HRP2 and pLDH-based rapid diagnostic tests, expert microscopy, and PCR for detection of malaria infection during pregnancy and at delivery in areas of varied transmission: A prospective cohort study in Burkina Faso and Uganda

Background: Intermittent screening and treatment (IST) of malaria during pregnancy has been proposed as an alternative to intermittent preventive treatment in pregnancy (IPTp), where IPTp is failing due to drug resistance. However, the antenatal parasitaemias are frequently very low, and the most appropriate screening test for IST has not been defined. Methodology/Principal Findings: We conducted a multi-center prospective study of 990 HIV-uninfected women attending ANC in two different malaria transmission settings at Tororo District Hospital, eastern Uganda and Colsama Health Center in western Burkina Faso. Women were enrolled in the study in the second or third trimester of pregnancy and followed to delivery, generating 2,597 blood samples for analysis. Screening tests included rapid diagnostic tests (RDTs) targeting histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH) and microscopy, compared to nPCR as a reference standard. At enrolment, the proportion of pregnant women who were positive for P. falciparum by HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT, microscopy and PCR was 38%, 29%, 36% and 44% in Uganda and 21%, 16%, 15% and 35% in Burkina Faso, respectively. All test positivity rates declined during follow-up. In comparison to PCR, the sensitivity of the HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT and microscopy was 75.7%, 60.1% and 69.7% in Uganda, 55.8%, 42.6% and 55.8% in Burkina Faso respectively for all antenatal visits. Specificity was greater than 96% for all three tests. Comparison of accuracy using generalized estimating equation revealed that the HRP2- detecting RDT was the most accurate test in both settings. Conclusions/Significance: The study suggests that HRP2-based RDTs are the most appropriate point-of-care test currently available for use during pregnancy especially for symptomatic women, but will still miss some PCR-positive women. The clinical significance of these very low density infections needs to be better defined. All participants provided their written informed consent to participate in the study before any study procedures were performed by the study staff member. When a participant could not read and write, an impartial adult witness confirmed that the mother had participated in the informed consent discussion, had understood the contents of the consent form, and freely agrees to participate. Additional consent was also obtained for future use of biological samples. The ethics committee approved this consent procedure. The study was approved by the following ethics review committees: a) World Health Organization Research Ethics Review Committee (protocol ID RPC390), b) Comité d’Ethique Institutionnel of the Centre Muraz of the Ministère de la Santé in Burkina Faso (reference number A23-2010/CE-CM), c) School of Medicine Research and Ethics Committee of Makerere University in Uganda (reference number 2011–046), and d) Uganda National Council of Science & Technology (reference number HS160)(S1 and S2 Files). This report conforms to STROBE (S1 Table) guidelines for reporting results of observational cohort studies[14] and STARD guidelines for studies of diagnostic accuracy[15]. The study was conducted at the Colsama health center in the District de Dô, a peri-urban area of Bobo-Dioulasso in southwestern Burkina Faso, and at the Tororo District Hospital in south-eastern Uganda. Both sites are government-sponsored health facilities that provide routine antenatal and delivery care, including: provision of LLINs, provision of IPTp-SP at least twice during pregnancy according to the standard of care at the time of the study [16], treatment of symptomatic malaria in pregnancy with quinine (Burkina Faso) or ACTs (Uganda), and testing and care for HIV-infected women. Malaria transmission in the region of Bobo-Dioulasso is highly seasonal peaking from June to October, with an estimated P. falciparum entomological inoculation rate of 300–500 infective bites per person per year [17]. Tororo region is holoendemic area for malaria, with an entomological inoculation rate (EIR) estimated at 310 infective bites per person per year [18]. The prevalence of malaria among pregnant women in the region was reported to be approximately 40% based on PCR-corrected microscopy[19]. In both sites, P. falciparum is the dominant malaria species. Specific participant inclusion criteria were: The target sample size at each study site was calculated to test hypotheses based on the positive and negative predictive values (PPV and NPV, respectively). The null hypothesis was: [PPV ≤ (disease prevalence + 0.4) or NPV ≤ (1-disease prevalence)] with 80% power and 5% significance at each study location. Previously published estimates of RDT sensitivity (65%) and specificity (98%) were used in these calculations and malaria prevalence was assumed to be 15% in each location. Based on these estimates, an enrollment target was set of 345 women in Uganda and 345 women in Burkina Faso, to ensure 90% confidence of obtaining the required number of positive and negative tests, and allowing for a 15% loss to follow-up between enrolment and delivery. To meet secondary study objectives involving clinical outcomes (not reported here), a larger target sample size was set in Burkina Faso. Study activities ran from November 2010 to April 2012 at the Burkina Faso site and from May 2011 to April 2012 at the Uganda site. Potential participants were referred to the study team by the antenatal clinic staff at each site; study staff completed screening and enrollment consecutively, according to the selection criteria shown in Fig 1. Subsequent antenatal visits were scheduled at least four weeks apart. Participants were encouraged to return to the study site in case of illness between scheduled visits, and to attend the study site for delivery. At each scheduled visit and at delivery, a focused physical examination was conducted