Measuring protective efficacy and quantifying the impact of drug resistance: A novel malaria chemoprevention trial design and methodology.

BACKGROUND: Recently revised WHO guidelines on malaria chemoprevention have opened the door to more tailored implementation. Countries face choices on whether to replace old drugs, target additional age groups, and adapt delivery schedules according to local drug resistance levels and malaria transmission patterns. Regular routine assessment of protective efficacy of chemoprevention is key. Here, we apply a novel modelling approach to aid the design and analysis of chemoprevention trials and generate measures of protection that can be applied across a range of transmission settings. METHODS AND FINDINGS: We developed a model of genotype-specific drug protection, which accounts for underlying risk of infection and circulating genotypes. Using a Bayesian framework, we fitted the model to multiple simulated scenarios to explore variations in study design, setting, and participant characteristics. We find that a placebo or control group with no drug protection is valuable but not always feasible. An alternative approach is a single-arm trial with an extended follow-up (>42 days), which allows measurement of the underlying infection risk after drug protection wanes, as long as transmission is relatively constant. We show that the currently recommended 28-day follow-up in a single-arm trial results in low precision of estimated 30-day chemoprevention efficacy and low power in determining genotype differences of 12 days in the duration of protection (power = 1.4%). Extending follow-up to 42 days increased precision and power (71.5%) in settings with constant transmission over this time period. However, in settings of unstable transmission, protective efficacy in a single-arm trial was overestimated by 24.3% if recruitment occurred during increasing transmission and underestimated by 15.8% when recruitment occurred during declining transmission. Protective efficacy was estimated with greater precision in high transmission settings, and power to detect differences by resistance genotype was lower in scenarios where the resistant genotype was either rare or too common. CONCLUSIONS: These findings have important implications for the current guidelines on chemoprevention efficacy studies and will be valuable for informing where these studies should be optimally placed. The results underscore the need for a comparator group in seasonal settings and provide evidence that the extension of follow-up in single-arm trials improves the accuracy of measures of protective efficacy in settings with more stable transmission. Extension of follow-up may pose logistical challenges to trial feasibility and associated costs. However, these studies may not need to be repeated multiple times, as the estimates of drug protection against different genotypes can be applied to different settings by adjusting for transmission intensity and frequency of resistance.

Uptake of intermittent preventive treatment of malaria in pregnancy and risk factors for maternal anaemia and low birthweight among HIV-negative mothers in Dschang, West region of Cameroon: a cross sectional study.

BACKGROUND: Approximately 32 million pregnant women are at risk of malaria with up to 10,000 maternal deaths and 200,000 neonates at risk annually. Intermittent Preventive Treatment (IPT) with sulfadoxine-pyrimethamine (SP) is recommended by the World Health Organization (WHO) to reduce disease in pregnancy and adverse maternal and newborn outcomes. At least three doses of SP should be taken by pregnant women during antenatal consultation (ANC) beginning from the thirteenth week of pregnancy till parturition. The aim of this study was to assess uptake of IPT during pregnancy and risk factors for maternal anaemia and infant birth weight in Dschang, West region of Cameroon. METHODS: A total of 380 consenting pregnant women at delivery were recruited in a cross- sectional prospective survey between January to December 2021. Data on ANC attendance, total dose of IPT and history of malaria were abstracted from hospital ANC records while socio-demographic characteristics, bed net use and obstetrics history of each participant were also recorded through an interview. Further, blood samples were collected from the intervillous space for assessment of maternal anaemia and microscopic parasitology. Nested PCR based on amplification of the Plasmodium 18S sRNA was carried out to detect submicroscopic infection. IPTp coverage was calculated per WHO recommendation and the prevalence of anaemia and low birth weight were estimated as proportions in the total sample of pregnant women and live births, respectively. Crude and adjusted odds ratios and their 95% confidence intervals were used to estimate associations between pregnancy outcomes considered and risk factors in specific and general models. A p < 0.05 was considered significant. The R software (V4.1.4) was used for all analyses. RESULTS: A majority of pregnant women was aged between 24 and 34 years old (59.2%) and had secondary education (58.8%). Uptake of ≥ 3 IPTp was 64.99% with 77.20% of all who received at least one IPTp doses taking a mix of SP and DP or DP alone in successive ANC contacts. Those with four or more ANC contacts (73.42%) were more likely to have received at least one IPTp. Furthermore, 13.9% of live births had low birthweights (BW < 2500 g) and one in four parturient women with moderate anaemia by WHO criteria. Microscopy (blood smear examination) and PCR-based diagnosis revealed between 0% and 1.57% of parasite-infected placental samples, respectively. Reported malaria in pregnancy predicted maternal anaemia at birth but not birth weight. Only gestational age (< 37 weeks) and bed net use (< 5 months) significantly predicted infant birth weight at delivery. CONCLUSION: The uptake of WHO recommended IPT doses during pregnancy was moderately high. Reported malaria in pregnancy, poor bed net coverage, gestational age less than 37 weeks adversely affect maternal haemoglobin levels at birth and infant birth weight. Asymptomatic and submicroscopic placental parasite infections was found at low prevalence. Together these results highlight the importance of maintaining aggressive measures to prevent malaria in pregnancy and protect the health of mother and baby.

