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.

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.

Drug resistance markers within an evolving efficacy of anti-malarial drugs in Cameroon: a systematic review and meta-analysis (1998-2020).

BACKGROUND: Malaria remains highly endemic in Cameroon. The rapid emergence and spread of drug resistance was responsible for the change from monotherapies to artemisinin-based combinations. This systematic review and meta-analysis aimed to determine the prevalence and distribution of Plasmodium falciparum drug resistance markers within an evolving efficacy of anti-malarial drugs in Cameroon from January 1998 to August 2020. METHODS: The PRISMA-P and PRISMA statements were adopted in the inclusion of studies on single nucleotide polymorphisms (SNPs) of P. falciparum anti-malarial drug resistance genes (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfatp6, Pfcytb and Pfk13). The heterogeneity of the included studies was evaluated using the Cochran's Q and I2 statistics. The random effects model was used as standard in the determination of heterogeneity between studies. RESULTS: Out of the 902 records screened, 48 studies were included in this aggregated meta-analysis of molecular data. A total of 18,706 SNPs of the anti-malarial drug resistance genes were genotyped from 47,382 samples which yielded a pooled prevalence of 35.4% (95% CI 29.1-42.3%). Between 1998 and 2020, there was significant decline (P < 0.0001 for all) in key mutants including Pfcrt 76 T (79.9%-43.0%), Pfmdr1 86Y (82.7%-30.5%), Pfdhfr 51I (72.2%-66.9%), Pfdhfr 59R (76.5%-67.8%), Pfdhfr 108 N (80.8%-67.6%). The only exception was Pfdhps 437G which increased over time (30.4%-46.9%, P < 0.0001) and Pfdhps 540E that remained largely unchanged (0.0%-0.4%, P = 0.201). Exploring mutant haplotypes, the study observed a significant increase in the prevalence of Pfcrt CVIET mixed quintuple haplotype from 57.1% in 1998 to 57.9% in 2020 (P < 0.0001). In addition, within the same study period, there was no significant change in the triple Pfdhfr IRN mutant haplotype (66.2% to 67.3%, P = 0.427). The Pfk13 amino acid polymorphisms associated with artemisinin resistance were not detected. CONCLUSIONS: This review reported an overall decline in the prevalence of P. falciparum gene mutations conferring resistance to 4-aminoquinolines and amino alcohols for a period over two decades. Resistance to artemisinins measured by the presence of SNPs in the Pfk13 gene does not seem to be a problem in Cameroon. Systematic review registration PROSPERO CRD42020162620.

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.

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.

Using a composite adherence tool to assess ART response and risk factors of poor adherence in pregnant and breastfeeding HIV-positive Cameroonian women at 6 and 12 months after initiating option B.

BACKGROUND: Antiretroviral therapy (ART) adherence in preventing HIV mother-to-child transmission in association with virological suppression and risk factors of low adherence in the Cameroon's Option B+ programme are poorly understood. We used a composite adherence score (CAS) to determine adherence and risk factors of poor adherence in association with virological treatment response in HIV-positive pregnant and breastfeeding women who remained in care at 6 and 12 months after initiating ART. METHODS: We prospectively enrolled 268 women after ART initiation between October 2013 and December 2015 from five facilities within the Kumba health district. Adherence at 6 and 12 months were measured using a CAS comprising of a 6-month medication refill record review, a four-item self-reported questionnaires and a 30-day visual analogue scale. Adherence was defined as the sum scores of the three measures and classified as high, moderate and low. Measured adherence levels were compared to virological suppression rates at month 12 and risk factors of poor adherence were determined. RESULTS: At 6 and 12 months, 217 (81.0%) and 185 (69.0%) women were available for adherence evaluation. Respectively. Of those, 128 (59.0%) and 68 (31.4%) had high or moderate adherence as per the CAS tool at month 6, and 116 (62.7%) and 48 (24.9%) at month 12, respectively. Viral loads were assessed in 165 women at months 12, and 92.7% had viral suppression (< 1000 copies/mL). Viral suppression was seen in 100% of women with high, 89.5% with moderate, and 52.9% with low adherence using the CAS tool. Virological treatment failure was significantly associated with low adherence [OR 7.6, (95%CI, 1.8-30.8)]. Risk factors for low adherence were younger age [aOR 3.8, (95%CI, 1.4-10.6)], primary as compared to higher levels of education [aOR 2.7, (95%CI, 1.4-5.2)] and employment in the informal sector compared to unemployment [aOR 1.9, (95%CI,1.0-3.6)]. CONCLUSIONS: During the first year of Option B+ implementation in Cameroon our novel CAS adherence tool was feasible, and useful to discriminate ART adherence levels which correlated with viral suppression. Younger age, less educated and informal sector employed women may need more attention for optimal adherence to reduce the risk of virological failure.

