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.

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.

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.

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.

Plasmodium falciparum histidine-rich protein 2 and 3 gene deletion in the Mount Cameroon region.

Objective: Plasmodium falciparum produces histidine-rich protein 2/3 (Pfhrp2/3) genes that accumulate to high levels in the bloodstream and serve as a diagnostic and prognostic marker for falciparum malaria. Pfhrp2/3 gene deletions may lead to false-negative rapid diagnostic test (RDT) results. We aimed to determine the prevalence of pfhrp2/3 gene deletions in P. falciparum isolates and the implications for RDT use in the Mount Cameroon region. Methods: A cross-sectional hospital-based study with malaria diagnosis performed using microscopy, RDT and nested polymerase chain reaction (nPCR). In total, 324 P. falciparum microscopy positive individuals were enrolled and their samples confirmed positive for P. falciparum using 18SrRNA PCR. Samples that gave false-negative RDT results were analyzed to detect pfhrp2/3 exon 2 deletions. Results: Of 324 positive microscopic and nPCR samples, 16 gave RDT false-negative results. Among the 324 P. falciparum positive isolates, exon 2 deletions were observed in 2.2% (7 of 324); 3 were negative for pfhrp2 gene, 2 for pfhrp3, and 2 for both pfhrp2 and pfhrp3 (double deletions). Conclusion: P. falciparum isolates with pfhrp2/3 gene deletion were present in the parasite populations and may contribute to the RDT false-negative results in the Mount Cameroon region.

Effectiveness and safety of artesunate-amodiaquine versus artemether-lumefantrine for home-based treatment of uncomplicated Plasmodium falciparum malaria among children 6-120 months in Yaoundé, Cameroon: a randomized trial.

BACKGROUND: Many studies have reported high efficacy and safety of artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) when administered under direct observation in Cameroon. There is paucity of data to support their continuous use in home-based treatment of uncomplicated Plasmodium falciparum malaria in Cameroon. Hence, this study aimed to assess the effectiveness and safety of AS-AQ versus AL for home-based treatment of uncomplicated P. falciparum malaria among children 6-120 months in Yaoundé, Cameroon. METHODS: A two-arm, open-label, randomized, controlled trial comparing the equivalence of AS-AQ (experimental group) and AL (control group) was carried out from May 2019 to April 2020 at two secondary hospitals in Yaoundé. Participants were randomized to receive either AS-AQ or AL. After the first dose, antimalarial drugs were given at home, rather than under direct observation by a study staff. The conventional on-treatment and post-treatment laboratory and clinical evaluations were not done until day 3 of the full antimalarial treatment course. The evaluation of effectiveness was mainly based on per protocol polymerase chain reaction adjusted adequate clinical and parasitological response (PP PCR adjusted ACPR) on day 28 post-treatment. Safety was based on assessment of adverse events (AEs) and severe adverse events (SAEs) from day 1 to day 28. RESULTS: A total of 242 children were randomized to receive AS-AQ (n = 114) and AL (n = 128). The PP PCR adjusted day 28 cure rates were [AS-AQ = 96.9% (95% CI, 91.2-99.4) versus AL = 95.5% (95% CI, 89.9-98.5), P = 0.797]. Expected mild to moderate adverse events were reported in both arms [AS-AQ = 83 (84.7%) versus AL = 99 (86.1%), P = 0.774]. The most common adverse events included: transient changes of hematologic indices and fever. CONCLUSIONS: This study demonstrated that AS-AQ and AL are effective and safe for home management of malaria in Yaoundé. The evidence from this study supports the parallel use of the two drugs in routine practice. However, the findings from this study do not describe the likely duration of antimalarial effectiveness in holoendemic areas where multiple courses of treatment might be required. TRIAL REGISTRATION: This study is a randomized controlled trial and it was retrospectively registered on 23/09/2020 at ClinicalTrials.gov with registration number NCT04565184.

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.

Comparison of the Accuracy of Four Malaria Diagnostic Methods in a High Transmission Setting in Coastal Cameroon.

