Partial spike gene sequencing for the identification of SARS-CoV-2 variants circulating in Cameroon in 2021.

INTRODUCTION: Global monitoring of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) genetic sequences and associated metadata is essential for coronavirus disease 2019 (COVID-19) response. Therefore, Sanger's partial genome sequencing technique was used to monitor the circulating variants of SARS-CoV-2 in Cameroon. METHODOLOGY: Nasopharyngeal specimen was collected from persons suspected of SARS-CoV-2 following the national guidelines between January and December 2021. All specimens with cycle threshold (Ct) below 30 after amplification were eligible for sequencing of the partial spike (S) gene of SARS-CoV-2 using the Sanger sequencing method. RESULTS: During the year 2021, 1481 real time reverse transcriptase polymerase chain reaction (RT-PCR) SARS-CoV-2 positive samples were selected for partial sequencing of the S gene of SARS-CoV-2. Amongst these, 878 yielded good sequencing products. A total of 231 probable variants (26.3%) were identified. The variants were mainly represented by Delta (70.6%), Alpha (15.6%), Omicron (7.4%), Beta (3.5%), Mu (1.7%) and Gamma (0.4%). Phylogenetic analysis of the probable variants from Cameroon with reference strains confirmed that all prior and current variants of concern (VOC) clustered with their respective reference sequences. CONCLUSIONS: The surveillance strategy implemented in Cameroon, based on partial sequencing of the S gene enabled identification of the major circulating variants and provided information on the distribution of these variants, which contributed to implementing public health measures to control disease spread in the country.

The Detection of Influenza Virus Before and During the COVID-19 Pandemic in Cameroon.

BACKGROUND: Influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are both respiratory viruses with similar clinical manifestations and modes of transmission. This study describes influenza data before and during the coronavirus disease pandemic (COVID-19) in Cameroon and SARS-CoV-2 data during the pandemic period. METHODS: The study ran from 2017 to 2022, and data were divided into two periods: before (2017-2019) and during (2020-2022) the COVID-19 pandemic. Nasopharyngeal samples collected from persons with respiratory illness were tested for influenza using the Centers for Disease Control and Prevention (CDC) typing and subtyping assays. During the COVID-19 pandemic, the respiratory specimens were simultaneously tested for SARS-CoV-2 using the DaAn gene protocol or the Abbott real-time SARS-CoV-2 assay. The WHO average curve method was used to compare influenza virus seasonality before and during the pandemic. RESULTS: A total of 6246 samples were tested. Influenza virus detection rates were significantly higher in the pre-pandemic period compared to the pandemic period (30.8% vs. 15.5%; p < 0.001). Meanwhile, the SARS-CoV-2 detection rate was 2.5%. A change in the seasonality of influenza viruses was observed from a bi-annual peak before the pandemic to no clear seasonal pattern during the pandemic. The age groups 2-4 and 5-14 years were significantly associated with higher influenza positivity rates in both pre-pandemic and pandemic periods. For SARS-CoV-2, all age groups above 15 years were the most affected population. CONCLUSION: The COVID-19 pandemic had a significant impact on the seasonal influenza by changing the seasonality of the virus and reducing its detection rates.

Impact of the 2022 African Cup of Nations mass-gathering event on the COVID-19 epidemic in Garoua, Cameroon.

BACKGROUND: The 2022 African Cup of Nations (AFCON) took place in Cameroon from January 9th to February 5th, 2022, including Garoua in the north. We aimed to measure the impact of this event on the local COVID-19 epidemic given the implementation of a preventive strategy based on a health pass. METHODS: All players, staff and fans involved in the AFCON event were screened with PCR tests. Symptomatic cases were also continuously monitored in the general population and screened for variants of concern. Daily numbers of confirmed cases were compared to neighboring countries numbers retrieved from a public domain source. RESULTS: In total, 1479 and 2481 tests were performed in the general population and on asymptomatic AFCON attendees, respectively. From the latter, 12.5% were PCR-positive; 97% were infected with Omicron, with no significant difference compared to the passive program (G-test, P value = 0.162). Surveillance indicated the AFCON did not increase the number of symptomatic PCR-positive cases in the general population compared to neighboring countries. CONCLUSIONS: Though the COVID-19 epidemic was fueled by asymptomatic cases infected with the Omicron variant at the time, the non-therapeutic preventive measures implemented for AFCON mitigated an increase in the epidemic in the local population.

