Genome-wide association study identifies novel candidate malaria resistance genes in Cameroon.

Recent data suggest that only a small fraction of severe malaria heritability is explained by the totality of genetic markers discovered so far. The extensive genetic diversity within African populations means that significant associations are likely to be found in Africa. In their series of multi-site genome-wide association studies (GWAS) across sub-Saharan Africa, the Malaria Genomic Epidemiology Network (MalariaGEN) observed specific limitations and encouraged country-specific analyses. Here, we present findings of a GWAS of Cameroonian participants that contributed to MalariaGEN projects (n = 1103). We identified protective associations at polymorphisms within the enhancer region of CHST15 [Benjamin-Hochberg false discovery rate (FDR) < 0.02] that are specific to populations of African ancestry, and that tag strong eQTLs of CHST15 in hepatic cells. In-silico functional analysis revealed a signature of epigenetic regulation of CHST15 that is preserved in populations in historically malaria endemic regions, with haplotype analysis revealing a haplotype that is specific to these populations. Association analysis by ethnolinguistic group identified protective associations within SOD2 (FDR < 0.04), a gene previously shown to be significantly induced in pre-asymptomatic malaria patients from Cameroon. Haplotype analysis revealed substantial heterogeneity within the beta-like globin (HBB) gene cluster amongst the major ethnic groups in Cameroon confirming differential malaria pressure and underscoring age-old fine-scale genetic structure within the country. Our findings revealed novel insights in the evolutionary genetics of populations living in Cameroon under malaria pressure with new significant protective loci (CHST15 and SOD2) and emphasized the significant attenuation of genetic association signals by fine-scale genetic structure.

Histidine-rich protein (hrp) 2-based RDT false-negatives and Plasmodium falciparum hrp 2 and 3 gene deletions in low, seasonal and intense perennial transmission zones in Cameroon: a cross - sectional study.

BACKGROUND: False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. METHODS: This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. RESULTS: A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. CONCLUSION: Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.

Long-lasting insecticidal nets use and the prevalence of Plasmodium falciparum infection among pregnant women attending antenatal care at the Bonassama District Hospital, Littoral Region of Cameroon: a cross-sectional study.

BACKGROUND: Malaria during pregnancy continues to be a significant cause of morbidity and mortality for both infants and mothers, particularly in sub-Saharan African (SSA) countries, despite increased efforts to control it. The utilization of long-lasting insecticide-treated nets (LLINs) during pregnancy is a well-established strategy to reduce the prevalence of malaria. Nonetheless, inadequate adherence remains a persistent challenge in certain regions with high malaria endemicity. This research aimed to assess the effectiveness of long-lasting insecticidal nets in preventing asymptomatic malaria infections among pregnant women attending antenatal care at the Bonassama District Hospital in the Littoral Region of Cameroon. METHODS: A hospital-based cross-sectional study was conducted from March to June 2022. Data on sociodemographic characteristics and LLIN usage were collected through a structured questionnaire, while asymptomatic malaria infections were identified using a PfHRP2/pLDH malaria qualitative rapid diagnostic kit. The relationship between categorical variables was analyzed using the chi-square test and logistic regression at a significance level of 5%. RESULTS: Out of the 411 pregnant women included in the study, 35.4% were diagnosed with malaria. The LLIN utilization rate was 65.1%. The risk of malaria infection was 2.7 times higher (AOR = 2.75, 95% CI = 1.83-4.14, p < 0.001) among women who did not consistently use LLINs compared to those who did. Pregnant women in their first trimester (AOR = 3.40, 95% CI = 1.24-4.64, p = 0.010) and second trimester (AOR = 1.90, 95%CI = 0.99-3.62, p = 0.055) were more likely to sleep under net when compared to those in the third trimester. Younger women 20-29 years (71.4%), those in the first trimester (69.6%) and those who had the nets before pregnancy (68.9%) were amongst those who frequently used use the nets. Among the reasons reported for not frequently using LLINs were heat (55.2%), suffocation (13.6%) and the smell of nets (8.4%). CONCLUSION: The use of LLIN was moderately high among the participants in this study, though still below national target. Age group, religion and gestation period were the major factors determining the use of LLINs. Considering the proven effectiveness of LLINs in reducing malaria morbidity and mortality, it is imperative for the National Malaria Control Programme (NMCP) to remain focused in promoting both LLIN ownership and utilization to achieve the national target of 100% and 80%, respectively.

