Immune responses to P falciparum antibodies in symptomatic malaria patients with variant hemoglobin genotypes in Ghana.

BACKGROUND: Haemoglobin (Hb) variants such as sickle cell trait (SCT/HbAS) play a role in protecting against clinical malaria, but little is known about the development of immune responses against malaria parasite (Plasmodium falciparum surface protein 230 (Pfs230) and Plasmodium falciparum erythrocyte binding antigen 175 region-3 (PfEBA175-3R)) and vector (on the An. gambiae Salivary Gland Protein-6 peptide 1 (gSG6-P1)) antigens in individuals with variants Hb genotypes. This study assessed antibody (IgG) responses against malaria parasite, Pfs230 and PfEBA175-3R and vector, gSG6-P1 in febrile individuals with variant Hb genotypes. METHODS: The study was conducted on symptomatic malaria patients attending various healthcare facilities throughout Ghana. Microscopy and ELISA were used to determine the natural IgG antibody levels of gSG6-P1, PfEBA175-3R & Pfs230, and Capillarys 2 Flex Piercing was used for Hb variants determination. RESULTS: Of the 600 symptomatic malaria patients, 50.0% of the participants had malaria parasites by microscopy. The majority 79.0% (398/504) of the participants had Hb AA, followed by HbAS variant at 11.3% (57/504) and HbAC 6.7% (34/504). There were significantly (p < 0.0001) reduced levels of gSG6-P1 IgG in individuals with both HbAC and HbAS genotypes compared to the HbAA genotype. The levels of gSG6-P1 IgG were significantly (p < 0.0001) higher in HbAS compared to HbAC. Similarly, Pfs230 IgG and PfEBA-175-3R IgG distributions observed across the haemoglobin variants were significantly higher in HbAC relative to HbAS. CONCLUSION: The study has shown that haemoglobin variants significantly influence the pattern of anti-gSG6-P1, Pfs230, and PfEBA-175 IgG levels in malaria-endemic population. The HbAS genotype is suggested to confer protection against malaria infection. Reduced exposure to infection ultimately reduces the induction of antibodies targeted against P. falciparum antigens.

Effectiveness of artemether-lumefantrine for treating uncomplicated malaria in low- and high-transmission areas of Ghana.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been effective in the supervised treatment of uncomplicated malaria in Ghana. Since ACT usage is primarily unsupervised, this study aimed to determine the effectiveness of artemether-lumefantrine (AL) for treating malaria patients in two transmission settings in Ghana. METHODS: Eighty-four individuals with uncomplicated Plasmodium falciparum malaria were recruited from Lekma Hospital (LH) in Accra (low-transmission area; N = 28), southern Ghana, and King's Medical Centre (KMC) in Kumbungu (high-transmission area; N = 56), northern Ghana. Participants were followed up for 28 days after unsupervised treatment with AL. The presence of asexual parasites was determined by microscopic examination of Giemsa-stained blood smears. Plasmodium species identification was confirmed using species-specific primers targeting the 18S rRNA gene. Parasite recrudescence or reinfection was determined by genotyping the Pfmsp 1 and Pfmsp 2 genes. RESULTS: After AL treatment, 3.6% (2/56) of the patients from KMC were parasitaemic on day 3 compared to none from the LH patients. One patient from KMC with delayed parasite clearance on day 3 remained parasite-positive by microscopy on day 7 but was parasite-free by day 14. While none of the patients from LH experienced parasite recurrence during the 28-day follow-up, three and two patients from KMC had recurrent parasitaemia on days 21 and 28, respectively. Percentage reduction in parasite densities from day 1, 2, and 3 for participants from the KMC was 63.2%, 89.5%, and 84.5%. Parasite densities for participants from the LH reduced from 98.2%, 99.8% on day 1, and 2 to 100% on day 3. The 28-day cumulative incidence rate of treatment failure for KMC was 12.8% (95% confidence interval: 1.9-23.7%), while the per-protocol effectiveness of AL in KMC was 89.47%. All recurrent cases were assigned to recrudescence after parasite genotyping by Pfmsp 1 and Pfmsp 2. CONCLUSION: While AL is efficacious in treating uncomplicated malaria in Ghana, when taken under unsupervised conditions, it showed an 89.4% PCR-corrected cure rate in northern Ghana, which is slightly below the WHO-defined threshold.

