The temporal dynamics of Plasmodium species infection after artemisinin-based combination therapy (ACT) among asymptomatic children in the Hohoe municipality, Ghana.

BACKGROUND: The routine surveillance of asymptomatic malaria using nucleic acid-based amplification tests is essential in obtaining reliable data that would inform malaria policy formulation and the implementation of appropriate control measures. METHODS: In this study, the prevalence rate and the dynamics of Plasmodium species among asymptomatic children (n = 1697) under 5 years from 30 communities within the Hohoe municipality in Ghana were determined. RESULTS AND DISCUSSION: The observed prevalence of Plasmodium parasite infection by polymerase chain reaction (PCR) was 33.6% (571/1697), which was significantly higher compared to that obtained by microscopy [26.6% (451/1697)] (P < 0.0001). Based on species-specific analysis by nested PCR, Plasmodium falciparum infection [33.6% (570/1697)] was dominant, with Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections accounting for 0.1% (1/1697), 0.0% (0/1697), and 0.0% (0/1697), respectively. The prevalence of P. falciparum infection among the 30 communities ranged from 0.0 to 82.5%. Following artesunate-amodiaquine (AS + AQ, 25 mg/kg) treatment of a sub-population of the participants (n = 184), there was a substantial reduction in Plasmodium parasite prevalence by 100% and 79.2% on day 7 based on microscopy and nested PCR analysis, respectively. However, there was an increase in parasite prevalence from day 14 to day 42, with a subsequent decline on day 70 by both microscopy and nested PCR. For parasite clearance rate analysis, we found a significant proportion of the participants harbouring residual Plasmodium parasites or parasite genomic DNA on day 1 [65.0% (13/20)], day 2 [65.0% (13/20)] and day 3 [60.0% (12/20)] after initiating treatment. Of note, gametocyte carriage among participants was low before and after treatment. CONCLUSION: Taken together, the results indicate that a significant number of individuals could harbour residual Plasmodium parasites or parasite genomic DNA after treatment. The study demonstrates the importance of routine surveillance of asymptomatic malaria using sensitive nucleic acid-based amplification techniques.

Plasmodium falciparum histidine-rich protein 2 diversity in Ghana.

BACKGROUND: In the absence of microscopy, Plasmodium falciparum histidine-rich proteins 2 (PfHRP2)-based rapid diagnostic tests (RDTs) are recommended for the diagnosis of falciparum malaria, particularly in endemic regions. However, genetic variability of the pfhrp2 gene threatens the usefulness of the test due to its impact on RDT sensitivity. This study aimed to investigate the diversity of pfhrp2 in malaria cases among children in Ghana. METHODS: A cross-sectional study was conducted at the Adidome Government Hospital in the Volta Region of Ghana. A total of 50 children with mean age of 6.6 ± 3.5 years and diagnosed falciparum malaria were included. Blood samples were collected for complete blood count, malaria parasite identification and counting using auto analyzer and microscopy, respectively. DNA was isolated from blood-spotted Whatman filters, amplified and sequenced. Nucleotide sequences were translated in silico to corresponding amino acids and the deduced amino acids sequences were analyzed for diversity using Mega X. RESULTS: The number of repeats and number of each repeat within PfHRP2 varied between isolates. Twelve rare PfHRP2 repeat types, two of which are previously unreported, were identified in this study. The HRP2 sequence obtained in this study shared high similarities with isolates from Kenya. Using Baker's regression model, Group B was the highest occurring type (58.0%). Screening of all sequences for epitopes recognized by PfHRP2-specific monoclonal antibodies (mAbs), the predominant motif was AHHAADAHH, which is recognized by the C1-13 mAbs. CONCLUSION: This study reports diversity of P. falciparum HRP2 in samples from Ghanaian children with symptomatic malaria. The findings of this study highlight the existence of extra amino acid repeat types which adds to the PfHRP2 antigenic variability.