Parasite clearance and protection from Plasmodium falciparum infection (PCPI): a three-arm, parallel, double-blinded, placebo-controlled, randomised trial of presumptive sulfadoxine-pyrimethamine versus sulfadoxine-pyrimethamine plus amodiaquine versus artesunate monotherapy among asymptomatic children 3-5 years of age in Cameroon.

BACKGROUND: The World Health Organization 2022 malaria chemoprevention guidelines recommend providing a full course of antimalarial treatment at pre-defined intervals, regardless of malaria status to prevent illness among children resident in moderate to high perennial malaria transmission settings as perennial malaria chemoprevention (PMC) with sulfadoxine-pyrimethamine (SP). The dhps I431V mutation circulating in West Africa has unknown effect on SP protective efficacy. METHODS: This protocol is for a three-arm, parallel, double-blinded, placebo-controlled, randomised trial in Cameroon among children randomly assigned to one of three directly-observed treatment groups: (i) Group 1 (n = 450) receives daily artesunate (AS) placebo on days - 7 to -1, then active SP plus placebo amodiaquine (AQ) on day 0, and placebo AQ on days 1 and 2; (ii) Group 2 (n = 250) receives placebo AS on days - 7 to -1, then active SP and AQ on day 0, and active AQ on days 1 and 2; and (iii) Group 3 (n = 200) receives active AS on days - 7 to -1, then placebo SP on day 0 and placebo AQ on days 0 to 2. On days 0, 2, 5, 7, and thereafter weekly until day 28, children provide blood for thick smear slides. Dried blood spots are collected on the same days and weekly from day 28 to day 63 for quantitative polymerase chain reaction (qPCR) and genotype analyses. DISCUSSION: Our aim is to quantify the chemopreventive efficacy of SP, and SP plus AQ, and measure the effect of the parasite genotypes associated with SP resistance on parasite clearance and protection from infection when exposed to SP chemoprevention. We will report unblinded results including: (i) time-to-parasite clearance among SP and SP plus AQ recipients who were positive on day 0 by qPCR and followed to day 63; (ii) mean duration of SP and SP plus AQ protection against infection, and (iii) mean duration of symptom-free status among SP and SP plus AQ recipients who were parasite free on day 0 by qPCR. Our study is designed to compare the 28-day follow-up of the new WHO malaria chemoprevention efficacy study protocol with extended follow-up to day 63. TRIAL REGISTRATION: ClinicalTrials.gov NCT06173206; 15/12/2023.

Diagnosis of malaria in pregnancy: accuracy of CareStart™ malaria Pf/PAN against light microscopy among symptomatic pregnant women at the Central Hospital in Yaoundé, Cameroon.