Mosquito bites and stage-specific antibody responses against Plasmodium falciparum in southern Ghana.

BACKGROUND: The human host elicits specific immune responses after exposure to various life stages of the malaria parasite as well as components of mosquito saliva injected into the host during a mosquito bite. This study describes differences in IgG responses against antigens derived from the sporozoite (PfCSP), asexual stage parasite (PfEBA175) and the gametocyte (Pfs230), in addition to an Anopheles gambiae salivary gland antigen (gSG6-P1), in two communities in Ghana with similar blood stage malaria parasite prevalence. METHODS: This study used archived plasma samples collected from an earlier cross-sectional study that enrolled volunteers aged from 6 months to 70 years from Simiw, peri-urban community (N = 347) and Obom, rural community (N = 291). An archived thick and thin blood smear for microscopy was used for the estimation of Plasmodium parasite density and species and DNA extraction from blood spots and P. falciparum confirmation was performed using PCR. This study used the stored plasma samples to determine IgG antibody levels to P. falciparum and Anopheles salivary antigens using indirect ELISA. RESULTS: Individuals from Simiw had significantly higher levels of IgG against mosquito gSG6-P1 [median (95%CI)] [2.590 (2.452-2.783) ng/mL] compared to those from Obom [2.119 (1.957-2.345) ng/mL], p < 0.0001. Both IgG responses against Pfs230proC (p = 0.0006), and PfCSP (p = 0.002) were significantly lower in volunteers from Simiw compared to the participants from Obom. The seroprevalence of PfEBA-175.5R (p = 0.8613), gSG6-P1 (p = 0.0704), PfCSP (p = 0.7798) IgG were all similar in Obom and Simiw. However, Pfs230 seroprevalence was significantly higher at Obom compared to Simiw (p = 0.0006). Spearman correlation analysis showed no significant association between IgG responses against gSG6-P1, PfCSP, Pfs230proC and PfEBA-175.5R and parasite density at both Obom and Simiw (p > 0.05). CONCLUSION: In conclusion, the study showed that participants from Simiw had higher concentrations of circulating gSG6-P1 IgG antibodies but lower concentrations of P. falciparum antibodies, PfCSP IgG and Pfs230proC IgG compared to participants from Obom.

Naturally acquired antibody response to a Plasmodium falciparum chimeric vaccine candidate GMZ2.6c and its components (MSP-3, GLURP, and Pfs48/45) in individuals living in Brazilian malaria-endemic areas.

BACKGROUND: The GMZ2.6c malaria vaccine candidate is a multi-stage Plasmodium falciparum chimeric protein which contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, a fusion protein of GLURP and MSP-3, that has been shown to be well tolerated, safe and immunogenic in clinical trials performed in a malaria-endemic area of Africa. However, there is no data available on the antigenicity or immunogenicity of GMZ2.6c in humans. Considering that circulating parasites can be genetically distinct in different malaria-endemic areas and that host genetic factors can influence the immune response to vaccine antigens, it is important to verify the antigenicity, immunogenicity and the possibility of associated protection in individuals living in malaria-endemic areas with different epidemiological scenarios. Herein, the profile of antibody response against GMZ2.6c and its components (MSP-3, GLURP and Pfs48/45) in residents of the Brazilian Amazon naturally exposed to malaria, in areas with different levels of transmission, was evaluated. METHODS: This study was performed using serum samples from 352 individuals from Cruzeiro do Sul and Mâncio Lima, in the state of Acre, and Guajará, in the state of Amazonas. Specific IgG, IgM, IgA and IgE antibodies and IgG subclasses were detected by Enzyme-Linked Immunosorbent Assay. RESULTS: The results showed that GMZ2.6c protein was widely recognized by naturally acquired antibodies from individuals of the Brazilian endemic areas with different levels of transmission. The higher prevalence of individuals with antibodies against GMZ2.6c when compared to its individual components may suggest an additive effect of GLURP, MSP-3, and Pfs48/45 when inserted in a same construct. Furthermore, naturally malaria-exposed individuals predominantly had IgG1 and IgG3 cytophilic anti-GMZ2.6c antibodies, an important fact considering that the acquisition of anti-malaria protective immunity results from a delicate balance between cytophilic/non-cytophilic antibodies. Interestingly, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity. CONCLUSIONS: The data showed that GMZ2.6c protein is widely recognized by naturally acquired antibodies from individuals living in malaria-endemic areas in Brazil and that these may contribute to parasite immunity. These data highlight the importance of GMZ2.6c as a candidate for an anti-malarial vaccine.