One hundred malaria attacks since birth. A longitudinal study of African children and young adults exposed to high malaria transmission.

Background: Despite significant progress in malaria control over the past twenty years, malaria remains a leading cause of child morbidity and mortality in Tropical Africa. As most patients do not consult any health facility much uncertainty persists about the true burden of the disease and the range of individual differences in susceptibility to malaria. Methods: Over a 25-years period, from 1990 to 2015, the inhabitants of Dielmo village, Senegal, an area of intense malaria transmission, have been monitored daily for their presence in the village and the occurrence of diseases. In case of fever thick blood films were systematically examined through microscopy for malaria parasites and patients received prompt diagnosis and treatment. Findings: We analysed data collected in 111 children and young adults monitored for at least 10 years (mean 17.3 years, maximum 25 years) enrolled either at birth (95 persons) or during the two first years of life. A total of 11,599 episodes of fever were documented, including 5268 malaria attacks. The maximum number of malaria attacks in a single person was 112. Three other persons suffered one hundred or more malaria attacks during follow-up. The minimum number of malaria attacks in a single person was 11. The mean numbers of malaria attacks in children reaching their 4th, 7th, and 10th birthdays were 23.0, 37.7, and 43.6 attacks since birth, respectively. Sixteen children (14.4%) suffered ten or more malaria attacks each year at ages 1-3 years, and six children (5.4%) each year at age 4-6 years. Interpretation: Long-term close monitoring shows that in highly endemic areas the malaria burden is higher than expected. Susceptibility to the disease may vary up to 10-fold, and for most children childhood is an endless history of malaria fever episodes. No other parasitic, bacterial or viral infection in human populations has such an impact on health. Funding: The Pasteur Institutes of Dakar and Paris, the Institut de Recherche pour le Développement, and the French Ministry of Cooperation provided funding.

Epidemiology of rotavirus in humans, animals, and the environment in Africa: a systematic review and meta-analysis.

INTRODUCTION: Globally, rotavirus infections are the most common cause of diarrhea-related deaths, especially among children under 5 years of age. This virus can be transmitted through the fecal-oral route, though zoonotic and environmental contributions to transmission are poorly defined. The purpose of this study is to determine the epidemiology of rotavirus in humans, animals, and the environment in Africa, as well as the impact of vaccination. METHODS: We searched PubMed, Web of Science, Africa Index Medicus, and African Journal Online, identifying 240 prevalence data points from 224 articles between 2009 and 2022. RESULTS: Human rotavirus prevalence among patients with gastroenteritis was 29.8% (95% CI, 28.1-31.5; 238710 participants), with similar estimates in children under 5 years of age, and an estimated case fatality rate of 1.2% (95% CI, 0.7-2.0; 10440 participants). Prevalence was estimated to be 15.4% and 6.1% in patients with non-gastroenteritis illnesses and apparently healthy individuals, respectively. Among animals, prevalence was 9.3% (95% CI, 5.7-13.7; 6115 animals), and in the environmental water sources, prevalence was 31.4% (95% CI, 17.7-46.9; 2530 samples). DISCUSSION: Our findings highlight the significant burden of rotavirus infection in Africa, and underscore the need for a One Health approach to limiting the spread of this disease.

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.

Improvement of the antibody-dependent respiratory burst assay for assessing protective immune responses to malaria.

