Plasmodium falciparum mutant haplotype infection during pregnancy associated with reduced birthweight, Tanzania

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is a key strategy in the control of pregnancy-associated malaria. However, this strategy is compromised by widespread drug resistance from single-nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. During September 2008-October 2010, we monitored a cohort of 924 pregnant women in an area of Tanzania with declining malaria transmission. P. falciparum parasites were genotyped, and the effect of infecting haplotypes on birthweight was assessed. Of the genotyped parasites, 9.3%, 46.3%, and 44.4% had quadruple or less, quintuple, and sextuple mutated haplotypes, respectively. Mutant haplotypes were unrelated to SP doses. Compared with infections with the less-mutated haplotypes, infections with the sextuple haplotype mutation were associated with lower (359 g) birthweights. Continued use of the suboptimal IPTp-SP regimen should be reevaluated, and alternative strategies (e.g., intermittent screening and treatment or intermittent treatment with safe and effective alternative drugs) should be evaluated. The study was conducted during September 2008–October 2010 in Korogwe District in the Tanga Region of northeastern Tanzania, where P. falciparum is the predominant malaria-causing species. A prospective cohort of 924 pregnant women was monitored from first attendance at the antenatal clinic through delivery. The study has been described in detail (22–24). In brief, pregnant women with a gestational age of <24 weeks, as estimated by using ultrasound, were enrolled if they had lived in Korogwe District for >6 months and were willing to give birth at Korogwe District Hospital. Study participants attended 3 additional prescheduled antenatal visits at weeks 26, 30, and 36 of pregnancy, and they were attended to by a study nurse/clinician. Obstetric history and maternal anthropometric measurements were recorded for all women (23). A venous blood sample was collected at each antenatal clinic visit, and venous and placental blood samples were collected at the time of delivery. All blood samples were tested for malaria parasites by using a rapid diagnostic test (RDT) (Parascreen, Zephyr Biomedicals, Goa, India; Paracheck Pf, Orchid Biomedical Systems, Mumbai, India; or ParaHIT-f, Span Diagnostics Ltd, Surat, India) and by microscopy. Blood smears for women with negative RDT results were examined retrospectively, whereas those for women with positive RDT results were examined immediately if deemed necessary by the study physician for a treatment decision (25). Parasite density was determined as the number of asexual stage parasites/200 leukocytes (500 leukocytes if <10 parasites) and converted to the number per microliter, as described (22,25); >100 fields were double-examined before a blood smear was declared negative. Women with positive RDT results were treated with the antimalarial drug artemether-lumefantrine (Coartem Dispersible, Norvatis Corporation, Suffern, New York, USA) or with quinine. For infections occurring during the first trimester of pregnancy, quinine sulfate–coated tablets (ELYS Chemical Industries Ltd, Nairobi, Kenya) were used, and for severe cases, quinine dihydrochloride injection (Vital Healthcare PVT Ltd, Mumbai, India) was used. Two doses of IPTp-SP (Sulphadar, Shelys Pharmaceutical Ltd, Dar es Salaam, Tanzania) were given >1 month apart as directly observed treatment; each dose contained 1,500 mg of sulfadoxine and 75 mg of pyrimethamine. Women with a gestational age of >20 weeks at enrollment were given the first IPTp-SP dose at the study-inclusion visit and the second dose during the third trimester. Women with a gestational age of <20 weeks at enrollment were given the first dose at 20 weeks of gestation. Women who had received IPTp-SP before study inclusion but earlier than recommended by the World Health Organization (i.e., after quickening in the second trimester) received a second dose after 20 weeks of gestation, and a third dose was given in the third trimester. Use of SP from private pharmacies/drug shops for malaria treatment before and after study inclusion was also documented. All study participants were provided with a voucher for procuring insecticide-treated nets. EDTA-preserved venous blood was used to estimate hemoglobin levels (KX-21N Automated Hematology Analyzer, Sysmex, Kobe, Japan). Live newborns whose birthweights were measured by using a spring scale (Fazzini, Vimodrone, Italy) with an accuracy of <50 g or a digital strain gauge scale (ADE, Hamburg, Germany) with an accuracy of <10 g within 24 h of delivery were included in the birthweight analysis. Newborns with severe malformations, twins, and those born to women with preeclampsia were excluded from analyses because these conditions can severely affect birthweight (23). EDTA-preserved blood (50 μL) was spotted on Whatman number 3 filter paper (VWR– Bie & Berntsen, Herlev, Denmark), dried at room temperature, and stored in separate zip-lock bags. DNA was extracted by using the Chelex 100 method, as described (26). The DNA supernatant was transferred to a 96-well PCR plate and stored at −20°C until use. The parasite DNA was amplified by outer and nested P. falciparum–specific PCRs, as described (27); the products were analyzed by electrophoresis in 1.5% ethidium bromide–stained gel, as described (22). To determine the multiplicity of infections, block 2 of the merozoite surface protein 2 domain was amplified by using fluorescent PCR (28). The results were analyzed by using GeneScan software, version 3.7 (Applied Biosystems, Naerum, Denmark). Parasite DNA was amplified by outer and nested PCR with specific primers targeting the Pfdhfr and Pfdhps genes, as described (29). Single-nucleotide polymorphisms (SNPs) in the Pfdhfr and Pfdhps genes were identified by using a sequence-specific oligonucleotide probe ELISA technique, as described (29) with minor modifications. In brief, we used sequence-specific oligonucleotide probes targeting Pfdhfr codons c50/51 CI/CN, c59 (C/R), c108 (S/N/T), and c164 (I/L) and Pfdhps codons c436/437 (AA/AG/SA/SG/FG), c540 (K/E), c581 (A/G), and c613 (A/S). Individual SNPs were combined to deduce the different infecting mutant haplotypes. We double-entered and validated data in Microsoft Access version 2007 (Redmond, WA, USA). Statistical analyses were conducted by using Stata version 10 (StataCorp, College Station, TX, USA) deploying parametric and nonparametric methods, as appropriate. The effect of infecting allelic haplotypes on birthweights was investigated by using multiple linear regression and dichotomized (as 6 and <6 SNPs) to infecting haplotypes; variables with a p<0.20 in univariate analysis were included in the multivariate models. By using a stepwise backward elimination approach, we obtained final models including only variables with a p<0.10. A 2-sided p-value of <0.05 was considered significant. Final models included only women without missing values.