Sub-microscopic Plasmodium falciparum parasitaemia, dihydropteroate synthase (dhps) resistance mutations to sulfadoxine-pyrimethamine, transmission intensity and risk of malaria infection in pregnancy in Mount Cameroon Region.

BACKGROUND: Plasmodium falciparum resistance to intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) continues to spread throughout sub-Saharan Africa. This study assessed the occurrence of microscopic and sub-microscopic P. falciparum parasitaemia, dihydropteroate synthase mutations associated with resistance to SP and maternal anaemia in the Mount Cameroon area. METHODS: Consenting pregnant women living in semi-rural and semi-urban/urbanized settings were enrolled in this cross-sectional study. Socio-demographic, antenatal and clinical data were documented. Microscopic and sub-microscopic parasitaemia were diagnosed using peripheral blood microscopy and nested polymerase chain reaction (PCR) respectively. The dhps mutations were genotyped by restriction fragment length polymorphism analysis. The presence of A437G, K540E, and A581G was considered a marker for high-level resistance. Haemoglobin levels and anaemia status were determined. RESULTS: Among the women, the prevalence of microscopic and sub-microscopic P. falciparum infection were 7.7% (67/874) and 18.6% (93/500) respectively. Predictors of microscopic infection were younger age (< 21 years) (AOR = 2.89; 95% CI 1.29-6.46) and semi-rural settings (AOR = 2.27; 95% CI 1.31-3.96). Determinants of sub-microscopic infection were the rainy season (AOR, 3.01; 95% CI 1.77-5.13), primigravidity (AOR = 0.45; 95% CI 0.21-0.94) and regular ITN usage (AOR = 0.49; 95% CI 0.27-0.90). Of the145 P. falciparum isolates genotyped, 66.9% (97) carried mutations associated with resistance to SP; 33.8% (49), 0%, 52.4% (76) and 19.3% (28) for A437G, K540E, A581G and A437G + A581G respectively. The A581G mutation was associated with ≥ 3 SP doses evident only among sub-microscopic parasitaemia (P = 0.027) and multigravidae (P = 0.009). Women with microscopic infection were more likely from semi-rural settings (AOR = 7.09; 95% CI 2.59-19.42), to report history of fever (AOR = 2.6; 95% CI 1.07-6.31), to harbour parasites with double resistant mutations (AOR = 6.65; 95% CI 1.85-23.96) and were less likely to have received 2 SP doses (AOR = 0.29; 95% CI 1.07-6.31). Microscopic infection decreased Hb levels more than sub-microscopic infection. CONCLUSION: The occurrence of sub-microscopic P. falciparum parasites resistant to SP and intense malaria transmission poses persistent risk of malaria infection during pregnancy in the area. ITN usage and monitoring spread of resistance are critical.

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

Coverage and effectiveness of intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) on adverse pregnancy outcomes in the Mount Cameroon area, South West Cameroon.