Plasmodium falciparum msp1 and msp2 genetic diversity in parasites isolated from symptomatic and asymptomatic malaria subjects in the South of Benin.

Symptomatic and asymptomatic malaria patients are considered as the reservoirs of human Plasmodium. In the present study, we have evaluated the Plasmodium falciparum merozoite surface protein-1 (Pfmsp1) and protein-2 (Pfmsp2) genetic diversity among the symptomatic and asymptomatic malaria infection from health facilities in Cotonou, Benin Republic. A cross-sectional study recruited 158 individuals, including 77 from the asymptomatic and 81 from the symptomatic groups. The parasites were genotyped using Nested Polymerase Chain Reaction. Samples identified as Plasmodium falciparum were genotyped for their genetic diversity. No significant difference was observed in the overall multiplicity of infection (MOI) between the asymptomatic and symptomatic groups. In the symptomatic group, the overall frequency of K1, MAD20, and RO33 allelic family was more predominant (98.5%) followed by 3D7 (87.3%) and FC27 (83.1%). However, in asymptomatic group, the K1 alleles were the most prevalent (100%) followed by FC27 (89.9%), 3D7 (76.8%), MAD20 (60.5%), and RO33 (35.5%). The frequency of multiple allelic types (K1+MAD20+RO33) at the Pfmsp1 loci in the symptomatic infections was significantly higher when compared to that of the asymptomatic ones (97% vs. 34%, p < 0.05), whereas no difference was observed in the frequency of multiple allelic types (3D7 and FC27) at the Pfmsp2 loci between the two groups. The high presence of msp1 multiple infections in the symptomatic group compared to asymptomatic ones suggests an association between the genetic diversity and the onset of malaria symptoms. These data can provide valuable information in the development of a vaccine that could reduce the symptomatic disease.

Population structure and diversity of Plasmodium falciparum in children with asymptomatic malaria living in different ecological zones of Ghana.

BACKGROUND: Genetic diversity in Plasmodium falciparum populations can be used to describe the resilience and spatial distribution of the parasite in the midst of intensified intervention efforts. This study used microsatellite analysis to evaluate the genetic diversity and population dynamics of P. falciparum parasites circulating in three ecological zones of Ghana. METHODS: A total of 1168 afebrile children aged between 3 to 13 years were recruited from five (5) Primary schools in 3 different ecological zones (Sahel (Tamale and Kumbungu), Forest (Konongo) and Coastal (Ada and Dodowa)) of Ghana. Asymptomatic malaria parasite carriage was determined using microscopy and PCR, whilst fragment analysis of 6 microsatellite loci was used to determine the diversity and population structure of P. falciparum parasites. RESULTS: Out of the 1168 samples examined, 16.1 and 39.5% tested positive for P. falciparum by microscopy and nested PCR respectively. The genetic diversity of parasites in the 3 ecological zones was generally high, with an average heterozygosity (He) of 0.804, 0.787 and 0.608 the rainy (peak) season for the Sahel, Forest and Coastal zones respectively. The mean He for the dry (off-peak) season were 0.562, 0.693 and 0.610 for the Sahel, Forest and Coastal zones respectively. Parasites from the Forest zone were more closely related to those from the Sahel than from the Coastal zone, despite the Coastal zone being closer in physical distance to the Forest zone. The fixation indexes among study sites ranged from 0.049 to 0.112 during the rainy season and 0.112 to 0.348 during the dry season. CONCLUSION: A large asymptomatic parasite reservoir was found in the school children during both rainy and dry seasons, especially those in the Forest and Sahel savannah zones where parasites were also found to be related compared to those from the Coastal zone. Further studies are recommended to understand why despite the roll out of several malaria interventions in Ghana, high transmission still persist.

Malaria parasitaemia and mRDT diagnostic performances among symptomatic individuals in selected health care facilities across Ghana.