The antibody-dependent respiratory burst (ADRB) assay is a sensitive isoluminol-based chemiluminescence (CL) functional assay designed to assess the capacity of opsonizing antibodies against merozoites to induce neutrophil respiratory burst. ADRB was shown to measure protective immunity against malaria in endemic areas, but the assay needed further improvement to ensure better sensitivity and reproducibility. Here, we adjusted parameters such as the freezing-thawing procedure of merozoites, merozoites's concentration and the buffer solution's pH, and we used the improved assay to measure ADRB activity of 207 sera from 97 and 110 individuals living, respectively, in Dielmo and Ndiop villages with differing malaria endemicity. The improvement led to increased CL intensity and assay sensitivity, and a higher reproducibility. In both areas, ADRB activity correlated with malaria endemicity and individual's age discriminated groups with and without clinical malaria episodes, and significantly correlated with in vivo clinical protection from Plasmodium falciparum malaria. Our results demonstrate that the improved ADRB assay can be valuably used to assess acquired immunity during monitoring by control programmes and/or clinical trials.

School-Based Serosurveys to Assess the Validity of Using Routine Health Facility Data to Target Malaria Interventions in the Central Highlands of Madagascar.

BACKGROUND: In low-malaria-transmission areas of Madagascar, annual parasite incidence (API) from routine data has been used to target indoor residual spraying at subdistrict commune level. To assess validity of this approach, we conducted school-based serological surveys and health facility (HF) data quality assessments in 7 districts to compare API to gold-standard commune-level serological measures. METHODS: At 2 primary schools in each of 93 communes, 60 students were randomly selected with parents and teachers. Capillary blood was drawn for rapid diagnostic tests (RDTs) and serology. Multiplex bead-based immunoassays to detect antibodies to 5 Plasmodium falciparum antigens were conducted, and finite mixture models used to characterize seronegative and seropositive populations. Reversible catalytic models generated commune-level annual seroconversion rates (SCRs). HF register data were abstracted to assess completeness and accuracy. RESULTS: RDT positivity from 12 770 samples was 0.5%. Seroprevalence to tested antigens ranged from 17.9% (MSP-1) to 59.7% (PF13). Median commune-level SCR was 0.0108 (range, 0.001-0.075). Compared to SCRs, API identified 71% (95% confidence interval, 51%-87%) of the 30% highest-transmission communes; sensitivity declined at lower levels. Routine data accuracy did not substantially affect API performance. CONCLUSIONS: API performs reasonably well at identifying higher-transmission communes but sensitivity declined at lower transmission levels.

Practical example of multiple antibody screening for evaluation of malaria control strategies.

BACKGROUND: Ongoing efforts to fight Plasmodium falciparum malaria has reduced malaria in many areas, but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Sero-surveillance is considered promising to monitor exposure, transmission and immunity. METHODS: IgG responses to three antigen biomarkers were evaluated in a retrospective study involving: (i) surveys of 798 asymptomatic villagers from 2 Senegalese endemic settings conducted before 2002 and after the 2013 intensification of control measures, and (ii) in 105 symptomatic individuals from different settings in Côte d'Ivoire. Response to up to eight P. falciparum antigens, including recombinant MSP1p9 antigen and LSA141 peptide, were analysed using multiplex technology and responses to whole P. falciparum schizont extract (SE, local strain adapted to culture) were measured by ELISA. RESULTS: MSP1p9 and LSA141 IgG responses were shown to be relevant indicators monitoring immune status in the different study sites both from Côte d'Ivoire and Senegal. Between 2002 and 2013, individuals participating in both studies showed higher decline of sero-positivity in young (< 15 years: range 12% to 50%) than older (> 15 years: no decline to 15%) individuals from Dielmo and Ndiop. A mathematical sero-catalytic model from the complete Dielmo/Ndiop survey was used to reconstruct declining levels of sero-positivity in more detail, demonstrating that anti-SE seroprevalence levels most accurately reflected malaria exposure in the two villages. CONCLUSION: For standard screening of population immune status at sites envisaging elimination, the use of ELISA-based assays targeting selected antigens can contribute to provide important epidemiologic surveillance data to aid malaria control programmes.