Inactivation of Plasmodium falciparum in whole blood by riboflavin plus irradiation.

BACKGROUND: Malaria parasites are frequently transmitted by unscreened blood transfusions in Africa. Pathogen reduction methods in whole blood would thus greatly improve blood safety. We aimed to determine the efficacy of riboflavin plus irradiation for treatment of whole blood infected with Plasmodium falciparum. STUDY DESIGN AND METHODS: Blood was inoculated with 10(4) or 10(5) parasites/mL and riboflavin treated with or without ultraviolet (UV) irradiation (40-160 J/mL red blood cells [mL(RBCs)]). Parasite genome integrity was assessed by quantitative amplification inhibition assays, and P. falciparum viability was monitored in vitro. RESULTS: Riboflavin alone did not affect parasite genome integrity or parasite viability. Application of UV after riboflavin treatment disrupted parasite genome integrity, reducing polymerase-dependent amplification by up to 2 logs (99%). At 80 J/mL(RBCs), riboflavin plus irradiation prevented recovery of viable parasites in vitro for 2 weeks, whereas untreated controls typically recovered to approximately 2% parasitemia after 4 days of in vitro culture. Exposure of blood to 160 J/mL(RBCs) was not associated with significant hemolysis. CONCLUSIONS: Riboflavin plus irradiation treatment of whole blood damages parasite genomes and drastically reduces P. falciparum viability in vitro. In the absence of suitable malaria screening assays, parasite inactivation should be investigated for prevention of transfusion-transmitted malaria in highly endemic areas.

Prevalence of Plasmodium falciparum gametocytaemia in asymptomatic school children before and after treatment with dihydroartemisinin-piperaquine (DP).

Background: Asymptomatic Plasmodium carriers form the majority of malaria-infected individuals in most endemic areas. A proportion of these asymptomatically infected individuals carry gametocytes, the transmissible stages of malaria parasites, that sustain human to mosquito transmission. Few studies examine gametocytaemia in asymptomatic school children who may form an important reservoir for transmission. We assessed the prevalence of gametocytaemia before antimalarial treatment and monitored clearance of gametocytes after treatment in asymptomatic malaria children. Methods: A total of 274 primary school children were screened for P. falciparum parasitaemia by microscopy. One hundred and fifty-five (155) parasite positive children were treated under direct observation with dihydroartemisinin-piperaquine (DP). Gametocyte carriage was determined by microscopy seven days prior to treatment, day 0 before treatment, and on days 7, 14 and 21 post initiation of treatment. Results: The prevalence of microscopically-detectable gametocytes at screening (day -7) and enrolment (day 0) were 9% (25/274) and 13.6% (21/155) respectively. Following DP treatment, gametocyte carriage dropped to 4% (6/135), 3% (5/135) and 6% (10/151) on days 7, 14 and 21 respectively. Asexual parasites persisted in a minority of treated children, resulting in microscopically detectable parasites on days 7 (9%, 12/135), 14 (4%, 5/135) and 21 (7%, 10/151). Gametocyte carriage was inversely correlated with the age of the participants (p = 0.05) and asexual parasite density (p = 0.08). In a variate analysis, persistent gametocytaemia 7 or more days after treatment was significantly associated with post-treatment asexual parasitaemia at day 7 (P = 0.027) and presence of gametocytes on the day of treatment (P < 0.001). Conclusions: Though DP provides both excellent cure rates for clinical malaria and a long prophylactic half-life, our findings suggest that after treatment of asymptomatic infections, both asexual parasites and gametocytes may persist in a minority of individuals during the first 3 weeks after treatment. This indicates DP may be unsuitable for use in mass drug administration strategies towards malaria elimination in Africa.

The global transcriptome of Plasmodium falciparum mid-stage gametocytes (stages II-IV) appears largely conserved and gametocyte-specific gene expression patterns vary in clinical isolates.