Retention in care and reasons for discontinuation of lifelong antiretroviral therapy in a cohort of Cameroonian pregnant and breastfeeding HIV-positive women initiating 'Option B+' in the South West Region.

OBJECTIVE: To assess linkage and retention in care along the PMTCT cascade in HIV-positive pregnant and breastfeeding women initiating Option B+ in Cameroon. METHODS: We prospectively determined uptake of HIV testing and counselling (HTC), uptake of ART and retention in care after Option B+ initiation between October 2013 and December 2014 in pregnant and breastfeeding women from five sites within the Kumba Health District. Retention in care was assessed over at least 12 months follow-up and estimated by Kaplan-Meier analysis. During follow-up, tracing outcomes and reasons for discontinuing treatment were documented. RESULTS: The uptake of HTC of 5813 women with unknown HIV status was 98.5%, 251 (4.4%) were newly diagnosed HIV positive, and ART uptake in women eligible to start Option B+ was 96.8%. We enrolled 268 women initiating lifelong ART in the follow-up. Overall, 65 (24.3%) discontinued treatment, either defined by loss to follow-up (44.6%) or actively stopped treatment (55.8%). Retention in care was 88.0% and 81.1% at 6 and 12 months, respectively. Discontinuation was significantly associated in multivariate analysis with small sites and high staff turnover [aOR 2.5 (95% CI 1.6, 3.9), P < 0.001]. Main reasons for stopping treatment were HIV status denial and stigma (52.8%), religious reasons (25.0%) and lack of transport fare (11.1%). CONCLUSION: We observed good uptake of HTC, ART and retention in care, which declined over time. Discontinuation of Option B+ was highest at small sites with a high staff turnover. Improved staffing, adequate task shifting and community interventions to track defaulters including reducing stigma and religious beliefs may improve Option B+ retention.

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.

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.

One year survey of human rotavirus strains suggests the emergence of genotype G12 in Cameroon.

In this study the emergence of rotavirus A genotype G12 in children <5 years of age is reported from Cameroon during 2010/2011. A total of 135 human stool samples were P and G genotyped by reverse transcriptase PCR. Six different rotavirus VP7 genotypes were detected, including G1, G2, G3, G8, G9, and G12 in combinations with P[4], P[6] and P[8] VP4 genotypes. Genotype G12 predominated in combination with P[8] (54.1%) and P[6] (10.4%) genotypes followed by G1P[6] (8.2%), G3P[6] (6.7%), G2P[4] (5.9%), G8P[6] (3.7%), G2P[6] (0.7%), G3P[8] (0.7%), and G9P[8] (0.7%). Genotype P[6] strains in combination with various G-types represented a substantial proportion (N=44, 32.6%) of the genotyped strains. Partially typed strains included G12P[NT] (2.2%); G3P[NT] (0.7%); G(NT)P[6] (1.5%); and G(NT)P[8] (0.7%). Mixed infections were found in five specimens (3.7%) in several combinations including G1+ G12P[6], G2+ G3P[6] + P[8], G3+ G8P[6], G3 + G12P[6] + P[8], and G12P[6] +P[8]. The approximately 10% relative frequency of G12P[6] strains detected in this study suggests that this strain is emerging in Cameroon and should be monitored carefully as rotavirus vaccine is implemented in this country, as it shares neither G- nor P-type specificity with strains in the RotaTeq® and Rotarix® vaccines. These findings are consistent with other recent reports of the global spread and increasing epidemiologic importance of G12 and P[6] strains.