BACKGROUND: Parasitological diagnosis generates data to assist malaria-endemic countries determine their status within the malaria elimination continuum and also inform the deployment of proven interventions to yield maximum impact. This study determined prevalence of malaria parasitaemia and mRDT performances among febrile patients in selected health care facilities across Ghana. METHODS: This study was a cross-sectional survey conducted in the previously 10 regions of Ghana from May to August 2018. Each patient suspected to have uncomplicated malaria was tested using microscopy and two malaria rapid diagnostic tests (mRDTs): routinely used CareStart™ Malaria HRP2 (Pf) and SD Bioline Malaria Ag Pf (HRP2/pLDH). Main outcome variables were malaria slide and CareStart™ Malaria HRP2 (Pf) positivity rates; and diagnostic accuracy of CareStart™ Malaria HRP2 (Pf) and SD Bioline Malaria Ag Pf (HRP2/pLDH) using microscopy as "gold standard". RESULTS: Overall parasite positivity rates were 32.3% (6266/19402) by mRDT and 16.0% (2984/18616) by microscopy, with Plasmodium falciparum mono-infection accounting for 98.0% of all infections. The odds of parasitaemia by microscopy was significantly lower among female patients compared with males (OR = 0.78; 95% CI: 0.66-0.91), and among patients with history of previous antimalarial intake compared with those with no such history (OR = 0.72; 95% CI: 0.54-0.95). Overall sensitivity of CareStart™ Malaria HRP2 (Pf) was statistically similar to that of the HRP2 band of SD Bioline Malaria Ag Pf (HRP2/pLDH) combo kit (95.4%; 95% CI: 94.6-96.1 vs 94.3%; 95% CI: 93.4-95.1; p = 0.065) but significantly higher than the pLDH band (89.3%; 95% CI: 88.1-90.4; p < 0.001). The same pattern was observed for negative predictive value. CONCLUSIONS: Malaria control interventions should be targeted at the general population, and history of antimalarial intake considered a key predictor of malaria slide negativity. Furthermore, HRP2-based mRDTs remain effective diagnostic tool in the management of suspected uncomplicated malaria in the country.

Comparison of leucocyte profiles between healthy children and those with asymptomatic and symptomatic Plasmodium falciparum infections.

BACKGROUND: The immune mechanisms that determine whether a Plasmodium falciparum infection would be symptomatic or asymptomatic are not fully understood. Several studies have been carried out to characterize the associations between disease outcomes and leucocyte numbers. However, the majority of these studies have been conducted in adults with acute uncomplicated malaria, despite children being the most vulnerable group. METHODS: Peripheral blood leucocyte subpopulations were characterized in children with acute uncomplicated (symptomatic; n = 25) or asymptomatic (n = 67) P. falciparum malaria, as well as malaria-free (uninfected) children (n = 16) from Obom, a sub-district of Accra, Ghana. Leucocyte subpopulations were enumerated by flow cytometry and correlated with two measures of parasite load: (a) plasma levels of P. falciparum histidine-rich protein 2 (PfHRP2) as a proxy for parasite biomass and (b) peripheral blood parasite densities determined by microscopy. RESULTS: In children with symptomatic P. falciparum infections, the proportions and absolute cell counts of total (CD3 +) T cells, CD4 + T cells, CD8 + T cells, CD19 + B cells and CD11c + dendritic cells (DCs) were significantly lower as compared to asymptomatic P. falciparum-infected and uninfected children. Notably, CD15 + neutrophil proportions and cell counts were significantly increased in symptomatic children. There was no significant difference in the proportions and absolute counts of CD14 + monocytes amongst the three study groups. As expected, measures of parasite load were significantly higher in symptomatic cases. Remarkably, PfHRP2 levels and parasite densities negatively correlated with both the proportions and absolute numbers of peripheral leucocyte subsets: CD3 + T, CD4 + T, CD8 + T, CD19 + B, CD56 + NK, γδ + T and CD11c + cells. In contrast, both PfHRP2 levels and parasite densities positively correlated with the proportions and absolute numbers of CD15 + cells. CONCLUSIONS: Symptomatic P. falciparum infection is correlated with an increase in the levels of peripheral blood neutrophils, indicating a role for this cell type in disease pathogenesis. Parasite load is a key determinant of peripheral cell numbers during malaria infections.

