Background

Malaria epidemiology and COVID-19

Over the last twenty years, tremendous progress has been made on malaria control, averting an estimate of 1.5 billion cases and saving 7.6 million lives. However, despite the astonishing results achieved in the last two decades, COVID-19 pandemic added a crucial challenge to the fight against the disease. On one side, malaria control relies heavily on individual choice to seek care, and early messaging targeted on reducing SARS-CoV2 transmission advised people to stay home in case of fever. On the other side, supply chains that allow the delivery of malaria commodities, such as insecticide-treated nets or antimalarial drugs, have been disrupted, and healthcare workforce constrained. All these factors contributed to a reverse of the reducing trend in malaria cases and deaths that shifted, respectively, from 227 million and 534,000 in 2019 to 241 million and 602,000 in 2020 [1].

According to the latest WHO World Malaria Report [1], Uganda accounts for 5% of all malaria cases reported globally. Oyam and Kole districts, selected for the study, are among the most affected areas in the country with, respectively, 407 and 361 new cases per 1000 inhabitants in 2019 [2]. In this context, particularly affected are children under five years of age (U5) and pregnant women. In the area where the project will operate, the rate of pregnant women that receives 3 or more doses of intermittent preventive treatment (IPTp) is less than 48% [2].

Concerning the impact of COVID-19 on malaria care in Uganda, a study conducted by Namuganga et al. [3]—except for a modest decrease in the proportion of malaria cases treated with ACT—documented no major effects on malaria disease burden. However, the study did not evaluate the impact of COVID-19 on antenatal care and malaria-related maternal outcomes. At this regard, in the pre-pandemic period (years 2018–2019) the mean antenatal care (ANC) attendance in the district of Oyam was of 1721 first visits per month, while an average of 472 women per month received at least 3 doses of IPTp.

Pregnancy associated malaria

Intermittent preventive treatment

In hyperendemic areas, pregnancy is a risk factor for both acquisition [4] of P. falciparum infection and for development of severe malaria [5]. Younger women, primi- or secundigravidae and HIV + women are particularly at risk [6]. Adverse outcomes for mothers and their infants include maternal anemia, low birthweight, prematurity, placental malaria, infant malaria, infant anemia and congenital malaria. Furthermore, adverse events are exacerbated by poor maternal nutritional status [7] and HIV coinfection [8].

Apart from consistent use of ITN, malaria control in this population is based on two pillars: intermittent preventive treatment of asymptomatic women and appropriate management in case of illness [9]. IPTp with sulfadoxine-pyrimethamine (SP) is still highly cost-effective in preventing the adverse consequences of malaria on maternal and foetal outcomes, even in areas with a high prevalence of quintuple mutant parasites [10], but coverage remains unacceptably low in several African countries [11]. For HIV-negative pregnant women, IPTp consists in the administration of at least three doses of SP (1500 mg sulfadoxine/75 mg pyrimethamine), in three antenatal care visits, starting early in the second trimester and at least four weeks apart [9].

In HIV negative women, a promising alternative to SP for IPTp is dihydroartemisinin-piperaquine (DHA-PPQ, 3 full strength tabs, 40 mg/320 mg, given once a day for 3 consecutive days), that showed to be more efficacious in reducing maternal malaria parasitemia and anemia at delivery, stillbirths and early infant mortality. In fact, SP efficacy may be decreased in areas with very high drug resistance and consistent presence of sextuple mutant haplotypes of P. falciparum [12]. However, there is no consensus as to the level of resistance at which SP-IPTp should be discontinued and an alternative regimen substituted.

For HIV positive patients, the current WHO guidelines recommend daily co-trimossazole prophylaxis.

Diagnosis and treatment

In most endemic countries, diagnosis heavily relies on the use of rapid diagnostic tests (RDT) which, however, are insufficiently sensitive in detecting the so-called subpatent infections—asymptomatic infections with low parasite densities—and infections due to parasites carrying the pfhrp2 and pfhrp3 gene deletions [13]. These limitations are partly overcome by microscopy and PCR-based tests, that can detect also low parasitemia [14], but the clinical impact of such infections is still matter of debate [15].

