Study design {8}
This is a phase IIIb open-label randomized controlled superiority trial of standard IPTp-SP vs. IPTp-SP plus screen-and-treat using DP (IPTp-SP+). Participants randomized to the IPTp-SP arm will receive care according to current national guidelines. Participants who are randomized to the IPTp-SP+ arm will receive standard-of-care with the addition of screen-and-treat using RDT and DP at the first ANC visit.
Study site {9}
The trial will take place in Nchelenge District, Luapula Province, Zambia. The district is located in the northern wetlands of Zambia alongside Lake Mweru, an area of hyperendemic malaria (17). Malaria transmission occurs throughout the year. The predominant vector is Anopheles funestus which peaks during the dry season (May-September) while both An. funestus and An. gambiae are found during the rainy season (18). The population of the district is estimated to be over 296,000 inhabitants consisting mostly of subsistence farmers and fishermen and women. Nchelenge District has one hospital, Saint Paul’s General Hospital (SPGH), twelve rural health centers, and two health posts. The trial will recruit and follow obstetric patients and their offspring at two of the rural health centers and SPGH. SPGH is equipped with an operation theater and labor-and-delivery ward for the provision of essential obstetric services.
Study population {9}
Pregnant volunteer will be recruited from routine ANC visits. Inclusion and exclusion criteria are listed in Table 2. All pregnant women presenting to the ANC for the first time of their current pregnancy will be assessed for eligibility according to the following criteria: age ≥15 years, estimated gestational age 16-26 weeks, hemoglobin concentration ≥7 g/dL, ability to provide informed consent, residence in the study area with intention to deliver at the health center, no evidence of clinical malaria or other acute illness, and ability to tolerate oral medication. Women will be deemed ineligible for any of the following: infection with human immunodeficiency virus (HIV) or unknown HIV status, prior IPTp-SP or other antimalarial use or antimicrobials with antimalarial activity during the current pregnancy, intolerance to either of the study drugs, existing or prior obstetric complications, prior enrollment in the study or concurrent enrollment in another study, concurrent medications with potential for drug-drug interactions or potentiation of cardiac arrhythmia, presence of chronic illness or other factor deemed likely to influence the pregnancy outcome, or other reason which is judged by the investigator to render the individual unsuitable for study participation.
Randomization and masking {16a, b, c and 17a, b}
The following procedures will be used to ensure an unbiased assessment of treatment safety and efficacy. A computer-generated randomization schedule will be generated prior to the start of the study and kept in a locked cabinet accessible only to the study coordinator. The schedule will comprise blocks of varying size to allocate, in a 1:1 ratio, eligible participants to either IPTp-SP or IPTp-SP+. The trial is open-label. However, study investigators, study clinicians, and statisticians are masked to treatment assignment. Investigators will be unmasked after all mothers have delivered. A Data and Safety Monitoring Board (DSMB) will be established to review safety data. Statisticians will remain masked until after the DSMB has approved the final analysis plan and the database is locked. All outcome assessors, including laboratory parasitologists and pathologists, will also be masked until the final database is locked.
Intervention {11a, b, c}
The intervention will consist of IPTp-SP with or without screen-and-treat using DP at the first ANC visit. Participants randomized to the IPTp-SP+ group who test positive by rapid diagnostic test (RDT) will be given a full treatment course of DP (D-ARTEPP® 40 mg/320 mg three tablets daily for three days; Guilin Pharmaceutical) under direct observation by a study nurse (Day 0) or community health worker (Days 1-2). All other participants will be treated with SP at the first visit, including those in the IPTp-SP+ arm with a negative RDT result, and all participants in both groups will be given SP at all subsequent visits (Day 35, Day 63, then monthly until delivery). IPTp-SP will be administered according to national guidelines (G-SCOPE® 1,500 mg sulfadoxine/75 mg pyrimethamine for one dose no fewer than 4 weeks between doses; Guilin Pharmaceutical). Participants will be observed for 1-hour post-dose to monitor for vomiting or other adverse reactions. If vomiting occurs within 30 minutes, the full dose will be readministered. If vomiting occurs after 30 minutes, half of the total dose will be readministered. In the case of persistent vomiting, the participant will be withdrawn from the study and referred for alternative treatment.
Clinical procedures {11d}, {13}, {15}, {26a}
Study procedures are outlined in Table 3. The informed consent will be administered by a study nurse in the local language (Bemba) and/or in English as preferred by the participant. Assent will be obtained from participants <18 years old, and informed consent will be obtained from those ≥18 or the legally authorized representative for minors. If the participant cannot provide a written signature then a thumbprint will be obtained. After collection of demographic data, provision of informed consent, baseline medical and obstetric history and physical examination by a study physician including eligibility assessment, participants will be randomized to either the IPTp-SP or IPTp-SP+ treatment groups. All participants will undergo hemoglobin measurement as part of the screening process. On enrollment, fingerstick blood will be drawn for preparation of dried blood spots (DBS) for subsequent molecular testing. In addition, participants randomized to the experimental group (IPTp-SP+) will undergo RDT.