BACKGROUND: Growing concerns about the waning efficacy of IPTp-SP warrants continuous monitoring and evaluation. This study determined coverage of IPTp-SP and compared the effectiveness of the 3-dose to 2-dose regimen on placental malaria (PM) infection and low birth weight (LBW) in the Mount Cameroon area. METHODS: Consenting pregnant women were enrolled consecutively through a cross-sectional survey at delivery at four antenatal clinics, two each from semi-rural and semi-urban settings from November 2016 to December 2017. Reported IPTp-SP use, demographic and antenatal clinic (ANC) data of the mothers and neonate birth weights were documented. Maternal haemoglobin concentration was measured using a haemoglobinometer and PM infection diagnosed by placental blood microscopy. Logistic regression analysis was used to model study outcomes. RESULTS: Among the 465 parturient women enrolled, 47.0% (203), 34.7% (150), 18.3% (79) and 7.1% (33) reported uptake of ≥ 3, 2.1 dose(s) and no SP, respectively. Uptake of ≥ 3 doses varied significantly (p < 0.001) according to type of medical facility, timing of ANC initiation and number of ANC visits. The prevalence of PM was 18.5% where uptake of ≥ 3 SP doses (AOR = 2.36: 95% CI 1.41-4.87), primiparity (AOR = 2.13: 95% CI 1.19-3.81), semi-rural setting (AOR = 1.85: 95% CI 1.12-3.04) increased odds of infection. Also, three or more dosing was associated (p < 0.001) with increased PM density notably among women from semi-urban areas. Compared with third trimester, ANC initiation in the second trimester (AOR: 0.39: 95% CI 0.20-0.74) lower odds of infection. The prevalence of LBW infants was 7.3% and were generally those of anaemic (AOR: 4.6: 95% CI 1.03-20.57) and semi-rural (AOR: 5.29: 95% CI 1.73-16.15) women. Although ≥ 3 (AOR: 0.31: 95% CI 0.11-0.87) and 2 (AOR: 0.32: 95% CI 0.11-0.93) doses of SP was associated with lower odds of LBW, ≥ 3 doses were not associated with additional increase in birth weight nor maternal haemoglobin levels when compared with 2 doses. CONCLUSION: In the Mount Cameroon area, reported uptake of IPTp with ≥ 3 SP doses did not provide observable prophylactic benefits. SP resistance efficacy studies are necessary.

Genetic diversity and drug resistance surveillance of Plasmodium falciparum for malaria elimination: is there an ideal tool for resource-limited sub-Saharan Africa?

The intensification of malaria control interventions has resulted in its global decline, but it remains a significant public health burden especially in sub-Saharan Africa (sSA). Knowledge on the parasite diversity, its transmission dynamics, mechanisms of adaptation to environmental and interventional pressures could help refine or develop new control and elimination strategies. Critical to this is the accurate assessment of the parasite's genetic diversity and monitoring of genetic markers of anti-malarial resistance across all susceptible populations. Such wide molecular surveillance will require selected tools and approaches from a variety of ever evolving advancements in technology and the changing epidemiology of malaria. The choice of an effective approach for specific endemic settings remains challenging, particularly for countries in sSA with limited access to advanced technologies. This article examines the current strategies and tools for Plasmodium falciparum genetic diversity typing and resistance monitoring and proposes how the different tools could be employed in resource-poor settings. Advanced approaches enabling targeted deep sequencing is valued as a sensitive method for assessing drug resistance and parasite diversity but remains out of the reach of most laboratories in sSA due to the high cost of development and maintenance. It is, however, feasible to equip a limited number of laboratories as Centres of Excellence in Africa (CEA), which will receive and process samples from a network of peripheral laboratories in the continent. Cheaper, sensitive and portable real-time PCR methods can be used in peripheral laboratories to pre-screen and select samples for targeted deep sequence or genome wide analyses at these CEAs.

Coverage and usage of insecticide treated nets (ITNs) within households: associated factors and effect on the prevalance of malaria parasitemia in the Mount Cameroon area.

BACKGROUND: Insecticide-treated nets (ITNs) are a widely used tool that has been proven to be effective in the prevention and control of malaria in malaria endemic countries. However, usage varies among households and can greatly affect the benefits of ITNs as a control tool for malaria transmission. This study determined the coverage and usage of ITNS as well as associated factors and the effect of coverage and usage on the prevalence of malaria parasitemia within households in the Mount Cameroon area. METHODS: A cross-sectional survey was conducted between August and September 2014 in six communities within the Mount Cameroon area. Households within the communities were enrolled through multistage sampling and household survey was done using a structured questionnaire. Capillary blood was collected for malaria parasite determination. Data was analysed using SPSS version 20 for windows. Differences in proportions were assessed using the Chi-square test while factors affecting ITNs usage were assessed in multivariate logistic regression at a statistical significance of P ≤ 0.05. RESULTS: A total of 504 households were surveyed, 1564 bed spaces reported while 915(58.5, 95% CI: 56.1-60.9) of the bed spaces had nets and 391(77.6, 95% CI, 74.0-80.2) of the households had at least one bed net. The odds of using ITNs was 2 folds higher (OR = 2.41; 95% CI 1.58-3.69 p = 0.001) and 3 folds higher (OR = 3.149, 95% CI 1.53-6.47 p = 0.002) among houses with 5 to 9 occupants and above 10 occupants respectively when compared to houses with less than 5 occupants. In addition, Individuals living in cement block houses were less likely to use ITNs. Compared to those living in wooden houses (OR = 0.488, 95% CI: 0.269-0.885; p = 0 .018). Rural communities had lower ITN coverage compared to semi-urban communities (p = 0.0001). Increase in ITNs coverage significantly reduces malaria prevalence (correlation - 0.899, p = 0.015). CONCLUSION: Despite the efforts made to scale up ITN distribution so that universal coverage can be attained, coverage remains low. Increasing coverage and putting in place a mechanism to replace torn nets will go a long way reduce the prevalence of malaria parasitemia.

