Performance of malaria rapid diagnostic test, microscopy, polymerase chain reaction, and histopathology to diagnose malaria among pregnant and parturient women using peripheral, placental, and cord blood, and placental biopsy in Majang Zone of Gambella Region, Southwest Ethiopia

Abstract

Background

Accurate, reliable, and timely diagnosis is essential for mitigating malaria in pregnancy (MiP) and its adverse outcomes. This study aimed to evaluate the accuracy of malaria diagnostic tests for detecting Plasmodium infection in peripheral, placental, and cord blood and placental biopsy in the Majang Zone of Gambella Region, Southwest Ethiopia.

Methods

A cross-sectional study involving 640 (460 pregnant and 180 parturient) women visiting five public health facilities for antenatal care and delivery services in Majang Zone was conducted from November 2022 to March 2023. Peripheral, placental, and cord blood were collected to detect Plasmodium infection by rapid diagnostic test (RDT), microscopy, and quantitative Polymerase Chain Reaction (qPCR). Placental biopsy was collected for placental malaria (PM) diagnosis by histopathology. Performance indices, Kappa Coefficient, and Receiver Operating Characteristic were determined using Statistical Package for Social Science Version 26.0, Microsoft Excel Version 19.0, and Stata Version 17.0.

Results

One thousand blood (640 peripheral, 180 placental, and 180 cord) and 180 placental biopsy specimens collected from pregnant and parturient women were analysed in this study. Malaria positivity rate among pregnant and parturient women was 21.1% and 28.9%, respectively. Considering peripheral blood qPCR as a reference, the sensitivity, specificity, accuracy, and agreement of RDT were (63.5%, 93.0%, 0.807, and 0.683), and microscopy were (73.1%, 98.0%, 0.855, and 0.764) to detect Plasmodium infection in combined peripheral blood of pregnant and parturient women, respectively. Considering placental blood qPCR as a reference, the sensitivity, specificity, accuracy, and agreement of RDT were (56.3%, 95.5%, 0.759, and 0.574), microscopy were (81.3%, 97.7%, 0.895, and 0.822), and histopathology (87.5%, 100.0%, 0.892, and 0.911) to detect Plasmodium infection in placental blood of parturient women, respectively. Considering placental histopathology a as reference, the sensitivity, specificity, accuracy, and agreement of RDT were (56.8%, 97.1%, 0.753, and 0.609), microscopy were (68.2%, 98.5%, 0.918, and 0.735), and qPCR (100.0%, 95.7%, 0.978, and 0.911) to detect Plasmodium infection in placental blood of parturient women, respectively.

Conclusion

Diagnostic performance of RDT and microscopy was sub-optimal to detect Plasmodium infection among pregnant and parturient women. More sensitive diagnostic tests are needed to mitigate MiP.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12936-025-05426-2.

Background

Early diagnosis followed by effective treatment is central to mitigating the burden of malaria in pregnancy (MiP) [1–3]. Accurate, reliable, and timely diagnosis of MiP helps to prevent its adverse outcome and interrupt transmission [1, 2, 4–7]. Diagnosis of MiP can be done from peripheral, placental, and cord blood using rapid diagnostic testing (RDT), microscopy, and quantitative Polymerase Chain Reaction (PCR), as well as from placental biopsy using histopathology. Peripheral blood specimens can be collected during pregnancy and delivery, but placental and umbilical cord specimens can only be collected after delivery. Peripheral blood malaria microscopy and RDT are commonly used for the diagnosis of MiP. Microscopy allows detection, identification, and quantification of the parasite, whereas RDT is chosen for its ease of operation within a short time [6, 8]. However, these tools fall short of detecting PM and low-density parasitaemia, which often remain asymptomatic [6–10]. On the other hand, molecular tools such as PCR and Loop-mediated Isothermal Amplification (LAMP) have good sensitivity to detect sub-microscopic Plasmodium infections from peripheral, placental, and cord blood. Yet, these molecular tools are relatively expensive, demand high expertise, and are scrutinized for the viability of the parasite material they detect [6, 8, 9, 11]. Histopathology is a very sensitive test for detecting placental malaria (PM), and it is considered the gold standard to characterize PM as acute, chronic, and past infection by allowing detection of sequestered parasites, haemozoin, and inflammatory cells in the intervillous space of the placenta [6, 8–10, 12]. But, the application of histopathology and PCR is limited to research purposes mainly due to their technical difficulty and high cost [6, 8].