Prophylactic effects of probiotic bacterium Latilactobacillus sakei on haematological parameters and cytokine profile of mice infected with Plasmodium berghei ANKA during early malaria infection.

Malaria is a deadly parasitic disease caused a by protozoan parasite of the genus plasmodium. The challenges facing by chemotherapy and vector control couple with the lack of vaccine against malaria necessitate an urgent need for the development of alternative treatment regimens to combat this disease. One possible antimalarial treatment regimen is the use of probiotic bacteria as dietary supplements. Traditionally fermented milk is a rich source of probiotic bacteria that up to date, very few studies have been carried out on their immunoprotective effects against early malaria infection in mice. This study sought to assess the prophylactic activities of a probiotic bacterium Latilactobacillus sakei on malaria and inflammation in Plasmodium berghei infected mice. The probiotic bacterium was isolated from the Fulani's traditionally fermented milk and identified using the sequencing of the 16S r RNA gene. The repository activity of L. sakei on malaria was assessed using the method described by Peters with slight modification. Eighty-four BALB/c mice were randomly divided into two sets of seven groups of six mice each. One set received orally different doses of L. sakei Chloroquine and Sulfadoxine/Pyrimethamine for seven days before infection while the other set received for fourteen days before infection with 0.1 mL of 107Plasmodium berghei. Parasitaemia density, haematological parameters and inflammatory cytokines profile were evaluated. Data were presented as Mean ± SEM and analysed using SPSS version 20.0. The results of this study revealed that L. sakei significantly (p < 0.05) reduced in dose dependent manner parasite load, body weight loss and reduction of body temperature in all the treated mice when compare to untreated mice. Leukocytopenia, thrombocytosis and inflammation were also found to be significantly (p < 0.05) prevented in treated mice as compared to untreated mice. This study suggested that L sakei possesses immunomodulation and protective effects on early malaria infection in Plasmodium berghei mice.

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.

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.

Diversity and behavioral activity of Anopheles mosquitoes on the slopes of Mount Cameroon.

BACKGROUND: Malaria remains endemic in Cameroon, with heterogeneous transmission related to eco-climatic variations, vector diversity and spatial distribution. The intensification of malaria prevention and control through the free distribution of insecticide-treated nets in recent years may have altered the composition, geographic distribution and natural infection rate of Anopheles species, with implications for malaria transmission dynamics. The present study seeks to assess the vectorial diversity, dynamics and infectivity across different seasons and altitudes in relationship to parasite prevalence around the slopes of Mount Cameroon, southwestern region. METHOD: Mosquitoes were sampled (indoors and outdoors) in 11 eco-epidemiological settings at low (18-197 m), intermediate (371-584 m) and high (740-1067 m) altitude by nightly human landing catches. The mosquitoes were identified morphologically and Anopheles gambiae sibling species identified by PCR. Parity status was ascertained by examining the ovaries and the entomological inoculation rates (EIR) determined by Plasmodium falciparum circumsporozoite antigen ELISA of the head-thorax. The prevalence of Plasmodium infection across target communities was assessed using rapid diagnostic tests. RESULTS: A total of 7327 (18.0 mosquitoes/trap/night) mosquitoes were trapped, mainly during the rainy season (5678, 77.5%) and at low altitude (3669, 50.1%). Anopheles spp. (5079, 69.3%) was the most abundant genera and An. gambiae complex (2691, 36.7%) the major vector, varying with altitude (χ2 = 183.87, df = 8, P < 0.001) and season (χ2 = 28.14, df = 4, P < 0.001). Only An. gambiae (s.s.) was identified following molecular analysis of An. gambiae complex siblings. The overall biting peak for An. gambiae complex was 2-3 a.m. Anopheles cinctus was the most abundant secondary vector in the area. The average EIR in the area was 2.08 infective bites per person per night (ib/p/n), higher at low (2.45 ib/p/n) than at intermediate altitude (1.39 ib/p/n) and during the rainy (1.76 ib/p/n) compared to the dry season (0.34 ib/p/n). Anopheles funestus was most infectious overall (28.1%, 16/57) while An. gambiae had the highest inoculation rates averaging 1.33 ib/p/n. Most Anopheles species across all altitudes and seasons were parous, highest in communities with the highest proportion of malaria parasite infections. CONCLUSION: Anopheles gambiae (s.s.) remains the major malaria vector in the area and An. cinctus possibly a secondary vector of the disease in the slopes of Mt. Cameroon. The seasonal and altitudinal effects on the distribution of these mosquitoes may have implications for the transmission of malaria and its control strategies in the area. Regular monitoring of the bionomics of local Anopheles vector species and targeted control interventions in the 'hotspots' is necessary to curb the prevalence of the infection and incidence of disease.