Treatment with either SP or DP will be administered as described above. Participants will return for follow-up on days 14, 28, 35, 42, 63 and then monthly thereafter until delivery. They will be instructed to report to the clinic for any illness between scheduled visits.
At each scheduled or unscheduled visit, the study clinician will perform an interval history and physical examination, including an updated medication history. During the active follow-up period (up to day 63) DBS will be collected and hemoglobin will be measured. SP will be given on days 35 and 63. Additional doses of SP will be given during monthly visits until delivery with a minimum of 4 weeks between doses and laboratory samples (e.g., blood slide for microscopy, urinary tests, full blood count) will be collected if clinically indicated according to signs and symptoms. Antimalarials or antibiotics with antimalarial activity (e.g. systemic erythromycin, macrolide antibiotics, trimethoprim-sulfamethoxazole or other sulphonamides, tetracyclines, quinolones, clindamycin) will be prohibited during the active follow-up period.
Participants who present during the follow-up period with any signs or symptoms of malaria will undergo testing by thick smear, and DBS will be collected from those with positive microscopy. The signs and symptoms that we will consider indicative of possible clinical malaria include fever, chills, weakness, fatigue, myalgia, arthralgia, headache, anorexia, nausea, vomiting, abdominal pain, or diarrhea. All confirmed uncomplicated malaria cases will be treated with artemether-lumefantrine according to national guidelines (Coartem™ 20 mg/120 mg four tablets taken at 0, 8, 24, 36, 48, and 60 hours). Participants who develop severe malaria defined according to WHO criteria will be referred to SPGH for inpatient management (19).
On the day of delivery, the participant and infant will be evaluated as soon as possible after delivery. The birthweight will be measured, and an assessment for congenital abnormalities will be done. Obstetric complications including but not limited to hemorrhage, premature rupture of membranes, Cesarean section, and others will be captured as adverse events. Congenital abnormalities, if present, will also be recorded. Thick and thin peripheral blood films, DBS, and hemoglobin measurement will be done on both the participant and infant. In addition, cord blood, placental blood films for thick and thin smear microscopy, placental DBS, and placental biopsy for histopathological analysis will be collected.
Infants will be reassessed at 1, 6, 9 and 12 months postpartum for focused history and physical examination, including interval medication and hospitalization history, growth, and assessment of developmental milestones. At the first monthly visit, hemoglobin measurement and malaria parasitology by thick smear and PCR testing using DBS prepared from heel stick blood will be performed.
Laboratory procedures {13}
Malaria microscopy
Thick blood smears will be stained with 3% Giemsa for 30 minutes and examined by trained microscopists. Parasite densities will be calculated by counting the number of asexual parasites per 200 leukocytes and the parasite density will be estimated assuming 8,000 leukocytes per microliter. A slide will be determined negative after counting 2,000 leukocytes. A thin blood smear will be made for species identification and quantification of high parasitemia (>16,000 parasites/µL). Non-falciparum species, gametocytes and other malaria pigments will be reported but not quantified. Each slide will be read separately by two independent experienced microscopists who will remain masked to treatment assignment.
Point-of-care testing
Malaria point-of-care diagnostic testing will be done via P. falciparum HRP2 antigen-based RDT (SD BIOLINE, Abbott, Illinois, USA). Hemoglobin concentrations will be measured using a point-of-care Hb 201 Hemocue® Analyser (Angelholm, Sweden).
Placental histopathology
At the time of delivery, a 1 cm3 biopsy specimen will be obtained from the maternal-facing side of the placenta. Biopsy specimens will be preserved in 10% neutral buffered formalin and embedded in paraffin wax. Pending histological evaluation, tissue will be kept at 4°C. Paraffin sections 4 mm thick will be stained with hematoxylin and eosin stain. Placental biopsies will be classified according to the following definitions: acute infection (parasites present, malaria pigment absent), chronic infection (both parasites and malaria pigment present), past infection (parasites absent, malaria pigment present), or no infection (both parasites and malaria pigment absent) (20).
Molecular assays
DBS collected onto filter paper cards (Whatman® 903 Protein Saver, Sigma-Aldrich) will be allowed to rack-dry overnight, and stored individually in opaque sealable plastic bags with desiccant before subsequent use in PCR assays. PCR detection of parasites will be done using cytochrome b. Recurrent episodes will be genotyped to distinguish recrudescence infections from new infections according to standard WHO protocols will be conducted with nested PCR of merozoite surface protein-1 and -2 (msp1, msp2) and glutamate-rich protein (glurp) genes for length polymorphisms (21). Infections will be classified as recrudescent infection if, for each marker, there is at least one identical allele between the initial and recurrent infection. Infections will be classified as reinfections if, for at least one marker, there is a different length polymorphism between the initial and recurrent infection. Infections will be classified as indeterminate in the case of low coverage or missingness due to amplification failure that precludes comparisons at all three genes. Drug resistance markers for artemisinins, SP, piperaquine, and related markers for chloroquine and amodiaquine cross-resistance will be evaluated by molecular inversion probe and amplicon deep sequencing based approaches (22, 23).