BACKGROUND: The need to start treatment early for pregnant women who present with clinical features of malaria usually conflicts with the need to confirm diagnosis by microscopy (MP) before treatment, due to delays in obtaining results. Parasite sequestration in the placenta is also a problem. Rapid diagnostic tests (RDT), which detect soluble antigens, are a valuable alternative. The objective of this study was to evaluate pretreatment parasite prevalence by microscopy and by RDT and to assess the accuracy of RDT with MP as reference. METHODS: A prospective cross-sectional study was carried out at the obstetrical unit of the Central Hospital in Yaoundé, during the period January-August 2015. Consenting patients with symptoms of suspected malaria in pregnancy were recruited and a blood sample taken for MP and RDT before treatment was started. The estimates of diagnostic performance (with 95% confidence interval) were calculated in OpenEpi online software using the Wilson's score. The agreement, as reflected by the Cohen's kappa, was calculated and interpreted using known intervals. RESULTS: The results showed that, out of the 104 patients recruited, 69.2% (95%CI: 59.1-77.5) were MP positive while 77.94% (95%CI: 63.1-80.9) were RDT positive. The sensitivity of the malaria RDT was 91.67% (95%CI: 83.69-96.77) while the specificity was 53.13% (95%CI: 31.39-65.57). The diagnostic accuracy of the RDT with MP as reference was 79.81% (95%CI: 70.0-86.1). All cases were due to Plasmodium falciparum. A Cohen's kappa of 0.45 (95%CI: 0.26-0.64) was obtained, consistent with a moderate agreement between the tests. CONCLUSIONS: The diagnostic accuracy of the CareStart™ malaria Pf/PAN compared to microscopy was high, but not as desirable, with a false negative RDT at very high parasitaemia. In tertiary facilities, RDTs appear to provide a better diagnostic solution compared to microscopy. However, future studies with larger sample sizes should make this observation more generalizable; as missing a case could have serious consequences on pregnancy outcome.

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.

Patterns of Antibiotic Resistance in Enterobacteriaceae Isolates from Broiler Chicken in the West Region of Cameroon: A Cross-Sectional Study.

Background: The emergence of multidrug-resistant food-borne pathogens of animal origin including Enterobacteriaceae is a growing concern. Identifying and monitoring resistance in isolates from human-related environments are of clinical and epidemiological significance in containing antimicrobial resistance. This study aimed to contribute towards the fight against antibiotic resistance and ameliorate the management/treatment of Enterobacteriaceae-linked diseases in Cameroon. Methods: Cloacal swabs from healthy broilers were enriched in buffered-peptone-water and cultured on EMB agar. Antibiotic susceptibility was tested on Mueller-Hinton-Agar by disc diffusion. Plasmid-borne genes for extended-spectrum beta lactamase (ESBL) and resistance to Quinolones (PMQR) and Aminoglycosides were detected by standard endpoint polymerase chain reaction (PCR). Results: A total of 394 isolates were identified belonging to 12 Enterobacteriaceae genera, the most prevalent were Escherichia coli (81/394 = 20.56%), Salmonella spp (74/394 = 18.78%), and Klebsiella spp (39/394 = 9.90%) respectively. Overall, 84/394 (21.32%) were ESBL producers, 164/394 (41.62%) were resistant to quinolones, 66/394 (16.75%) resistant to aminoglycosides with 44.0% (173/394) expressing MDR phenotype. Poor hygiene practice (OR 2.55, 95% CI: 1.67, 3.89, p=0.001) and rearing for >45 days, (OR = 7.98, 95% CI: 5.05, 12.6, p=0.001) were associated with increased carriage of MDR. Plasmid-borne resistance genes were detected in 76/84 (90.48%) of ESBL-producing isolates, 151/164 (92.07%) quinolone resistant isolates and 59/66 (89.39%) aminoglycoside resistant isolates with co-occurrence of two or more genes per isolate in 58/84 (69.05%) of ESBLs, 132/164 (80.49%) of quinolone resistant isolates and 28/66 (42.42%) of aminoglycoside resistant isolates. Conclusion: This study found high carriage and widespread distribution of Enterobacteriaceae with ESBL and MDR in broiler chicken in the West Region of Cameroon. Most PMQR genes in bacteria were found at levels higher than is seen elsewhere, representing a risk in the wider human community.

