ADQ+ART; 97.5% for coartem, 60% for SP and 25% for CHQ. The artemisinin-based combinations effected rapid fever and parasite clearance. Prevalence of gametocytaemia was highest in the SP group whilst the CHQ group did not show any significant changes in haemoglobin levels during the follow-up period. The findings are in agreement with current recommendations for using artemisinin-based combinations for treating uncomplicated malaria in areas of high CHQ failure such as Ghana

Mutations in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance genes, and treatment outcomes in Ghanaian children with uncomplicated malaria.

The association between the clinical outcome of chloroquine treatment and mutations in the putative Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene at codon 76 and multidrug resistance gene 1 (Pf mdr1) at codon 86 were investigated among 406 children with uncomplicated malaria presenting at five sentinel health centres in Ghana. Presence of mutations in isolates taken at pre-treatment and on day of recurrence of parasites was detected using PCR followed by RFLP techniques. The prevalence of Pfcrt T76 mutants was 80% at Hohoe, 46% at Navrongo, 98% at Tarkwa, 61% at Sunyani and 46% at Yendi. The prevalence of the mutant Pfmdr1 at Hohoe, Navrongo, Tarkwa, Sunyani and Yendi were 78, 58, 95, 53 and 42%, respectively. Significant association between the Pfcrt mutation and treatment outcome was observed at Hohoe and Sunyani (p 0.05). Similarly, a statistical significant association between Pfmdr1 86 and treatment failures was observed at Hohoe and Sunyani (p < 0.05) but not at the other three sites. A positive correlation was found between mutant Pfcrt prevalence only and treatment failures with a Spearman's rho-value of 0.872 and a p-value = 0.027. All parasite isolates from samples taken at recrudescence from patients with chloroquine treatment failures were found to have both Pfcrt and Pfmdr mutations