Our overall understanding of the developmental biology of malaria parasites has been greatly enhanced by recent advances in transcriptomic analysis. However, most of these investigations rely on laboratory strains (LS) that were adapted into in vitro culture many years ago, and the transcriptomes of clinical isolates (CI) circulating in human populations have not been assessed. In this study, RNA-seq was used to compare the global transcriptome of mid-stage gametocytes derived from three short-term cultured CI, with gametocytes derived from the NF54 reference laboratory strain. The core transcriptome appeared to be consistent between CI- and LS-derived gametocyte preparations, but some important differences were also observed. A majority of gametocyte-specific genes (43/53) appear to have relatively higher expression in CI-derived gametocytes than in LS-derived gametocytes, but a K-means clustering analysis showed that genes involved in flagellum- and microtubule-based processes (movement/motility) were more abundant in both groups, albeit with some differences between them. In addition, gametocytes from one CI described as CI group II gametocytes (CI:GGII) showed gene expression variation in the form of reduced gametocyte-specific gene expression compared to the other two CI-derived gametocytes (CI gametocyte group I, CI:GGI), although the mixed developmental stages used in our study is a potential confounder, only partially mitigated by the inclusion of multiple replicates for each CI. Overall, our study suggests that there may be subtle differences in the gene expression profiles of mid-stage gametocytes from CI relative to the NF54 reference strain of Plasmodium falciparum. Thus, it is necessary to deploy gametocyte-producing clinical parasite isolates to fully understand the diversity of gene expression strategies that may occur during the sequestered development of parasite sexual stages. IMPORTANCE Maturing gametocytes of Plasmodium falciparum are known to sequester away from peripheral circulation into the bone marrow until they are mature. Blocking gametocyte sequestration can prevent malaria transmission from humans to mosquitoes, but most studies aim to understand gametocyte development utilizing long-term adapted laboratory lines instead of clinical isolates. This is a particular issue for our understanding of the sexual stages, which are known to decrease rapidly during adaptation to long-term culture, meaning that many LS are unable to produce transmissible gametocytes. Using RNA-seq, we investigated the global transcriptome of mid-stage gametocytes derived from three clinical isolates and a reference strain (NF54). This identified important differences in gene expression profiles between immature gametocytes of CI and the NF54 reference strain of P. falciparum, suggesting increased investment in gametocytogenesis in clinical isolates. Our transcriptomic data highlight the use of clinical isolates in studying the morphological, cellular features and molecular biology of gametocytes.

Expression of a type B RIFIN in Plasmodium falciparum merozoites and gametes.

BACKGROUND: The ability of Plasmodium falciparum to undergo antigenic variation, by switching expression among protein variants encoded by multigene families, such as var, rif and stevor, is key to the survival of this parasite in the human host. The RIFIN protein family can be divided into A and B types based on the presence or absence of a 25 amino acid motif in the semi-conserved domain. A particular type B RIFIN, PF13_0006, has previously been shown to be strongly transcribed in the asexual and sexual stages of P. falciparum in vitro. METHODS: Antibodies to recombinant PF13_0006 RIFIN were used in immunofluorescence and confocal imaging of 3D7 parasites throughout the asexual reproduction and sexual development to examine the expression of PF13_0006. Furthermore, reactivity to recombinant PF13_0006 was measured in plasma samples collected from individuals from both East and West African endemic areas. RESULTS: The PF13_0006 RIFIN variant appeared expressed by both released merozoites and gametes after emergence. 7.4% and 12.1% of individuals from East and West African endemic areas, respectively, carry plasma antibodies that recognize recombinant PF13_0006, where the antibody responses were more common among older children. CONCLUSIONS: The stage specificity of PF13_0006 suggests that the diversity of RIFIN variants has evolved to provide multiple specialized functions in different stages of the parasite life cycle. These data also suggest that RIFIN variants antigenically similar to PF13_0006 occur in African parasite populations.