Study To Evaluate the Performance of a Point-of-Care Whole-Blood HIV Viral Load Test (SAMBA II HIV-1 Semi-Q Whole Blood).

Remote areas of many low and middle income (LMI) countries have poor access to HIV viral load (HIV VL) testing. The SAMBA II (simple amplification-based assay) Semi-Q whole-blood test (Diagnostics for the Real World [DRW], Cambridge, UK) is a point-of-care assay, which uses leucodepletion technology to allow whole-blood testing in remote settings. A total of 1,540 consecutive HIV-positive clinic patients in Cameroon (250), United Kingdom (633), Ukraine (412), and Zimbabwe (245) donated venous blood (all countries) and finger prick blood (all except UK) for testing on SAMBA II. SAMBA II results were compared with simultaneous plasma results on the Abbott RealTime HIV-1 (Abbott Molecular, Des Plaines, IL) viral load assay and interpreted as either <1,000 RNA copies/ml or ≥1,000 RNA copies/ml. For 1,528 venous whole-blood samples tested on SAMBA II, overall percent agreement with the reference test at a cutoff HIV VL of ≥1,000 copies/ml was 96.9% (1,480/1,528; 95% confidence interval [CI], 95.9% to 97.7%), negative percent agreement was 97.7% (1,259/1,289; 95% CI, 96.7% to 98.4%), and positive percent agreement was 92.5% (221/239; 95% CI, 88.4% to 95.5%). For 854 finger prick samples, there was 95.0% (811/854; 95% CI, 93.3% to 96.3%) overall percent agreement, 98.0% (625/638; 95% CI, 96.5% to 98.9%) negative percent agreement, and 86.1% (186/216; 95% CI, 80.8% to 90.4%) positive percent agreement. These rose to 93.5% (82.1% to 98.6%), 97.6% (95.6% to 98.8%), and 95.6% (93.3% to 97.3%) after exclusion of aberrant results from the Ukraine center. These results show a high level of agreement between SAMBA-II and a laboratory-based assay. SAMBA-II has a performance that is suitable to use as a VL point-of-care assay in remote settings.

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.

A Cross-Sectional Comparative Study of the Performance of the Widal Test and the Typhidot Immunoassay for Typhoid Fever Diagnosis in the West Region of Cameroon.

BACKGROUND: The diagnosis of typhoid fever based on the Widal slide agglutination test remains a major hurdle in developing countries due to varied perceptions of the value of the Widal test in determining clinical decision-making. We undertook a study to evaluate the diagnostic performance of the Widal test and the Typhidot immunoassay in patients suspected of having typhoid fever in the Menoua division, West Region of Cameroon. METHODS: Blood and stool samples were collected from 558 consenting febrile patients on the basis of suspicion of typhoid fever. These patients attended three district health services of the Menoua division between April 2018 and September 2019. These patients had clinical symptoms suggestive of typhoid fever as determined by their consultant. Serum was used for the Widal slide agglutination test and for the Typhidot rapid immunoassay test based on manufacturer's guidelines. A composite reference of fever plus positive coproculture for Salmonella typhi and Salmonella paratyphi was used as the reference. The sensitivity, specificity, and predictive values of the positive and negative tests were calculated as well as Cohen's kappa for agreement between the two tests. RESULTS: Of 558 patients, 12.90% tested positive for the reference method, 57.17% tested positive for the Widal slide agglutination test, while 15.59% were positive for Typhidot-IgM. The overall sensitivity, specificity, and predictive values of the positive and negative tests were 80.56%, 94.03%, 66.6%, and 97.03% for Typhidot-IgM and 94.44%, 48.35%, 21.32%, and 98.33% for the Widal slide agglutination test, respectively. Cohen's kappa estimates were 0.1660 (0.121-0.211) and 0.386 (0.312-0.460) for the Widal test and Typhidot immunoassay for 53.6% and 76.16% agreements of all observations, respectively. CONCLUSION: The Widal test was found to have a lower predictive value for the diagnosis of typhoid fever in our setting. However, the Typhidot test, although better, was not ideal. Diagnosis of typhoid fever should therefore rely on adequate clinical suspicion and a positive Typhidot test to improve the clinical management of typhoid fever in our setting.