Intermittent preventive treatment with Sulphadoxine-Pyrimethamine (IPTp-SP) is associated with protection against sub-microscopic P. falciparum infection in pregnant women during the low transmission dry season in southwestern Cameroon: A Semi - longitudinal study.

The current guidelines for malaria prevention and control during pregnancy in Africa is predicated on the prevention of infection and/or disease through intermittent preventive treatment in pregnancy (IPTp), insecticide-treated nets (ITNs) and effective malaria case diagnosis and management. Concerns that increasing SP resistance in some areas of SSA may have compromised IPTp-SP efficacy prompted this contemporaneous study, designed to assess the prevalence and risk factors of sub-microscopic infection in parturient women during the low transmission season in Mutengene, a rapidly growing semi-urban area in Southwest Region, Cameroon. Pregnant women originally reporting for the establishment of antenatal clinic care during the dry season were followed-up to term and their pregnancy outcomes recorded. About 2 ml of venous blood was collected for malaria diagnosis using PfHRP2/pLDH malaria rapid diagnostic kit and light microscopy. DNA was extracted from dried blood spots by the Chelex-100 method and the Plasmodium falciparum status detected by nested PCR amplification of the 18SrRNA gene using specific predesigned primers. Of the 300 women enrolled, the proportion of malaria parasite infected as determined by microscopy, RDT and PCR was 12.9%, 16.4% and 29.4% respectively, with 39.9% overall infected with P. falciparum by microscopy and/or RDT and/or PCR and a very low-density infection, averaging 271 parasites per microliter of blood. About 25.0% (68/272) of women who were negative by microscopy were positive by PCR (submicroscopic P. falciparum infection), with primigravidae and IPTp-SP non usage identified as independent risk factors for submicroscopic P. falciparum parasitaemia while fever history (aOR = 4.83, 95% CI = 1.28-18.22, p = 0.020) was associated with risk of malaria parasite infection overall. IPTp-SP use (p = 0.007) and dosage (p = 0.005) significantly influenced whether or not the participant will be malaria parasite negative or carry submicroscopic or microscopic infection. Although Infant birthweight and APGAR score were independent of the mother's P. falciparum infection and submicroscopic status, infant's birthweight varied with the gravidity status (p = 0.001) of the mother, with significantly lower birthweight neonates born to primigravidae compared to secundigravidae (p = 0.001) and multigravidae (p = 0.003). Even in holo-endemic dry season, there exists a large proportion of pregnant women with very low density parasitaemia. IPTp-SP seems to be relevant in controlling submicroscopic P. falciparum infections, which remains common in pregnant women, and are hard to diagnose, with potentially deleterious consequences for maternal and fetal health. Future studies should be carried out in hyperendemic malaria foci where the parasitemia levels are substantially higher in order to confirm the efficacy of IPTp-SP.