Generation of Plasmodium falciparum Gametocytes In Vitro with Specific Considerations for Field Isolates.

Reproducible induction of gametocytes of Plasmodium falciparum in vitro is crucial for performing various experimental analyses to understand gametocyte cellular and molecular biology and immunology, and for the evaluation of antigametocidal agents and vaccine development. In this protocol, we present specific procedures for the enrichment, synchronous production and separation of developmental stages of P. falciparum gametocytes from culture-adapted field isolates.

Parasite clearance dynamics in children hospitalised with severe malaria in the Ho Teaching Hospital, Volta Region, Ghana.

Background: Over 90% of severe malaria (SM) cases occur in African children. Parenteral artesunate is currently the recommended treatment for SM. Studies of parasite clearance in paediatric SM cases are needed for assessment of therapeutic outcomes but are lacking in Africa. Methods: Severe malaria patients were recruited in the children's emergency ward at Ho Teaching Hospital, Ghana, in 2018. Blood samples were taken upon admission, every 24 h for 3 days and 1 week after treatment, and DNA extracted. Parasitaemia and parasite densities were performed by microscopy at enrolment and the follow-up days wherever possible. Relative parasite density was measured at each timepoint by duplex qPCR and parameters of parasite clearance estimated. Results: Of 25 evaluable SM patients, clearance of qPCR-detectable parasites occurred within 48 h for 17 patients, but three out of the remaining eight were still qPCR-positive on day 3. Increased time to parasite clearance was seen in children ≥5 years old, those with lower haemoglobin levels and those with a high number of previous malaria diagnoses, but these associations were not statistically significant. Conclusion: We examined parasite clearance dynamics among paediatric cases of SM. Our observations suggest that daily sampling for qPCR estimation of P. falciparum peripheral density is a useful method for assessing treatment response in hospitalised SM cases. The study demonstrated varied parasite clearance response, thus illuminating the complex nature of the mechanism in this important patient group, and further investigations utilizing larger sample sizes are needed to confirm our findings.

Targeting the Plasmodium falciparum proteome and organelles for potential antimalarial drug candidates.

There is an unmet need to unearth alternative treatment options for malaria, wherein this quest is more pressing in recent times due to high morbidity and mortality data arising mostly from the endemic countries coupled with partial diversion of attention from the disease in view of the SARS-Cov-2 pandemic. Available therapeutic options for malaria have been severely threatened with the emergence of resistance to almost all the antimalarial drugs by the Plasmodium falciparum parasite in humans, which is a worrying situation. Artemisinin combination therapies (ACT) that have so far been the mainstay of malaria have encountered resistance by malaria parasite in South East Asia, which is regarded as a notorious ground zero for the emergence of resistance to antimalarial drugs. This review analyzes a few key druggable targets for the parasite and the potential of specific inhibitors to mitigate the emerging antimalarial drug resistance problem by providing a concise assessment of the essential proteins of the malaria parasite that could serve as targets. Moreover, this work provides a summary of the advances made in malaria parasite biology and the potential to leverage these findings for antimalarial drug production.

Immune Responses to the Sexual Stages of Plasmodium falciparum Parasites.

Malaria infections remain a serious global health problem in the world, particularly among children and pregnant women in Sub-Saharan Africa. Moreover, malaria control and elimination is hampered by rapid development of resistance by the parasite and the vector to commonly used antimalarial drugs and insecticides, respectively. Therefore, vaccine-based strategies are sorely needed, including those designed to interrupt disease transmission. However, a prerequisite for such a vaccine strategy is the understanding of both the human and vector immune responses to parasite developmental stages involved in parasite transmission in both man and mosquito. Here, we review the naturally acquired humoral and cellular responses to sexual stages of the parasite while in the human host and the Anopheles vector. In addition, updates on current anti-gametocyte, anti-gamete, and anti-mosquito transmission blocking vaccines are given. We conclude with our views on some important future directions of research into P. falciparum sexual stage immunity relevant to the search for the most appropriate transmission-blocking vaccine.

