1. Introduction

Sub-Saharan Africa represents the region most heavily affected by both malaria and HIV ^[1] The overlap of these two infections is common and it would be important to understand their interactions and their correct management in order to limit their clinical burden ^[2] Today, approximately 50% of the world’s population, or 3.3 billion people, are at varying degrees of risk of malaria. During the acute bouts of clinical malaria, plasma HIV RNA level rise ^{[3, 4]} and CD4 cells decline by approximately 40 cells/µL/year with each malaria episodes ^[5]. The interaction between falciparum malaria infection with HIV and the involvement of the cellular immune system in determining disease pathogenesis is still far from being elucidated. Malaria infection is also associated with strong CD4⁺ cell activation and up-regulation of pro-inflammatory cytokines, providing an ideal micro-environment for the spread of the virus among CD4⁺ cells and thus, for rapid HIV replication ^[6]. Therefore, the need to determine the impact of P. falciparum and HIV co-infection on the number of CD4⁺ cells and levels of cytokines produced in pregnant women. Some early studies found no association between HIV and malaria disease severity in adults ^[7]. More recent research has shown that HIV infection predispose to more frequent episodes of symptomatic malaria ^[8] and more episodes of severe malaria including death adult ^[9].

An inverse relationship was found between incidence of severe malaria and CD4⁺ T lymphocyte counts. It is known that co-infected pregnant women are particularly at risk of complications due to interactions between malaria and HIV; pregnant women are more likely susceptible to malaria disease than non-pregnant women ^[10]. High levels of parasitaemia and chronic parasite infection in placental blood can lead to consumption of nutritive blood substances, to a worsening of perinatal outcomes and to increased rates of maternal morbidity. Malaria and HIV infections were associated with several negative outcomes in newborns of co-infected mothers. ^[11] demonstrated an association between co-infection and an increased risk of stillbirth and preterm delivery. The two infections were independently associated with an increased risk of low birth weight and foetal growth retardation compared to HIV-uninfected women and malaria infected women ^[11]. A study conducted by ^[12] in Malawi reported significantly higher mortality rates among children born to HIV-sero-positive compared to HIV seronegative women, and the risk of neonatal mortality was greater in co-infected mothers compared to mono-infected mothers. As previously mentioned, malaria promotes HIV RNA replication: a study showed that women had about 2-fold increase in HIV RNA both in peripheral and placental blood, with a consequent higher possibility of mother-to-child transmission of HIV ^[3].

P. falciparum infections promote macrophages and CD4⁺ cells to activate viral transcription. A high parasite density elicits a strong immune response, leading to a high turnover of HIV RNA and fever; this then indicate cytokine response that raise HIV RNA concentrations ^[13]. HIV infection targets the immune system leading to a state of immunodeficiency in a setting of immune activation.

2. Methodology

Saki is a border town in Oyo State Nigeria bounded by Kwara State in the North and to the West by Cotonou (Republic of Benin) and Lome (Togo). It comprises of three Local Government Areas, viz: Saki West, Saki East and ATISBO (Ago-Are, Tede, Irawo, Sabe, Baasi, and Oje-Owode). The selected hospitals are located in Saki in the region with mesoendemic P. falciparum infection. According to the 2006 National Census figures, Saki is the third largest city in Oyo state with a population density of 189,700. The town is heterogeneous comprising the main people of the Yoruba ethnic group who speak the Yoruba language and in the minority; other ethnic groups which include the Fulani, Togolese, Beninois, and the Tangitas. The study design was of longitudinal study which lasted for 18-month period (August, 2009- February, 2011).

Subjects included in the study gave informed verbal consent and the study was approved by the Joint Ethical Committee UI/UCH and the concerned hospitals. Other ethical considerations as contained in the National Code of Health Research Ethics ^[14] were followed. Each respondent was pre- and post-counseled after informed consent was given. The questionnaire was drafted and given to an expert in the Faculty of Education at the University of Ibadan for content and face validities. A pilot test was carried out on fifty pregnant volunteers after necessary corrections had been effected, in order to establish the reliability of the instrument. Structured questionnaires were used to collect baseline demographic data (e.g. age, sex, household size etc), clinical data/history, anthropometric data, and head circumference, Last menstrual date, estimated date of delivery, use of IPT since gestation, number of previous pregnancies (gravidity), gestation, history, income status, educational status, history of previous pregnancies. The target population was pregnant women who reported for routine ante-natal check-up. Pregnant mothers who consented to participate in the study after being adequately informed of the project objectives, protocol and benefits were then enrolled. All subjects were encouraged to deliver their babies at the selected hospitals of the project.

