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.

Recognition of Plasmodium falciparum mature gametocyte-infected erythrocytes by antibodies of semi-immune adults and malaria-exposed children from Gabon.

BACKGROUND: Transmission of malaria from man to mosquito depends on the presence of gametocytes, the sexual stage of Plasmodium parasites in the infected host. Naturally acquired antibodies against gametocytes exist and may play a role in controlling transmission by limiting the gametocyte development in the circulation or by interrupting gamete development and fertilization in the mosquito following ingestion. So far, most studies on antibody responses to sexual stage antigens have focused on a subset of gametocyte-surface antigens, even though inhibitory Ab responses to other gametocyte antigens might also play a role in controlling gametocyte density and fertility. Limited information is available on natural antibody response to the surfaces of gametocyte-infected erythrocytes. METHODS: Ab responses to surface antigens of erythrocytes infected by in vitro differentiated Plasmodium falciparum mature gametocytes were investigated in sera of semi-immune adults and malaria-exposed children. In addition, the effect of immunization with GMZ2, a blood stage malaria vaccine candidate, and the effect of intestinal helminth infection on the development of immunity to gametocytes of P. falciparum was evaluated in malaria-exposed children and adults from Gabon. Serum samples from two Phase I clinical trials conducted in Gabon were analysed by microscopic and flow-cytometric immunofluorescence assay. RESULTS: Adults had a higher Ab response compared to children. Ab reactivity was significantly higher after fixation and permeabilization of parasitized erythrocytes. Following vaccination with the malaria vaccine candidate GMZ2, anti-gametocyte Ab concentration decreased in adults compared to baseline. Ab response to whole asexual stage antigens had a significant but weak positive correlation to anti-gametocyte Ab responses in adults, but not in children. Children infected with Ascaris lumbricoides had a significantly higher anti-gametocyte Ab response compared to non-infected children. CONCLUSION: The current data suggest that antigens exposed on the gametocyte-infected red blood cells are recognized by serum antibodies from malaria-exposed children and semi-immune adults. This anti-gametocyte immune response may be influenced by natural exposure and vaccination. Modulation of the natural immune response to gametocytes by co-infecting parasites should be investigated further and may have an important impact on malaria control strategies.

The effect of immunization schedule with the malaria vaccine candidate RTS,S/AS01E on protective efficacy and anti-circumsporozoite protein antibody avidity in African infants.

BACKGROUND: The malaria vaccine RTS,S induces antibodies against the Plasmodium falciparum circumsporozoite protein (CSP) and the concentration of Immunoglobulin G (IgG) against the repeat region of CSP following vaccination is associated with protection from P. falciparum malaria. So far, only the quantity of anti-CSP IgG has been measured and used to predict vaccination success, although quality (measured as avidity) of the antigen-antibody interaction shall be important since only a few sporozoites circulate for a short time after an infectious mosquito bite, likely requiring fast and strong binding. METHODS: Quantity and avidity of anti-CSP IgG in African infants who received RTS,S/AS01E in a 0-1-2-month or a 0-1-7-month schedule in a phase 2 clinical trial were measured by enzyme-linked immunosorbent assay. Antibody avidity was defined as the proportion of IgG able to bind in the presence of a chaotropic agent (avidity index). The effect of CSP-specific IgG concentration and avidity on protective efficacy was modelled using Cox proportional hazards. RESULTS: After the third dose, quantity and avidity were similar between the two vaccination schedules. IgG avidity after the last vaccine injection was not associated with protection, whereas the change in avidity following second and third RTS,S/AS01E injection was associated with a 54% risk reduction of getting malaria (hazard ratio: 0.46; 95% confidence interval (CI): 0.22-0.99) in those participants with a change in avidity above the median. The change in anti-CSP IgG concentration following second and third injection was associated with a 77% risk reduction of getting malaria (hazard ratio: 0.23, 95% CI: 0.11-0.51). CONCLUSIONS: Change in IgG response between vaccine doses merits further evaluation as a surrogate marker for RTS,S efficacy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT00436007 .

Humoral immune response to Plasmodium falciparum vaccine candidate GMZ2 and its components in populations naturally exposed to seasonal malaria in Ethiopia.