Treatment of malaria differs according to gestational age. For women in their first trimester with uncomplicated P. falciparum malaria, WHO recommends 7 days of quinine + clindamycin. From the second trimester on, experience with artemisinin derivatives is increasingly reassuring: no adverse effects on the mother or foetus have been reported. Thus, treatment of uncomplicated P. falciparum malaria consists of three-day course of oral artemisinin-based combination therapy (ACT). On the other side, treatment of severe malaria does not differ from the one prescribed to non-pregnant women. Parenteral artesunate is the treatment of choice in all trimesters.

Resistance to sulfadoxine-pyrimethamine and artemisinin derivatives

Another substantial challenge for malaria case-management is resistance to first line drugs, namely artesunate and ACT. A recent paper published by Balikagala et al. documented, for the first time in African history, the presence of artemisinin resistance in a longitudinal study conducted in Gulu, Uganda [16]. According to this study, single-nucleotide polymorphisms haplotypes associated with artemisinin resistance (i.e., mutations involving the gene locus kelch13) clearly showed the substantial difference of haplotypes between A675V isolates in Uganda and in Southeast Asia, which suggested that the mutation probably emerged independently in Africa and Southeast Asia. In Africa, potential factors that may contribute to a delayed emergence and spread of artemisinin resistance are the extent of acquired immunity, the rate of polyclonal infections and of chronic asymptomatic infections [17]. However, the constant selective pressure exerted by the widespread use of ACT pose a substantial threat for the emergence of clinically relevant forms of resistance.

Furthermore, a study conducted by Mbonye et al. [18] in 2015 documented a baseline prevalence of Pfdhfr and Pfdhps mutations—conferring resistance to SP in P. falciparum—to be 89% for the quintuple mutated haplotype and 3.9% for the sextuple mutated haplotype, reaching 16.7% after one dose of SP. Today’s prevalence of the sextuple mutated haplotypes, potentially impairing the effectiveness of SP-IPTp is not known. Conceptual framework of the study is provided in Fig. 1.

Fig. 1
figure 1

Conceptual framework showing the possible impact of COVID-19 and antimalarial resistance on malaria care among pregnant women. Dashed line (green): antimalarial resistance; Dotted line (blue): COVID-19; Continuous line (yellow): both COVID-19 and antimalarial resistance

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Methods

Study design

This will be a facility-based, prospective observational study, using quantitative methods of data collection. Semi-structured questionnaires will be administered to collect the data. The study will be conducted on three separate populations, that is: “Cohort of pregnant women”, “Antimalarial resistance sub-population”, and “COVID-19 impact population” for which study methods are described separately.

Cohort of pregnant women

The data will be collected following a cohort of pregnant women presenting to antenatal care visits. We shall have both a retrospective cohort for the period January 2018 to December 2021 to determine the Impact of COVID-19 pandemic on malaria control and a prospective cohort for the period July 2022 to June 2024 to determine the incidence of malaria related adverse maternal and foetal outcomes. For the prospective cohort, recruitment will take place at ANC clinic. Collected data will explore the practices towards malaria prevention during the COVID-19 pandemic, malaria and COVID risk-perception and use of insecticide-treated nets, while and follow up will investigate access to antenatal care visits, administration of IPTp, healthcare seeking behaviour in case of fever. Follow-up will end at delivery, when maternal and foetal outcomes will be collected.

Antimalarial resistance population

To estimate the epidemiological burden of resistance to first-line drugs for treatment and prevention of malaria in pregnant women, a separate sub-population of women diagnosed with positive malaria parasitaemia will be recruited. Prevalence of antimalaria resistance will be evaluated with a cross-sectional analysis of genetic polymorphisms in plasmodium parasites isolated from blood samples collected for the period July 2022 to June 2024. Individuals eligible for this subpopulation will be all pregnant women presenting with microscopically confirmed P. falciparum malaria in the study sites. For individuals included in this population, clinical data will be collected, and blood samples to be sent to Italian National Institute of Health, Rome, for detection of genetic markers of resistance to artemisinin derivatives and sulfadoxine-pyrimethamine.