Field evaluation of the 22 rapid diagnostic tests for community management of malaria with artemisinin combination therapy in Cameroon.

BACKGROUND: All suspected cases of malaria should receive a diagnostic test prior to treatment with artemisinin-based combinations based on the new WHO malaria treatment guidelines. This study compared the accuracy and some operational characteristics of 22 different immunochromatographic antigen capture point-of- malaria tests (RDTs) in Cameroon to inform test procurement prior to deployment of artemisinin-based combinations for malaria treatment. METHODS: One hundred human blood samples (50 positive and 50 negative) collected from consenting febrile patients in two health centres at Yaoundé were used for evaluation of the 22 RDTs categorized as "Pf Only" (9) or "Pf + PAN" (13) based on parasite antigen captured [histidine rich protein II (HRP2) or lactate dehydrogenase (pLDH) or aldolase]. RDTs were coded to blind technicians performing the tests. The sensitivity, specificity, and predictive values of the positive and negative tests (PPV and NPV) as well as the likelihood ratios were assessed. The reliability and some operational characteristics were determined as the mean values from two assessors, and the Cohen's kappa statistic was then used to compare agreement. Light microscopy was the referent. RESULTS: Of all RDTs tested, 94.2 % (21/22) had sensitivity values greater than 90% among which 14 (63.6%) were 'Pf + PAN' RDTs. The specificity was generally lower than the sensitivity for all RDTs and poorer for "Pf Only" RDTs. The predictive values and likelihood ratios were better for non-HRP2 analytes for "Pf + PAN" RDTs. The Kappa value for most of the tests obtained was around 67% (95% CI 50-69%), corresponding to a moderate agreement. CONCLUSION: Overall, 94.2% (21/22) of RDTs tested had accuracy within the range recommended by the WHO, while one performed poorly, below acceptable levels. Seven "Pf + PAN" and 3 "Pf Only" RDTs were selected for further assessment based on performance characteristics. Harmonizing RDT test presentation and procedures would prevent mistakes of test performance and interpretation.

Delayed entry into HIV care after diagnosis in two specialized care and treatment centres in Cameroon: the influence of CD4 count and WHO staging.

BACKGROUND: Delayed entry into HIV care has complicated the challenges faced in sub-Saharan Africa due to the high HIV burden. A clear knowledge of the factors affecting delayed entry will be essential in directing interventions towards reducing delayed entry into HIV care. There exist very limited data on delayed entry in Cameroon despite its relevance; hence this study was conducted to determine the rate of delayed entry and its associated factors in HIV programmes in Cameroon. METHODS: Data used for this study was routine data obtained from the files of HIV patients who were diagnosed between January 1, 2015 and June 30, 2015 at Limbe and Buea regional hospital HIV centers in the South West region of Cameroon. Data analysis was done using SPSS version 20. RESULTS: Of the 223 patients included in the study, nearly one-quarter of patients (22.4 %) delayed to enter HIV care within 3 months. Those who delayed to enter care were less likely to present at first diagnosis (using HIV rapid test) with symptoms such as fever > 1 month (5 % versus 30 %, p = 0.01) and weight loss > 10 % (13 % versus 48 %, p < 0.001). Alcohol consumption, WHO stage and CD4 count levels were also associated with delayed entry in bivariate analysis. In multivariate analysis only CD4 count greater than 500cells/µl and WHO stages I and II were independently associated with delayed entry into HIV care within 3 months. CONCLUSION: In the South West region of Cameroon, approximately 1 out of 4 patients delay to enter HIV care. This high proportion of patients who delay to enter care correlates to the findings recorded by other studies in sub Saharan Africa. Interventions tackling delayed entry into HIV care might need to be favorably directed towards patients that have high CD4 counts and are at very early WHO clinical stages.

