Study design, period and area
Health facility based cross-sectional study was conducted from February to March 2019 at Fendeka town Jawi district Northwest Ethiopia (Fig 1). Jawi district is one of the districts with endemic malaria transmission in Amhara region. The mean temperature varies between 16.68 0c-37.6 0c and the altitude ranges from 648-1300m above sea level. The number of malaria reported cases in 2018/19 were 24,434 (Jawi district office malaria cases report). Moreover, Tana Beles integrated sugar development project is found in the study area and there is year-round transmission of malaria. According to 2007 national central statistical census report, the total population of the district was 79,090; of whom 41,407 were men and 37,683 women [19]. The district has long summer rain fall (June-September) and a winter dry season (October-May) with mean annual rain fall of 1569.4mm [20]. Moreover, Tana Beles integrated sugar development project is found at the study area.
Sample size determination and sampling techniques
Sample size was calculated by Buderer’s formula using 39% prevalence 56.4% sensitivity, 90% specificity of RDT [21]; and 12% error rate. A total of 166 pregnant women were participated in the study. Using proportionate allocation, equal 83 participants were involved in both Jawi hospital and Jawi health centre. Convenient sampling technique was implemented.
Data collection methods and recruitment of pregnant women
Socio-demographic and other characteristics of pregnant women
When pregnant women attending their follow up at the antenatal care clinic (ANC), the midwife gave their identity card and examined the status of their pregnancy. After checking their status and if they were normal to malaria infection, the pregnant women were recruited regardless of their appointment period. Then, the midwife requested their willingness to participate in the study. On volunteered pregnant women, socio-demographic characteristics like sex, residence, age, educational level, occupation, marital status, family size, and other characteristics like gravidity, gestational age, ownership and utilization of ITNs and use of IRS, previous use of antimalarials and clinical questions like fever, shivering and sweating were collected via structured questionnaire by the trained midwives.
Blood sample collection: Blood sample was collected by pricking at their ring finger for RDTs, thick and thin blood films and DBS using Whatman filter paper. For RDTs, 5 microliters of capillary blood were added using micropipette to the sample well and 2 drops (60μl) of buffer solution to the buffer well and then after waiting for 20 minutes, the results were reported [23] according to the manufacturer’s instruction. Thin and thick blood films were prepared by dropping the required amount of blood sample on a single slide and spreading on it. In addition, each of the thick and thin films was air dried, and the thin films were fixed by dipping in absolute methanol for 10–20 seconds. Then, slides were stained with 3% Giemsa for 30–45 minutes [22]. For DBS, drops of blood were spotted on Whatman filter paper (DBS card) and allow them to dry. After the DBS cards were dried, they were packaged in to each sealed plastic bags with desiccant; and it was stored at - 20 0c in the lab. Later, it was transferred to Amhara Public Health Institute for molecular analysis using RT-PCR.
Laboratory diagnosis: Parasite detection by CareStart™ Malaria Pf / Pv (HRP2/pLDH) Ag Combo RDTs was done based on manufacturer instructions. Thin and thick stained smears were examined under light microscope (Olympus CX-21) with 100x magnification for detection and identification of Plasmodium parasites by experienced laboratory professionals working at Jaw health centre and hospital. Thick blood films were considered positive when sexual and asexual forms were detected and considered as negative after observing 200 high power fields without detecting any parasite.
Finally, for molecular analysis, 3-mm punches of the DBS were punched out and placed in to 1.5-ml micro-centrifuge tubes. The punches inside the tube were treated with ATL buffer, proteinase K, AL buffer and 96% ethanol, mixing thoroughly by vortexing, brief centrifuge and incubation with consecutive additions. The mixtures were transferred into QIAamp Mini spin column and with subsequent addition of wash buffer AW1 and AW2, DNA was extracted. The DNA was eluted in 100μl of elution buffer, aliquoted and stored at -20 0C until running PCR assay [24]. The PCR assay was run using Agilent Technologies strata gene Mx3005p PCR machine.
Photo-induced electron transfer PCR (PET-PCR) Primer sequence.
Original Genus 18sFor, 5’-GGC CTA ACA TGG CTA TGA CG-3’
Original Genus FAM 18sRev, 5’-agg cgc ata gcg cct ggC TGC CTT CCT TAG ATG TGG TAG CT-3’
Falciparum For, 5’-ACC CCT CGC CTG GTG TTT TT-3’
Falciparum Rev, HEX-5’-agg cgg ata ccg cct ggT CGG GCC CCA AAA ATA GGA A-3’
P. vivax For, 5’-GTA GCC TAA GAA GGC CGT GT-3’
P. vivax Rev, HEX-5’- agg cgc ata gcg cct ggC CTG GGG GAT GAA TAT CTC TAC AGC ACT GT-3’
Amplification of Plasmodium Genus and P. falciparum or P. vivax was performed in a 20µl reaction containing 2X TaqMan Environmental Master Mix buffer, forward and reverse primers for the Genus and P. falciparum multiplex assays and P. vivax singleplex assay, and 5µl of DNA samples. Samples were run in duplicate on manually loaded 96-well PCR plates. The reactions performed under the following cycling conditions: initial hot-start at 95 0C for 15 minutes, followed by 45 cycles of denaturation at 95 0C for 20 seconds and annealing at 60 0C for 40 seconds for Genus and P. falciparum multiplex assays.
For P. vivax single plex assay; initial hot-start at 95 0C for 15 minutes, followed by 45 cycles of denaturation at 95 0C for 20 seconds and annealing at 60 0C for 40 seconds and then at 72 0C for 30 seconds. The correct fluorescence channel was selected for each fluorescently labeled primer set and the cycle threshold (CT) values recorded at the end of annealing step. Any sample with a CT value of 40.5 or below was considered positive [24]. The Genus and P. falciparum multiplex assays were performed for all samples. P. vivax singleplex assay was performed for Genus positive and P. falciparum negative samples.
Data quality control and management
Before the data collection, training was given to the data collectors by the principal investigator (PI) about how to collect the sample and fill the questionnaires. Socio-demographic and other characteristics were collected by midwives; while experienced laboratory professionals were involved for RDTs, thin and thick blood film preparation, DBS sample collection and Plasmodium species identification. Moreover, the same microscope mark (CX21) was used to minimize the possible instrumental error. The questionnaires were prepared in clear and understandable way. The clarity, understandability, and flow of each question were assessed properly. Data were collected under intensive supervision by PI. Finally, the discordant results were rechecked by the PI along with other experienced laboratory professionals who did not participate in the initial identification and preparation process.
Data analysis
Data was analyzed using SPSS version 20 statistical package. Descriptive statistics was used to assess the prevalence of API among pregnant women. Cohen’s kappa coefficient (k) was used to determine the degree of agreement among the diagnostic methods.