This will be a hospital-based cross-sectional study that will run from March 2022 to February 2023. It will enroll pregnant women with and without HIV who are in the third trimester of pregnancy (26-32 weeks) and have willingly accepted to participate in the study by signing a consent form. During delivery, samples will be collected and questionnaires will be appropriately filled.

The study will be conducted in Kumba, which is a metropolitan city in Meme Division of the southwest region (SWR) of Western Cameroon. Meme Division is 1 of the 6 divisions of the SWR of Cameroon. It is made up of 5 subdivisions: Kumba I, II, and III (largely urban) and Mbonge and Konye (dominantly rural) [28].

Ethical clearance for this study has been obtained from the Institute for Advanced Medical Research and Training (IAMRAT), College of Medicine, University of Ibadan, Nigeria (ID NHREC/05/01/2008a). However, ethical clearance from Cameroon has been obtained from the Institutional Review Board of the Faculty of Health Sciences at the University of Buea (ID 2022/1655-02/UB/SG/IRB/FHS). Administrative clearance has been obtained from the Ministry of Public Health, Regional Delegation of Public Health for the SWR (ID R11 /MINSANTE/SWR/RDPH/PS/276/281). Further authorization has been obtained from the Meme Health District Health service (ID 12/022/MINSANTE/RDPHSW/KHD/DMO/048). Directors of the health facilities involved have also provided an authorization letter for the study to take place in their various facilities. Participants who wish to participate in the study will first sign an informed consent form. For participants who are unable to read or write, the information will be read out and explained to them and their thumbprints will be taken. The study will consider pregnant women with and without HIV who are in the third trimester of pregnancy and are fully aware of their viral load counts.

The estimated sample size for the study was calculated using the Cochrane formula [29]. The estimated prevalence of malaria among pregnant women with HIV is 81%, as obtained from a study looking at malaria prevalence in pregnant women with and without HIV in neighboring Nigeria [30]. The critical value and standard value for the corresponding level of confidence were 1.96, and the margin of error was 0.05. The estimated sample size is 236. The ratio of pregnant women with to those without HIV will be 1:1, which therefore implies that the total estimated sample size is 472. That is, 236 (50%) pregnant women with and 236 (50%) pregnant women without HIV will be enrolled in the study.

A probability proportionate to size sampling will be used to determine the number of participants to be recruited in each health institution (Table 1). A simple random sampling method will be used each day to recruit participants as they come for their ANC visits to the selected health facilities.

A well-structured semiquestionnaire will be used to collect demographic and clinical parameters from study participants. The questionnaire will be pretested at District Hospital Kumba-town before being issued to participants for data collection.

The result of the rapid diagnostic test and microscopy for each participant will be made available to them. This is to ensure that those positive for malaria should be placed immediately on treatment.

Briefly, 3 mL of the mother’s peripheral (venous) blood, placental blood, and baby cord blood will be collected into EDTA, heparinized, and sodium-citrated tubes, respectively. A capillary puncture will be made to collect the sample, and a thick-film smear will be prepared for microscopy. Smears will be allowed to air-dry and then carefully placed in well-cleaned slide boxes to be stored for further analysis.

Data will be entered into Microsoft Excel, cleaned, and transformed for the different analyses to be performed. SPSS Statistics (IBM Corp) and STATA will be used for statistical analysis. A normality test will be performed to determine the different parametric and nonparametric tests to be used for the different analyses.

Descriptive analysis of the general study population will be conducted, and both the mean and median of each parameter will be considered. The chi-square test will be performed to analyze categorical variables, and the Student t-test/Mann-Whitney U test will be performed to analyze continuous and categorical variables. The Kruskal-Wallis test/ANOVA will be conducted to analyze continuous and more than 2 categorical variables, while linear regression/Spearman correlation will be performed for multivariate analysis to determine the strength of association between different factors of the study and our dependent variables. All P values <.05 will be considered statistically significant. BioEdit 7.0.0 software will be used to align the nucleotide sequences of different genes after sequencing. Phylogenetic tree searching will be performed using the maximum-likelihood (ML) method in MEGA V6.0.

Immune development during pregnancy is important as it determines the resistance of pregnant women to malaria transmission. Few studies have shown that women develop protective immunity against pregnancy-associated malaria (PAM), which is thought to be due to the acquisition of antibodies to the parasite variant surface antigen 2 chondroitin sulphate A (VAR2CSA) [38]. This immunity becomes stronger, especially in multigravidae [38]. However, this does not appear to be the case with pregnant women with HIV, as studies have shown that HIV alters the immunity against malaria, making both primigravidae and multigravidae vulnerable to PAM [11]. Although studies looking at malaria prevalence have been carried out in Kumba, studies evaluating the immune response of pregnant women with and without HIV to malaria have not been conducted. This study will attempt to determine the prevalence of malaria infection, assess the occurrence of P. falciparum genetic diversity, and evaluate the antibody (IgG and IgM: AMA1, MSP1, MSP2, MSP3, and EBA175) and cytokine (IL-10, TNF-α, and IFNγ) response to malaria infection among pregnant women with and without HIV in Kumba.