Association of high Plasmodium falciparum parasite densities with polyclonal microscopic infections in asymptomatic children from Toubacouta, Senegal.

BACKGROUND: Malaria is a leading cause of mortality and morbidity in tropical countries, especially in sub-Saharan Africa. In Senegal, a control plan implemented in the beginning of the 2000s has enabled a substantial reduction of mortality and morbidity due to malaria. However, eradication of malaria requires a vaccine that protects against Plasmodium falciparum the deadliest species of the parasite that causes this disease. Plasmodium falciparum is characterized by an extensive genetic diversity that makes vaccine development challenging. In this study, the diversity of P. falciparum isolates was analysed from asymptomatic children residing in the district of Toubacouta, Senegal. METHODS: A nested PCR approach was used to perform genotyping of the msp-1 and msp-2 loci in samples from 87 asymptomatic children infected with P. falciparum, collected during a cross sectional survey in November and December 2010. Parasite densities in blood samples were determined by microscopic examination and statistical analyses were used to identify association of parasite genotype and parasitaemia. RESULTS: Genotyping was successful in 84/87 and 82/87 samples for msp-1 and msp-2, respectively. A strong genetic diversity was found with a total of 15 and 21 different alleles identified for msp-1 and msp-2, respectively. RO33 was the most frequent allelic family of msp-1 followed by MAD20, then by K1. Regarding msp-2 allelic families, 3D7 was more common than FC27. Multiple infections were predominant, since 69% and 89% of the samples genotyped for msp-1 and msp-2 showed more than one clone of P. falciparum with complexity of infection (COI) of 2.5 and 4.7, respectively. Expected heterozygosity (HE) was 0.57 and 0.55 for msp-1 and msp-2, respectively. Interestingly, polyclonal infections were significantly associated with higher parasitaemia. CONCLUSIONS: The strong genetic diversity of P. falciparum clones and the association of polyclonal infection with high parasitaemia call for a multi-allelic approach in the design of vaccine candidates for efficient malaria eradication.

Optimization of a magnetic bead-based assay (MAGPIX®-Luminex) for immune surveillance of exposure to malaria using multiple Plasmodium antigens and sera from different endemic settings.

BACKGROUND: Serological markers are potentially useful tools for monitoring the progress of malaria control programs, but a better understanding of antibody response dynamics is necessary. The use of a magnetic bead-based immunoassay (MBA) is advantageous compared to ELISA, due to its multiplexing capacity, but limited information is available on the standardization and validation of this assay. METHODS: Several parameters for multiplex testing of antibodies to Plasmodium antigens were analysed using a set of 4 antigens and 98 sera from Senegalese rural asymptomatic and urban symptomatic individuals. The 4 antigens included Plasmodium falciparum CSP and PfAMA1 peptides, recombinant P. falciparum MSP4p20 and a Plasmodium malariae CSP (PmCSP) peptide. Comparisons with ELISA were done using MSP4p20 and whole schizont extract (SE) antigens. RESULTS: The use of fewer beads (1000 beads per well instead of 2000) and 5 µg of antigen per 106 bead were validated as lower amounts. The use of a carrier protein (BSA) was shown to be critical when using peptides and the effect of a 24 h delayed measures was evaluated (5-25% signal decrease). Analysis of Ab responses showed almost equally high levels and prevalence in all transmission settings. Clear distinctions between rural and urban malaria were noted using PmCSP and SE antigens. CONCLUSIONS: This study underlines the importance of further optimization of the MBA technique and highlights the interest of using multistage/multispecies antigens for surveillance of malaria in endemic settings.

Unexpected high circulation of Plasmodium vivax in asymptomatic children from Kédougou, southeastern Senegal.