and 2 mL venous blood sample was drawn for measurement of hemoglobin (Hb; HemoCue, Quest Diagnostics, Ängelholm, Sweden), preparation of RDTs and blood smears, and storage in a microtainer at -20°C and on filter paper. After delivery, placental blood and a placental biopsy were collected; the complete methods and the resulting findings will be reported separately. If a participant missed a visit then a home visit was arranged, and study staff attempted to visit the homes of those who delivered elsewhere within 24 hours of the birth. At enrollment and at each scheduled visit, RDT results were used to guide management. When all RDT test bands were negative, the participant took a directly observed scheduled dose of SP. When any RDT test band was positive, the participant received treatment with quinine (600mg, eight hourly for 7 days) in Burkina Faso or artemether-lumefantrine (AL) (80/480 mg twice daily for three days) in Uganda; the initial dose was directly observed, and the remaining doses were given to the participant with clear instructions for completing the treatment at home. If quinine or AL was administered, study staff visited the woman at home two weeks later to administer a dose of SP as IPTp. If a participant presented with symptoms suggestive of malaria, microscopy was performed immediately by study staff and microscopy results (rather than RDT) were used for clinical management according to national policy. All study participants received routine antenatal care according to national guidelines, including receipt of a LLIN, iron and folate supplementation, and management of any other symptoms at the treating clinician’s discretion. RDT kits for this study were selected on the basis of their performance in WHO malaria RDT product testing and the target antigens detected[20]. The study selected the three most commonly used antigen-detection targets: HRP2, pan-specific pLDH (pan-pLDH) and P. falciparum-specific pLDH (Pf-pLDH). Two combination tests comprising four test bands were used: CareStart™ Malaria pLDH (Pf/PAN) Test, product G0121, lot E10IL, expiry April 2012 (here referred a pLDH); and CareStart™ Malaria HRP2/pLDH (Pf/PAN) COMBO Test, product G0131, lot E10IR, expiry April 2012 (here referred a HRP2). RDTs were procured directly from the manufacturer, Access Bio, Inc. (Monmouth Jct, New Jersey, USA). Before the study began, RDTs passed lot testing according to WHO-FIND guidelines[20] at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. At the study sites, temperature and humidity of the storage areas for RDTs were not controlled but were monitored and remained within the manufacturer’s recommended storage conditions of 4°C to 40°C. After the completion of participant follow-up, RDTs were retrieved from both sites and again subjected to lot performance testing at the CDC. RDTs were performed on maternal venous blood according to manufacturers’ instructions. Results of all four test bands were recorded. Invalid RDT results were repeated using the same blood sample. RDT results were reported to the treating clinician to guide participant management according to the study protocol. Thick and thin blood smears were prepared and stained with 2% Giemsa for 30 minutes. A smear was declared negative if examination of 100 high power fields did not reveal asexual parasites or gametocytes. For positive smears, parasite density was calculated by counting the number of asexual parasites per 200 leukocytes (or per 500, if the count is <10 asexual parasites/200 leukocytes), assuming a leukocyte count of 8,000/μl. When a thick smear was positive, the corresponding thin blood smear was read to determine the parasite species. Each smear was read by two microscopists who were blinded to the RDT results and any previous microscopy. Results were considered discordant if one result was negative and the other positive for malaria infection; for positive smears, if the density estimates differed by >50% of the maximum reading; or if the two microscopists reported different species present. Discordant results were resolved by a third microscopist, taking into account concordance with the initial readings[21]. Before the study began, all microscopists were pre-qualified using WHO slide banks and procedures [22] and required to reach Level 1 or 2 expertise level. In addition, a random sample of 10% of slides from each site was sent for review at University of Lagos by Level 1 expert microscopists. For all routine antenatal visits, 2.5 mL of peripheral blood was collected into an EDTA microtainer, labeled and stored at -20°C. Stored samples were transferred to a central laboratory (at Institut de Recherche en Sciences de la Santé [IRSS] in Bobo-Dioulasso, Burkina Faso) and analyzed by nested PCR to confirm presence or absence of parasites, and parasite species. PCR procedures were performed according to standardized protocols and published methods[23]. PCR technologists were blinded to RDT and microscopy results. Before the study began, proficiency testing of the IRSS molecular laboratory was performed using reference samples from the CDC. In addition, a stratified random sample of 5% of samples were re-tested at the Institut Pasteur du Cambodge laboratory in Phnom Penh, Cambodia, as a means of external quality control. Test outcomes and clinical data were collected on case record forms and were double-entered in an electronic database using Epi-Info version 6.04 (Centers for Disease Control and Prevention, Atlanta, GA). Data were subsequently cleaned and analyzed using SPSS version 16.0. Data from the antenatal visits (655 samples from Uganda and 1087 samples from Burkina Faso) were used to calculate sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for each diagnostic test. Generalized Estimating Equations (GEEs) were used to compare the sensitivity, specificity, PPV and NPV between diagnostics, assuming that PCR is the gold standard for the presence of P. falciparum. Explanatory variables including age, gravidity, prior antimalarial treatment or IPTp, fever symptoms within last 24 hours, were tested for their influence on diagnostic performance.