Probing the composition of Plasmodium species contained in malaria infections in the Eastern region of Ghana.

BACKGROUND: Asymptomatic falciparum and non-falciparum malaria infections are major challenges to malaria control interventions, as they remain a source of continual infection in the community. This becomes even more important as the debate moves towards elimination and eradication. This study sought to quantify the burden of Plasmodium malaria infection in seven communities in the Eastern Region of Ghana. METHODS: The cross-sectional study recruited 729 participants aged 85 years old and below from 7 closely linked communities. Finger pricked blood was used to prepare thick and thin blood smears as well as spot filter paper and an histidine rich protein 2 (HRP2) rapid diagnostic test kit (RDT). Genomic DNA was extracted from the filter paper dry blood spot (DBS) and used in PCR to amplify the Plasmodium 18S rRNA gene using species specific PCR. RESULTS: 96.6% of the participants were identified as afebrile, with axillary temperatures below 37.5 °C. PCR identified 66% of the participants to harbor malaria parasites, with 9 P. malariae and 7 P. ovale mono-infections accounting for 2.2% and P. falciparum combined with either 36 P. malariae or 25 P. ovale infections, accounting for 13.3%. Parasite prevalence by microscopy (32%) was similar to the RDT positivity rate (33%). False positive RDT results ranged from 64.6% in children aged between 5 and 9 years to 10% in adults aged 20 years and above. No significant differences were observed in falciparum and non-falciparum parasite carriage at the community level, however young adults aged between 15 and 19 years had the highest prevalence (34.8% (16/46)) of P. falciparum and P. malariae parasite carriage whilst children aged between 5 and 9 years had the highest level (11.4% (14/123)) of P. ovale carriage. CONCLUSION: The high rate of misidentification of non-falciparum parasites and the total absence of detection of P. ovale by microscopy suggests that more sensitive malaria diagnostic tools including molecular assays are required to accurately determine the prevalence of carriers of non-falciparum parasites and low density P. falciparum infections, especially during national surveillance exercises. Additionally, malaria control interventions targeting the non-falciparum species P. malariae and P. ovale parasites are needed.

Antibody responses to two new Lactococcus lactis-produced recombinant Pfs48/45 and Pfs230 proteins increase with age in malaria patients living in the Central Region of Ghana.

BACKGROUND: Recent advances in malaria control efforts have led to an increased number of national malaria control programmes implementing pre-elimination measures and demonstrated the need to develop new tools to track and control malaria transmission. Key to understanding transmission is monitoring the prevalence and immune response against the sexual stages of the parasite, known as gametocytes, which are responsible for transmission. Sexual-stage specific antigens, Pfs230 and Pfs48/45, have been identified and shown to be targets for transmission blocking antibodies, but they have been difficult to produce recombinantly in the absence of a fusion partner. METHODS: Regions of Pfs48/45 and Pfs230 known to contain transmission blocking epitopes, 6C and C0, respectively, were produced in a Lactococcus lactis expression system and used in enzyme linked immunosorbent assays to determine the seroreactivity of 95 malaria patients living in the Central Region of Ghana. RESULTS: Pfs48/45.6C and Pfs230.C0 were successfully produced in L. lactis in the absence of a fusion partner using a simplified purification scheme. Seroprevalence for L. lactis-produced Pfs48/45.6C and Pfs230.C0 in the study population was 74.7 and 72.8%, respectively. CONCLUSIONS: A significant age-dependent increase in antibody titers was observed, which suggests a vaccine targeting these antigens could be boosted during a natural infection in the field.

Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2: based malaria rapid diagnostic tests in Ghana.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) play a key role in malaria management and control. The PfHRP-2 based RDT is the most widely used RDT for malaria diagnosis in Ghana. Deletion of pfhrp2 in Plasmodium falciparum parasites affect the diagnostic accuracy of PfHRP-2 based RDT kits. Identifying the prevalence and distribution of P. falciparum parasites with deleted pfhrp2 is important for malaria control. AIM: The purpose of this study was to identify and confirm the prevalence of pfhrp2 deletant P. falciparum parasites circulating within different regions of Ghana. METHODS: DNA was extracted from the membrane of spent CareStart™ PfHRP-2 RDT kits and dried filter paper blood blots using Chelex-100. Exon 2 of pfhrp2 and pfhrp3 genes were amplified by polymerase chain reaction (PCR), resolved by agarose gel electrophoresis and visualized under UV light. RESULTS: Microscopic analysis of blood smears from samples that were PfHRP-2 RDT positive revealed a parasite prevalence of 54/114 (47.4 %) and 2/26 (7.7 %) in Accra and Cape Coast, respectively. PCR analysis increased parasite prevalence in the RDT positive samples to 94/114 (82.5 %) and 6/26 (23.1 %) in Accra and Cape Coast respectively. The exon 2 of the pfhrp2 gene was deleted in 18/54 (33.3 %) of the microscopy confirmed and 36.2 % (34/94) of the PCR confirmed RDT positive samples collected in Accra. No RDT sample, confirmed to contain parasites by either PCR or microscopy was negative by pfhrp2 exon 2 PCR in Cape Coast. A survey of an additional 558 DBS revealed that 22.4 % (46/205) and 40 % (44/110) of PCR positive samples in Accra and Cape Coast, respectively, lacked the exon 2 region of pfhrp2 and possibly the entire pfhrp2 gene. CONCLUSIONS: A high number of P. falciparum parasites, which lack pfhrp2 exon 2 gene have been identified in two communities in Ghana. Continuous nationwide monitoring of the prevalence of pfhrp2 deletant parasites would be essential to malaria control. The use of RDT kits that are effective at malaria diagnosis despite deletion of pfhrp2, such as the PfHRP-2/PfLDH combo RDT kit could enhance the diagnosis of clinical malaria in Ghana.

Prevalence of G6PD deficiency and Plasmodium falciparum parasites in asymptomatic school children living in southern Ghana.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that results in impaired enzyme activity. Although G6PD deficiency is globally distributed it is more prevalent in malaria-endemic countries. Several mutations have been identified in the G6PD gene, which alter enzyme activity. The G6PD genotype predominantly found in sub-Saharan Africa is the G6PDB (G6PD376A) with (G6PD376G) and G6PDA- (G6PD376G/202A, G6PD376G/542T, G6PD376G/680T and G6PD376G/968C) occurring at lower frequencies. AIM: The aim of this study was to identify the prevalence of G6PD deficiency and asymptomatic Plasmodium falciparum carriage in children living in southern Ghana and determine whether G6PD deficiency influences asymptomatic carriage of P. falciparum parasites. METHODS: Blood samples were obtained once a month from 170 healthy Ghanaian school children aged between 5 and 12 years from Basic schools in two communities Obom and Abura with similar rainfall patterns and malaria peak seasons. G6PD enzyme activity was assessed using the qualitative G6PD RDT kit (AccessBIO). G6PD genotyping and asymptomatic parasite carriage was determined by PCR followed by restriction fragment length polymorphism (RFLP) of DNA extracted from dried blood spots. RESULTS: The only sub-Saharan G6PD A- allele detected was the A376G/G202A found in 12.4 % (21/170), of the children and distributed as 4.1 % (7/170) A-, 1.8 % (3/170) A-/A- homozygous deficient males and females and 6.5 % (11/170) A/A- and B/A- heterozygous deficient females. Phenotypically, 10.6 % (15/142) of the children were G6PD deficient. The asymptomatic carriage of P. falciparum by PCR was 50, 29.4, 38.2 and 38.8 % over the months of February through May 2015, respectively, and 28.8, 22.4, 25.9 and 5.9 % by microscopy during the same periods. CONCLUSIONS: G6PD deficiency was significantly associated with a lowered risk of PCR-estimated asymptomatic P. falciparum carriage in children during the off peak malaria season in Southern Ghana.

Plasmodium falciparum genotype and gametocyte prevalence in children with uncomplicated malaria in coastal Ghana.