From enrollment until delivery, each woman enrolled was followed-up until few months after delivery. All the enrollees were subsequently reached through medical staff of each Hospital. At each antenatal and post delivery visit, information on occurrence of symptoms, illness, and treatment taken since the last visit to the project was collected on standardized questionnaires. All women recruited received Insecticide Treated Mosquito Nets (LLINs) and free SP (IPT) according to the WHO recommendation.

Blood of (5mL) was collected from each patient, 2mL into sterile EDTA bottles which were taken to the Laboratory and another 2 mL into vacutainer bottles. The vacutainer tubes were immediately transported to the General Hospital for CD4⁺ analysis. The EDTA samples were moved under ice to the Department of Science Laboratory Technology Laboratory. The remaining blood was used for thin and thick blood smear for malaria parasitemia, PCV and filter paper preparation. Within six hours of sample collection CD4⁺ total enumeration was performed on the whole blood transported to General Hospital using BD FACS Count CD4 technique. At SLT Department, the EDTA blood sample was centrifuged at 400 rpm in the Laboratory in order to obtain the plasma which was subsequently used to run the ELISA assay. The plasma samples remained stored at -70C until analysis. In the babies, cord blood samples were collected at delivery for parasitological studies.

After cleaning the upper surface of the arm with cotton wool moistened with methylated spirit, peripheral blood samples were collected in sterile containers. Thin and thick blood smears were made from each of these samples. A staining protocol using 30% Giemsa stain for thick smear for 15 minutes and Leishman stain was used for thin smear. Parasite counts standardized per 200 leukocytes were performed on thick/thin blood films. The number of parasites per microliter of blood was calculated by assuming an average white blood cell count of 8,000/µL. The preparation of both thick and thin blood films for confirmation of parasitemia followed the methods described ^[15]. The degree of parasitemia was graded by modification of ^[16] thus (1-999/µL) as mild Low and High above (>1000-9999/µL). A drop of immersion oil was placed on the stained thin film and another drop on thick film. They were systematically examined using x 100 objectives (oil immersion). A negative result was recorded after thorough examination of 100 fields without any parasite. Quality control was ensured, by using freshly reconstituted filter Giemsa stains for parasitemia.

All participants who consented to participate were screened for the presence of HIV antibody by using Determine HIV rapid test kits and/or UNI-Gold Rapid Test grouped into positive reactive and negative samples according to ^[17]. All those tested positive to RDT kits were later subjected to a confirmatory test at UCH through the Baptist Medical Centre, Saki. Infection status was determined according to the method of ^[18].

Peripheral plasma concentrations of IL-10, IL-2, IFN-γ and TNF-α were measured in all the samples of 179 participants by using cytokine ELISA techniques. The cytokine detection kits purchased from (Mabtech, Stockholm, Sweden) were used for this assay. ELISA plates (Corning Incorporated, Nunc Maxicorb) were coated with the purified anti-cytokine capture monoclonal antibody to (1µg/ml) in a coating buffer (Sodium bicarbonate). Monoclonal antibodies used were TNF-α, I-D1K, 9D7 and IL-2-I for TNF-α, IFN-γ, IL-10 and IL-2 respectively. The average ODs were calculated and its corresponding concentrations on the x-axis of the standard curve. Each final value was presented in the nearest x10^-3 pg/µL.

FACS technique is designed to be used with human whole blood samples. The whole blood samples was collected in vacutainer tubes and stored no longer than 6 hours of room temperature at 20-25 ^[19].