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.

Population genomics of Plasmodium ovale species in sub-Saharan Africa.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) are relapsing malaria parasites endemic to Africa and Asia that were previously thought to represent a single species. Amid increasing detection of ovale malaria in sub-Saharan Africa, we performed a population genomic study of both species across the continent. We conducted whole-genome sequencing of 25 isolates from Central and East Africa and analyzed them alongside 20 previously published African genomes. Isolates were predominantly monoclonal (43/45), with their genetic similarity aligning with geography. Pow showed lower average nucleotide diversity (1.8×10-4) across the genome compared to Poc (3.0×10-4) (p < 0.0001). Signatures of selective sweeps involving the dihydrofolate reductase gene were found in both species, as were signs of balancing selection at the merozoite surface protein 1 gene. Differences in the nucleotide diversity of Poc and Pow may reflect unique demographic history, even as similar selective forces facilitate their resilience to malaria control interventions.

Population Genomics of Plasmodium malariae from Four African Countries.

Plasmodium malariae is geographically widespread but neglected and may become more prevalent as P. falciparum declines. We completed the largest genomic study of African P. malariae to-date by performing hybrid capture and sequencing of 77 isolates from Cameroon (n=7), the Democratic Republic of the Congo (n=16), Nigeria (n=4), and Tanzania (n=50) collected between 2015 and 2021. There is no evidence of geographic population structure. Nucleotide diversity was significantly lower than in co-localized P. falciparum isolates, while linkage disequilibrium was significantly higher. Genome-wide selection scans identified no erythrocyte invasion ligands or antimalarial resistance orthologs as top hits; however, targeted analyses of these loci revealed evidence of selective sweeps around four erythrocyte invasion ligands and six antimalarial resistance orthologs. Demographic inference modeling suggests that African P. malariae is recovering from a bottleneck. Altogether, these results suggest that P. malariae is genomically atypical among human Plasmodium spp. and panmictic in Africa.

Molecular characterization of antimicrobial resistance related genes in E. coli, Salmonella and Klebsiella isolates from broilers in the West Region of Cameroon.

BACKGROUND: Antibiotic resistance has become an enduring threat to human health. This has prompted extensive research to identify the determinants responsible in a bid to fight the spread of resistance and also develop new antibiotics. However, routine procedures focus on identifying genetic determinants of resistance only on phenotypically resistant isolates. We aimed to characterise plasmid mediated resistance determinants in key Enterobacteriaceae isolates with differential phenotypic susceptibility profiles and evaluated the contribution of resistance genes on phenotypic expression of susceptibility. METHODS: The study was carried out on 200 Enterobacteriaceae isolates belonging to the genera E. coli, Salmonella, and Klebsiella; 100 resistant and 100 susceptible to quinolones, aminoglycosides, and ESBL-producing as determined by disk diffusion. Reduced susceptibility in susceptible isolates was determined as an increased MIC by broth microdilution. Plasmid-borne resistance genes were sought in all isolates by endpoint PCR. We performed correlations tests to determine the relationship between the occurrence of resistance genes and increased MIC in susceptible isolates. We then used the notion of penetrance to show adequacy between resistance gene carriage and phenotypic resistance as well as diagnostic odds ratio to evaluate how predictable phenotypic susceptibility profile could determine the presence of resistant genes in the isolates. RESULTS: Reduced susceptibility was detected in 30% (9/30) ESBL negative, 50% (20/40) quinolone-susceptible and 53.33% (16/30) aminoglycoside-susceptible isolates. Plasmid-borne resistance genes were detected in 50% (15/30) of ESBL negative, 65% (26/40) quinolone susceptible and 66.67% (20/30) aminoglycoside susceptible isolates. Reduced susceptibility increased the risk of susceptible isolates carrying resistance genes (ORs 4.125, 8.36, and 8.89 respectively for ESBL, quinolone, and aminoglycoside resistance genes). Resistance gene carriage correlated significantly to reduced susceptibility for quinolone and aminoglycoside resistance genes (0.002 and 0.015 at CI95). Gene carriage correlated with phenotypic resistance at an estimated 64.28% for ESBL, 56.90% for quinolone, and 58.33% for aminoglycoside resistance genes. CONCLUSIONS: A high carriage of plasmid-mediated genes for ESBL, quinolone, and aminoglycoside resistance was found among the Enterobacteriaceae tested. However, gene carriage was not always correlated with phenotypic expression. This allows us to suggest that assessing genetic determinants of resistance should not be based on AST profile only. Further studies, including assessing the role of chromosomal determinants will shed light on other factors that undermine antimicrobial susceptibility locally.