BACKGROUND: Plasmodium falciparum gametocytes are vital to sustaining malaria transmission. Parasite densities, multiplicity of infection as well as asexual genotype are features that have been found to influence gametocyte production. Measurements of the prevalence of Plasmodium sp. gametocytes may serve as a tool to monitor the success of malaria eradication efforts. METHODS: Whole blood was collected from 112 children aged between 6 months and 13 years with uncomplicated P. falciparum malaria attending three health facilities in southern Ghana from June to August, 2014 before (day 0) and 4 days after completion of anti-malaria drug treatment (day 7). Malaria parasites were observed by microscopy and polymerase chain reaction (PCR); submicroscopic gametocyte carriage was measured by a Pfs25 (PF3D7_1031000) mRNA real time reverse transcriptase polymerase chain reaction (RT-PCR). Parasite genotyping was performed on gDNA extracted from dried filter paper blood blots by amplification of the polymorphic regions of msp1 (PF3D7_0930300) and msp2 (PF3D7_0206800) using PCR. RESULTS: Microscopy estimated 3.1% (3/96) of the total population to carry gametocytes on day 0, which decreased to 2.1% (2/96) on day 7. In contrast, reverse transcriptase-real time PCR (RT-PCR) analysis of a subset of 35 samples estimated submicroscopic gametocyte carriage to be as high as 77% (27/35) using primers specific for Pfs25 (CT < 35) on day 0 and by day 7 this only declined to 60% (21/35). Genotyping the msp2 gene identified higher levels of MOI than the msp1 gene. CONCLUSIONS: Although below detection by microscopy, gametocyte prevalence at submicroscopic levels are high in this region and emphasize the need for more effective elimination approaches like the development of transmission-blocking vaccines and safer gametocytocidal drugs.

Cytokine gene polymorphisms implicated in the pathogenesis of Plasmodium falciparum infection outcome.

Cytokines play a critical role in the immune mechanisms involved in fighting infections including malaria. Polymorphisms in cytokine genes may affect immune responses during an infection with Plasmodium parasites and immunization outcomes during routine administration of malaria vaccines. These polymorphisms can increase or reduce susceptibility to this deadly infection, and this may affect the physiologically needed balance between anti-inflammatory and pro-inflammatory cytokines. The purpose of this review is to present an overview of the effect of selected cytokine gene polymorphisms on immune responses against malaria.

Asymptomatic malaria parasitaemia and virological non-suppression among children living with HIV in Accra, Ghana: a cross-sectional study.

Background: Human Immunodeficiency Virus (HIV) and malaria are two major diseases in sub-Saharan Africa, with coinfections having an impact on the outcomes of both. We assessed the association between asymptomatic malaria parasitaemia and virological non-suppression among children living with HIV attending a clinic at the Korle Bu Teaching Hospital (KBTH) and the Princess Marie Louis Hospital (PML) in the city of Accra, Ghana. Methods: This was a cross-sectional study of asymptomatic malaria in children receiving care at paediatric HIV clinics at KBTH and PML conducted from September to November 2022. Patients who had been on ART for at least 6 months were eligible to participate. Structured questionnaires were used to collect socio-demographic, malaria prevention behaviors, and ART-related data using in-person interviews. Microscopy and PCR were used to screen for malaria and GeneXpert to determine viral load. To examine the determinants of malaria PCR positivity and virological non-suppression, Chi-square tests and logistic regression were utilized. Results: The participants' median age was 9 years with a range of 6 to 12 years. Males made up 57% of the population. We detected 3.6% (10 of 277) and 7.6% (21 of 277) cases of malaria using microscopy and PCR, respectively. Virological non-suppression (VL > 1000 copies/ml) was seen in 82 (29.6%) of the 277 participants. Among the suppressed individuals, 62 (22.4%) exhibited low-level viraemia (VL level 40-1000 copies/ml) and 133 (48%) had non-detectable viral load levels. There were no factors associated with malaria PCR positivity carriage. Poor adherence to antiretroviral therapy was associated with a fivefold increase in the risk of viral load non-suppression (AOR = 4.89 [CI = 2.00-11.98], p = 0.001). Conclusion: The study showed that the proportion of children living with HIV with asymptomatic malaria parasitaemia was low, with about one third of the study population having virological non suppression. The interaction between malaria parasitemia and viral replication may not be the main culprit for virological non suppression.

Correction: Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

[This corrects the article DOI: 10.1371/journal.pntd.0008919.].

Sensitive Detection of Asymptomatic and Symptomatic Malaria with Seven Novel Parasite-Specific LAMP Assays and Translation for Use at Point-of-Care.