Several studies assessed the diagnostic performance of malaria tests using various specimen types (peripheral, placental, and cord blood, and placental biopsy) to detect Plasmodium infection among pregnant and parturient women [13–19]. According to the report of a recently published systematic review and meta-analysis (SRMA), highly sensitive RDTs (hsRDTs) were slightly more sensitive than conventional RDTs (cRDTs) to detect MiP [14]. Consistent finding was recorded from a study conducted among pregnant women in Benin, where hsRDTs showed better sensitivity (60.5%) than cRDTs (44.2%) [13]. In a study conducted in the Southwest part of Ethiopia, the sensitivity of RDT (66.7%) was better than microscopy (55.6%) to detect MiP [20]. Similarly, RDT and PCR outperformed microscopy in detecting asymptomatic malaria in pregnancy (AMiP) in Burkina Faso [15, 16]. Furthermore, in a recently published study from Colombia, LAMP showed the highest sensitivity, including low-density parasitaemia among pregnant women, indicating its promising relevance for efficient diagnosis of MiP during antenatal care (ANC) visits in malaria-endemic areas [17]. Studies conducted in Sudan and Papua New Guinea revealed lower accuracy of RDT, microscopy, and PCR than placental histopathology to detect PM [18, 19]. A contradictory finding was reported from a study conducted in Mozambique, where the sensitivity of histopathology to detect PM was 41.8% (considering only active infection), and 72.4% (considering active and past infections) using placental blood PCR as reference [21].

Considering the potential sequestration of the parasite in the placenta, and the associated clinical and public health importance of MiP, it is crucial to ensure that malaria among pregnant and parturient women is diagnosed by an accurate test that can detect low parasitaemia [7, 9]. According to the report of a study conducted in Mozambique, microscopy, histopathology, and HRP2-based plasma diagnostic tests missed the majority of the Plasmodium falciparum infections detected by qPCR in peripheral and placental blood. The reported association of these undetected infections with maternal anaemia rendered the adequacy of these tools for effective mitigation of MiP questionable [14, 21]. As missed infections entail many adverse outcomes of MiP, it is worth noting that false-positive infections render wastage of expensive resources and potential development of drug resistance [15]. The diagnostic performance of malaria tests to detect MiP depends on various factors, including gravidity, gestational age, symptoms, and parasite density [9, 13, 14]. Thus, it is conceivable that the current RDT and microscopy used in malaria-endemic settings are inadequate to show the true prevalence of MiP, partly due to their inherent problems, and capacity issues, as well as the changing characteristics of the parasite and the human [6, 8, 9].

Despite their good stability and sensitivity, HRP2-based malaria RDTs are criticized for antigen persistence and hrp2 gene deletion-related poor accuracy [7, 15, 22, 23]. To overcome the limitation of conventional diagnostic tests and improve their performance in detecting MiP and PM, various strategies have been used, including the use of hsRDTs and the use of microscopy and RDT in combination [13, 19, 24]. In a study conducted in Uganda, peripheral blood HRP2-based RDT and microscopy detected a substantial portion of PM, and also showed a better performance when used in combination [24]. However, given the low parasitaemia in peripheral blood due to placental sequestration, and partially acquired immunity combined with limited accuracy of the routinely used conventional diagnostic tests in malaria-endemic settings, PM and AMiP remain a formidable challenge to mitigation of MiP, and thus elimination of malaria. This signifies the importance of continuously monitoring the accuracy of the existing malaria tests while searching for more accurate and reliable tests [7, 8, 11, 20, 25–27].