Predictors of the use of interventions to prevent malaria in pregnancy in Cameroon.

BACKGROUND: Malaria in pregnancy is common in sub-Saharan Africa where it contributes to perinatal morbidity and mortality. Use of insecticide-treated bed nets and intermittent preventive therapy with sulfadoxine-pyrimethamine during pregnancy are effective but underutilized interventions to prevent infection. Factors associated with bed net ownership and usage, and use of prophylaxis among recently pregnant women in Cameroon were investigated. METHODS: National data from the 2011 Cameroon Demographic Health Survey was used to identify women with a pregnancy within the previous 5 years. Logistic regression models were created to assess for independent predictors of reported bed net ownership, bed net usage, and the use of malaria prophylaxis medications during pregnancy. RESULTS: Nearly one in two women surveyed had a recent pregnancy (n = 7647). In this group, bed net ownership and usage rates were low (33.7 and 16.9%, respectively); 61.6% used medication for malaria prophylaxis during pregnancy. Bed net ownership and usage were associated with maternal literacy (aOR 1.4 for net usage, 95% CI 1.1-1.8) and the presence of children under age 5 in the home (aOR 2.3 for net usage, 95% CI 1.6-3.3). The use of malaria prophylaxis medication was associated with measures of healthcare access (aOR 17.8, 95% CI 13-24.5 for ≥4 antenatal care visits), higher maternal education (aOR 1.5, 95% CI 1.1-2.1) and maternal literacy (aOR 1.4, 95% CI 1.1-1.7). CONCLUSIONS: Women in Cameroon and their antenatal providers missed many opportunities to prevent malaria in pregnancy. Efforts toward ensuring universal bed net provision, consistent antenatal care and the education of girls are likely to improve birth outcomes attributable to malaria infection.

Malaria, helminths, co-infection and anaemia in a cohort of children from Mutengene, south western Cameroon.

BACKGROUND: Malaria and helminthiases frequently co-infect the same individuals in endemic zones. Plasmodium falciparum and helminth infections have long been recognized as major contributors to anaemia in endemic countries. Several studies have explored the influence of helminth infections on the course of malaria in humans but how these parasites interact within co-infected individuals remains controversial. METHODS: In a community-based longitudinal study from March 2011 to February 2012, the clinical and malaria parasitaemia status of a cohort of 357 children aged 6 months to 10 years living in Mutengene, south-western region of Cameroon, was monitored. Following the determination of baseline malaria/helminths status and haemoglobin levels, the incidence of malaria and anaemia status was determined in a 12 months longitudinal study by both active and passive case detection. RESULTS: Among all the children who completed the study, 32.5 % (116/357) of them had at least one malaria episode. The mean (±SEM) number of malaria attacks per year was 1.44 ± 0.062 (range: 1-4 episodes) with the highest incidence of episodes occuring during the rainy season months of March-October. Children <5 years old were exposed to more malaria attacks [OR = 2.34, 95 % CI (1.15-4.75), p = 0.019] and were also more susceptible to anaemia [OR = 2.24, 95 % CI (1.85-4.23), p = 0.013] compared to older children (5-10 years old). Likewise children with malaria episodes [OR = 4.45, 95 % CI (1.66-11.94), p = 0.003] as well as those with asymptomatic parasitaemia [OR = 2.41, 95 % CI (1.58-3.69) p < 0.001] were susceptible to anaemia compared to their malaria parasitaemia negative counterparts. Considering children infected with Plasmodium alone as the reference, children infected with helminths alone were associated with protection from anaemia [OR = 0.357, 95 % CI (0.141-0.901), p = 0.029]. The mean haemoglobin level (g/dl) of participants co-infected with Plasmodium and helminths was higher (p = 0.006) compared to participants infected with Plasmodium or helminths alone. CONCLUSION: Children below 5 years of age were more susceptible to malaria and anaemia. The high prevalence of anaemia in this community was largely due to malaria parasitaemia. Malaria and helminths co-infection was protective against anaemia.

