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.

Fear and depression during the COVID-19 outbreak in Cameroon: a nation-wide observational study.

BACKGROUND: The COVID-19 pandemic has been associated with significant psychological and social distress worldwide. We investigated fear and depression among adults in Cameroon during different phases of the COVID-19 outbreak. METHODS: An online survey was conducted in Cameroon from June-December 2020 using a structured questionnaire. Socio-demographic data and information regarding COVID-19 history were obtained. Fear and depressive symptoms were assessed using the Fear of COVID-19 score (FCV-19S) and the Patient Health Questionnaire (PHQ-9), respectively. Responses were clustered in weeks to better appreciate their evolution over time. RESULTS: Overall, 7381 responses from all ten regions of Cameroon were analysed (median age: 30 years, 73.3% male). The prevalence of depression (PHQ-9 score ≥ 10) was 8.4%, and that of high fear of COVID-19 (FCV-19S scores ≥19) was 57.4%. These rates were similar across genders, age-groups, and region of residence. While mean weekly PHQ-9 scores remained fairly stable throughout the study period (range: 2.53-3.21; p = 0.101), mean FCV-19S scores were highest during the early weeks but decreased significantly thereafter (from 20.31 to 18.34; p <  0.001). Multivariate analyses revealed that having a postgraduate degree, a history of quarantine, flu-like symptoms during the past 14 days, and higher FCV-19S scores were associated with more severe depressive symptoms, while obtaining COVID-19 information from various sources reduced the odds for depression. CONCLUSION: Depression amidst the COVID-19 crisis is less prevalent in Cameroon than in other countries. Prompt and widespread dissemination of adequate COVID-19 information may reduce the risks for depression by dispelling fear and anxiety among Cameroonians.

Systematic review of artemisinin embryotoxicity in animals: Implications for malaria control in human pregnancy.

Pregnant women are one of the most susceptible and vulnerable groups to malaria, the most important parasitic disease worldwide. Artemisinin-based combination therapies (ACTs) are recommended for the treatment of uncomplicated malaria in all population groups including pregnant women. However, due to the embryotoxicity observed in animal studies, ACTs have long been contraindicated during the first trimester in pregnant women. Despite the safety concerns raised in pre-clinical studies, recent findings on ACTs's use in pregnant women appear to be reassuring regarding safety and have prompted a revision of malaria treatment guidelines for first trimester of pregnancy. To contribute to the risk-benefit assessment of ACTs, we conducted a systematic literature review of animal studies published between 2007 and 2019, which evaluated the embryotoxic effects of artemisinin and its derivatives among pregnant mammals. Eighteen experimental studies fitted the inclusion criteria. These studies confirmed and further characterized the severe embryolethal and embryotoxic dose-dependent effects of artemisinin and its derivatives when administered during the organogenesis period in rats, rabbits and monkeys. Timing of administration and dosage of the drug were found to be key factors in the appearance of embryo damage. Overall, the translation of the findings of artemisinin derivatives use in animal studies to pregnant women remains disturbing. Thus, a policy change in the use of ACTs during the first trimester in pregnant women for the treatment of uncomplicated malaria does not seem pertinent and if implemented, it should be accompanied by solid pharmacovigilance systems, which are challenging to establish in malaria endemic countries.

The effect of improved housing on indoor mosquito density and exposure to malaria in the rural community of Minkoameyos, Centre Region of Cameroon.