In Nigeria, the prevalence of malaria infection among pregnant mothers with HIV was 81% compared to 75% among pregnant women without HIV [30]. When compared to mothers without HIV, mothers with HIV have more moderate parasitemia [30]. This finding is in line with a similar study from Kumba, Cameroon, which reported a 61.5% prevalence of malaria among pregnant women with HIV [39]. Even though the prevalence of malaria in Kumba is high, it would have been much higher if more sensitive diagnostic techniques, such as PCR, had been used for diagnosis in this study rather than traditional microscopy, whose sensitivity is dependent on technician expertise [40], and RDTs, which lack specificity due to the high possibility of cross-reaction of antibodies [39]. Kenji et al [40] discovered that PCR could diagnose 64 misdiagnosed cases that were missed by RDTs and microscopy. In addition, in the Republic of Congo, it was discovered that the prevalence of malaria infection is high in pregnant women attending antenatal facilities or hospitalized and that it increases when HIV infection is present [41]. However, in Jos, Nigeria, the prevalence of malaria infection reduced from 34.1% to 7.2% among women administered cotrimozaxole and from 25% to 10% among women administered SP-IPT for malaria prophylaxis [42]. According to some studies, HIV and AIDS cause immunosuppression, which reduces the overall immune response to malaria parasitemia, increasing the frequency of clinical malaria attacks [43,44]. Effective implementation of IPT strategies among pregnant women reduces their likelihood of malaria infection [42]. Nevertheless, studies in Ghana have also shown that HIV infection increases the rate of vertical transmission of malaria to children born to a mother with HIV [45]. This study was able to show that HIV-malaria coinfection is a significant predictor of adverse perinatal outcomes and neonatal morbidity [45]. However, although national guidelines in Cameroon recommend the use of cotrimozaxole for pregnant women with HIV and SP for pregnant women without HIV at all ANC visits [46], most health facilities suffer from drug shortages, forcing women to attend some ANC visits without medication. The effect can be seen in a study carried out in Cameroon, where not taking Fansidar for IPT was found to be associated with the development of malaria among pregnant women [1].

Studies have shown that P. falciparum, the most virulent species, has a wide genetic variety at all degrees of endemicity, which is not surprising, given that genetic diversity has been shown to be a function of transmission in specific regions [47,48]. Oboh et al [49] discovered that the prevalence of the FC27 allele type in pregnant women isolates is higher (33.3%) than that in nonpregnant women (29.2%) when looking at the genetic diversity of P. falciparum (MSP1 and MSP2) infection among pregnant women in Lagos, Southwest Nigeria [49]. This finding is in line with a similar study carried out in Yaoundé, Cameroon [50]. Although no study has been conducted to assess the diversity of P. falciparum among pregnant women with and without HIV as 2 distinct entities, a couple of studies have evaluated the diversity of P. falciparum among individuals with HIV. In Mutengene, Cameroon, it was reported that the MSP1 allelic variants detected among patients with HIV were KI (65.22%), MAD20 (27.54%), and RO33 (27.54%), while the MSP2 allelic variants were FC27 (30.43%) and 3D7/IC (31.88%), which was high and associated with anemia [51]. A study in Senegal reported no difference in genetic diversity between P. falciparum isolates from patients with malaria and concurrent malaria-arbovirus in Kedougou [52]. However, the increased number of genotypes observed in the malaria group was attributed to the larger number of samples rather than the extensive genetic diversity of P. falciparum isolates from patients with malaria [52]. The frequency of all MSP1 allelic families (K1, MAD20, and RO33) and the IC/3D7 allelic family of MSP2 was high (>70%) and comparable between the 2 study groups in this study [52]. Nevertheless, clonal P. falciparum population dominance is expected to be observed in regions with declining endemicity as a result of genetic drift caused by scale-up interventions [53]. In other words, in parasite populations where control efforts have left behind lineages that create clusters of highly related parasites, the persistence of a specific haplotype throughout the year is expected [53,54]. As a result, unrelated parasites are unlikely to coexist in the same mosquito blood meal [53].

In addition, the protective role of antibodies to malaria cannot be overemphasized, as studies have shown that the antibody response plays a vital role in the protection of pregnant women against PAM infection [55,56]. A sero-surveillance study in Thai-Myanmar at the Shoklo Malaria Research Centre reported a significant variation in the antibody profiles of PfAMA₁, PfEBA175, PfMSP₂, and PfVAR₂CSA. Most controls’ antibody profiles for PfMSP₃ and PvAMA₁ remained low throughout gestation regardless of malaria exposure, whereas the antibody response varied more among the cases and the control group of pregnant women with and without infection [57]. This study is in line with a study in Yaoundé that instead looked at the antibody response of malaria vaccine candidates among children [58]. This confirms that during a malaria infection, antibody levels should rise as an indication of immune protection. Alistair et al [59] even demonstrated that babies born to women with PAM with high levels of VAR2CSA antibodies are protected against the vertical transmission of malaria and have a higher mean birth weight than those with low VAR2CSA antibody levels [59].