Human malaria is a life-threatening parasitic disease with high impact in the sub-Saharan Africa region, where 95% of global cases occurred in 2021. While most malaria diagnostic tools are focused on Plasmodium falciparum, there is a current lack of testing non-P. falciparum cases, which may be underreported and, if undiagnosed or untreated, may lead to severe consequences. In this work, seven species-specific loop-mediated isothermal amplification (LAMP) assays were designed and evaluated against TaqMan quantitative PCR (qPCR), microscopy, and enzyme-linked immunosorbent assays (ELISAs). Their clinical performance was assessed with a cohort of 164 samples of symptomatic and asymptomatic patients from Ghana. All asymptomatic samples with a parasite load above 80 genomic DNA (gDNA) copies per µL of extracted sample were detected with the Plasmodium falciparum LAMP assay, reporting 95.6% (95% confidence interval [95% CI] of 89.9 to 98.5) sensitivity and 100% (95% CI of 87.2 to 100) specificity. This assay showed higher sensitivity than microscopy and ELISA, which were 52.7% (95% CI of 39.7 to 67%) and 67.3% (95% CI of 53.3 to 79.3%), respectively. Nine samples were positive for P. malariae, indicating coinfections with P. falciparum, which represented 5.5% of the tested population. No samples were detected as positive for P. vivax, P. ovale, P. knowlesi, or P. cynomolgi by any method. Furthermore, translation to the point-of-care was demonstrated with a subcohort of 18 samples tested locally in Ghana using our handheld lab-on-chip platform, Lacewing, showing comparable results to a conventional fluorescence-based instrument. The developed molecular diagnostic test could detect asymptomatic malaria cases, including submicroscopic parasitemia, and it has the potential to be used for point-of-care applications. IMPORTANCE The spread of Plasmodium falciparum parasites with Pfhrp2/3 gene deletions presents a major threat to reliable point-of-care diagnosis with current rapid diagnostic tests (RDTs). Novel molecular diagnostics based on nucleic acid amplification are needed to address this liability. In this work, we overcome this challenge by developing sensitive tools for the detection of Plasmodium falciparum and non-P. falciparum species. Furthermore, we evaluate these tools with a cohort of symptomatic and asymptomatic malaria patients and test a subcohort locally in Ghana. The findings of this work could lead to the implementation of DNA-based diagnostics to fight against the spread of malaria and provide reliable, sensitive, and specific diagnostics at the point of care.

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.].

Immunodominant T cell peptides from four candidate malarial antigens as biomarkers of protective immunity against malaria.

A malaria vaccine with high efficacy and capable of inducing sterile immunity against malaria within genetically diverse populations is urgently needed to complement ongoing disease control and elimination efforts. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection and the rapid identification of malaria antigen targets that elicit these responses will fast-track the development of simpler, cost-effective interventions. This study extends our previous work which used peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites to identify immunodominant antigen-specific peptide pools composed of overlapping 15mer sequences spanning full length proteins of four malarial antigens. Our current study aimed to identify CD8 + T cell epitopes within these previously identified positive peptide pools. Cryopreserved PBMCs from 109 HLA-typed subjects were stimulated with predicted 9-11mer CD8 + T cell epitopes from P. falciparum circumsporozoite protein (CSP), apical membrane antigen 1 (AMA1), thrombospondin related anonymous protein (TRAP) and cell traversal for ookinetes and sporozoites (CelTOS) in FluoroSpot assays. A total of 135 epitopes out of 297 tested peptides from the four antigens were experimentally identified as positive for IFN-γ and/or granzyme B production in 65 of the 109 subjects. Forty-three of 135 epitopes (32 %) were promiscuous for HLA binding, with 31 of these promiscuous epitopes (72 %) being presented by HLA alleles that fall within at least two different HLA supertypes. Furthermore, about 52 % of identified epitopes were conserved when the respective sequences were aligned with those from 16 highly diverse P. falciparum parasite strains. In summary, we have identified a number of conserved epitopes, immune responses to which could be effective against multiple P. falciparum parasite strains in genetically diverse populations.

B-Cell Epitope Mapping of the Plasmodium falciparum Malaria Vaccine Candidate GMZ2.6c in a Naturally Exposed Population of the Brazilian Amazon.

The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.