Accurate and reliable data are central to mitigating MiP in particular, and the elimination of malaria in general. In the quest to find a robust tool for the diagnosis of PM, and hence, mitigate its adverse outcome, it is important to evaluate the accuracy of peripheral and placental blood RDT, microscopy, and PCR against a reference test [6, 8, 9]. Only a few studies assessed the performance of diagnostic tests to detect MiP, particularly PM, using histopathology as reference, in Ethiopia. Among those studies, only a few studies provided comprehensive data involving many tests and specimens (peripheral, placental, and cord blood, and biopsy) collected from symptomatic and asymptomatic pregnant and parturient women using diversified statistical analysis that can show the disaggregated and overall performance of each tool and agreement among one another [28–31]. Diagnostic accuracy studies involving diverse statistical analyses to generate performance metrices (such as sensitivity and specificity), agreement among results of tests, and operational utility through a Receiver Operating Characteristic (ROC) analysis (which is less susceptible to prevalence of the disease under study) data of many malaria tests in many specimen types are central to provide comprehensive evidence to inform practice and policy. Such data can show a wide-ranging picture of each tool and the overall situation of MiP diagnosis that can guide policy-makers to design, introduce, and monitor interventions. Besides, evaluating the diagnostic performance of many tools using peripheral, placental, and cord blood could indicate the potential tools and specimens suitable for timely, accurate, and cost-effective diagnosis of MiP and hence, timely prevent its adverse outcome [6, 8, 9, 32, 33]. However, there is a paucity of such data, particularly in this malaria-endemic part of the country, despite the recently reported significant prevalence of AMiP and PM in the study area [27, 34]. Therefore, this study aimed to evaluate the diagnostic performance of RDT, microscopy, histopathology, and qPCR to detect Plasmodium infection in peripheral, placental, and cord blood, and placental biopsy among pregnant and parturient women in the Majang Zone of the Gambella Region, Southwest Ethiopia.

Methods

Study setting, period, design, and population

A facility-based prospective cross-sectional study was conducted from November 2022 to March 2023 among pregnant and parturient women in Majang Zone. Majang Zone is located in the Southwestern part of the country in the Gambella Peoples’ National Regional State of Ethiopia. This Zone has two woredas (Mengesh and Godere woreda). It has one hospital, four health centres, and over 30 health posts providing healthcare service for over 87,374 population [27, 35]. The altitude of the Zone ranges from 750 to 1800 m above sea level. The mean annual temperature and rainfall range from 17.6 to 27.5 °C and 1401 to 1800 mm, respectively [36–38]. Majang Zone is among malaria-endemic areas, where Plasmodium falciparum and Plasmodium vivax are co-endemic, with recently increasing prevalence of AMiP and PM. The health institutions (with their geographic location) involved in this study were: Godere Mission HC (7° 27′ 13′′ N 35^o 02′ 52′′ E), Fejeji HC (7^o 21′ 54′′ N 35^o 05′51′′ E), Kumi Primary Hospital (7^o 16′ 27′′ N 35^o 09′ 10′′ E), Metti HC (7^o 13′ 43′′ N 35^o 19′ 16′′ E), and Dunchay HC (7^o 08′ 28′′ N 35^o 19′ 07′′ E) [27, 34, 38, 39].

Sample size and sampling technique

The required sample size was calculated using the single population proportion formula described below. A 95% confidence level, 5% margin of error, and 12.72% prevalence of MiP (from SRMA in Ethiopia) were considered [40]. Buderer’s formula [41, 42] was applied to calculate sample size considering 96.05% sensitivity and 99.0% specificity reported from a previous study conducted in Southern Ethiopia [43]. A minimum sample size of 452 pregnant women was attained by considering 96.05% sensitivity, 10% non-response rate, and 12.72% prevalence. A minimum sample size of 172 parturient women was attained by considering a 96.05% sensitivity, 10% non-response rate, and 50.0% prevalence. A systematic sampling technique with an interval of three was applied to select a representative and proportional sample of pregnant women from the ANC logbook in the five health facilities of the Zone. On the other hand, eligible and consenting parturient women attending the delivery ward of Metti HC during the data collection period were consecutively included in the study until the final sample size was met (Supplement Eq. 1).