Plasma piperaquine concentrations
Participants who are randomized to the IPTp-SP+ who screen positive and are administered DP treatment will have an additional 2 mL blood collection for measurement of piperaquine plasma concentrations on days 0, 14, 28, 35, and 42. Piperaquine quantitation will be done using liquid chromatography tandem mass spectrometry as previously described (24). The terminal elimination half-life will be estimated using noncompartmental analysis, and the effect of age, body surface area, body mass index, and gestational age on drug concentrations will be explored in mixed effects models using WinNonlin software (Certara, Princeton, NJ).
Safety assessments {22}
Drug safety will be monitored at every visit during the course of the study in compliance with International Conference on Harmonization Good Clinical Practice guidelines for adverse events (AEs) and serious adverse events (SAEs) (25). All SAEs will be reported to the sponsor and the ethics committee within 24 working hours of the study staff first becoming aware. Investigators will determine the relationship between safety signals and the study drugs, and define outcomes, according to standard classifications (25).
Sample size calculation {14}
The sample size was determined based on available data and with the objective of achieving a detectable difference of a 50% reduction in the incidence of MIP. In 2019, Nchelenge District recorded 7,791 live births (unpublished data). Prior studies in the same population found PCR prevalence of P. falciparum parasitemia to be 22-26% in pregnant women (14, 15). A sample size of 324 pregnant women (n=162 per arm) will afford 80% power to detect a 50% difference of effect between study arms with a two-tailed alpha of 0.05. To allow for loss to follow-up of up to 20%, a total of 392 (n=196 per arm) will be recruited.
Data management {18a} and {19}
Source data will be recorded using paper case report forms (CRFs) which will then be double-entered into an electronic database. The CRFs and database will be routinely checked for accuracy by the investigators during the data collection period. The final database will be locked after resolution of all queries. All paper CRFs will be filed and kept in lockable cabinets in offices accessible only to study personnel. At the conclusion of the study, the files will be transferred to the Tropical Diseases Research Centre where they will be stored for a period of 5 years after study completion.
Participant retention and withdrawal {18b}
To facilitate retention in the study, participants will be issued study visit cards that include the dates of their follow-up visits. We will collect participants’ telephone numbers and physical addresses to allow the study team to contact participants in case of missed visits, advising them to report to the clinic for their scheduled visit within the window period of 3 days. Participants will be withdrawn from the study only if they withdraw informed consent.
Statistical Analysis {20a, 20b}
Longitudinal data will be displayed using the nonparametric Kaplan-Meier estimations of the survival function. The primary analysis will be comparison of the 42-day incidence of MIP using multivariate models to estimate the relative hazard between the IPTp-SP and IPTp-SP+ groups. Testing for proportionality of hazards will be done using formal statistical testing with Schoenfeld residuals as well as visual inspection of the nonparametric survival estimates. For non-proportional hazards an extended Cox model will be applied. The relative hazard will be estimated in parametric models accounting for recurrent events by using a robust variance estimator. Treatment assignment of individuals will be according to intention-to-treat (ITT) for the primary analysis. For the primary safety analysis, we will compare the proportions of serious and non-serious AEs using logistic regression models and relative hazards of AEs using Cox regression methods as above. Secondary analyses of continuous outcomes (hemoglobin concentration, birthweight) will be examined in linear regression models and in logistic regression models for binary outcomes (prevalence of low birth weight, neonatal mortality, placental malaria, congenital malaria, maternal anemia, congenital anemia, drug resistance allele frequency). Prespecified subgroup analyses will be done according to the following strata: primigravidae, multigravidae, gestational age at enrollment, delivery before or after the day 63 visit, treatment dose on a mg/kg basis, positive P. falciparum PCR on enrollment, and presence or absence of quintuple and sextuple mutations. We will compare the proportions of treatment failures and prevention failures according to the antimalarial drug given at the first encounter (SP or DP) in a modified ITT analysis using logistic regression. Treatment failures will be defined as recurrent infections that are determined by genotyping to be recrudescent parasites from the initial infection in participants who tested positive by PCR at the initial visit. Prevention failures will be defined as PCR-confirmed parasitemia in participants who initially tested negative by PCR at the first visit, or who tested positive by PCR at the first visit but who experienced reinfection with a new parasite as determined by genotyping.
Ethical approval {24}
This protocol has been approved by the European and Developing Countries Clinical Trial Partnership Ethics Review Committee (ERC) and locally by the Tropical Diseases Research Centre ERC, the Zambia Medicine Regulatory Authority and the National Health Research Ethics Board of the Ministry of Health of the Government of the Republic of Zambia.
Quality assurance and data and safety monitoring board {21a}
Four site visits will be conducted by an independent external monitor who will carry out a minimum of 20% source data verification. A data and safety monitoring board (DSMB) composed of four members will be established. The DSMB will meet quarterly to review progress and provide independent assessments of the quality of the data produced and the safety of study treatments.