K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa.

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa.

Plasmodium falciparum parasitaemia and malaria among pregnant women at first clinic visit in the mount Cameroon Area.

BACKGROUND: Pregnant women in malaria endemic areas are at high risk of P. falciparum infection and its complications. This study investigated the prevalence and risk factors for P. falciparum infection and malaria among pregnant women reporting for first antenatal care (ANC) clinic visit in the mount Cameroon area. METHODS: Venous blood samples from consented pregnant women were screened for malaria parasitaemia by light microscopy. Haemoglobin levels, white blood cell (WBC) counts, lymphocyte counts and percentage were determined using an automated haematology analyser. Socio-demographic/economic data, environmental factors and use of malaria control measures were documented. Univariate and multivariate statistical analyses were used. RESULTS: Sixty-eight (22.4%; N = 303) of the women enrolled were positive for P. falciparum parasitaemia. Malaria parasitaemia was significantly (P < 0.001) associated with febrile illness. The overall prevalence of malaria and asymptomatic infection was 16.0% (95% CI = 11-20%) and 10.5% (95% CI = 7.3-15%) respectively. A greater proportion of the malaria cases (61%) reported at the clinic during unscheduled days meanwhile women with asymptomatic parasitaemia mostly (92.8%) seek for ANC during scheduled clinic days. Lower lymphocyte percentage was significantly associated with increase parasite density (r = - 0.34; P = 0.011) and febrile status (MU = 2.46; P = 0.014). While age and gravidity were significant factors associated with P. falciparum infection and/or malaria, the presence of bush and/or standing water around human residence was an independent risk factor of P. falciparum parasitaemia (OR = 3.3: 95% CI = 1.6-7.0; P = 0.002) and malaria (OR = 5.2: 95% CI = 2.0-14; P = 0.001). Being unmarried was significantly associated with increase risk (OR = 2.6:95% CI = 1.1-6.0; P = 0.032) of P. falciparum parasitaemia. Similarly, single women (938) had a significantly higher (t = 2.70; P = 0.009) geometric mean parasite density (GMPD) compared with married women (338). CONCLUSION: Marital status and human residence in areas with bushes and/or standing water modify risk of P. falciparum infection and malaria. Education on early ANC attendance and environmental sanitation are important public health targets for malaria control in pregnancy in this setting.

Plasmodium falciparum merozoite surface protein 1 block 2 gene polymorphism in field isolates along the slope of mount Cameroon: a cross - sectional study.

BACKGROUND: Malaria remains a major global health burden despite the intensification of control efforts, due partly to the lack of an effective vaccine. Information on genetic diversity in natural parasite populations constitutes a major impediment to vaccine development efforts and is limited in some endemic settings. The present study characterized diversity by investigating msp1 block 2 polymorphisms and the relationship between the allele families with ethnodemographic indices and clinical phenotype. METHOD: Individuals with asymptomatic parasitaemia (AP) or uncomplicated malaria (UM) were enrolled from rural, semi-rural and semi-urban localities at varying altitudes along the slope of mount Cameroon. P. falciparum malaria parasitaemic blood screened by light microscopy was depleted of leucocytes using CF11 cellulose columns and the parasite DNA genotyped by nested PCR. RESULTS: Length polymorphism was assessed in 151 field isolates revealing 64 (5) and 274 (22) distinct recombinant and major msp1 allelic fragments (genotypes) respectively. All family specific allelic types (K1, MAD20 and RO33) as well as MR were observed in the different locations, with K1 being most abundant. Eighty seven (60 %) of individuals harbored more than one parasite clone, with a significant proportion (p = 0.009) in rural compared to other settings. AP individuals had higher (p = 0.007) K1 allele frequencies but lower (p = 0.003) mean multiplicity of genotypes per infection (2.00 ± 0.98 vs. 2.56 ± 1.17) compared to UM patients. CONCLUSIONS: These results indicate enormous diversity of P. falciparum in the area and suggests that allele specificity and complexity may be relevant for the progression to symptomatic disease.