Plasmodium falciparum malaria cases detected for prompt treatment by rapid diagnostic tests in the Ho Teaching Hospital of the Volta Region of Ghana.

BACKGROUND: Prompt diagnosis and effective treatment of malaria cases with efficacious drugs is an important strategy in the management and control of malaria in endemic populations. As part of a study investigating the factors modulating the development of Plasmodium falciparum gametocytes in the human host, we assessed the rate of RDT positivity of patients in different departments of the Ho Teaching Hospital and the relation with age and anaemia. MATERIALS AND METHODS: Eight-hundred and ten individuals attending clinic at various departments within the Ho Teaching Hospital were screened for malaria antigenaemia using RDT as a point-of-entry investigation. RDT positive individuals were immediately treated for malaria whereas RDT negative individuals were treated for other ailments. Haematological analyses were performed for 69 of these patients and the relationship between RDT results and haemoglobin levels were investigated. RESULTS: The overall RDT positivity rate was 19.8% (160/810) of all individuals screened. There was no significant difference in the haemoglobin levels of RDT-positive and RDT-negative individuals (p value = 0.272). The highest number of attendees screened was children in the paediatric outpatient department and paediatric ward, 62% (507/810), with RDT positivity rate of 17% (91/507). We found the highest RDT positivity rate of 51% (19/37) in the male medical ward. CONCLUSIONS: This study shows that RDT is a useful tool in promoting prompt diagnosis and management of malaria and though children form a majority of hospital attendees and malaria infections, the frequency of malaria detection may be higher in adults as compared to children.

Gametocyte Development and Carriage in Ghanaian Individuals with Uncomplicated Plasmodium falciparum Malaria.

Plasmodium falciparum gametocytes develop over 9-12 days while sequestered in deep tissues. On emergence into the bloodstream, they circulate for varied amounts of time during which certain host factors might influence their further development. We aimed to evaluate the potential association of patient clinical parameters with gametocyte development and carriage via in vivo methods. Seventy-two patients were enrolled from three hospitals in the Volta region of Ghana in 2016. Clinical parameters were documented for all patients, and gametocyte prevalence by microscopy was estimated at 12.5%. By measuring RNA transcripts representing two distinct gametocyte developmental stages using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), we obtained a more precise estimate of gametocyte carriage while also inferring gametocyte maturation. Fifty-three percent of the study participants harbored parasites expressing transcripts of the immature gametocyte-specific gene (PF3D7_1477700), whereas 36% harbored PF3D7_1438800 RNA-positive parasites, which is enriched in mid and mature gametocytes, suggesting the presence of more immature stages. Linear logistic regression showed that patients older than 5 years but less than 16 years were more likely to carry gametocytes expressing both PF3D7_1477700 and PF3D7_1438800 compared with younger participants, and gametocytemia was more likely in mildly anemic individuals compared with those with severe/moderate anemia. These data provide further evidence that a greater number of malaria patients harbor gametocytes than typically estimated by microscopy and suggest a possible association between age, fever, anemia, and gametocytemia.

Low Levels of Human Antibodies to Gametocyte-Infected Erythrocytes Contrasts the PfEMP1-Dominant Response to Asexual Stages in P. falciparum Malaria.