Inclusion and exclusion criteria

Pregnant women who resided in the Zone for at least six months, attending ANC clinics of the five health facilities during the study period, were included. Besides, singleton parturient women delivering at Metti HC during the study period were included. Whereas, those who took antimalarial drugs within the previous 14 days before the data collection date, and those with complicated pregnancy and delivery were excluded.

Socio-demographic, obstetric, and anti-malarial intervention data collection

Socio-demographic (age, education, marital status, residence), obstetric (gestational age, gravidity, parity), and anti-malarial intervention (IRS, and ITN) use data of pregnant and parturient women were collected by two trained midwives using questionnaire adapted from Ethiopian Demographic Health Survey (EDHS 2016), Malaria indicator Survey (MIS-2015) and related literature [36, 44].

Blood and biopsy specimen collection

From pregnant women (only peripheral blood) and parturient women (peripheral, placental, and cord blood and placental biopsy) were collected. Approximately 212 µl of peripheral blood (capillary) was obtained by pricking the middle or ring finger of the pregnant and parturient women by a Medical Laboratory Technologist using a disposable sterile lancet. Approximately 212 µl of cord blood was collected by aspirating the umbilical cord blood vessels using a 5 cc syringe (Jiangsu Kanghua Medical Equipment Co., China). Approximately 212 µl of placental blood was collected by a Medical Laboratory Technologist through aspirating (using a Pasteur pipette) the blood pooled in the well formed after removing the placental biopsy with a surgical blade. Four dried blood spots (DBSs) per filter paper per blood specimen type (i.e., peripheral, placental, and cord blood) for each participant were prepared by disposing of four independent drops of the blood on Whatman filter paper and air-drying it overnight. Then, each DBS-holding filter-paper was independently packed in a plastic bag containing desiccant, and refrigerated at − 20 °C until transported to the Molecular Biology Laboratory of Tropical and Infectious Diseases Research Centre (TIDRC) at Sokoru, Jimma Zone, Southwest Ethiopia [45–47]. For histopathological analysis, two placental biopsies (2 cm³ size each from the left and right side of the cord insertion) were collected through incision by surgical blade at a position of mid-distance between the cord insertion and the periphery of the placenta on its maternal side. Then, each biopsy was separately preserved in a screw-capped cup containing 40 ml of 10% Neutral Buffered Formalin (NBF) until shipped to Jimma University Medical Centre Pathology Laboratory for histopathological assay within two weeks [45] (Fig. 1).

Fig. 1.

Flowchart showing the process of specimen collection and laboratory tests employed to evaluate the diagnostic accuracy of malaria diagnostic tests in detecting Plasmodium infection among pregnant and parturient women in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023

Malaria rapid diagnostic testing from blood

Onsite diagnosis of Plasmodium infection in each blood specimen type of the pregnant and parturient women was done by Bioline™ Malaria Ag P.f/Pan RDT [Catalogue number: 05 FK60, Lot number of the cassette and the assay buffer were 05EDG040 A and 05BDDG105, respectively: (Abbott Diagnostics, Republic of Korea)] strictly following manufacturer’s instruction. Bioline™ Malaria Ag P.f/Pan RDT is a qualitative lateral-flow immunochromatographic cassette-based test that detects HRP2 antigen of P. falciparum and pLDH (Pan) antigen of the human malaria-causing Plasmodium species from blood based on the capture of a labelled antibody-parasite-antigen-complex resulting in the formation of a visible band at the result window [48, 49]. Detail of this assay procedure was published elsewhere [27, 34].