Association of candidate gene polymorphisms and TGF-beta/IL-10 levels with malaria in three regions of Cameroon: a case-control study.

BACKGROUND: Plasmodium falciparum malaria is one of the most widespread and deadliest infectious diseases in children under five years in endemic areas. The disease has been a strong force for evolutionary selection in the human genome, and uncovering the critical host genetic factors that confer resistance to the disease would provide clues to the molecular basis of protective immunity and improve vaccine development initiatives. METHODS: The effect of single nucleotide polymorphisms (SNPs) and plasma transforming growth factor beta (TGF-ß) and interleukin 10 (IL-10) levels on malaria pathology was investigated in a case-control study of 1862 individuals from two major ethnic groups in three regions with intense perennial P. falciparum transmission in Cameroon. Thirty-four malaria candidate polymorphisms, including the sickle cell trait (HbS), were assayed on the Sequenom iPLEX platform while plasma TGF-ß and IL-10 levels were measured by sandwich ELISA. RESULTS: The study confirms the known protective effect of HbS against severe malaria and also reveals a protective effect of SNPs in the nitrogen oxide synthase 2 (NOS2) gene against malaria infection, anaemia and uncomplicated malaria. Furthermore, ADCY9 rs10775349 (additive G) and ABO rs8176746 AC individuals were associated with protection from hyperpyrexia and hyperparasitaemia, respectively. Meanwhile, individuals with the EMR1 rs373533 GT, EMR1 rs461645 CT and RTN3 rs542998 (additive C) genotypes were more susceptible to hyperpyrexia while both females and males with the rs1050828 and rs1050829 SNPs of G6PD, respectively, were more vulnerable to anaemia. Plasma TGF-ß levels were strongly correlated with heterozygosity for the ADCY9 rs2230739 and HBB rs334 SNPs while individuals with the ABO rs8176746 AC genotype had lower IL-10 levels. CONCLUSION: Taken together, this study suggests that some rare polymorphisms in candidate genes may have important implications for the susceptibility of Cameroonians to severe malaria. Moreover using the uncomplicated malaria phenotype may permit the identification of novel pathways in the early development of the disease.

Antenatal care visit attendance, intermittent preventive treatment during pregnancy (IPTp) and malaria parasitaemia at delivery.

BACKGROUND: The determinants and barriers for delivery and uptake of IPTp vary with different regions in sub-Saharan Africa. This study evaluated the determinants of ANC clinic attendance and IPTp-SP uptake among parturient women from Mount Cameroon Area and hypothesized that time of first ANC clinic attendance could influence uptake of IPTp-SP/dosage and consequently malaria parasite infection status at delivery. METHODS: Two cross sectional surveys were carried out at the Government Medical Centre in the Mutengene Health Area, Mt Cameroon Area from March to October 2007 and June 2008 to April 2009. Consented parturient women were consecutively enrolled in both surveys. In 2007, socio-demographic data, ANC clinic attendance, gestational age, fever history and reported use/dosage of IPTp-SP were documented using a structured questionnaire. In the second survey only IPT-SP usage/dosage was recorded. Malaria parasitaemia at delivery was determined by blood smear microscopy and placental histology. RESULTS AND DISCUSSION: In 2007, among the 287 women interviewed, 2.2%, 59.7%, and 38.1% enrolled in the first, second and third trimester respectively. About 90% of women received at least one dose SP but only 53% received the two doses in 2007 and by 2009 IPTp-two doses coverage increased to 64%. Early clinic attendance was associated (P = 0.016) with fever history while being unmarried (OR = 2.2; 95% CI: 1.3-3.8) was significantly associated with fewer clinic visits (<4visits). Women who received one SP dose (OR = 3.7; 95% CI: 2.0-6.8) were more likely not to have attended ≥ 4visits. A higher proportion (P < 0.001) of women with first visit during the third trimester received only one dose, meanwhile, those who had an early first ANC attendance were more likely (OR = 0.4; 95% CI = 0.2 - 0.7) to receive two or more doses. Microscopic parasitaemia at delivery was frequent (P = 0.007) among women who enrolled in the third trimester and had received only one SP dose than in those with two doses. CONCLUSION: In the study area, late first ANC clinic enrolment and fewer clinic visits may prevent the uptake of two SP doses and education on early and regular ANC clinic visits can increase IPTp coverage.