BACKGROUND: This study evaluated the effectiveness of improved housing on indoor residual mosquito density and exposure to infected Anophelines in Minkoameyos, a rural community in southern forested Cameroon. METHODS: Following the identification of housing factors affecting malaria prevalence in 2013, 218 houses were improved by screening the doors and windows, installing plywood ceilings on open eaves and closing holes on walls and doors. Monthly entomological surveys were conducted in a sample of 21 improved and 21 non-improved houses from November 2014 to October 2015. Mosquitoes sampled from night collections on human volunteers were identified morphologically and their parity status determined. Mosquito infectivity was verified through Plasmodium falciparum CSP ELISA and the average entomological inoculation rates determined. A Reduction Factor (RF), defined as the ratio of the values for mosquitoes collected outdoor to those collected indoor was calculated in improved houses (RFI) and non-improved houses (RFN). An Intervention Effect (IE = RFI/RFN) measured the true effect of the intervention. Chi square test was used to determine variable significance. The threshold for statistical significance was set at P < 0.05. RESULTS: A total of 1113 mosquitoes were collected comprising Anopheles sp (58.6%), Culex sp (36.4%), Aedes sp (2.5%), Mansonia sp (2.4%) and Coquillettidia sp (0.2%). Amongst the Anophelines were Anopheles gambiae sensu lato (s.l.) (95.2%), Anopheles funestus (2.9%), Anopheles ziemanni (0.2%), Anopheles brohieri (1.2%) and Anopheles paludis (0.5%). Anopheles gambiae sensu stricto (s.s.) was the only An. gambiae sibling species found. The intervention reduced the indoor Anopheles density by 1.8-fold (RFI = 3.99; RFN = 2.21; P = 0.001). The indoor density of parous Anopheles was reduced by 1.7-fold (RFI = 3.99; RFN = 2.21; P = 0.04) and that of infected Anopheles by 1.8-fold (RFI = 3.26; RFN = 1.78; P = 0.04). Indoor peak biting rates were observed between 02 a.m. to 04 a.m. in non-improved houses and from 02 a.m. to 06 a.m. in improved houses. CONCLUSION: Housing improvement contributed to reducing indoor residual anopheline density and malaria transmission. This highlights the need for policy specialists to further evaluate and promote aspects of house design as a complementary control tool that could reduce indoor human-vector contact and malaria transmission in similar epidemiological settings.

A comparison of thick-film microscopy, rapid diagnostic test, and polymerase chain reaction for accurate diagnosis of Plasmodium falciparum malaria.

BACKGROUND: Accurate diagnosis of malaria is important for effective disease management and control. In Cameroon, presumptive clinical diagnosis, thick-film microscopy (TFM), and rapid diagnostic tests (RDT) are commonly used to diagnose cases of Plasmodium falciparum malaria. However, these methods lack sensitivity to detect low parasitaemia. Polymerase chain reaction (PCR), on the other hand, enhances the detection of sub-microscopic parasitaemia making it a much-needed tool for epidemiological surveys, mass screening, and the assessment of interventions for malaria elimination. Therefore, this study sought to determine the frequency of cases missed by traditional methods that are detected by PCR. METHODS: Blood samples, collected from 551 febrile Cameroonian patients between February 2014 and February 2015, were tested for P. falciparum by microscopy, RDT and PCR. The hospital records of participants were reviewed to obtain data on the clinical diagnosis made by the health care worker. RESULTS: The prevalence of malaria by microscopy, RDT and PCR was 31%, 45%, and 54%, respectively. However, of the 92% of participants diagnosed as having clinical cases of malaria by the health care worker, 38% were malaria-negative by PCR. PCR detected 23% and 12% more malaria infections than microscopy and RDT, respectively. A total of 128 (23%) individuals had sub-microscopic infections in the study population. The sensitivity of microscopy, RDT, and clinical diagnosis was 57%, 78% and 100%; the specificity was 99%, 94%, and 17%; the positive predictive values were 99%, 94%, and 59%; the negative predictive values were 66%, 78%, and 100%, respectively. Thus, 41% of the participants clinically diagnosed as having malaria had fever caused by other pathogens. CONCLUSIONS: Malaria diagnostic methods, such as TFM and RDT missed 12-23% of malaria cases detected by PCR. Therefore, traditional diagnostic approaches (TFM, RDT and clinical diagnosis) are not adequate when accurate epidemiological data are needed for monitoring malaria control and elimination interventions.

Association of Antibodies to VAR2CSA and Merozoite Antigens with Pregnancy Outcomes in Women Living in Yaoundé, Cameroon.