Calcium supplementation in pregnancy: An analysis of potential determinants in an under-resourced setting.

INTRODUCTION: Despite the evidence that calcium supplementation in pregnancy improves maternofoetal outcomes, many women still do not take calcium supplements during pregnancy in Cameroon. This study identifies factors that influence calcium supplementation during pregnancy in a low resource setting. METHODS: We conducted a cross-sectional hospital-based study (from November 2020 to September 2021) targeting 1074 healthy women in late pregnancy at the maternities of four major health facilities in the Nkongsamba Health District, Cameroon. Data were collected using an interview-administered semi-structured questionnaire and analysed using Epi Info version 7.2.4.0, and the statistical threshold for significance set at p-value = 0.05. RESULTS: The mean age of the participants was 28.20±6.08 years, with a range of 15-47 years. The proportion of women who reported taking any calcium supplements in pregnancy was 72.62 [69.85-75.22]%. Only 12% of calcium-supplemented women took calcium supplements throughout pregnancy, while a majority (50%) took calcium supplements just for 4-5 months. Women believe that taking calcium supplements is more for foetal growth and development (37.12%) and prevention of cramps (38.86%), than for the prevention of hypertensive diseases in pregnancy (2.84%). About all pregnant women (97.65%) took iron and folic acid supplements during pregnancy, and 99.24% took these supplements at least once every two days. Upon control for multiple confounders, the onset of antenatal care before 4 months of pregnancy (AOR = 2.64 [1.84-3.78], p-value = 0.000), having had more than 3 antenatal care visits (AOR = 6.01 [3.84-9.34], p-value = 0.000) and support/reminder from a partner on the necessity to take supplements in pregnancy (AOR = 2.00 [1.34-2.99], p-value = 0.001) were significantly associated with higher odds of taking any calcium supplements in pregnancy. CONCLUSION: Calcium supplementation practices in pregnancy remain poor in this population and far from WHO recommendations. Early initiation of antenatal care, a high number of antenatal visits and reminders or support from the partner on supplement intake significantly increase the odds of taking any calcium supplements in pregnancy. In line with WHO recommendations, women of childbearing age should be sensitised to initiate antenatal care earlier and attain as many visits as possible. Male involvement in prenatal care might also boost the likelihood of these women taking calcium supplements.

Association of hypertension in pregnancy with serum electrolyte disorders in late pregnancy among Cameroonian women.

