High Prevalence of Polyclonal Plasmodium falciparum Infections and Association with Poor IgG Antibody Responses in a Hyper-Endemic Area in Cameroon.

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p < 0.0001) but decreased with participant age (r = -0.4860, p < 0.0001). Parasitaemia and the number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.

Chemokine modulation in microscopic and submicroscopic Plasmodium falciparum malaria infection in women at delivery in Yaoundé, Cameroon.

In pregnancy-associated malaria, chemokines such as CXCL-4, CXCL-13, CXCL-16, and CCL-24 play critical roles in leucocyte trafficking to tissue sites in the infected placenta where inflammatory reactions are active. However, how plasma levels of these chemokines associate with Plasmodium falciparum placental malaria and pregnancy outcomes remains not well understood. The present study analyzed the plasma levels of CXCL-4, CXCL-13, CXCL-16, and CCL-24 chemokines in matched peripheral, placental and cord blood in relation with placental malaria (PM), and with submicroscopic parasitaemia. This was a retrospective case-control study (1:3 ratio) involving samples from 134 women (34 PM+ and 100 PM-) enrolled at delivery at the Marie Reine Health Center in Yaoundé, Cameroon between June 2013 and October 2018. Samples were collected just after delivery and used to diagnose microscopic and submicroscopic Plasmodium falciparum infections. Submicroscopic infections were detected by reverse transcription LAMP whereas chemokine levels were determined by Magnetic Luminex Screening Assay. Overall, PM was associated with increased plasma levels of CXCL-13 and CXCL-16 and low levels of CXCL-4 and CCL-24 in both peripheral and placental blood (0.0002 ≤ p ≤ 0.042). Similarly, CCL-24 levels in peripheral and placental blood samples were significantly lower in submicroscopically infected women compared to healthy controls (p = 0.04 and 0.02, respectively). Maternal hemoglobin levels increased with peripheral plasma levels of CXCL-4 (p = 0.005), CXCL-16 (p = 0.03), and CCL-24 (p = 0.002) while birth weight was lower for babies born from women with high levels of peripheral CXCL-13 (p = 0.0006) and low levels of cord CXCL-4 and CCL-24 (p = 0.02 and 0.08, respectively). Together the data suggest that low levels of CXCL-4 and CCL-24 coupled with high plasma levels of CXCL-13 and for a lesser extend CXCL-16 represent signatures of PM in the study population. These findings are relevant for understanding the immunopathogenesis of PM and developing new therapeutic or preventive strategies against severe PM outcomes.

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.

High Prevalence of Asymptomatic Malarial Anemia and Association with Early Conversion from Asymptomatic to Symptomatic Infection in a Plasmodium falciparum Hyperendemic Setting in Cameroon.

Asymptomatic malarial parasitemia is highly prevalent in Plasmodium falciparum endemic areas and often associated with increased prevalence of mild to moderate anemia. The aim of this study was to assess the prevalence of anemia during asymptomatic malaria parasitemia and its interplay with persistent infection in highly exposed individuals. A household-based longitudinal survey was undertaken in a malaria hyperendemic area in Cameroon using multiplex nested polymerase chain reaction to detect plasmodial infections. Residents with P. falciparum asymptomatic parasitemia were monitored over a 3-week period with the aid of structured questionnaires and weekly measurements of axillary temperatures. Of the 353 individuals included (median age: 26 years, range 2-86 years, male/female sex ratio 0.9), 328 (92.9%) were positive for malaria parasitemia of whom 266 (81.1%) were asymptomatic carriers. The prevalence of anemia in the study population was 38.6%, of which 69.2% were asymptomatic. Multivariate analyses identified high parasitemia (> 327 parasites/µL) and female gender as associated risk factors of asymptomatic malarial anemia in the population. Furthermore, risk analyses revealed female gender and anemia at the time of enrolment as key predictors of early development of febrile illness (< 3 weeks post enrolment) among the asymptomatic individuals. Together, the data reveal an extremely high prevalence of asymptomatic malaria parasitemia and anemia in the study area, unveiling for the first time the association of asymptomatic malarial anemia with early clinical conversion from asymptomatic to symptomatic infection. Furthermore, these findings underscore the negative impact of asymptomatic malaria parasitemia on individual health, necessitating the development of appropriate control and preventive measures.

Diagnostic performance of a rapid whole blood-based RT-LAMP method for malaria diagnosis among apparently healthy blood donors and febrile neonates in Cameroon.