Malaria microscopy from blood

For each blood specimen type collected from each participant (pregnant and parturient woman), thick (6 µl) and thin (2 µl) blood films were prepared on glass slides in duplicate and air-dried (as previously published [27, 34]. After fixing the thin film in absolute methanol for 10–15 s, the adequately air-dried blood films were stained with 3% Giemsa for 35–45 min, rinsed in water, air-dried, and then examined under 1000× magnification using Olympus CX23 bright-field light microscope (Olympus, Japan) by two trained Medical Laboratory Technologists (who are WHO level one experts) at Metti HC. The examination involved assessing the blood film for the presence of the Plasmodium parasite, identifying the species, and developmental stage, and quantifying the parasites detected. Sexual and asexual parasite quantities were determined by counting against 500 and 200 White blood cells (WBCs), respectively. The obtained parasite number is converted into the number of parasites/µl, assuming 8.0 × 10³ WBCs/µl of blood. A blood film was declared negative if no Plasmodium parasite was seen after examining 200 OIFs [50–52].

Real-time quantitative polymerase chain reaction

The molecular detection and identification of the P. falciparum and P. vivax involved genomic deoxyribonucleic acid (DNA) extraction by the Chelex-100 method and target amplification by qPCR in the molecular laboratory at Sokoru TIDRC, Jimma Zone, Southwest Ethiopia. The genomic DNA of the Plasmodium species from the DBSs of capillary, placental, and cord blood collected on a Whatman filter paper was extracted using the Chelex-100 method. The PCR assay targeting species-specific 18S rRNA gene in the extracted DNA was done by real-time qPCR using QuantStudio™ 3 System instrument (Applied Biosystems Inc., USA). The qPCR amplification process involved the use of forward and reverse primer pair sequences and probes that were precisely designed to amplify the 18S rRNA genes for simultaneous detection and identification of both P. falciparum and P. vivax [53, 54]. For quality control purposes, a DNA of P. falciparum isolates (MR4) as a positive, and molecular grade water as a negative control were included in the run. The qPCR amplification reaction was done on a total reaction volume of 12 µl in a 96-well PCR plate. The qPCR mix was prepared by adding 6 µl (× 2) of PerfeCTa master mix [PerfeCTa^® qPCR ToughMix^® Low ROX™, Lot number: 66181991 (Quantabio)], 0.5 µl (× 2) each of the Pf-Fam and Pv-NED probes, 0.4 µl (× 4) each of the forward and reverse primers, 1.4 µl of molecular grade water, and a 2 µl of the extracted target DNA into each PCR plate well. The thermal cycling conditions involved a hold stage at 50 °C for 2 min and initial denaturation at 95 °C for 2 min, followed by an amplification stage at 95 °C for 3 s and 60 °C for 30 s for 45 cycles. A Ct value between 12 to 40 with a sigmoidal curve was considered positive [27, 34, 55].

Diagnosis of placental malaria from placental biopsy by histopathology

Diagnosis of PM using histopathological method involved a microscopic examination of an H&E-stained 5 µm thick section of neutral-buffered formalin-fixed paraffin-embedded (NBFFPE) placental biopsy smear [45, 56–58]. Briefly, the histopathological diagnosis of PM involved collecting 2 cm³ tissue biopsy from two places in the middle of the distance from cord insertion to the periphery of the fresh placenta on its maternal side → fixing it with 10% NBF → grossing it → processing it (through fixing, dehydrating, clearing and impregnating) → embedding it in a paraffin wax → sectioning it into 5 µm slices using microtome → attaching/smearing it onto a labeled glass-slide → staining it with H&E stains → examining it at 400X magnification under a bright-field microscope for the presence of Plasmodium-iRBCs and/or haemozoin pigment and/or inflammatory cells (especially monocytes and macrophages) and/or fibrinoid region as well as syncytial knotting by pathologists and haematologist [56–59].