Plasmodium falciparum infections are serious in pregnant women, because VAR2CSA allows parasitized erythrocytes to sequester in the placenta, causing placental malaria (PM). In areas of endemicity, women have substantial malarial immunity prior to pregnancy, including antibodies to merozoite antigens, but produce antibodies to VAR2CSA only during pregnancy. The current study sought to determine the importance of antibodies to VAR2CSA and merozoite antigens in pregnant women in Yaoundé, Cameroon, where malaria transmission was relatively low. A total of 1,377 archival plasma samples collected at delivery were selected (at a 1:3 ratio of PM-positive [PM+] to PM-negative [PM-] women) and screened for antibodies to full-length VAR2CSA and 7 merozoite antigens. Results showed that many PM+ women and most PM- women lacked antibodies to VAR2CSA at delivery. Among PM+ women, antibodies to VAR2CSA were associated with a reduced risk of having high placental parasitemia (odds ratio [OR], 0.432; confidence interval [CI], 0.272, 0.687; P = 0.0004) and low-birth-weight (LBW) babies (OR = 0.444; CI, 0.247, 0.799; P = 0.0068), even during first pregnancies. Among antibodies to the 7 merozoite antigens, i.e., AMA1, EBA-175, MSP142, MSP2, MSP3, MSP11, and Pf41, only antibodies to MSP3, EBA-175, and Pf41 were associated with reduced risk for high placental parasitemias (P = 0.0389, 0.0291, and 0.0211, respectively) and antibodies to EBA-175 were associated with reduced risk of premature deliveries (P = 0.0211). However, after adjusting for multiple comparisons significance declined. Thus, in PM+ women, antibodies to VAR2CSA were associated with lower placental parasitemias and reduced prevalence of LBW babies in this low-transmission setting.

Individuals living in a malaria-endemic area of Cameroon do not have an acquired antibody response to Plasmodium falciparum histidine-rich protein 2.

BACKGROUND: Diagnosis of Plasmodium falciparum is often based on detection of histidine-rich protein 2 (HRP2) in blood. Most HRP2-based assays have high sensitivity and specificity; however, authors have suggested that antibodies (Ab) to HRP2 could reduce assay sensitivity. This study sought to characterize the antibody response to HRP2 with respect to prevalence, class, subclass, affinity, and age distribution in Cameroonian children and adults residing in an area with high P. falciparum transmission. METHODS: Plasma samples from 181 Cameroonian children and adults who had been repeatedly exposed to P. falciparum and 112 samples from American adults who had never been exposed were tested for IgG Ab to HRP2. For comparison, Ab to the merozoite antigens MSP1, MSP2, MSP3 and the pregnancy-associated antigen VAR2CSA were measured using a multiplex bead-based assay. In addition, 81 plasma samples from slide-positive individuals were screened for IgM Ab to HRP2. RESULTS: As expected, children and adults had IgG Ab to MSP1, MSP2 and MSP3, antibody levels increased with age, and only women of child-bearing age had Ab to VAR2CSA; however, no convincing evidence was found that these individuals had an acquired antibody response to HRP2. That is, using two sources of recombinant HRP2, identical results were obtained when plasma from 110 Cameroonian adults and 112 US adults were screened for IgG Ab. Further studies showed that antibody prevalence and levels did not increase with age in Cameroonians between ages 5 and >80 years. Although a few samples from slide-positive Cameroonians had IgM values slightly above the American cut-off, it was unclear if the individuals had a true IgM response to HRP2 or if the values were due to non-specific binding from elevated immunoglobulin levels associated with infection. Data from prediction models showed a paucity of Class II T cell epitopes in HRP2. CONCLUSIONS: These data support the conclusion that most individuals in malaria-endemic areas do not produce an acquired humoral response to HRP2. The absence of Ab helps explain why HRP2-based assays are able to detect nanogram amounts of HRP2 and why HRP2 continues to circulate for a long time after parasite clearance.

Statistical prediction of immunity to placental malaria based on multi-assay antibody data for malarial antigens.