Trimethoprim-Sulfamethoxazole Plus Azithromycin to Prevent Malaria and Sexually Transmitted Infections in Pregnant Women With HIV (PREMISE): A Randomized, Double-Masked, Placebo-Controlled, Phase IIB Clinical Trial.

Background: This trial tested the effectiveness of a novel regimen to prevent malaria and sexually transmitted infections (STIs) among pregnant women with HIV in Cameroon. Our hypothesis was that the addition of azithromycin (AZ) to standard daily trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis would reduce malaria and STI infection rates at delivery. Methods: Pregnant women with HIV at gestational age <28 weeks were randomized to adjunctive monthly oral AZ 1 g daily or placebo for 3 days and both groups received daily standard oral TMP-SMX through delivery. Primary outcomes were (1) positive peripheral malaria infection by microscopy or polymerase chain reaction and (2) composite bacterial genital STI (Chlamydia trachomatis, Neisseria gonorrhoeae, or syphilis) at delivery. Relative risk and 95% confidence intervals were estimated using 2 × 2 tables with significance as P < .05. Results: Pregnant women with HIV (n = 308) were enrolled between March 2018 and August 2020: 155 women were randomized to TMP-SMX-AZ and 153 women to TMP-SMX-placebo. Groups were similar at baseline and loss to follow up was 3.2%. There was no difference in the proportion with malaria (16.3% in TMP-SMX-AZ vs 13.2% in TMP-SMX; relative risk, 1.24 [95% confidence interval, .71-2.16]) or STI at delivery (4.2% in TMP-SMX-AZ vs 5.8% in TMP-SMX; relative risk, 0.72 [95% confidence interval, .26-2.03]). Adverse birth outcomes were not significantly different, albeit lower in the TMP-SMX-AZ arm (preterm delivery 6.7% vs 10.7% [P = .3]; low birthweight 3.4% vs 5.4% [P = .6]). Conclusions: The addition of monthly azithromycin to daily TMP-SMX prophylaxis in pregnant women living with HIV in Cameroon did not reduce the risk of malaria or bacterial STI at delivery.

Status of the Multidrug Resistance-1 Gene of Plasmodium falciparum in Four Malaria Epidemiological Strata, Two Decades after the Abolition of Chloroquine as First-Line Treatment for Uncomplicated Malaria in Cameroon.

Drug-resistant malaria parasites pose a threat to global malaria control efforts, and it is important to know the extent of these drug-resistant mutations in each region to determine appropriate control measures. Chloroquine (CQ) was widely used in Cameroon for decades, but its declining clinical efficacy due to resistance prompted health authorities in 2004 to resort to artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. Despite numerous efforts to control malaria, it persists, and the emergence and spread of resistance to ACTs make the development of new drugs or the possible reintroduction of discontinued drugs increasingly urgent. Malaria-positive blood samples were collected from 798 patients on Whatman filter paper to determine the status of resistance to CQ. DNA was extracted by boiling in Chelex and analysis of Plasmodium species. Four hundred P. falciparum monoinfected samples, 100 per study area, were amplified by nested PCR, and allele-specific restriction analysis of Pfmdr1 gene molecular markers was performed. Fragments were analyzed using a 3% ethidium bromide-stained agarose gel. P. falciparum was the most abundant Plasmodium species, accounting for 87.21% of P. falciparum monoinfections only. No infection with P. vivax was detected. The majority of samples contained the wild type for all 3 SNPs evaluated on the Pfmdr1 gene with N86, Y184, and D1246 accounting for 45.50%, 40.00%, and 70.00%, respectively. The most abundant haplotype observed was the Y184D1246 double wild type at 43.70%. The results suggest that P. falciparum is the major infecting species and that P. falciparum species with the susceptible genotype are gradually recapturing the parasite population.

