This study was conducted at the Centre Hospitalier de Kingasani II (CHK), commonly known as Maternité des Soeurs. With twenty to thirty deliveries daily, it is the most frequented Centre in Kinshasa and countrywide. Located in the heart of the semi-rural areas of the south-eastern suburbs of Kinshasa, the maternity provides invaluable health care at an accessible rate.
In a cross-sectional survey, the prevalence of asymptomatic P. falciparum infection was determined in apparently healthy pregnant women, going to the CHK for their first antenatal care (ANC) visit. Data and blood samples were collected prior to any routine administration of intermittent preventive treatment (IPT). Participants were recruited from July 27th, 2012 to August 27th, 2012. Women not providing written informed consent or presenting fever, muscle aches or other symptoms suggestive for malaria were excluded. Women who received anti-malarial treatment within the past two weeks were also excluded from the study. A structured questionnaire was used to obtain information on age, parity, and gestational age, level of education, previous or current use of anti-malarial drugs and the use of bed nets.
Blood sample collection
Blood samples were collected from a finger prick for laboratory analysis which included: thick blood smears for microscopy, RDT performance, determination of haemoglobin concentration and molecular analysis. For molecular analysis, blood was collected on a filter paper (Whatmann 3 MM), dried thoroughly, put in individual zip lock plastic bags containing desiccant and stored at room temperature (< 25°C) until completion of the study and then transported to the Tropical Diseases Research Centre (TDRC) in Zambia for molecular analyses.
Microscopy, RDTs and PCR were used to detect P. falciparum infection. Giemsa-stained thick blood smears (TBS) were used for microscopy. Blood slides were examined using light microscopy at 1,000 × magnification. Hundred microscopic fields were examined in the thick smear before concluding that a blood slide was negative. All slides were read twice by experienced microscopists. If the discrepancy was greater than 15%, a third reader was used to confirm diagnosis. The parasite density per microlitre of blood was computed using the following formula: (Number of trophozoites × 6,000)/Number of leucocytes. Besides microscopy, rapid diagnostic tests (RDTs) were also performed. In this study the SD Bioline Malaria Ag Pf® detecting HRP2 was used.
Plasmodium species-specific diagnostic PCR assay
As microscopy in pregnancy has a lower sensitivity due to placental sequestering of parasites and RDTs have internal validity problems, species specific PCR was conducted on all microscopy negative samples but RDT positive samples. In addition, 166 randomly chosen samples were analysed using nested Plasmodium species diagnostic PCR assay. Molecular analysis was performed at TDRC Ndola, Zambia.
The blood spot samples of 5 mm diameter size were soaked in 0.5% saponin in phosphate-buffered saline (PBS), incubated for 10 minutes at room temperature in a 1.5-ml tube, and centrifuged at 14000 rpm. The supernatant was discarded and blood spots were washed in 1 ml of PBS. One hundred Fifty microlitres of a 2% Chelex-100 resin work solution (Bio-Rad Richmond, CA) and 50 μl of water (pH 9.5) were added to the sample in a1.5-ml tube and incubated at 100°C for 10 minutes. After centrifugation at 10,000 g for 1 min, the supernatant was collected and stored at -20°C, prior to the PCR assay.
Polymerase chain reaction investigation
Nested PCR assay was performed as a two-step procedure. Firstly, amplification of Plasmodium genus specific fragment was carried out as follow: PCR mixture containing buffer, dNTPs, MgCl2, primers, Taq polymerases and sterile water. All PCR reactions were carried out in a total volume of 20 μl. One μl of the purified template DNA was used for the first reaction, in which the fragment spanned by rPLU5 (5' CCTGTT GTTGCCTTAAACTTC 3') and rPLU6 (5' TTAAAATTGTT GCAGTTAAAACG 3') was amplified. Secondly, a 1 μl aliquot from the product of the first PCR reaction was subsequently used as a template for Plasmodium falciparum-specific fragment amplification using FAL1 (5' TTAAACTGGTTTGGGAAAACCAAATATATT3') and FAL2 (5’ ACACAATGAACTCAA TCATGACTACCCGTC 3') specific primers
. Positive and negative controls were always included in the assays. A negative control without DNA template and a positive control with appropriate template (3D7) were always included.
PCR products were detected by running 20 μl of DNA product on a 3% agarose gel, small fragments (Eurogentec®), which was subsequently stained with a 0.5 μg/ml ethidium bromide solution and visualized under ultraviolet transillumination. The specific size of the PCR product (second amplification) was 205 bp for P. falciparum.
Determining the concentration of haemoglobin was performed with a portable HemoControl® device. Anaemia was defined by a haemoglobin concentration <11 g/dL. Anaemia was classified as severe anaemia Hb <7 g/dl, moderate anaemia Hb: 7–9.9 g/dl and mild anaemia Hb: 10-10.9 g/dl.
The population of pregnant women in the health zone of Kingasani was estimated at 7,187
. With an expected malaria prevalence of 30%, a desired accuracy set at 5% and a confidence interval of 95%, the minimum sample size was calculated at 309 pregnant women.
Data was entered and stored in Epi info™7. Descriptive statistics were employed for the analysis of socio-demographic data. Frequencies were used to assess the prevalence of asymptomatic malaria and anaemia in pregnant women. The χ2 test was used to investigate associations between categorical variables. Odds ratios (ORs), 95% confidence intervals (CIs) were calculated and p < 0.05 values were considered to be statistically significant. Multivariate logistic regression models were constructed to identify factors associated with asymptomatic malaria or anaemia during pregnancy. Based on a priori knowledge and a P value less than 0.05 considered as significant, the following variables: Age, sex, parity, marital status, ownership of bed net, presence of lattice on windows, malaria infection and geophagia were included in the model. Backward regression technique was used to construct the model. Statistical analyses were performed using SPSS statistical program, version 17 (SPSS, Chicago, IL, USA).
The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of microscopy and RDT (SD Bioline malaria Ag Pf®) were determined with PCR as gold standard, for the 166 samples (50%) of which PCR results were available.
Collection of data to estimate the cost of P. falciparum infection diagnosis
Financial data were obtained by conducting interviews with Hospital managers and laboratory staff in Kingasani health Zone. Costs were collected in Congolese Francs (CDF) and converted to US dollars (exchange rate US$1 = CDF 920, July 2012). The cost for one thick blood smear includes: a glass slide, cotton, lancet, Giemsa and other stains, immersion oil, alcohol and gloves. The personnel cost for the service provided corresponded to the time spent from drawing blood samples, preparing the thick and thin smears, staining, reading and reporting the results. At CHK II as in all public maternities in Kinshasa, RDT kits are donated and the personnel service is free of charge. In the present study, for RDT diagnostic strategy, the effective contact time with those seeking care, which accounted for the personnel cost, and the cost of RDT kit were the main input parameters to figure out the amount of money a pregnant women would pay. The effective contact time was comprised of: drawing blood samples from patients, applying samples onto the test, test reading and reporting of results. The time was recorded on a “time sheet” by laboratory personnel for every RDT and microscopy service provided. Laboratory staff work eight hours from Monday to Friday and 5 hours on Saturday, thus 68 hours (4,080 minutes) weekly. Assuming a month of four weeks; laboratory staff work 272 hours (16,320 minutes) monthly. Staff salary data were provided by the staff manager.
This study was approved by the Ethical Committee board of the University of Kinshasa. Test results of the thick blood smear were revealed to all participants three days after sample collection. All women were given mebendazole, iron and folic acid on a routine basis and were treated with SP regardless of the laboratory test results.