BACKGROUND: Plasmodium falciparum infections are especially severe in pregnant women because infected erythrocytes (IE) express VAR2CSA, a ligand that binds to placental trophoblasts, causing IE to accumulate in the placenta. Resulting inflammation and pathology increases a woman's risk of anemia, miscarriage, premature deliveries, and having low birthweight (LBW) babies. Antibodies (Ab) to VAR2CSA reduce placental parasitaemia and improve pregnancy outcomes. Currently, no single assay is able to predict if a woman has adequate immunity to prevent placental malaria (PM). This study measured Ab levels to 28 malarial antigens and used the data to develop statistical models for predicting if a woman has sufficient immunity to prevent PM. METHODS: Archival plasma samples from 1377 women were screened in a bead-based multiplex assay for Ab to 17 VAR2CSA-associated antigens (full length VAR2CSA (FV2), DBL 1-6 of the FCR3, 3D7 and 7G8 lines, ID1-ID2a (FCR3 and 3D7) and 11 antigens that have been reported to be associated with immunity to P. falciparum (AMA-1, CSP, EBA-175, LSA1, MSP1, MSP2, MSP3, MSP11, Pf41, Pf70 and RESA)). Ab levels along with clinical variables (age, gravidity) were used in the following seven statistical approaches: logistic regression full model, logistic regression reduced model, recursive partitioning, random forests, linear discriminant analysis, quadratic discriminant analysis, and support vector machine. RESULTS: The best and simplest model proved to be the logistic regression reduced model. AMA-1, MSP2, EBA-175, Pf41, and MSP11 were found to be the top five most important predictors for the PM status based on overall prediction performance. CONCLUSIONS: Not surprising, significant differences were observed between PM positive (PM+) and PM negative (PM-) groups for Ab levels to the majority of malaria antigens. Individually though, these malarial antigens did not achieve reasonably high performances in terms of predicting the PM status. Utilizing multiple antigens in predictive models considerably improved discrimination power compared to individual assays. Among seven different classifiers considered, the reduced logistic regression model produces the best overall predictive performance.

Detection of Plasmodium falciparum DNA in saliva samples stored at room temperature: potential for a non-invasive saliva-based diagnostic test for malaria.

BACKGROUND: Current malaria diagnostic methods require blood collection, that may be associated with pain and the risk of transmitting blood-borne pathogens, and often create poor compliance when repeated sampling is needed. On the other hand, the collection of saliva is minimally invasive; but saliva has not been widely used for the diagnosis of malaria. The aim of this study was to evaluate the diagnostic performance of saliva collected and stored at room temperature using the OMNIgene®â€¢ORAL kit for diagnosing Plasmodium falciparum malaria. METHODS: Paired blood and saliva samples were collected from 222 febrile patients in Cameroon. Saliva samples were collected using the OMNIgene®â€¢ORAL (OM-501) kit and stored at room temperature for up to 13 months. Thick blood film microscopy (TFM) was used to detect P. falciparum blood-stage parasites in blood. Detection of P. falciparum DNA in blood and saliva was based on amplification of the multi-copy 18 s rRNA gene using the nested-polymerase chain reaction (nPCR). RESULTS: Prevalence of malaria detected by TFM, nPCR-saliva and nPCR-blood was 22, 29, and 35%, respectively. Using TFM as the gold standard, the sensitivity of nPCR-saliva and nPCR-blood in detecting P. falciparum was 95 and 100%, respectively; with corresponding specificities of 93 and 87%. When nPCR-blood was used as gold standard, the sensitivity of nPCR-saliva and microscopy was 82 and 68%, respectively; whereas, the specificity was 99 and 100%, respectively. Nested PCR-saliva had a very good agreement with both TFM (kappa value 0.8) and blood PCR (kappa value 0.8). At parasitaemia > 10,000 parasites/µl of blood, the sensitivity of nPCR-saliva was 100%. Nested PCR-saliva detected 16 sub-microscopic malaria infections. One year after sample collection, P. falciparum DNA was detected in 80% of saliva samples stored at room temperature. CONCLUSIONS: Saliva can potentially be used as an alternative non-invasive sample for the diagnosis of malaria and the OMNIgene®â€¢ORAL kit is effective at transporting and preserving malaria parasite DNA in saliva at room temperature. The technology described in this study for diagnosis of malaria in resource-limited countries adds on to the armamentarium needed for elimination of malaria.

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.

Antibody responses to the full-length VAR2CSA and its DBL domains in Cameroonian children and teenagers.