BACKGROUND: Malaria in Senegal is due essentially to infections by Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. By the use of molecular methods, detection of Plasmodium vivax has been recently reported in the region of Kedougou, raising the question of appraisal of its potential prevalence in this setting. METHODS: A retrospective serological study was carried out using 188 samples taken from 2010 to 2011 in a longitudinal school survey during which 48 asymptomatic children (9-11 years) were recruited. Four collections of samples collected during two successive dry and rainy seasons were analysed for antibody responses to P. vivax and P. falciparum. Recombinant P. falciparum and P. vivax MSP1 antigens and total P. falciparum schizont lysate from African 07/03 strain (adapted to culture) were used for ELISA. Nested PCR amplification was used for molecular detection of P. vivax. RESULTS: A surprising high prevalence of IgG responses against P. vivax MSP1 was evidenced with 53% of positive samples and 58% of the individuals that were found positive to this antigen. There was 77% of responders to P. falciparum outlined by 63% of positive samples. Prevalence of responders did not differ as function of seasons. Levels of antibodies to P. falciparum fluctuated with significant increasing between dry and rainy season (P < 0.05), contrary to responses to P. vivax. There was a significant reciprocal relationship (P < 10-3) between antibody responses to the different antigens, but with weak coefficient of correlation (Rho around 0.3) underlining a variable profile at the individual level. Clear molecular signature was found in positive IgG to P. vivax msp1 samples by PCR. CONCLUSION: This cross-sectional longitudinal study highlights the unexpected high circulation of P. vivax in this endemic area. Sero-immunology and molecular methods are powerful additive tools to identify endemic sites where relevant control measures have to be settled and monitored.

Modelling dynamic change of malaria transmission in holoendemic setting (Dielmo, Senegal) using longitudinal measures of antibody prevalence to Plasmodium falciparum crude schizonts extract.

BACKGROUND: Evaluation of local Plasmodium falciparum malaria transmission has been investigated previously using the reversible catalytic model based on prevalence of antibody responses to single antigen to estimate seroconversion rates. High correlations were observed between seroconversion rates and entomological inoculation rates (EIR). However, in this model, the effects of malaria control interventions and clinical episodes on serological measurements were not assessed. This study monitors the use of antibody responses to P. falciparum crude extracts for assessing malaria transmission, compares seroconversion rates estimated from longitudinal data to those derived from cross-sectional surveys and investigates the effects of malaria control interventions on these measures in an area of declining malaria transmission. In addition, the validity of this model was evaluated by comparison with the alternative model. METHODS: Five cross-sectional surveys were carried out at the end of the wet season in Dielmo, a malaria-endemic Senegalese rural area in 2000, 2002, 2008, 2010 and 2012. Antibodies against schizonts crude extract of a local P. falciparum strain adapted to culture (Pf 07/03) were measured by ELISA. Age-specific seroprevalence model was used both for cross-sectional surveys and longitudinal data (combined data of all surveys). RESULTS: A total of 1504 plasma samples obtained through several years follow-up of 350 subjects was used in this study. Seroconversion rates based on P. falciparum schizonts crude extract were estimated for each cross-sectional survey and were found strongly correlated with EIR. High variability between SCRs from cross-sectional and longitudinal surveys was observed. In longitudinal studies, the alternative catalytic reversible model adjusted better with serological data than the catalytic model. Clinical malaria attacks and malaria control interventions were found to have significant effect on seroconversion. DISCUSSION: The results of the study suggested that crude extract was a good serological tool that could be used to assess the level of malaria exposure in areas where malaria transmission is declining. However, additional parameters such as clinical malaria and malaria control interventions must be taken into account for determining serological measurements for more accuracy in transmission assessment.

Association of antibodies to Plasmodium falciparum merozoite surface protein-4 with protection against clinical malaria.