COVID-19 impact population

To investigate the impact of COVID-19 pandemic on malaria care, we will conduct a retrospective time-interrupted series. Data will be collected at multiple and equally spaced time points (monthly) comparing trends in two different time periods: “pre-pandemic”, from January 2018 to December 2019, and “during pandemic”, from January 2020 to December 2021. Facility-based aggregate data will be extracted about the following indicators: total admissions in maternity ward, total deliveries in maternity unit, women presenting to first ANC contact, women presenting to fourth ANC contact, administration of at least one dose of IPTp, administration of at least three doses of IPTp, number of pregnant women presenting to outpatient visits, number of pregnant women diagnosed of malaria during outpatient clinic, number of pregnant women diagnosed of severe malaria, total number of stillbirths.

Eligibility criteria

Cohort of pregnant women


Inclusion criteria:


All pregnant women at any gestational age presenting to the study sites, both at the emergency department, outpatient or ANC clinic will be eligible to participate in this study. Inclusion criteria will be:

  1. (a)

    Pregnancy confirmed by obstetrical visit, positive urine pregnancy test or intrauterine pregnancy by ultrasound

  2. (b)

    Provision of informed consent

  3. (c)

    Plan to deliver in the hospital


Exclusion criteria:

  1. a.

    Ongoing labor or delivery

Antimalarial resistance population

All pregnant women presenting to Aber Hospital and selected healthcare facilities with microbiologically confirmed malaria will be eligible for recruitment.


Inclusion criteria:

  1. a.

    Microscopically-confirmed diagnosis of malaria

  2. b.

    Pregnancy confirmed by obstetrical visit, positive urine pregnancy test or intrauterine pregnancy by ultrasound

  3. c.

    Provision of informed consent

  4. d.

    Agreement to avoid antimalarial medications given outside the Uganda Clinical Guidelines [19]


Exclusion criteria:

  1. a.

    Too ill to participate in the study according to clinician judgment

  2. b.

    Intake of any antimalarial medication before recruitment

Sample size estimation

Sample size estimation

  • COVID-19 impact population

For the retrospective cohort investigating the impact of COVID-19, we shall use a facility-based census to include all pregnant mothers who sought care at the study sites for the period January 2018 to December 2021.

  • Prospective çohort of pregnant women

For the prospective cohort, the sample size was estimated using the sample size estimation function in STATA12 for two-sample comparison of proportions. Null hypothesis: p1 = p2, (no difference in preterm birth rates), where p1 is the preterm birth rate among pregnant mothers diagnosed with malaria (exposed group) and p2 is the preterm birth rate among pregnant mothers with no diagnosis of malaria (non-exposed group). Assuming a type I error, alpha, of 0.050 for two-sided hypothesis, power of the study at 0.80, p1 = 0.075 and p2 = 0.039 based on the preterm birth rates reported in the Uganda Birth Cohort Study conducted from 2014–2016 in 12 districts in rural northern and southwestern Uganda [20]. Assuming equal number of participants in both groups, the required sample sizes in each of the two groups is 705. And after factoring in 10% for non-response in both groups, the total minimum required sample size is 1552 pregnant women (776 in each of the 2 groups).

  • Antimalarial resistance population

Given the total population of 779,600 in Oyam and Kole districts. Given that, based on Uganda bureau of statistics (UBOS), the total number of pregnant women in the two districts is expected to be 5% of the total population (n = 38,980); the expected rate of parasitemia among pregnant mother is expected to be 27% (n = 10,525) [21]; the expected resistance to sulfadoxine-pirimethamine and artemisinin-derivatives are, respectively, 16% and 20% [16, 18]; using the sample size calculation formula developed by Daniel and colleagues [22] and a margin of error, alpha = 5% the minimum required number of pregnant women diagnosed with malaria is 203. Applying 10% correction factor and assuming an increasing trend the required minimum sample size for this population is 224 cases of microscopically confirmed malaria among pregnant women.

Sampling procedure and selection of participants

Sampling of health Units: The study will use purposive sampling to include Aber Hospital, Aboke HCIV and Atipe HCIII as the site for enrollment. This is based on the following criteria:

  • Already existent good laboratory capacity for diagnosis of malaria.

  • Presence of an experienced microscopist.

  • Antenatal care visit volumes of at least 50 visits per month.

  • Good quality of routine service offered to patients with malaria as per periodic quality of care assessment.

Participants coming to the sampled in healthcare facilities that will meet the eligibility criteria will be included in the study. Consecutive enrollment of participants will be undertaken up to when the minimum sample size required for the study will be met.