BACKGROUND: Antigenic variation of Plasmodium falciparum erythrocyte membrane protein 1 is a key parasite mechanism for immune evasion and parasite survival. It is assumed that the number of parasites expressing the same var gene must reach high enough numbers before the host can produce detectable levels of antibodies (Ab) to the variant. VAR2CSA is a protein coded for by one of 60 var genes that is expressed on the surface of infected erythrocytes (IE) and mediates IE binding to the placenta. The idea that Ab to VAR2CSA are pregnancy-associated was challenged when VAR2CSA-specific Ab were reported in children and men. However, the frequency and conditions under which Ab to VAR2CSA are produced outside pregnancy is unclear. This study sought to determine frequency, specificity and level of Ab to VAR2CSA produced in children and whether children with hyperparasitaemia and severe malaria are more likely to produce Ab to VAR2CSA compared to healthy children. METHODS: Antibody responses to a panel of recombinant proteins consisting of multiple VAR2CSA Duffy-binding-like domains (DBL) and full-length VAR2CSA (FV2) were characterized in 193 1-15 year old children from rural Cameroonian villages and 160 children with severe malaria from the city. RESULTS: Low Ab levels to VAR2CSA were detected in children; however, Ab levels to FV2 in teenagers were rare. Children preferentially recognized DBL2 (56-70%) and DBL4 (50-60%), while multigravidae produced high levels of IgG to DBL3, DBL5 and FV2. Sixty-seven percent of teenage girls (n = 16/24) recognized ID1-ID2a region of VAR2CSA. Children with severe forms of malaria had significantly higher IgG to merozoite antigens (all p < 0.05), but not to VAR2CSA (all p > 0.05) when compared to the healthy children. CONCLUSION: The study suggests that children, including teenage girls acquire Ab to VAR2CSA domains and FV2, but Ab levels are much lower than those needed to protect women from placental infections and repertoire of Ab responses to DBL domains is different from those in pregnant women. Interestingly, children with severe malaria did not have higher Ab levels to VAR2CSA compared to healthy children.

Comparison of the specificity of antibodies to VAR2CSA in Cameroonian multigravidae with and without placental malaria: a retrospective case-control study.

BACKGROUND: Antibodies (Ab) to VAR2CSA prevent Plasmodium falciparum-infected erythrocytes from sequestrating in the placenta, i.e., prevent placental malaria (PM). The specificity of Ab to VAR2CSA associated with absence of PM is unknown. Accordingly, differences in the specificity of Ab to VAR2CSA were compared between multigravidae with and without PM who had Ab to VAR2CSA. METHODS: In a retrospective case-control study, plasma collected from Cameroonian multigravidae with (n = 96) and without (n = 324) PM were screened in 21 assays that measured antibody levels to full length VAR2CSA (FV2), individual VAR2CSA DBL domains, VAR2CSA domains from different genetic backgrounds (variants), as well as proportion of high avidity Ab to FV2. RESULTS: Multigravidae with and without PM had similar levels of Ab to FV2, the six VAR2CSA DBL domains and different variants, while the proportion of high avidity Ab to FV2 was significantly higher in women without PM at delivery (p = 0.0030) compared to women with PM. In a logistic regression model adjusted for gravidity and age, the percentage of high avidity Ab to FV2 was associated with reduced likelihood of PM in multigravidae. A 5 % increase in proportion of high avidity Ab to FV2 was associated with a nearly 15 % lower likelihood of PM. CONCLUSION: Ab avidity to FV2 may be an important indicator of immunity to PM.

Cord blood Vγ2Vδ2 T cells provide a molecular marker for the influence of pregnancy-associated malaria on neonatal immunity.

BACKGROUND: Plasmodium falciparum placental infection primes the fetal immune system and alters infant immunity. Mechanisms leading to these outcomes are not completely understood. We focused on Vγ2Vδ2 cells, which are part of the immune response against many pathogens, including P. falciparum. These unconventional lymphocytes respond directly to small, nonpeptidic antigens, independent of major histocompatibility complex presentation. We wondered whether placental malaria, which may increase fetal exposure to P. falciparum metabolites, triggers a response by neonatal Vγ2Vδ2 lymphocytes that can be a marker for the extent of fetal exposure to malarial antigens. METHODS: Cord blood mononuclear cells were collected from 15 neonates born to mothers with P. falciparum infection during pregnancy (8 with placental malaria) and 25 unexposed neonates. Vγ2Vδ2 cell phenotype, repertoire, and proliferative responses were compared between newborns exposed and those unexposed to P. falciparum. RESULTS: Placental malaria-exposed neonates had increased proportions of central memory Vγ2Vδ2 cells in cord blood, with an altered Vγ2 chain repertoire ex vivo and after stimulation. CONCLUSION: Our results suggest that placental malaria affects the phenotype and repertoire of neonatal Vγ2Vδ2 lymphocytes. Placental malaria may lower the capacity for subsequent Vγ2Vδ2 cell responses and impair the natural resistance to infectious diseases or the response to pediatric vaccination.