Identification of parasite antigens targeted by immune effector mechanisms that confer protection against malaria is important for the design of a multi-component malaria vaccine. Here, the association of antibodies reacting with the Plasmodium falciparum merozoite surface protein-4 (MSP4) with protection against clinical malaria was investigated in a Senegalese community living in an area of moderate, seasonal malaria transmission. Blood samples were collected at the end of an 8-month long dry season without any recorded parasite transmission from 206 residents enrolled in a prospective follow-up study. Active daily clinical monitoring was implemented during the subsequent five months. Entomologic monitoring documented parasite transmission during the first three months of follow-up. Serum IgG levels were determined by ELISA against three MSP4 baculovirus-encoded recombinant protein constructs, namely the full-length MSP4p40, MSP4p30 devoid of a highly polymorphic sequence stretch and the conserved C-terminal EGF-containing MSP4p20, as well as against a merozoite crude extract. Community seroprevalence against all three constructs was quite high, the lowest being against MSP4p30. Seroprevalence and antibody levels against the three MSP4 constructs were age-dependent. IgG1 dominated the anti-MSP4p20 responses, while both IgG1 and IgG3 were observed against MSP4p40. Anti-MSP4 antibodies were associated with the antibody-dependent respiratory burst (ADRB) activity in a functional assay of merozoite phagocytosis by polymorphonuclear cells. Importantly, high antibody levels against each of the three MSP4 constructs at the end of the dry season were associated with reduced morbidity during the subsequent transmission season in an age-adjusted Poisson regression model (IRR = 0.65 [0.50-0.83], P<0.001 for responses over the median values). These data are consistent with a protective role for the naturally acquired anti-MSP4 antibodies and support further development of MSP4 as a candidate component of malaria vaccine.

Temporal analysis of IgG antibody responses to Plasmodium falciparum antigens in relation to changing malaria epidemiology in a West African setting.

BACKGROUND: Coordinated scaled-up malaria control interventions have substantially contributed to the dramatic decrease of malaria-related morbidity and mortality in several endemic countries, including Senegal. However, the impacts of a given malaria control intervention on vector and parasite populations, acquired immunity, and disease burden remain very poorly documented largely due to the lack of continuous surveys. This study took advantage of the sera bank established as part of the Dielmo longitudinal project to investigate the dynamics of IgG antibody responses that accompanied the epidemiological changes resulting from malaria control interventions. Schizonts crude extract of a local strain of Plasmodium falciparum (Pfsch07/03) was used in ELISA to measure and compare seroprevalence and magnitude of IgG antibody responses from 2000 to 2012. RESULTS: The prevalence of Pfsch07/03 IgG antibody responses progressively decreased from 97.25% in 2000 to 57.3% in 2012. The prevalence of Pfsch07/03 antibodies categorized between three different age groups (<7, 7-15, and >15 years) revealed increased seroprevalence with age ranging from 47.19 to 62.67 and 89.45%, respectively in (<7, 7-15, and >15 years) old age groups. A marked drop in seroprevalence was observed after 2008 and was significant in the younger (<7 years) and intermediate (7-15 years) age groups, unlike older individuals aged >15 years (p = 1.00). CONCLUSIONS: The study revealed a substantial contribution of all malaria control interventions to the decrease of IgG antibodies responses to Pfsch07/03 throughout prevention of human-mosquitos contacts, or reduction of parasite biomass. The present study demonstrates the wider potential of sero-epidemiological analysis in monitoring changes in malaria transmission resulting from a given malaria control intervention.

Serological signatures of declining exposure following intensification of integrated malaria control in two rural Senegalese communities.