After the additions of CD4⁺ and CD3⁺ monoclonal antibody (10µL), the samples were incubated and analyzed; immediately it was read on FACS Count Machine according to the manufacturer’s instructions (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) and a standard six-tube, two-color monoclonal antibody panel (Becton Dickinson). Controls were prepared by adding normal whole blood, and then fixative solution was added to the CD4 reagents tubes followed by addition of control beads. For this study, absolute CD4⁺ T lymphocytes counts were used to classify patients into three categories: patients with less than 100 cells/µL of blood were classified as seriously immunocompromised, those with between 100-350 cells/µL of blood were immunosuppressed, the other category were those whose CD4 counts fall above 350 cells/ µL of blood were regarded as stable ^{[20, 21]}.

Subjects were grouped according to infection status: HIV negative, malaria negative (no-infection); HIV positive, malaria negative (HIV only); HIV negative, malaria positive (Malaria only) and HIV positive, malaria positive (co-infection) adapting a similar procedure of ^[22]. All data collected were analyzed using SPSS version 15 according to the protocol described by ^[23]. Demographic characteristics at baseline and the variations in the CD4 cell counts in the sample cohort were analyzed. The mean CD4 cell count (and its variance) by co-infected participants were then compared using Students t-tests (for characteristics with two categories) and ANOVA tests. The significance limit was p=0.05

3. Results

A total of 149 pregnant women participated in the study. The general descriptions and schematic description of categories of the study participants were as shown in (Figure 1). Among the participants, several groups were identified based on their infection status. There was the group infected with HIV only (30.2%), and that infected with P. falciparum (57.1%). Participants with co-infection of P. falciparum and HIV made up another group 39 (21.8 %). (Figure 1). Majority (70%) of the participating pregnant women were in their second trimester and 69 (46.3%) were multigravidae. All the participating women were further stratified based on their gravidity and their P. falciparum and HIV infections status. The highest prevalence of P. falciparum infection (21.5%) was observed among multigravidae. Malaria infection was found in all gravid states of pregnancy, but seems to be more in the secungravidae. The highest prevalence of HIV infection was observed among the primigravidae than both the secundigravidae and multigravidae and there was a statistically significant association between parity and prevalence of HIV infection.

Figure 1. schematic representation of the study

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Table 1. Medical Characteristics of Participating Mothers by Gravidity

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Figure 2. Mean plasma cytokine concentration and the infection status of participants

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Figure 3. Infection categories and frequency of CD4⁺ lymphocytes counts among the women participants

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4. Haematological parameters among co-infected participants

The mean Hb level of P. falciparum and HIV co-infected babies was 8.73 ± 3.13 g/dl and that of the pregnant mothers was 7.49 ± 3.34 g/dl. There was no significant difference between the means of CD4⁺, haemoglobin level and P. falciparum density in women (p=0.335) (Table 2). There was a significant difference between the means of CD4⁺, haemoglobin levels and P. falciparum density in babies (p=0.005). The mean CD4⁺T cell count for the co-infected group was 195 ± 23 cells/μl and that of the babies was 220 ± 140 cells/μl. However, the mean parasite density in the HIV and P. falciparum co-infected pregnant women was 32,362 ± 3.43 parasites/μl and that of the babies was 4, 863 ± 5.23 parasites/μl (Table 2). The CD4⁺ lymphocyte count in all respondents were grouped based on categories of infection; the mean CD4 cell counts in all respondents was classified and then compared with the recommended standard (WHO, 2004). The percentage of CD4⁺ counts in HIV infected, P. falciparum infected and the P. falciparum and HIV co-infected were 46.9% (Figure 3), 53.4% and 47.1% respectively; pregnant women with less than 350 cells/µl below the WHO standard.

Table 2. Relationship between mean haemoglobin levels, CD4⁺ T cell count and parasite density in HIV and malaria co-infected mothers and co-infected babies

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In Figure 2, higher mean plasma concentration of TNF-α was observed from 18.0 x 10^-3pg/μL in un-infected to 28.2 x 10^-3 pg/μL in co-infected and 32.3 x 10^-3 pg/μL in mothers infected with HIV only, the levels of IFN-γ was low from 50.0 x 10^-3 pg/μL in un-infected to 32.0 x 10^-3 pg/μL and 28.3 x 10^-3 pg/μL in both co-infected and those infected with HIV only. However, low level of TNF-α was observed from 30.0 x 10^-3 pg/μL in un-infected to 17.0 x 10^-3 pg/μL, 20.0 x 10^-3 pg/μL and 0.8 x 10^-3 pg/μL in co-infected HIV only and those infected with P. falciparum only. The IL-2 levels reduced from (78.0 x 10^-3 pg/µL to 45 x 10^-3 pg/µL) in both co-infected and HIV infected mothers then high (82.0 x 10^-3 pg/µL) in malaria infected mothers. Also, there were strong significant associations between the plasma IL-2 levels and infections status among the study participants (Table 3).

Table 3. Cytokines levels and infection status of pregnant women

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In pregnant mothers, the plasma level of IL-10 in the co-infected women was low when compared to the un-infected and those infected with P. falciparum only. The concentration of IL-10 in co-infected mothers was slightly lower (10.0 10^-3 pg/µL) than (25.3 10^-3 pg/µL). There was no significant relationship between the plasma concentration of IL-10 and HIV status (Table 3). The concentration of IFN-γ in was highest in un-infected (48.0 10^-3 pg/µ). Also, the concentration of IFN-γ co-infected women (22.2 10^-3 pg/µL) Figure 3) was higher than in the un-infected (22.0 10^-3 pg/µL), lowest in HIV infected (0.8 10^-3 pg/µL). In general, there was significant association between the plasma levels of TNF-α, IL-2 and IFN-γ and the HIV status of the pregnant women. There was significant association between the plasma concentration of IFN-γ and HIV status (Table 3) and the infection status.

5. Discussion

Both malaria and HIV infection during pregnancy increase the susceptibility of pregnant women to the negative effects of malaria, and also makes them more vulnerable to infections regardless of parity and impaired immune state ^[23]. The mechanism governing this increased susceptibility of pregnant women to falciparum malaria is not well understood ^[25]. It is presumed to be related to probable serious modifications of both the systemic and immunological placental parameters.

In this study, there was a high prevalence of HIV and malaria co-infection, both infections have been shown to be associated with serious immune disturbance ^[26] among pregnant mothers. The finding in this study that malaria infection was higher in HIV positive pregnant women than in the non HIV positive women was supported by earlier reports ^[27]. The low mean birth weight observed in this study is in agreement with studies that predicted poor perinatal outcomes such as low birth weight as consequences of co-infection of HIV and Plasmodium falciparum in pregnant women ^[28]

The underlying cellular immune disruptions that make all parities equally susceptible to infections have not been elucidated. Even though opinions were divided on the true immune state of pregnant mothers as to guarantee successful pregnancy, previous papers ^[38] hypothesized on the mechanism responsible for cellular consequences of the co-infections during pregnancy and that the increased susceptibility of co-infected individuals is due to excessive destruction of the IL-12-mediated IFN-γ pathway due to HIV infection ^[25].

Co-infected pregnant mothers had lower level of haemoglobin which was an indication of anaemia than women with single infection of malaria or HIV (Table 2). Clearly, increased parasitemia can lead to greater destruction of red blood cells ^[29], dramatic decrease in CD4⁺ T cells which can also lead to early progression of HIV to AIDS ^[30]. The observed relationship between mean haemoglobin level and the CD4⁺ cell reported among the co-infected participants was similar to ^[30] who reported a statistically significant association between CD4⁺ and haematological parameters. Co-infected women are more likely to be immunosuppressed with lower CD4⁺ especially during the second and third trimester. HIV and malaria co-infected multigravid women showed better improvement towards the treatment than the primigravid ones, although further studies are required to investigate the findings by ^[31]. Several factors which include nutrition, age, time of sample collection had been documented as factors that could be responsible for the reduced CD4 counts among the un-infected groups ^{[32, 33]}. Both malaria and HIV cause immunodeficiency by depletion, lysis and infection of CD4⁺ T-lymphocytes ^[34]. ^[32] obtained a similar result that led to the geometric increase in parasite density in patients with CD4 count <200/µL and 50% higher at CD4 count 200-499/µL than uninfected participants.

The high in TNF-α level among those infected with HIV and the co-infected women compared to their uninfected peers (Figure 2), contradict the recent data ^[36] Ordinarily high concentrations of TNF-α as reported by ^[37] was an indication of increased HIV-1 expression in the materno-foetal milieu and facilitation of MTCT of HIV-1. Also, ^[38] indicated that increase in TNF-α cytokine was deleterious to both the mothers and her baby post-partum ^{[38, 39]}.

The reduction in the plasma levels of IL-2 in co-infected and HIV infected when compared to uninfected pregnant women contradicted the work of earlier authors ^{[40, 41]} on the high concentration of IL-2 in their subjects. Also, the observed high IL-2 levels agree with similar study conducted by ^[42], where high levels of IL-2 was observed among malaria infected cohort (Figure 2). The protective role of IL-2 as being indispensable in P. falciparum has been extensively documented by ^[36].

IL-10; an anti-inflammatory cytokine inhibits Th-1 stimulating cytokine secretion that will ensure successful pregnancy ^[39]. The cytokine is thought to play a key role in the regulation of biological parameters in pregnant mothers. Since, no case of spontaneous abortion was reported among groups, it is expected that its level will predominate in this study. Its continuous production could also be associated with falciparum placenta infection and anaemia in newborn but on the contrary, the level of IL-10 was reduced among the pregnant women.

The pattern observed in the level of IFN-γ in this study contradicts studies of ^{[42, 43]}; the concentration of IFN-γ was low upon infection. Low circulating levels of IFN-γ have been previously associated with severe malaria and mortality in children especially infants with cerebral malaria. The concentration of IFN-γ followed the same trend that was observed in the levels of IL-2 in this study. IFN-γ was expected to have played a crucial role in the clearance of intracellular parasites, and associates with high in malaria severity in young African children ^[44]. Its reduction in all infectious cases (especially in co-infected and HIV infected only) in this study could be regarded as being pathological (biomarker) of possible impaired resistance against infection which confirmed the result obtained in similar study by ^[45].

^[46] established that in newborns of HIV infected mothers, HIV and falciparum co-infection was a strong lead predictor of adverse perinatal outcomes (anaemia), and could be indicative of impaired immunity and susceptibility of newborn to infections. The general reduction in TNF-α level that was observed in this study was a confirmation of that assertion. As suggested earlier, the similar low level observed in newborns of HIV infected could be an indication of vertical transmission of impaired immunity from their HIV infected mothers. This might have led to increased IFN-γ concentration among co-infected babies and malaria only infected. The high level of IFN-γ in non-infected mothers compared to the levels of IL-10 (2) was supposed to be deleterious to successful pregnancy and a comparative indication of severe perinatal outcome and high parasite density which would be potentially harmful for the pregnancy ^{[44, 47]}.

There was no statistical association between CD4⁺ lymphocytes, and IFN-γ levels and HIV infection status of pregnant mothers, and supports the work of [39,47,] who reported that successful pregnancy is dependent of the dis-equilibrium between Th 1 and Th 2 cells. Also in the study, only IFN-γ was significantly associated with the infection status of the women, this was consistent with the work of ^[36] The appropriate immune reaction associated with protection in pregnancy is believed to be provided by Th2 cells which inhibit the development of Th1 cells, and hence prevent macrophage from mounting massive immune reactions against pregnancy.

The current study was characterized by low levels of IL-10; this contradicts ^[48] who associated high levels of IL-10 and low levels of the other cytokines IFN-γ, IL-2 and TNF-α with severe infection and poor pregnancy outcome. Whether protective or not, the levels of IFN-γ, TNF-α and IL-10 in new born of HIV infected mothers should be applied with caution because the switch (Th-1 to Th-2) might not entirely explain HIV progression. Early stage HIV infection as a Th1-predominant profile (characterized by a high production of IL-2 and IFN-γ, while the late HIV infection as a Th-2 predominant profile characterized by IL-4 and IL-10 production. There has been a strong association between levels of pro-inflammatory cytokines and spontaneous abortion ^{[49, 50]}.

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