Multiple electrolyte disorders, including sodium, potassium and calcium disorders, have been associated with hypertension in pregnancy. Most of these studies failed to evaluate the combined effect of low and high sodium, potassium, calcium and chloride ion concentrations on hypertension in pregnancy. This study evaluates the combined effect of these ion categories (low, normal, high) on hypertension in pregnancy. Biochemical ion assays and blood pressure measurements were carried out on 1074 apparently healthy pregnant women in late third trimester. Serum potassium, sodium, chloride, and ionised calcium were measured by ion-selective electrode potentiometry, while total plasma calcium was measured by absorption spectrophotometry. Hypertension in pregnancy was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg. The prevalence of hyponatraemia, hypokalaemia, hypochloraemia, ionised hypocalcaemia and total hypocalcaemia in late pregnancy was 1.30 [0.78-2.18]%, 3.55 [2.60-4.84]%, 1.96 [1.28-2.97]%, 1.49 [0.92-2.21]% and 43.58 [40.64-46.56]%, respectively. Hypernatraemia, hyperkalaemia, hyperchloraemia, ionised hypercalcaemia and total hypercalcaemia were found in 1.49 [0.92-2.41]%, 2.34 [1.59-3.43]%, 4.38 [3.31-5.77]%, 39.94 [37.06-42.90]%, 2.79 [1.96-3.96]% of the participants, respectively. The prevalence of hypertension in pregnancy was 7.17 [5.77-8.87]%. When ion categories were considered in multiple logistic regression, only ionised and total calcium had significant associations with hypertension in pregnancy. Women with ionised hypercalcaemia had lower odds of hypertension in pregnancy (AOR = 0.50 [0.29-0.87], p-value = 0.015), and women with total hypocalcaemia had higher odds of hypertension in pregnancy (AOR = 1.99 [1.21-3.29], p-value = 0.007), compared to women with ionised and total normocalcaemia, respectively. Increasing kalaemia was associated significantly with higher odds of hypertension in pregnancy; however, kalaemia below and above the normal concentrations had no significant association with hypertension. Nonetheless, participants with kalaemia ≤ 3.98 mmol/L, had lower odds of hypertension in pregnancy compared with those with higher kalaemia (OR = 0.40 [0.24-0.66], p-value = 0.0003). Calcium disorders remain the most frequent electrolyte disorders in pregnancy. When normal cut-offs are considered for calcium and other ions, only ionised and total calcium influence the occurrence of hypertension in pregnancy. Kalaemia seems to affect hypertension in pregnancy but primarily within its normal concentrations. Serum electrolyte follow-up is indispensable for a proper pregnancy follow-up.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI 2.7-18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri species infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining malaria species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI( 2.7- 18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 10° copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to 14 oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovate-infected persons, mixed Poc/Pow infections were detected in 11 (79%). Based on these results, 8/9 P. ovate carriers transmitted both P. ovate species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Evolution of Plasmodium falciparum antimalarial drug resistance markers post-adoption of artemisinin-based combination therapies in Yaounde, Cameroon.

OBJECTIVES: This study aimed to investigate the evolution of Plasmodium falciparum antimalarial drug resistance markers by comparing the pre- and post-adoption of artemisinin-based combination therapies (ACTs) in Yaounde, Cameroon. METHODS: The molecular characterization of known antimalarial drug resistance markers (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and Pfk13) in P. falciparum-positive samples collected in 2014 and 2019-2020 was achieved using nested polymerase chain reaction, followed by targeted amplicon deep sequencing on the Illumina MiSeq platform. Data derived were compared with those published during the pre-ACT adoption period from 2004 to 2006. RESULTS: A high prevalence of Pfmdr1 184F, Pfdhfr 51I/59R/108N, and Pfdhps 437G mutant alleles was observed during the post-ACT adoption period. The Pfcrt 76T and Pfmdr1 86Y mutant alleles significantly declined between 2004 and 2020 (P <0.0001). Conversely, the resistance markers to antifolates, Pfdhfr 51I/59R/108N and Pfdhps 437G, significantly increased during the same study period (P <0.0001). We identified nine mutations in the propeller domains of Pfk13; although they were all present in single parasite isolates, none of them are known to confer artemisinin resistance. CONCLUSION: This study documented a near-complete reversion to sensitive parasites for markers conferring resistance to the 4-aminoquinolines and arylamino alcohols in Yaounde. In contrast, the Pfdhfr mutations associated with pyrimethamine resistance are moving toward saturation.