Study procedures

Microscopy

Thick and thin blood smears will be stained with 2% Giemsa and read by experienced laboratory technologists. Parasite densities will be calculated by counting the number of asexual parasites per 200 leukocytes (or per 500 leukocytes, if the count is < 10 asexual parasites/200 leukocytes), assuming a leukocyte count of 8000/µl. A blood smear will be considered negative when the examination of 100 high power fields does not reveal asexual parasites. Gametocytemia will also be determined from thick smears. Thin smears will be used for parasite species identification. At the time of delivery, recruited women will be screened for parasitemia on placental blood. This will be done by microscopy, with the same methods described above.

Molecular diagnosis and Plasmodium species confirmation

The blood samples of the patients will be collected using filter paper (Whatman 3 MM) during admission to the healthcare facility. The dried blood spots (DBSs) will be collected through a finger prick (three drops of blood per participant) on filter papers which will be dried and kept in plastic bags with desiccant and stored in boxes in a cool dry place at room temperature before being transferred at the ISS for molecular diagnosis and drug resistance analysis.

Shipment of the blood samples

The collected blood samples will be shipped to Italy for advanced polymorphism analysis. During shipment, all the samples will be stored in a dry, cool place at room temperature to the Italian National Institute of Health (Istituto Superiore di Sanità, ISS). To enhance local capacity building, one laboratory person from Aber hospital will attend a two weeks exposure at the reference laboratory in Italy.

Advanced analysis of polymorphism

Total genomic DNA will be extracted from filter blots (3MM Whatman) using the PureLink Genomic DNA Kits-Invitrogen, according to the manufacturer’s recommendation. Parasite identification is based on nested PCR assay targeting the 18S rRNA gene [23]. The 18S rRNA gene is used as a target since it contains both highly conserved and variable regions for each Plasmodium species. The genus-specific PCR will be followed by Plasmodium species-specific PCR amplification. Amplicons from the second PCR will be separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide for visualization using ultraviolet trans-illumination. The presence of parasitaemia will be confirmed when the expected band size corresponding to P. falciparum, P. vivax, P. malariae and or P. ovale will be identified.

Assessment of Plasmodium falciparum drug resistance.

Target P. falciparum drug resistance genes: Pfk13 propeller, Pfdhfr and Pfdhps.

The polymorphisms analysis of the propeller domain of the Pfk13 gene will be performed by PCR amplifications and subsequent sequencing. Analysis of Pfdhfr gene at codons 51, 59, 108 and Pfdhps gene at codon positions 436, 437, 540, 581, 613 will be done by means of amplifications and subsequent Sanger sequencing. Commercial oligonucleotide primer pairs for Pfk13 will be obtained based on the published article by Taylor et al. [24], whereas for the analysis of dhfr and dhps genes primer pairs will be obtained based on the published article by Menegon et al. [25]. The obtained sequences will be compiled and analyzed by Accelrys DS Gene software. PlasmoDB gene identification no. PF3D7_1343700 (P. falciparum 3D7 strain) will be used as reference in the numbering of nucleotide and amino acid positions. Molecular studies will be performed only for research purposes and will have no impact on the clinical management of study patients.

HIV and syphilis screening

HIV and Syphilis will be measured according to the Uganda National Guidelines [19]

Blood glucose

Blood glucose will be measured by Glucometer “Accu-Chek Active”, Narang Medical LTD.

Hemoglobin levels

Blood haemoglobin levels will be measured by Hemoglobin Testing System “Mission Ultra Hb”, Narang Medical LTD.

Data analysis

For descriptive purposes continuous and ordinal variables data will be expressed as median with interquartile range. For categorical variables, percentages are calculated. Student’s t-test or analysis of variance (ANOVA) will be used to compare normally distributed numerical variables. Mann Whitney U-tests and Kruskal–Wallis tests will be used to compare numerical variables when normality cannot be assumed, while chi-squared tests will be used to compare categorical variables.

Association analysis will be carried out to identify risk factors for Plasmodium infection and adverse maternal or foetal outcomes. We will compare behavioural factors and adherence to IPTp (and type of IPTp regimen) to the incidence of symptomatic/severe malaria and adverse neonatal or foetal outcomes (miscarriage, stillbirth, low birthweight). Multivariable logistic regression models will be used to identify independent risk factors for the same clinical outcomes. A forward and backwards stepwise approach will used to include variables into the models, with a limit of P < 0.2.