BACKGROUND: In Ethiopia, the general population is vulnerable to unpredictable epidemics of Plasmodium falciparum malaria. However, there is little information on the anti-malaria immune profile of the population in the endemic regions of the country. METHODS: The study was designed to investigate the nature of humoral immune response to malaria in two ethnic groups in two endemic localities: Shewa Robit in north, and Boditi in south Ethiopia which are characterized by varying levels of malaria transmission and altitude. In a cross-sectional study, the study participants were diagnosed for malaria infection microscopically and by the rapid diagnostic test (RDT). Sera were tested by using enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin (Ig) G against P. falciparum blood-stage vaccine candidate GMZ2 and its subunits (Glutamate-rich protein (GLURP-R0), merozoite surface protein 3 (MSP3); as well as IgG subclasses against GLURP-R0 and MSP3. RESULTS: Whereas 23(8.6%) blood smear-positive cases for P. falciparum were detected in Boditi, all Shewa Robit study participants had no detectable P. falciparum infection. In both localities, total IgG prevalence and levels to GMZ2 were significantly higher than the response to the component domains indicating the strong recognition of GMZ2 by antibodies acquired through natural exposure. Total IgG and subclass prevalence and levels were higher in Shewa Robit than Boditi, suggesting difference in the intensity of malaria transmission in the two localities and/or genetic differences between the two populations in their response to the antigens. In both study sites, IgG subclass levels to GLURP-R0 were significantly higher than that to MSP3 for all corresponding subclasses in most individuals, indicating the higher relative antigenicity and probably protective potential of GLURP-R0 compared to MSP3. Against both GLURP-R0 and MSP3, the ratio of cytophilic to noncytophilic antibodies was >1 in the majority of the study participants, in both study sites, suggesting the induction of protective (cytophilic) antibodies against the two antigens. Analysis of age-related pattern in antibody levels against the antigens showed a positive association with increasing age. CONCLUSIONS: P. falciparum GLURP-R0 and MSP3 separately as well as in a fused form in GMZ2 are readily recognized by the sera of the study populations. The significantly higher antibody prevalence and level detected against GMZ2 compared to either of its subunits separately, in naturally exposed populations, suggests the synergistic effect of GLURP-R0 and MSP3 and that GMZ2 could be a more relevant blood-stage malaria vaccine candidate than the individual components. Detection of high-level antibody responses in non-febrile, smear-negative individuals may possibly be an indication of a low-grade, asymptomatic sub-microscopic infection in the induction and maintenance of high-level malaria immunity.

Amodiaquine-Artesunate versus Artemether-Lumefantrine against Uncomplicated Malaria in Children Less Than 14 Years in Ngaoundere, North Cameroon: Efficacy, Safety, and Baseline Drug Resistant Mutations in pfcrt, pfmdr1, and pfdhfr Genes.

Background. In Cameroon, both Artesunate-amodiaquine (AS/AQ) and artemether-lumefantrine (AL) are used as first-line treatment against uncomplicated malaria in line with the WHO recommendations. We compared the efficacy and safety of both therapeutic combinations and determined the prevalence of drug resistance conferring mutations in three parasite genes. Methods. One hundred and fifty acute malaria patients between six months and 14 years of age were randomized to receive standard doses of either AS/AQ (73) or AL (77) and followedup for 28 days. Outcome of treatment was according to the standard WHO classification. DNA samples from pretreatment parasite isolates were used to determine the prevalence of resistant mutations in the pfcrt, pfmdr1, and dhfr genes. Results. Both drug combinations induced rapid clearance of parasites and malaria symptoms. PCR-corrected cure rates were 100% and 96.4% for AL. The combinations were well tolerated. Major haplotypes included CVIET (71%), CVMNT (25%) for the pfcrt; SND (100%) for the pfmdr1; IRN (79, 8%), NCS (8.8%), and mixed haplotype (11, 8%) for the dhfr. Conclusion. Both AS/AQ and AL were highly effective and well tolerated for the treatment of uncomplicated falciparum malaria in Ngaoundere, Cameroon. High prevalence of mutant pfcrt alleles confirms earlier observations. Long-term monitoring of safety and efficacy and molecular markers is highly solicited.

A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals.

BACKGROUND: Antibodies play a central role in naturally acquired immunity against Plasmodium falciparum. Current assays to detect anti-plasmodial antibodies against native antigens within their cellular context are prone to bias and cannot be automated, although they provide important information about natural exposure and vaccine immunogenicity. A novel, cytometry-based workflow for quantitative detection of anti-plasmodial antibodies in human serum is presented. METHODS: Fixed red blood cells (RBCs), infected with late stages of P. falciparum were utilized to detect malaria-specific antibodies by flow cytometry with subsequent automated data analysis. Available methods for data-driven analysis of cytometry data were assessed and a new overlap subtraction algorithm (OSA) based on open source software was developed. The complete workflow was evaluated using sera from two GMZ2 malaria vaccine trials in semi-immune adults and pre-school children residing in a malaria endemic area. RESULTS: Fixation, permeabilization, and staining of infected RBCs were adapted for best operation in flow cytometry. As asexual blood-stage vaccine candidates are designed to induce antibody patterns similar to those in semi-immune adults, serial dilutions of sera from heavily exposed individuals were compared to naïve controls to determine optimal antibody dilutions. To eliminate investigator effects introduced by manual gating, a non-biased algorithm (OSA) for data-driven gating was developed. OSA-derived results correlated well with those obtained by manual gating (r between 0.79 and 0.99) and outperformed other model-driven gating methods. Bland-Altman plots confirmed the agreement of manual gating and OSA-derived results. A 1.33-fold increase (p=0.003) in the number of positive cells after vaccination in a subgroup of pre-school children vaccinated with 100 µg GMZ2 was present and in vaccinated adults from the same region we measured a baseline-corrected 1.23-fold, vaccine-induced increase in mean fluorescence intensity of positive cells (p=0.03). CONCLUSIONS: The current workflow advances detection and quantification of anti-plasmodial antibodies through improvement of a bias-prone, low-throughput to an unbiased, semi-automated, scalable method. In conclusion, this work presents a novel method for immunofluorescence assays in malaria research.