Finally, microscopic examination findings were reported as follows:

1. Negative: No evidence of Plasmodium-iRBCs or inflammatory cells or haemozoin pigment in the intervillous space after examining 40 fields;

2. Active-acute infection: presence of Plasmodium-iRBCs and few haemozoin pigments in the iRBCs, and inflammatory cells in the intervillous space, but no haemozoin pigment or inflammatory cells trapped inside fibrin deposit in the absence of iRBCs;

3. Active-chronic infection: presence of Plasmodium-iRBCs, and inflammatory cells in the intervillous space, and large amount of haemozoin pigment inside inflammatory cells and/or inside fibrin;

4. Past infection: presence of haemozoin pigment and inflammatory cells in fibrin deposits with no Plasmodium-iRBCs in the intervillous space. Past infection was considered as negative for this diagnostic accuracy study [34, 45, 56–59].

Data quality control

Before data collection, training on blood specimens and data collection was provided to the data collectors by the research team. All laboratory tests were performed strictly following standard operating procedures, job aids, and manufacturers’ instructions, ensuring acceptable quality control results and under close supervision of the research team. Two trained Medical Laboratory Technologists were blinded for the results of both index and reference tests when conducting microscopic examination. Any discrepant results in microscopy were resolved by a third examiner.

Statistical analysis

The collected data were appropriately cleaned, coded, and entered into Epidata version 3.1. Then, data were exported into Statistical Package for Social Science Version 26.0 (SPSS, IL, USA), Microsoft Excel Version 19.0 (Microsoft Corp., USA), and STATA Version 17.0 (Stata Corp., TX, USA), by which statistical analyses were conducted after checking for completeness and consistency. Diagnostic performance metrics such as sensitivity, specificity, positive and negative predictive values with their respective 95% CIs, were calculated using Buderer’s formula [41, 42]. (Supplement Eq. 2).

Cohen’s Kappa Coefficient and ROC analysis were conducted to determine the agreement and accuracy of tests, respectively of tests. Kappa values ranged from 0.0 to 1.0, showing the pattern of agreement between the index and the reference tests [60]. Besides, the area under the ROC curve (AUC) with its 95% confidence interval (CI) was determined by a non-parametric method. The AUC of a diagnostic test must be ˃0.5 and ≥ 0.8 to be considered a clinically meaningful and acceptable test, respectively [32, 33]. Blood film light microscopy, blood qPCR, and histopathology were interchangeably used as an index test and reference test to generate multidimensional comprehensive data. Missing data on index and reference tests (particularly for qPCR) were handled by analyzing only available cases [61, 62]. A p < 0.05 was considered statistically significant, and findings were illustrated in texts, tables, and graphs, mainly tabular presentation of diagnostic performance metrics followed by graphical depiction by ROC curve.

Ethical consideration

The research protocol was reviewed and approved by the Institutional Review Board (IRB) of the Institute of Health, Jimma University (Ref: JUIH/IRB/59/22). Besides, support and permission letters to conduct this study were collected from Jimma University, Gambella Peoples’ National Regional State Bureau of Health, and Majang Zone Health Department, and accordingly delivered to the included health institutions. After explaining the purpose and process of the study, a written informed consent was obtained from each pregnant and parturient woman before starting specimen and data collection. All participants found positive for malaria were linked to their respective health facility for appropriate treatment. Finally, data were anonymized through coding to ensure their confidentiality.

Operational definitions

• AUC value of: < 0.50, 0.50–0.59, 0.60–0.69, 0.70–0.79, 0.80–0.89, and ≥ 0.90 was considered as not suitable for utility, fail, poor, fair, good, and excellent accuracy, respectively [32, 33].

• Kappa value of: ≤ 0.0, 0.10–0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80, and 0.81–1.00 was considered as no, poor, fair, moderate, good, and very good agreement, respectively [60].