Recent control scale-up has reduced malaria in many areas but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Although serology is considered a promising approach in this regard, the serological impact of control interventions has been so far studied using indirect quantification of exposure. Cohort surveys concomitantly recording entomological and malariometric indices have been conducted in two Senegalese settings where supervised control intensification implemented in 2006 shifted malaria from historically holoendemic in Dielmo and mesoendemic in Ndiop to hypoendemic in both settings by 2013. We analyse here serological signatures of declining transmission using archived blood samples. Responses against ten pre-erythrocytic and erythrocytic antigens from Plasmodium falciparum and P. malariae alongside an Anopheles gambiae salivary gland antigen were analysed. Cross-sectional surveys conducted before (2002) and after (2013) control intensification showed a major impact of control intensification in both settings. The age-associated prevalence, magnitude and breadth of the IgG responses to all antigens were village-specific in 2002. In 2013, remarkably similar patterns were observed in both villages, with marginal responses against all parasite antigens in the 0-5y children and reduced responses in all previously seropositive age groups. Waning of humoral responses of individuals who were immune at the time of control intensification was studied from 2006 to 2013 using yearly samplings. Longitudinal data were analysed using the Cochran-Armittage trend test and an age-related reversible catalytic conversion model. This showed that the antigen-specific antibody declines were more rapid in older children than adults. There was a strong association of antibody decline with the declining entomological inoculation rate. We thus identified serological markers of declining exposure to malaria parasites that should help future monitoring of progress towards malaria elimination.

Longitudinal analysis of antibody responses in symptomatic malaria cases do not mirror parasite transmission in peri-urban area of Cote d'Ivoire between 2010 and 2013.

BACKGROUND: In the agenda towards malaria eradication, assessment of both malaria exposure and efficacy of anti-vectorial and therapeutic strategies is a key component of management and the follow-up of field interventions. The simultaneous use of several antigens (Ags) as serological markers has the potential for accurate evaluation of malaria exposure. Here we aimed to measure the longitudinal evolution of the background levels of immunity in an urban setting in confirmed clinical cases of malaria. METHODS: A retrospective serological cross-sectional study on was carried out using 234 samples taken from 2010 to 2013 in peri-urban sentinel facility of Cote d'Ivoire. Antibody responses to recombinant proteins or BSA-peptides, 8 Plasmodium falciparum (PfAMA1, PfMSP4, PfMSP1, PfEMP1-DBL1α1-PF13, PfLSA1-41, PfLSA3-NR2, PfGLURP and PfCSP), one P. malariae (PmCSP) and one Anopheles gambiae salivary (gSG6-P1) antigens were measured using magnetic bead-based multiplex immunoassay (MBA). Total anti- P. falciparum IgG responses against schizont lysate from african 07/03 strain (adapted to culture) and 3D7 strain was measured by ELISA. RESULTS: High prevalence (7-93%) and levels of antibody responses to most of the antigens were evidenced. However, analysis showed only marginal decreasing trend of Ab responses from 2010 to 2013 that did not parallel the reduction of clinical malaria prevalence following the implementation of intervention in this area. There was a significant inverse correlation between Ab responses and parasitaemia (P<10-3, rho = 0.3). The particular recruitment of asymptomatic individuals in 2011 underlined a high background level of immunity almost equivalent to symptomatic patients, possibly obscuring observable yearly variations. CONCLUSION: The use of cross-sectional clinical malaria surveys and MBA can help to identify endemic sites where control measures have unequal impact providing relevant information about population immunity and possible decrease of transmission. However, when immunity is substantially boosted despite observable clinical decline, a larger cohort including asymptomatic recruitment is needed to monitor the impact of control measures on level of immunity.

Relationship between Antibody Levels, IgG Binding to Plasmodium falciparum-Infected Erythrocytes, and Disease Outcome in Hospitalized Urban Malaria Patients from Dakar, Sénégal.

Background. Management of clinical malaria requires the development of reliable diagnostic methods and efficient biomarkers for follow-up of patients. Protection is partly based on IgG responses to parasite antigens exposed at the surface of infected erythrocytes (iRBCs). These IgG responses appeared low during clinical infection, particularly in severe disease. Methods. We analyzed the IgG binding capacity to the surface of live erythrocytes infected by knob positive FCR3 strain. Sera from 69 cerebral malaria (CM) and 72 mild malaria (MM) cases were analyzed by ELISA for IgG responses to five antigens from iRBC and by flow cytometry for IgG binding as expressed in labeling index ratio (LIR). The relationship between IgG levels, LIR, parasitemia, age, and the clinical outcomes was evaluated. Results. We found a significant decrease of LIR in adult CM fatal cases compared to surviving patients (p = 0.019). In MM, LIRs were correlated to IgG anti-iRBC and anti-PfEMP3/5 levels. In CM, no correlation was found between LIR, IgG levels, and parasitemia. Conclusion. The IgG binding assay was able to discriminate outcome of cerebral malaria cases and it deserves further development as a potential functional-associated assay for symptomatic malaria analysis.

Inflammatory cytokine and humoral responses to Plasmodium falciparum glycosylphosphatidylinositols correlates with malaria immunity and pathogenesis.

Pro-inflammatory cytokines induced by glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum contribute to malaria pathogenesis and hence, the naturally acquired anti-GPI antibody thought to provide protection against severe malaria (SM) by neutralizing the stimulatory activity of GPIs. In previous studies, the anti-GPI antibody levels increased with age in parallel with the development of acquired immunity, and high levels of anti-GPI antibodies were associated with mild malaria (MM) cases. In the present study, the relationship between the levels of pro-inflammatory cytokines and anti-GPI IgG antibody responses, parasitemia, and the clinical outcomes were evaluated in SM and mild malaria (MM) patients. Sera from a total of 110 SM and 72 MM cases after excluding of ineligible patients were analyzed for the levels of anti-GPI antibodies, IgG subclasses, and cytokine responses by ELISA. While the total anti-GPI antibody levels were similar in overall SM and MM groups, they were significantly higher in surviving SM patients than in fatal SM cases. In the case of cytokines, the TNF-α and IL-6 levels were significantly higher in SM compared to MM, whereas the IL-10 levels were similar in both groups. The data presented here demonstrate that high levels of the circulatory pro-inflammatory, TNF-α, and IL-6, are indicators of malaria severity, whereas anti-inflammatory cytokine IL-10 level does not differentiate SM and MM cases. Further, among SM patients, relatively low levels of anti-GPI antibodies are indicators of fatal outcomes compared to survivors, suggesting that anti-GPI antibodies provide some level of protection against SM fatality.

Analysis of antibody profiles in symptomatic malaria in three sentinel sites of Ivory Coast by using multiplex, fluorescent, magnetic, bead-based serological assay (MAGPIX™).

BACKGROUND: Advances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead-based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast. METHODS: Recombinant proteins or peptides derived from liver or blood stage antigens of Plasmodium falciparum (CSP, LSA141, LSA3, SALSA, PF13-DBL1α1, GLURP, AMA1, MSP1p19, MSP4p20), the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour-coded microsphere (Luminex™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings (rural and periurban) were analysed. RESULTS: Highly variable seroprevalence of antibody responses against parasite antigens was found ranging from 3 (gSG6) to 97% (MSP4p20). A marked prevalence and significantly higher level of antibodies was found in patients from the rural site (Korhogo), those harbouring the lowest level of parasitaemia. The use of whole schizont extract could not discriminate immunity level, contrary to parasite-derived recombinant proteins or peptides. Prevalence of responders to LSA141 and levels of antibodies to PF13 were significantly different between the three settings. Moreover, the post-treatment clearance of parasites was clearly associated with a significantly higher level of antibody response for almost 50% of the parasite antigens tested. CONCLUSION: The multiplex MBA-Magpix technology assay provides an accurate high throughput monitoring of parasite-specific antibodies during symptomatic malaria. The levels of antibody responses may provide a risk criterion with respect to the degree of parasitic infection. Additionally, they can be used as an indicator in the implementation of malaria prevention and local control strategies.