Effect of IL-15 on IgG versus IgE antibody-secreting cells in vitro.

Immunoglobulin E (IgE) antibodies are major contributors to the pathology of atopic and allergic diseases as well as to immune response to helminth infections. Development of an adequate immunoglobulin G (IgG) immune response against infectious agents and vaccine antigens is considered in most cases as crucial for protection from disease. In vivo and in vitro production of IgE and IgG depends on cytokines and other soluble factors. Recently it has been shown that IgG antibody secreting cells (ASCs) can be generated by in vitro maturation of blood cells with Interleukin- (IL-)15 and CpG DNA or other stimulation cocktails, while IgE-ASCs develop upon cultivation with anti-CD40 and IL-4. In the present study we employed an enzyme linked immunospot assay (ELISPOT) to assess the capacity of individuals to develop into either IgE-ASCs or IgG-ASCs upon stimulation with different combinations of stimulation cocktails in order to investigate the influence of cytokines that are dysregulated in IgE-mediated immune reactions on ASC generation. Furthermore, we modified the method to assess IgG- and IgE-ASCs specific for two model antigens causing allergic rhinitis in humans. We demonstrate that IL-15, which is important for development of IgG-ASCs, decreases the number of IgE-ASCs when added to media commonly used for in vitro development of IgE-ASCs. We show that our method is suitable for the detection of specific and non-specific IgE-ASCs and IgG-ASCs and allows the investigation of the interplay between IgG-ASCs and IgE-ASCs in different populations.

Efficacy of amodiaquine, sulphadoxine-pyrimethamine and their combination for the treatment of uncomplicated Plasmodium falciparum malaria in children in Cameroon at the time of policy change to artemisinin-based combination therapy.

BACKGROUND: The efficacy of amodiaquine (AQ), sulphadoxine-pyrimethamine (SP) and the combination of SP+AQ in the treatment of Cameroonian children with clinical malaria was investigated. The prevalence of molecular markers for resistance to these drugs was studied to set the baseline for surveillance of their evolution with time. METHODS: Seven hundred and sixty children aged 6-59 months with uncomplicated falciparum malaria were studied in three ecologically different regions of Cameroon - Mutengene (littoral equatorial forest), Yaoundé (forest-savannah mosaic) and Garoua (guinea-savannah). Study children were randomized to receive either AQ, SP or the combination AQ+SP. Clinical outcome was classified according to WHO criteria, as either early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR). The occurrence of mutations in pfcrt, pfmdr1, dhfr and dhps genes was studied by either RFLP or dot blot techniques and the prevalence of these mutations related to parasitological and therapeutic failures. RESULTS: After correction for the occurrence of re-infection by PCR, ACPRs on day 28 for AQ, SP and AQ+SP were 71.2%, 70.1% and 80.9%, in Garoua, 79.2%, 62.5%, and 81.9% in Mutengene, and 80.3%, 67.5% and 76.2% in Yaoundé respectively. High levels of Pfcrt 76T (87.11%) and Pfmdr1 86Y mutations (73.83%) were associated with quinoline resistance in the south compared to the north, 31.67% (76T) and 22.08% (86Y). There was a significant variation (p < 0.001) of the prevalence of the SGK haplotype between Garoua in the north (8.33%), Yaoundé (36.29%) in the savannah-forest mosaic and Mutengene (66.41%) in the South of Cameroon and a weak relation between SGK haplotype and SP failure. The 540E mutation on the dhps gene was extremely rare (0.3%) and occurred only in Mutengene while the pfmdr1 1034K and 1040D mutations were not detected in any of the three sites. CONCLUSION: In this study the prevalence of molecular markers for quinoline and anti-folate resistances showed high levels and differed between the south and north of Cameroon. AQ, SP and AQ+SP treatments were well tolerated but with low levels of efficacy that suggested alternative treatments were needed in Cameroon since 2005.