Light microscopy and rapid diagnostic tests are the two commonly used methods for malaria diagnosis that rely on the direct use of unprocessed blood samples. However, both methods do not have the level of sensitivity required for malaria diagnosis in cases of low density parasitaemia. We report here the diagnostic performance of a whole blood-based reverse transcription loop-mediated isothermal amplification method for Plasmodium falciparum malaria diagnosis in apparently healthy blood donors and febrile neonates in Cameroon. The presence of malaria parasites in whole blood samples was determined by light microscopy, antigen-based rapid diagnostic test (RDT), and by RT-LAMP using a "lyse and amplify" experimental protocol. Of the 256 blood donors tested, 36 (14.1%) were positive for malaria parasites by light microscopy, 38 (14.8%) were positive by RDT whereas 78 (30.5%) were positive by RT-LAMP. Only light microscopy and RT-LAMP detected infection among the febrile neonates (279 neonates, median age: 2 days, range: 1-9 days), with positivity rates of 8.6% and 12.2%, respectively. The overall concordance between the three methods were 75.9% for RT-LAMP and light microscopy, 75.1% for RT-LAMP and RDT, and 83.9% for light microscopy and RDT. Blood parasite densities were significantly lower in the neonates (mean: 97.6, range: 61-192 parasites/µL) compared to the blood donors (mean: 447.8, range: 63-11 000 parasites/µL). Together, the study demonstrates the usefulness of whole blood RT-LAMP for use in rapid pre-screening of blood donors and suspected neonates to avert severe consequences of P. falciparum infections.

Preliminary validation of the use of IgG antibody response to Anopheles gSG6-p1 salivary peptide to assess human exposure to malaria vector bites in two endemic areas of Cameroon in Central Africa.

The specific immune response to the Anopheles salivary peptide could be a pertinent and complementary tool to assess the risk of malaria transmission and the effectiveness of vector control strategies. This study aimed to obtain first reliable data on the current state of the Anopheles gSG6-P1 biomarker for assess the level of exposure to Anopheles bites in high malaria endemic areas in Cameroon. Blood smears were collected from people living in the neighborhoods of Youpwe (suburban area, continental) and Manoka (rural area, Island), both areas in the coastal region of Cameroon. Malaria infection was determined using thick blood smear microscopy, whereas the level of specific IgG response to gSG-P1 peptide was assessed by enzyme-linked immunosorbent assay from the dried blood spots. Of 266 (153 from Youpwe, 113 from Manoka) malaria endemic residents (mean age: 22.8±19.8 years, age range: 6 months-94 years, male/female sex ratio: 1/1.2, with Manoka mean age: 23.71±20.53, male/female sex ratio:1/1.13 and Youpwe mean age: 22.12±19.22, male/female sex ratio 1/0.67) randomly included in the study, Plasmodium infection prevalence was significantly higher in Manoka than in Youpwe (64.6% vs 12,4%, p = 0.0001). The anti-gSG6-P1 IgG response showed a high inter-individual heterogeneity and was significantly higher among individuals from Manoka than those from Youpwe (p = 0.023). Malaria infected individuals presented a higher anti-gSG6-P1 IgG antibody response than non-infected (p = 0.0004). No significant difference in the level of specific IgG response to gSG-P1 was observed according to long lasting insecticidal nets use. Taken together, the data revealed that human IgG antibody response to Anopheles gSG-P1 salivary peptide could be also used to assess human exposure to malaria vectors in Central African region. This finding strengthens the relevance of this candidate biomarker to be used for measuring human exposure to malaria vectors worldwide.

Demographical, hematological and serological risk factors for Plasmodium falciparum gametocyte carriage in a high stable transmission zone in Cameroon.

Presence of mature gametocyte forms of malaria parasites in peripheral blood is a key requirement for malaria transmission. Yet, studies conducted in most malaria transmission zones report the absence of gametocyte in the majority of patients. We therefore sought to determine the risk factors of both all-stage and mature gametocyte carriage in an area with high stable transmission of Plasmodium falciparum in Cameroon. Gametocyte positivity was determined using three complementary methods: thick blood smear microscopy, RT-PCR and RT-LAMP, whereas exposure to the infection was assessed by enzyme-linked immunosorbent assay. Of 361 malaria endemic residents randomly included in the study (mean age: 28±23 years, age range: 2-100 years, male/female sex ratio: 1.1), 87.8% were diagnosed with P. falciparum infection, of whom 45.7% presented with fever (axillary body temperature ≥37.5°C). Mature gametocyte positivity was 1.9% by thick blood smear microscopy and 8.9% by RT-PCR targeting the mature gametocyte transcript, Pfs25. The gametocyte positivity rate was 24.1% and 36.3% by RT-PCR or RT-LAMP, respectively, when targeting the sexual stage marker, Pfs16. Multivariate analyses revealed anemia as a common independent risk factor for both mature and all-stage gametocyte carriage, whereas fever and low anti-gametocyte antibody levels were independently associated with all-stage gametocyte carriage only. Taken together, the data suggest important differences in risk factors of gametocyte carriage depending on stage analyzed, with anemia, fever and low antiplasmodial plasma antibody levels representing the major contributing risk factors.

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.

Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ µL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ µL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ µL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar's test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar's test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings.