Study design and setting

This was an analytic cross-sectional study conducted at the ANC clinic of Arua Regional Referral Hospital between October and December 2021. Arua hospital is located in Arua city and is the regional referral hospital for 12 other districts in north-western Uganda. The hospital serves about 3.5 million people in the region, including some parts of the neighboring Democratic Republic of Congo and South Sudan. The ANC clinic operates five days a week, Monday to Friday, from 8:00 am to 5:00 pm. On average, the clinic provides ANC services to approximately 120 women daily.

Mothers receive a focused antenatal care package which includes counseling, provision of ITNs, IPTp with sulfadoxine-pyrimethamine nutritional and personal hygiene education, obstetric scan, and sexually transmitted infections (STIs), malaria, anemia screening, ferrous and folic acid supplementation, and immunization. According to the Annual Health Sector Performance Report 2020/2021, only 31% of women attended the recommended four ANC visits, and IPTp coverage was below 43.2% in Arua [29].

Arua sits at an average elevation of 1310 meters above sea level, located between the drier East African plateau and the wetter Congo basin [30]. The climate is generally warm, with an average annual temperature of 23.0 ºC [31]. The warmest month of the year is February, with an average temperature of 26.0ºC, and the lowest average temperature occurs in August around 21.2ºC [31]. The rainfall pattern is bimodal, with a dry season starting from mid-December to mid-March and a less predictable rainfall season occurring around March to June of every year [31]. The more reliable rainfall season begins in late July and until November each year [31]. The warm and relatively humid conditions favor the life cycle of Anopheles gambiae, the primary malaria vector in the region [32]. Arua district experiences perennial intense malaria transmission with asymptomatic parasite prevalence rates of 40–60% in school children [33, 34]. The annual entomological inoculation rate in the region is estimated at 397 infective mosquito bites per person per year, with Anopheles gambiae responsible for >80% of infective bites [35]. The peak transmissions occur around August to September and the lowest transmission intensities are observed yearly between January and March [35].

Study population and sampling

All the pregnant women attending routine ANC clinics at Arua Regional Referral Hospital during the study period were eligible to participate in the study. We enrolled women attending any of the four ANC visits. At the same visit, blood samples were collected and analyzed. Pregnant women were excluded if they were critically ill or required hospitalization. We used a combination of systematic and simple random sampling to select the participants. Upon arrival, pregnant women were registered by the midwife, and basic biodata were collected. This information was used to construct a sampling interval. Using previous ANC pattern, we estimated that we would need ten days to enroll 238 participants at an average of twenty-four per day. We divided the number of women recorded in the ANC register by the number of women required for the interview on each day to obtain a sampling interval of five and henceforth, selected every 5^th person recorded in the ANC register. Using the registration serial numbers, we then employed a simple random sampling method using a lottery approach to select the first and subsequent participants. This process was repeated daily until the required sample size was reached.

Data collection and measurements

We enrolled participants in the second year (2021) of the Coronavirus disease (COVID-19) pandemic when all COVID-19 pandemic restrictions were lifted. Therefore, prenatal care and health service delivery was not affected. All the participants received a rapid malaria diagnostic test as a standard-of-care blood screening test. In this setting, rapid diagnostic tests (RDTs) are used as a point of care test partly because of the high clinic volumes but also for screening purposes. Therefore, no cases were tested by microscopy or other diagnostic methods. We used a paper-based structured questionnaire administered face-to-face by two trained research assistants. The questionnaire was a modified version of the malaria indicator survey tool, developed by the Monitoring and Evaluation Working Group of Roll Back Malaria. This international partnership coordinates global efforts to fight malaria [36]. The rationale for adopting this tool includes its replicability, and excellent psychometric properties, and exceptionally high reliability.

The outcome variable was malaria in pregnancy, measured by a positive malaria test result following a rapid malarial antigen test (RDT) as a standard of care at the hospital. The tests were performed by a laboratory technician with ≥ 10 years of work experience. Briefly, about 5μL of capillary or venous EDTA blood was introduced into the sample well using the sample transfer device that came with the RDT kit. This was followed by two drops of the assay buffer to the assay buffer well. The timer was set for 20 minutes before a test result could be read. The test results were interpreted anytime from 20 but before 30 minutes to minimize inaccurate test results that occur due to reaction product deterioration. A positive result was considered if two lines appeared on the cassette, otherwise it was negative if one control line appeared. In case a test line appeared without a control line or nothing at all appeared, the test was regarded invalid and a repeat test performed on a new device. Standard laboratory practices including aseptic procedures for obtaining samples and infection control measures were observed at all times by the laboratory personnel testing the samples. The performance of the test kits, SD Bioline Malaria Ag Pf (SD Bioline Inc. Korea) were as follows; sensitivity 98.2%, and specificity 91.6%, with positive and negative predictive values of 81.8% and 99.2% respectively [37].

The independent variables included age measured in absolute years later categorized as <25 versus ≥25 years, marital status namely married versus single, level of education categorized as none/primary versus secondary and beyond, and occupation measured as none/peasant versus employed/business. The age cutoff of 25 years was taken as a borderline to compare malaria in pregnancy between early/young women and mature/older women. The other variables included gestational age categorized as the first trimester (≤12 weeks) versus second/third trimester (>12 weeks), gravidity measured as ≤2 versus ≥3, time of first ANC visit measured as first trimester (≤12) versus second/third trimester (>12 weeks), and time of intermittent preventive treatment of malaria in pregnancy start measured as early second trimester (≤14 weeks) versus late trimester (>14 weeks).

We also collated data regarding knowledge of mothers about the intermittent preventive treatment of malaria in pregnancy doses (no versus yes), sleeping under insecticide-treated nets on daily basis (no versus yes), history of seeking obstetric care from a traditional birth attendant during the current pregnancy (no versus yes) and distance from home to a health facility measured as ≤5 km versus >5kms. Data on gestational age, use of insecticide-treated nets, use of intermittent preventive treatment of malaria in pregnancy, and recent ANC visits were verified using the ANC card at the time of the interview.

Quality control measures

To ensure data quality, the questionnaire was pretested on 10 participants outside the study area in August 2021 at another health facility located about two kilometers away from Arua Regional Referral Hospital. We trained the research assistants for two days regarding the study processes, responsible conduct of research, and questioning techniques. The regional laboratory participates in the ongoing national malaria external quality assessment (EQA) scheme and every quarter, a batch testing of RDTs is conducted prior to their issue for laboratory use. The regional external quality assurance team samples RDT cassettes from each batch and are tested using a pair of known positive and negative blood samples confirmed by expert microscopy and PCR. Therefore, the batch used for malaria testing in the present study passed QC testing. The lab technician performed initial testing of the patients and results were reviewed by the research assistant and entered into appropriate data collection forms. The research assistants were supervised by a research team lead who reviewed the filled questionnaires daily for completeness and accuracy. Concerning data on obstetric history, the completed questionnaire was verified against the ANC register or card.

Ethical considerations

This study received approval from the Lacor Hospital Institutional Research Ethics Committee (LHIREC#2021–61), Uganda National Council of Science and Technology (HS2210ES). It was further granted administrative approval by the Arua Hospital administration. We sought voluntary informed written consent from pregnant women aged ≥18 years. For participants below the age of 18 years, an adult either a spouse or guardian provided written informed consent in addition to assent from the participant. The research assistant explained the study’s purpose, risks, and benefits in the local language and provided ample time for participants to ask questions/clarifications. The participants were further assured of anonymity and confidentiality of the data collected. Pregnant women were free to refuse to participate or withdraw from the study without any prejudice to services offered at the clinic.

Background characteristics of pregnant women and prevalence of malaria in pregnancy

We studied 238 women with a mean age of 25.32±5.79 years who had attended the ANC clinic, and all were not malaria symptomatic cases. Of these, 207 (87.0%) resided within a 5 km radius of the health facility, 191 (80.3%) were married, 149 (62.6%) attained at least a secondary level of education, and 183 (76.9%) were unemployed. Also, 173 (72.7%) were in their second or third trimester, and 117 (49.2%) were pregnant for the first or second time. The majority of the women, 169 (71.0%), started ANC in the first trimester, 71 (30.1%) began IPTp late (> 14 weeks of pregnancy), 137 (57.6%) had adequate knowledge about the correct IPTp doses, 212 (89.1%) reported sleeping under ITNs every day, and 111 (46.6%) reported seeking obstetric care from a traditional birth attendant (TBA) during the current pregnancy. Overall, 62 (26.1%) of the participants had malaria in pregnancy by rapid diagnostic tests (RDT), with all the malaria-positive cases (62 or 100.0%) due to P. falciparum infection.

In Table 1, our data show that malaria in pregnancy was prevalent in the first trimester compared to the second or third trimester (35.4% versus 22.5%), and among women who attended less than four ANC visits compared to those who had attended ≥4 ANC visits (74.4% versus 71.0%, respectively). However, malaria in pregnancy was less common among pregnant women aged <25 years compared to those aged ≥25 years (25.8% versus 26.3%, respectively), those in the category of one or two pregnancies versus three or more (24.8% versus 27.3%, respectively), those that had ANC visit in the first trimester (≤12 weeks) versus later trimester (>12 weeks) (23.7% versus 31.9%, respectively) and those that had early IPTp initiation (≤14 weeks) versus late IPTp initiation (>14 weeks) (23.6% versus 32.4%, respectively). Being in the first trimester (p = 0.044), daily ITN use (p<0.001), and seeking obstetric care from a traditional birth attendant (p = 0.036) showed significant differences in malaria in pregnancy in the bivariate analysis.

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Table 1. Background characteristics of pregnant women and prevalence of malaria in pregnancy.

https://doi.org/10.1371/journal.pone.0283755.t001

Factors associated with malaria in pregnancy

The unadjusted and adjusted analysis results are presented in Table 2. In the unadjusted analysis, malaria in pregnancy was less likely among women who everyday slept under insecticide-treated bed nets (unadjusted prevalence ratio (PR), 0.38; 95% CI, 0.25–0.58) and those in the second or third trimester (PR 0.64; 95% CI, 0.41–0.98). But it was more likely among women who had initiated ANC late (>12 weeks) (PR1.35; 95% CI, 0.87–2.09), and among women who sought obstetric care from a traditional birth attendant during the current pregnancy (PR, 1.58; 95% CI, 1.02–2.45).

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Table 2. Modified Poisson regression analysis of factors associated with malaria in pregnancy.

https://doi.org/10.1371/journal.pone.0283755.t002

In the multivariable or adjusted analysis, the factors that remained statistically significant were: sleeping daily under ITNs (adjusted prevalence ratio (aPR), 0.41; 95% CI 0.28, 0.62), gestational age (aPR 0.45, 95% CI 0.26, 0.76), and time of first ANC visit (aPR, 1.78; 95% CI 1.05, 3.03). The relationship between the variables seeking obstetric care from a TBA and malaria in pregnancy was confounded by maternal age in the multivariable analysis. The other variables namely: knowledge about IPTp doses, time of IPTp started, number of ANC visits, and gravidity were not statistically significant in the final model (Table 2).

Study strengths and limitations

Our study has several strengths and limitations. First, we selected a random sample from a large tertiary care facility which is more likely to be heterogeneous. Hence, our results can be generalized to the population under study. Second, we controlled for interviewee response bias by verifying self-reported data against ANC records. Third, we used the malaria indicator survey toolkit, a validated tool with excellent psychometric properties, to collect all the data in the study. Our study had limitations that should be considered in the interpretation of the findings, and they include 1) A relatively small sample, which might have reduced the statistical power to detect a true association in the population; 2) The study recruitment period was relatively short (October to December or three months), and it coincided with moderate malaria transmission intensity [35]. The malaria transmission intensity in our setting peaks around August and September every year [35], so the study might have underestimated the burden of malaria in pregnancy; 3) The ANC coverage in our setting was approximately 30.0%, which is lower than the recommended ≥4 ANC visits [29], implying that a high percentage of potentially malaria-infected pregnant women had missed the opportunity to participate in the study. This might have led to an inaccurate estimate of the prevalence of malaria in pregnancy in the study; 4) The study did not include symptomatic malaria cases, and more importantly so there is the possibility that the pregnant women who sought ANC are likely those most protected from malaria and other diseases; 5) The study lacked explanatory variables, such as the reasons for not sleeping under an insecticide-treated bednets daily among the participants as these were not within the scope of the study; and, 6) malaria in pregnancy was diagnosed with a low-sensitivity method so some cases might have been erroneously classified as uninfected if the parasite densities were below the detection limits. Rapid diagnostic assays for malaria based on the histidine-rich protein (HRP-2) have sensitivity and specificity that vary from 95% to 98.2% and 59.2 to 86.3%, respectively, in investigations including general populations [37, 64–66]. However, due mostly to placental parasite sequestration, the tests are typically less sensitive when used for malaria diagnosis in pregnant women [67, 68]. We admit that despite the fact that it was outside the scope of our study to evaluate the performance of histidine rich protein (HRP2)-based rapid diagnostic tests (RDTs), this may have contributed to the study’s low rate of malaria in pregnant women. Not to mention, we recognize the limitations brought on by the choice of our cut-off points for variables like age and gravidity. They further restrict the comparability of our findings with previous studies investigating teenage pregnancy and primigravida as potential risk factors for malaria in pregnancy. Nevertheless, our findings are insightful and should be carefully interpreted to direct malaria interventions among pregnant women.

Implications of the study findings

Our findings have ramifications for clinical care, public health prevention, and control, including surveillance and future research. Our study has shown that malaria in pregnancy is prevalent, necessitating routine surveillance for malaria at the individual and population levels. On the clinical front, our findings emphasize a need to routinely test pregnant women for malaria to enable early malaria diagnosis and treatment, thereby preventing adverse pregnancy and birth outcomes. Our study also provides a benchmark for future research about malaria in pregnancy in the region. Few studies have focused on malaria in pregnancy in the West Nile region, so our findings might inform the design of future observational and interventional studies. Policy-wise, the enforcement of malaria prevention and control measures at the family and community levels is important to reduce the burden of malaria in the region. The use of cost-effective and proven malaria prevention measures such as insecticide-treated bed nets (ITNs) and intermitted preventive therapy in pregnancy (IPTp) should be promoted. Environmental control measures such as the draining of stagnant waters on the compound, cleaning/slushing busy areas, and discarding broken/empty containers that provide breeding grounds for mosquitoes have to be enforced.