Human cord blood γδ T cells expressing public Vγ2 chains dominate the response to bisphosphonate plus interleukin-15.

Compared with adults, the circulating Vγ2Vδ2 T-cell population in cord blood is present at low levels and does not show the strong bias for Vγ2-Jγ1.2 rearrangements. These features may be a result of limited exposure to stimulatory phosphoantigens, lack of T-cell-derived interleukin-2 (IL-2) or both. In cord blood mononuclear cell cultures, a single round of stimulation, using aminobisphosphonates to elevate phosphoantigen levels, resulted in expansion of adult-like Vγ2 chains and accumulation of memory cells with cytotoxic potential. Selection was similar using IL-2 or myeloid-derived IL-15. The Vγ2Vδ2 T cells present in neonates are capable of generating potent immune responses even when relying on IL-15.

High levels of antibodies to multiple domains and strains of VAR2CSA correlate with the absence of placental malaria in Cameroonian women living in an area of high Plasmodium falciparum transmission.

Placental malaria, caused by sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, is associated with increased risk of maternal morbidity and poor birth outcomes. The parasite antigen VAR2CSA (variant surface antigen 2-chondroitin sulfate A) is expressed on infected erythrocytes and mediates binding to chondroitin sulfate A, initiating inflammation and disrupting homeostasis at the maternal-fetal interface. Although antibodies can prevent sequestration, it is unclear whether parasite clearance is due to antibodies to a single Duffy binding-like (DBL) domain or to an extensive repertoire of antibodies to multiple DBL domains and allelic variants. Accordingly, plasma samples collected longitudinally from pregnant women were screened for naturally acquired antibodies against an extensive panel of VAR2CSA proteins, including 2 to 3 allelic variants for each of 5 different DBL domains. Analyses were performed on plasma samples collected from 3 to 9 months of pregnancy from women living in areas in Cameroon with high and low malaria transmission. The results demonstrate that high antibody levels to multiple VAR2CSA domains, rather than a single domain, were associated with the absence of placental malaria when antibodies were present from early in the second trimester until term. Absence of placental malaria was associated with increasing antibody breadth to different DBL domains and allelic variants in multigravid women. Furthermore, the antibody responses of women in the lower-transmission site had both lower magnitude and lesser breadth than those in the high-transmission site. These data suggest that immunity to placental malaria results from high antibody levels to multiple VAR2CSA domains and allelic variants and that antibody breadth is influenced by malaria transmission intensity.

Elevated levels of soluble TNF receptors 1 and 2 correlate with Plasmodium falciparum parasitemia in pregnant women: potential markers for malaria-associated inflammation.

Plasmodium falciparum-infected erythrocytes (IEs) sequester in the intervillous space (IVS) of the placenta causing placental malaria (PM), a condition that increases a woman's chances of having a low-birth-weight baby. Because IEs sequester, they frequently are not observed in peripheral blood smears, resulting in women with PM being misdiagnosed and thus not treated. Because sequestered IEs induce inflammation in the IVS, detection of inflammatory mediators in the peripheral blood may provide an approach for diagnosing PM. Two counterregulatory molecules, TNF-αR (TNFR) 1 and TNFR2, modulate the pathological effects of TNF-α. Levels of these soluble TNFRs (sTNFRs) are reported to be elevated in children with severe malaria, but it is unclear if they are increased in the peripheral blood of PM-positive women with asymptomatic infections. In this study, sTNFR levels were measured throughout the course of pregnancy, as well as at delivery, in women with asymptomatic infections and those who remained uninfected. Results showed that both sTNFRs were significantly increased in the peripheral blood of women with asymptomatic malaria (p < 0.0001) and were positively correlated with parasitemia (p < 0.0001 for sTNFR1 and p = 0.0046 for sTNFR2). Importantly, levels of sTNFR2 were elevated in the peripheral blood of women who were PM-positive but peripheral blood-smear negative (p = 0.0017). Additionally, sTNFR2 levels were elevated in the blood of malaria-positive women who delivered low-birth-weight babies. In vitro studies demonstrated that syncytiotrophoblasts were not a major source of sTNFR. These data suggest that sTNFR2 may be a valuable biomarker for detection of malaria-associated inflammation.

Positive selection of Plasmodium falciparum parasites with multiple var2csa-type PfEMP1 genes during the course of infection in pregnant women.

Placental malaria infections are caused by Plasmodium falciparum-infected red blood cells sequestering in the placenta by binding to chondroitin sulfate A, mediated by VAR2CSA, a variant of the PfEMP1 family of adhesion antigens. Recent studies have shown that many P. falciparum genomes have multiple genes coding for different VAR2CSA proteins, and parasites with >1 var2csa gene appear to be more common in pregnant women with placental malaria than in nonpregnant individuals. We present evidence that, in pregnant women, parasites containing multiple var2csa-type genes possess a selective advantage over parasites with a single var2csa gene. Accumulation of parasites with multiple copies of the var2csa gene during the course of pregnancy was also correlated with the development of antibodies involved in blocking VAR2CSA adhesion. The data suggest that multiplicity of var2csa-type genes enables P. falciparum parasites to persist for a longer period of time during placental infections, probably because of their greater capacity for antigenic variation and evasion of variant-specific immune responses.

Transplacental transfer of total immunoglobulin G and antibodies to Plasmodium falciparum antigens between the 24th week of gestation and term.

Full-term newborns have antibody (Ab) repertoires and levels similar to their mothers to help protect them from environmental pathogens. Unfortunately, preterm babies, especially those born < 34 weeks, have reduced levels of protective antibodies. In Africa, antibodies to Plasmodium falciparum are important in protection from malaria. This study investigated the transfer of total IgG and antibodies to 9 P. falciparum antigens and tetanus toxoid between 24 weeks and term. Paired maternal and cord samples from 166 preterm (24-37 weeks) and 154 term deliveries were used. Transfer efficiency was expressed as the ratio of Ab levels in cord to maternal plasma (CMR). At 24-25 weeks, CMR ranged from 0.31 to 0.94 for the different antigens; the rate of transfer was similar for all antigens between 24 and 40 weeks; resulting in median CMR of 0.49-0.95 at term. Babies of mothers with hypergammaglobulinemia and normal IgG levels had similar amounts of IgG, supporting data that saturation of the neonatal Fc-receptor occurs at ~ 16 mg IgG/ml. Thus, babies born prior to 34-35 weeks in Africa are likely to have reduced Ab levels to some, but not all antigens. Since IgG transfer is Fc-mediated, why differences exist in CMR among the antigens warrants further investigation.

COVID-19 Preventive Behaviours in Cameroon: A Six-Month Online National Survey.

Since March 2020, the Cameroonian government implemented nationwide measures to stall COVID-19 transmission. However, little is known about how well these unprecedented measures are being observed as the pandemic evolves. We conducted a six-month online survey to assess the preventive behaviour of Cameroonian adults during the COVID-19 outbreak. A five-point adherence score was constructed based on self-reported observance of the following preventive measures: physical distancing, face mask use, hand hygiene, not touching one's face, and covering the mouth when coughing or sneezing. Predictors of adherence were investigated using ordinal logistic regression models. Of the 7381 responses received from all ten regions, 73.3% were from male respondents and overall mean age was 32.8 ± 10.8 years. Overall mean adherence score was 3.96 ± 1.11 on a scale of 0-5. Mean weekly adherence scores were initially high, but gradually decreased over time accompanied by increasing incidence of COVID-19 during the last study weeks. Predictors for higher adherence included higher age, receiving COVID-19 information from health personnel, and agreeing with the necessity of lockdown measures. Meanwhile, experiencing flu-like symptoms was associated with poor adherence. Continuous observance of preventive measures should be encouraged among Cameroonians in the medium- to long-term to avoid a resurgence in COVID-19 infections.