• MiP: Detection of Plasmodium infection in a pregnant and/or parturient woman from peripheral and/or placental and/or cord blood by malaria RDT and/or malaria microscopy and/or malaria qPCR and/or from placental biopsy by malaria histopathology test [3, 57, 58].

• PM: Detection of Plasmodium infection in a parturient woman from placental blood by malaria RDT and/or malaria microscopy and/or malaria qPCR and/or detection (from placental biopsy) of Plasmodium parasite-iRBCs and/or haemozoin pigment in inflammatory immune cells and/or haemozoin in fibrin deposits in the intervillous space of the placenta by malaria histopathology test [56–58].

• Primigravida: Pregnant for the first time [63].

• Primipara: Gave birth only once [63].

• Past infection in placental histopathology was considered negative for this particular study.

• Parasitaemia: Presence of asexual-stage Plasmodium parasite in thick and/or thin blood films [50–52, 64].

• RDT and microscopy combined: The result of participant was considered as negative, if both these tests were negative. But, if both tests or either of these tests were positive, then the final result was considered as positive.

• Mixed species infection with RDT: RDT result with three bands was considered as a mixed species infection, if it was confirmed as a mixed species infection by microscopic examinations two independent of two readers.

Results

Background characteristics of pregnant and parturient women

Four hundred and sixty pregnant women participated in this study. The median age of the pregnant women was 26 years [inter-quartile range (IQR): 22–30] with a minimum and maximum of 18 and 42 years, respectively. The majority of them were housewives (60.4%), urban residents (43.0%), and attended formal education (73.7%). The median gestational age of the pregnant women was 23 weeks (IQR: 16–29) with a minimum and maximum of 6 and 41 weeks, respectively. Half (50.7%) of the participants had at least two ANC contacts with a maximum of seven contacts. The majority of the participants owned at least one ITN (67.6%), and their home was sprayed with IRS (54.6%). Axial body temperature of pregnant women ranged from 35.6 °C to 38.2 °C with a mean of 36.8 °C (± 1.6). The majority (87.0%) of them were asymptomatic for malaria (Table 1).

Table 1.

Socio-demographic, obstetric, and anti-malarial intervention data of pregnant women in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 460)

Variable	Frequency	%
Age
≤ 20	73	15.9
21–25	138	30.0
26–30	151	32.8
≥ 31	98	21.3
Educational status
Unable to read and write	121	26.3
Primary	130	28.3
Secondary	164	35.7
College and above	45	9.7
Marital status
Single	70	15.2
Married	390	84.8
Occupation
Student	42	9.1
Housewife	278	60.4
Employed	140	30.5
Residence
Rural	262	57.0
Urban	198	43.0
ITN availability with quantity
0	149	32.4
1	235	51.1
≥ 2	76	16.5
Slept under ITN during the previous night
No	246	53.5
Yes	214	46.5
House sprayed with IRS within the previous year
No	209	45.4
Yes	251	54.6
Had a history of malaria within the previous year
No	256	55.7
Yes	204	44.3
Trimester
1 st	69	15.0
2nd	253	55.0
3rd	138	30.0
Gravidity
Primigravida	177	38.5
Secundigravida	82	17.7
Multigravida	201	43.8
Parity
Nullipara	194	42.3
Primipara	83	18.0
Multipara	183	39.7
Frequency of an ANC contact during the index pregnancy
1	227	49.3
≥ 2	233	50.7

One hundred and eighty parturient women participated in this study. The minimum and maximum of the parturient women’s age were 18 and 42 years, respectively, with the median of 26 years (IQR: 21–30). The majority of the parturient women were housewives (65.6%) and urban residents (59.4%). Approximately 37.8% and 47.8% of the parturient women were primigravida and primipara, respectively. The majority of them had at least one ANC contact (73.9%), and at least one ITN (72.8%). About half of the parturient women spent the previous night under ITN, and their home was sprayed with IRS. The axial body temperature of the parturient women ranged from 35.6 °C to 38.4 °C with a mean of 37.1 °C (± 0.46). (Detail was published elsewhere) [34].