Vaccines that target Plasmodium falciparum gametocytes have the potential to reduce malaria transmission and are thus attractive targets for malaria control. However, very little is known about human immune responses to gametocytes present in human hosts. We evaluated naturally-acquired antibodies to gametocyte-infected erythrocytes (gametocyte-IEs) of different developmental stages compared to other asexual parasite stages among naturally-exposed Kenyan residents. We found that acquired antibodies strongly recognized the surface of mature asexual-IEs, but there was limited reactivity to the surface of gametocyte-IEs of different stages. We used genetically-modified P. falciparum with suppressed expression of PfEMP1, the major surface antigen of asexual-stage IEs, to demonstrate that PfEMP1 is a dominant target of antibodies to asexual-IEs, in contrast to gametocyte-IEs. Antibody reactivity to gametocyte-IEs was similar to asexual-IEs lacking PfEMP1. Significant antibody reactivity to the surface of gametocytes was observed when outside of the host erythrocyte, including recognition of the major gametocyte antigen, Pfs230. This indicates that there is a deficiency of acquired antibodies to gametocyte-IEs despite the acquisition of antibodies to gametocyte antigens and asexual IEs. Our findings suggest that the acquisition of substantial immunity to the surface of gametocyte-IEs is limited, which may facilitate immune evasion to enable malaria transmission even in the face of substantial host immunity to malaria. Further studies are needed to understand the basis for the limited acquisition of antibodies to gametocytes and whether vaccine strategies can generate substantial immunity.

Comparison of malaria diagnostic methods in four hospitals in the Volta region of Ghana.

Background: Rapid diagnostic tests (RDTs) and microscopy are routinely used for the diagnosis of malaria in Ghana. DNA-based polymerase chain reaction (PCR) is not yet used routinely. We compared diagnostic methods and tested the sensitivities of different malaria diagnostic methods against PCR. Materials and methods: Study participants from four hospitals with a suspicion of malaria donated finger -prick blood for RDT and blood film examination. In addition, a blood spot was collected for PCR analysis, prior to treatment. Retrospective species-specific PCR was performed on all samples collected. Results: Using PCR we found an overall malaria prevalence of 39% among the 211 evaluable blood spots (83/211) and this ranged between 6-61% across the four hospitals. Of the 164 participants with RDT data, malaria prevalence was 57% (94/164), ranging from 3-100% from the four hospitals. Microscopy was the least sensitive with a parasite prevalence of 21% (25/119) of the evaluable 119 participants, varying from 9 to 35% across three health facilities. By comparison, we found the sensitivities and specificities of RDT results when compared to PCR to be slightly higher than microscopy compared to PCR. These were 56.4% versus 41.7% and 90% versus 81.9%, respectively, but generally lower than expected. Ninety-five percent of the PCR-detected infections were P. falciparum, while 4% were mixed species infections of P. falciparum and P. malariae, with the remaining being a mono-infection of P. malariae. Conclusions: While using PCR as a gold standard, we found RDT to be more reliable in diagnosing malaria than microscopy. In addition, a majority of malaria-treated cases were not supported by PCR diagnosis, leading to possible overtreatment. Pragmatic strategies are needed to ensure suspected malaria cases are accurately diagnosed before treatment.

Persistent detection of Plasmodium falciparum, P. malariae, P. ovale curtisi and P. ovale wallikeri after ACT treatment of asymptomatic Ghanaian school-children.

Two hundred and seventy four asymptomatic Ghanaian school-children aged 5 to 17 years were screened for malaria parasites by examination of blood films. One hundred and fifty five microscopically-positive individuals were treated with dihydroartemisinin-piperaquine and followed for 3 weeks. Retrospective species-specific PCR of all 274 screened samples identified an additional 60 children with sub-patent parasitaemia, and a substantial proportion of co-infections with Plasmodium malariae, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. One hundred individuals harboured at least one non-falciparum parasite species. Using standard double-read microscopy, the 21-day efficacy of treatment against Plasmodium falciparum was 91.4% among the 117 children seen at all 5 visits. Using nested PCR to test 152 visit 5 blood samples, 22 were found to be parasite-positive. Twenty individuals harboured P. falciparum, four harboured P. ovale spp. and two P. malariae, with four of these 22 isolates being mixed species infections. The persistent detection of low density Plasmodium sp. infections following antimalarial treatment suggests these may be a hitherto unrecognised obstacle to malaria elimination.