A P-value of < 0.05 will be considered statistically significant. Final analyses will be conducted after the end of patient recruitment while interim analyses are planned at half 7 months from the incipit. Statistical analysis will be performed with R-software (R Foundation for Statistical Computing, Vienna, Austria).

Discussion

According to the 2021 Essential Maternal and Newborn Clinical Care Guidelines for Uganda, for a woman with a normally progressing pregnancy the standard recommendation is a minimum of eight antenatal visits [26].

  • Contact 1: Anytime ≤ 12 weeks.

  • Contact 2: 13–20 weeks of gestation.

  • Contact 3: 21–28 weeks of gestation.

  • Contact 4: 30 weeks of gestation.

  • Contact 5: 34 weeks of gestation.

  • Contact 6: 36 weeks of gestation.

  • Contact 7: 38 weeks of gestation.

  • Contact 8: 40 weeks of gestation.

In addition, participants will be instructed to come to the clinic every time they are ill and will be evaluated at this point too. Outcomes will be assessed at the delivery or/and at the discharge if admitted to the hospital for any other causes related with the pregnancy or malaria (Table 1).

Table 1 Study time points
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Data will be collected using semi-structured questioners and managed using REDCap electronic data capture tools hosted at “Catholic University of the Sacred Heart”, Rome, Italy [28], and will be recorded on standard study data collection forms and will be reviewed for accuracy and completion. Upon resolution of data forms errors/missing values, the form will be ready for data entry. The obtained results will be entered into a database. A database will be developed to accommodate data entry and management of the study's data. The database will be created with a standard data management software package, such as Microsoft Office. A file for each study form will be created.

Ethical considerations

Pregnant women will be asked for written informed consent to participation to the study. In line with the Ugandan National Guidelines for Research involving Humans as Research Participants [29], women below the age of 18 will be considered emancipated minors. Every clinical outcome will be managed as per National Clinical Guidelines for the care of pregnant mothers. Counselling and related clinical support will be offered to participants who get pregnancy losses. This study will not introduce any clinical management strategies outside the national clinical guidelines.

We shall administer consent at two levels; first we shall administer and obtain consent for participation in the study and publication of future results and thereafter consent for collection and transportation of blood samples to Italy. The blood samples collected will be used only for the purpose of this study and will not be stored beyond the current study project. All unprocessed blood samples will be destroyed from the department of infectious disease, Italian National Institute of Health as biological sanitary waste, at the end of the project. The same standard of care will be guaranteed to all individuals irrespectively of participation. The dignity of study participants will be guaranteed by the investigators, as well as data confidentiality and the right to withdraw data and/or biological samples at any time. Consent to pregnant women and to mature and emancipated minors will be obtained according to Uganda Human Subjects Protection Guidelines [29].

For all malaria positive samples processed for molecular analysis in the present study, identifying information will be removed to provide appropriate protection of medical confidentiality and privacy. In this way, sensitive data cannot be linked or re-linked with identifiable human subjects, making anonymous each sample processed.

The polymorphisms analysis of the propeller domain of the Pfk13, Pfdhfr and Pfdhps genes will be performed by advanced amplifications techniques and subsequent Sanger sequencing in a highly specialized scientific laboratory. The blood samples collected in this study will be sent to a laboratory in the Italian National Institute of Health, Italy, in order to have access to the latest gene amplification techniques. To enhance local capacity building, one laboratory staff from Aber Hospital will be supported to attend a two weeks exposure at the reference laboratory in Italy.

The present study has been approved by the Lacor Hospital Institutional Research and Ethics Committee (prot. no LACOR-2022-95).

Dissemination of results

Results from this research will be disseminated at various fora, including local and international scientific conferences, the Ministry of Health, technical working groups and meetings at district offices in Lango region involving participation of local leaders (Cultural, technical, and Political leaders). To better reach enrolled communities, the findings of the study will also be disseminated in places of worship and community health service points using the community outreaches being organized by CUAMM and the district local governments in Oyam and Kole districts. Findings will also be published in a peer reviewed journal.