BACKGROUND: Despite recommendation from the World Health Organization that all malaria suspected patients undergo a parasitological confirmation using rapid diagnostic test or light microscopy prior to treatment, health facilities in remote malaria endemic settings sometimes resort to presumptive diagnosis of malaria for clinical management for various reasons. Following observation of this practice, we undertook a cross-sectional study aimed at comparing presumptive diagnosis based on axillary temperature, SD Bioline™ rapid test, and light microscopy as strategies for malaria diagnosis in the coastal region of Mutengene in the South West of Cameroon with the overall goal of supporting improved malaria diagnosis at local levels. METHODOLOGY: Venous blood from 320 participants was used to detect the presence of malaria parasite using SD Bioline™ mRDT and Giemsa stained microscopy or spotted on filter paper for PCR amplification of the 18s rRNA gene of Plasmodium sp following standard procedures. The axillary temperature of each participant was also measured. The sensitivity, specificity, and predictive values and their confidence intervals were determined for each of the methods with PCR as the reference. The area under the curve was used to estimate accuracy of diagnostic method and compared between test method using the X2 test with P<0.05 considered significant. RESULTS: The overall diagnostic sensitivities of presumptive diagnosis using axillary temperature, light microscopy, and SD Bioline™ were observed to be 74.30% (95%CI: 67.90-80.01), 94.86% (95%CI: 90.99-97.41), and 95.33% (95%CI: 91.57-97.74), respectively, and their respective diagnostic specificities were 53.77% (95%CI: 43.82-63.51), 94.34% (95%CI: 88.09-97.87), and 94.34%(95%CI: 88.09-97.89). SD Bioline™ had a diagnostic sensitivity of 91.80% [95%CI: 81.90-97.28] at a parasitaemia of less than 500 parasites/µl of blood but a sensitivity of 100% for parasite counts above 500 parasites/µl of blood. The predictive values of the positive test were highly comparable between light microscopy (90.09%, [95%CI: 83.61-94.18]) and SD Bioline™ mRDT (90.91%, [95%CI: 84.50-94.83]), P=0.98 with kappa values of 0.898 but lower for presumptive diagnosis (50.89%, [95%CI: 43.72-58.03]), P<0.0001, and kappa value of 0.277. Perfect agreement was observed between SD Bioline™ mRDT and light microscopy (Cohen kappa= 0.924). CONCLUSIONS: The study showed that SD Bioline™ was as good as light microscopy in the diagnosis of malaria in remote areas of perennial transmission in South West Cameroon. This study equally revealed the limitations of presumptive diagnosis of malaria (as opposed to the use of RDTs or microscopy). Efforts should be made in such areas to promote parasitological confirmation of malaria using quality assured rapid tests or light microscopy for case management of malaria. The presence of nonnegligible levels of Plasmodium ovale in this study area indicate that treatment guidelines may require revision if same trend is proven in several other areas of same ecology.

Effects of Drug Policy Changes on Evolution of Molecular Markers of Plasmodium falciparum Resistance to Chloroquine, Amodiaquine, and Sulphadoxine-Pyrimethamine in the South West Region of Cameroon.

BACKGROUND: As a result of the spread of parasites resistant to antimalarial drugs, Malaria treatment guidelines in Cameroon evolved from nonartemisinin monotherapy to artemisinin-based combination therapy. The aim of this study was to assess the effect of these therapy changes on the prevalence of molecular markers of resistance from 2003 to 2013 in Mutengene, Cameroon. METHODOLOGY: Dry blood samples (collected in 2003-2005 and 2009-2013) were used for parasite DNA extraction. Drug resistance genes were amplified by PCR and hybridized with oligonucleotide probes or subjected to restriction digestion. The prevalence of individual marker polymorphisms and haplotypes was compared in these two study periods using the Chi square test. RESULTS: Alleles conferring resistance to 4-aminoquinolines in the Pfcrt 76T and Pfmdr1 86Y, 184F, and 1246Y genotypes showed a significant reduction of 97.0% to 66.9%, 83.6% to 45.2%, 97.3% to 56.0%, and 3.1% to 0.0%, respectively (P < 0.05). No difference was observed in SNPs associated with antifolate drugs resistance 51I, 59R, 108N, or 540E (P > 0.05). Haplotype analysis in the Pfmdr1 gene showed a reduction in the YFD from 75.90% to 42.2%, P < 0.0001, and an increase in the NYD (2.9% to 30.1%; P < 0.0001). CONCLUSIONS: The results indicated a gradual return of the 4-aminoquinoline sensitive genotype while the antifolate resistant genotypes increased to saturation.

Randomized non-inferiority and safety trial of dihydroartemisin-piperaquine and artesunate-amodiaquine versus artemether-lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in Cameroonian children.

BACKGROUND: Artemether-lumefantrine and artesunate-amodiaquine are first-line treatment for uncomplicated malaria in Cameroon. No study has yet compared the efficacy of these drugs following the WHO recommended 42-day follow-up period. The goal of this study was to compare the clinical efficacy, tolerability and safety of artesunate-amodiaquine (ASAQ), artemether-lumefantrine (AL) and dihydroartemisinin piperaquine (DHAP) among children aged less than ten years in two malaria-endemic ecological regions of Cameroon. METHODS: A three-arm, randomized, controlled, non-inferiority trial was conducted among children of either gender aged six months (>5 kg) to ten years (n = 720) with acute uncomplicated Plasmodium falciparum infection. Parents/guardians of children provided consent prior to randomization to receive ASAQ, DHAP or AL in the ratio of 2:2:1, respectively. Treatment outcome was assessed based on standard WHO 2003 classification after 42 days of follow-up. The primary outcome was PCR-corrected day-42 cure rates. The non-inferiority, one-sided, lower limit asymptotic 97.5% confidence interval (CI) on the difference in PCR-corrected cure rates of ASAQ and DHAP when compared to AL was accepted if the lower limit of the CI was greater than -10%. Secondary outcomes were parasite and fever clearances and day 7 haemoglobin changes. RESULTS: PCR-corrected PP cure rates of 96.7, 98.1 and 96.3, respectively, for AL, ASAQ and DHAP was observed. The lower bound of the one-sided 97.5% CI calculated around the difference between day-42 cure rate point estimates in AL and ASAQ groups, AL and DHAP groups were, -6% and -4% respectively. There were no statistical significant differences in parasite or fever clearance times between treatments, although fever clearance pattern was different between ASAQ and DHAP. No statistical significant differences were observed in the occurrence of adverse events among treatment groups. CONCLUSION: ASAQ and DHAP are considered safe and tolerable and are not inferior to AL in the treatment of uncomplicated P. falciparum malaria in Cameroonian children. TRIAL REGISTRATION: NCT01845701.