Knowledge, attitude, and adherence to malaria control guidelines and the prevalence of Plasmodium species infection in localities across transmission and ecological zones in Cameroon.

Background: Despite a scale up of control interventions over the years, malaria remains a major public health and economic concern in Cameroon, contributing considerably to hospitalization and deaths. The effectiveness of control strategies depends on the extent of adherence by the population to national guidelines. This study assessed the influence of human knowledge, attitudes, and practices related to malaria and its control on the prevalence of malaria parasite infection, with implications for the elimination of the disease. Methodology: This is a cross-sectional community and hospital-based study, covering the five ecological and three malaria transmission zones in Cameroon. A pre-tested semi-structured questionnaire was used to document socio-demographic and clinical parameters as well as knowledge, attitudes, and practices toward malaria control and management. Consenting participants were screened for malaria parasite with rapid diagnostic test (mRDT) of the peripheral blood. Association between qualitative variables was determined using the chi-square test and logistic regression analysis. Results: A total of 3,360 participants were enrolled, 45.0% (1,513) of whom were mRDT positive, with 14.0% (451/3,216) and 29.6% (951/3,216) having asymptomatic parasitaemia and malaria, respectively. Although most participants knew the cause, symptoms, and control strategies, with 53.6% (1,000/1,867) expertly knowledgeable about malaria overall, only 0.1% (2/1,763) individuals were fully adherent to malaria control measures. Conclusion: The risk of malaria in Cameroon remains high, with the population considerably knowledgeable about the disease but poorly adherent to national malaria control guidelines. Concerted and more effective strategies aimed at improving knowledge about malaria and adherences to control interventions are necessary to ultimately eliminate the disease.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Severe and uncomplicated falciparum malaria in children from three regions and three ethnic groups in Cameroon: prospective study.

BACKGROUND: To identify the factors that account for differences in clinical outcomes of malaria as well as its relationship with ethnicity, transmission intensity and parasite density. METHODS: A prospective study was conducted in nine health facilities in the Centre, Littoral and South West regions of Cameroon, and in three ethnic groups; the Bantu, Semi-Bantu and Foulbe. Children aged one month to 13 years, with diagnosis suggestive of malaria, were recruited and characterized using the WHO definition for severe and uncomplicated malaria. Malaria parasitaemia was determined by light microscopy, haematological analysis using an automated haematology analyser and glucose level by colorimetric technique. RESULTS: Of the febrile children screened, 971 of the febrile children screened fulfilled the inclusion criteria for specific malaria clinical phenotypes. Forty-nine (9.2%) children had cerebral malaria, a feature that was similar across age groups, ethnicity and gender but lower (P < 0.004) in proportion in the Centre (3.1%, 5/163) compared to the Littoral (11.3%, 32/284) and South West (13.6%, 12/88) regions. Severe anaemia was the most frequent severe disease manifestation, 28.0% (248/885), which was similar in proportion across the three ethnic groups but was more prevalent in females, less than 60 months old, and the Centre region. About 20% (53/267) of the participants presented with respiratory distress, a clinical phenotype independent of age, gender and ethnicity, but highest (P < 0.001) in the Centre (55%, 11/20) compared to the Littoral (27.3%, 3/11) and South West (16.5%, 39/236) regions. Uncomplicated malaria constituted 27.7% (255/920) of hospital admissions and was similar in proportion with gender and across the three ethnic groups but more prevalent in older children (≥ 60 months) as well as in the South West region. The density of malaria parasitaemia was generally similar across clinical groups, gender and ethnicity. However, younger children and residents of the Centre region carried significantly higher parasite loads, with the burden heavier in the Semi-Bantu compared to their Bantu (P = 0.009) and Foulbe (P = 0.026) counterparts in the Centre region. The overall study case fatality was 4.8 (47/755), with cerebral malaria being the only significant risk factor associated with death. Severe anaemia, though a common and major clinical presentation, was not significantly associated with risk of death. CONCLUSION: About half of the acutely febrile children presented with severe malaria, the majority being cases of severe malaria anaemia, followed by respiratory distress and cerebral malaria. The latter two were less prevalent in the Centre region compared to the other regions. Cerebral malaria and hyperpyrexia were the only significant risk factors associated with death.