In contrast, studies have shown that HIV infection amplifies the effect of malaria, especially among pregnant women [39,43,44]. One of these effects is the dysregulation of immunity to malaria in mothers and even their children exposed in utero [12,60]. In Yaoundé, it was reported that IgG levels to CSP and TCC were lower in mothers with HIV, as were cord IgG levels to CSP, MSP1, and TCC in neonates born to mothers with HIV [12]. This was in line with a study in Kenya that reported that HIV infection is associated with a decrease in the magnitude and breadth of IgG responses to merozoite antigens, as well as a delay in the age-related acquisition of the IgG antibody response to schizont extract [9]. The study also revealed that children with HIV are less likely to be high responders to AMA1 [9]. This effect can be seen in children, as studies have reported that children exposed to HIV are more susceptible to infections than children not exposed to HIV [60]. However, there is still a lack of understanding of why children born to these women are more susceptible to infections [60]. To better understand why children exposed to HIV are more susceptibility to infections, such as malaria, studies evaluating the effect of HIV infection on the vertical transfer of mononuclear WBCs, as well as a longitudinal study monitoring the immune response of these children exposed over time, are needed.

Cytokines and chemokines are elevated in peripheral blood during an acute malaria infection and contribute to parasite clearance, but they are also likely to be responsible for many of the symptoms and pathological changes seen during malaria [61,62]. The balance of pro- and anti-inflammatory signals appears to play a significant role in determining the outcome of an infection (ie, whether it leads to protection or immunopathology) [62]. Eduard et al [63] reported that an imbalanced pro-inflammatory cytokine response may exacerbate infection severity. Among pregnant women in Cameroon, it was realized that placental malaria reduces maternal plasma levels of IL-17E, IL-27, and IL-28A, as well as neonatal plasma levels of IL-17E and IL-28A, which may aid in parasite clearance and increase child birth weight [14]. This is in line with a similar study carried out by Megnekou et al [64]. Nonetheless, Djontu an coworkers [65] showed that IL-28A, IL-27, and IL-17E levels are higher in cord plasma than in maternal peripheral plasma and that they correlate positively with increasing baby birth weight. This positive relationship between birth weight and both Th1- and Th2-promoting cytokines (IL-27, IL-28A) is consistent with the findings of Chêne et al [65], who found a link between low birth weight and lower levels of IFNγ and IL-5 in intervellous plasma samples from Beninese women. A study carried out in Abia State, Nigeria, showed that cytokine levels in the same woman's peripheral and placental blood may differ [66]. It was realized that IFNγ is significantly higher in peripheral blood than in placental blood. IL-4 and IL-10 levels were significantly lower in peripheral blood than in placental blood [66].

Among pregnant women with HIV, it was discovered that cytokine activity during pregnancy, as measured by plasma cytokine concentrations, varies with antiretroviral therapy and P. falciparum coinfection [67]. In Mozambique, it was reported that among adults with HIV, IL-8 and IFNγ-induced protein 10 (IP-10) play a complex role in inflammation during P. falciparum infection and a potential pathogenic role [67]. Another study reported that patients with HIV and cerebral malaria have lower median plasma levels of TNF-α and IL-10 than patients without HIV [68]. This goes to confirm that HIV affects the cytokine response to malaria infection. Nevertheless, information about the cytokine response to malaria among pregnant women with HIV is scanty. However, studies comparing the levels of cytokine response in pregnant women with HIV and with and without malaria, as well as pregnant women without HIV and with and without malaria, are required to better understand the role of cytokines in the pathogenesis of malaria.

There is a need for research into the prevalence of malaria among pregnant women in Kumba, considering all trimesters. It is also necessary to conduct a study on P. falciparum resistance to SP-IPT as well as a longitudinal study on children born to mothers with HIV in order to understand why these children are more vulnerable to infections.

The results of previous studies depict that HIV affects the prevalence of malaria among pregnant women irrespective of gravidity; malaria prevalence turns out to be much higher among pregnant women with than pregnant women without HIV. It was also revealed that more P. falciparum MSP1 alleles are associated with malaria infection among individuals with HIV and that genetic diversity is a function of malaria transmission in specific regions. In addition, HIV infection lowers the level of IgG response of antibodies to malaria infection for both mother and newborn babies. The likelihood of children with HIV to develop an immune response to AMA1 is low. HIV infection also alters the levels of the pro- and anti-inflammatory cytokine response, contributing to the lack of immune parasite clearance and parasite pathogenicity.

The findings of this work will be published in peer review journals and will be presented in seminars as well as conferences. Copies of the work will be submitted at the Pan-African University of Life and Earth Sciences Institute, University of Ibadan, Nigeria; the University of Buea; and the hospitals where the samples were collected. A copy will also be sent to the Union for African Population Studies.