Malaria positivity rate among pregnant women by different tests

The overall malaria test positivity rate among pregnant women was 21.1% (97/460, 95% CI 17.4–25.1). From the 97 malaria-positive pregnant women, 57, 32, and 8 have P. falciparum, P. vivax, and mixed infection, respectively. The asexual stage Plasmodium parasite density ranged from 64 p/µl to 127,360 p/µl, with a geometric mean density of 4,083.9 p/µl (95% CI 1351.4–6815.7). The positivity rate of RDT, microscopy and qPCR to detect Plasmodium infection in peripheral blood of the pregnant women was 17.2% (79/460, 95% CI 13.8–20.9), 18.0% (83/460, 95% CI 14.6–21.9), and 24.6% (35/142, 95% CI 17.8–32.6), respectively. Based on available results for all tests (n = 142), the concurrently positive and negative result rate among the three tests was 12.6% (n = 18) and 73.9% (n = 105), respectively, while the discordance rate was 13.5% (n = 19) (Fig. 2).

Fig. 2.

Venn diagram showing malaria positive results by RDT, microscopy, and qPCR in peripheral blood of pregnant women in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 142)

Malaria positivity rate among parturient women by different tests

The overall malaria test positivity rate among parturient women was 28.9% (52/180, 95% CI 22.4–36.1). From the 52 malaria-positive parturient women, 44 have peripheral blood infection (P. falciparum = 27, P. vivax = 11 and mixed = 6), and 38 have placental blood infection (P. falciparum = 25, P. vivax = 8 and mixed = 5). The asexual stage Plasmodium parasite density in peripheral blood ranged from 480 p/µl to 114,600 p/µl, with a geometric mean density of 6,790.2 p/µl (95% CI 4508.7–10,226.1). The asexual stage Plasmodium parasite density in placental blood ranged from 192 p/µl to 102,840 p/µl, with a geometric mean density of 5007.2 p/µl (95% CI 2927.5–8564.5). The positivity rate of malaria RDT, microscopy and qPCR to detect Plasmodium infection in the peripheral blood of the parturient women was 17.8% (32/180, 95% CI 12.1–23.4), 21.2% (38/180, 95% CI 15.1–27.1), and 28.3% (17/60, 95% CI 16.6–40.1), respectively. The positivity rate of malaria RDT, microscopy, qPCR, and histopathology to detect Plasmodium infection in the placental samples was 16.1% (29/180, 95% CI 11.4–22.3), 17.8% (32/180, 95% CI 12.8–24.1), 26.7% (16/60, 95% CI 16.7–39.6), and 24.5% (44/180, 95% CI 18.4–31.4), respectively. The positivity rate of malaria RDT, microscopy, and qPCR to detect Plasmodium infection in the cord blood was 2.8% (5/180, 95% CI 1.1–6.0), 3.9% (7/180, 95% CI 1.7–7.4), and 5.0% (3/60, 95% CI 1.3–12.5), respectively. In 5.0% (n = 9), 4.5% (n = 8), and 1.7% (n = 3) of the parturient women, the Plasmodium infection was detected in all, only placental, and only peripheral specimens, respectively (Supplement Fig. 1).

Based on the available results for all tests of all specimen types (n = 60), the concurrently positive and negative result rate was 16.6% (n = 10) and 66.7% (n = 40) among the three tests in peripheral blood, 13.4% (n = 8) and 66.6% (n = 40) among the four tests in placental specimens, and 1.6% (n = 1) and 95.0% (n = 57) among the three tests in cord blood, respectively. The discordance rate among these tests was 16.6% (n = 10), 20.0% (n = 12), and 3.4% (n = 2) in peripheral blood, placental specimen, and cord blood, respectively (Fig. 3).

Fig. 3.

Venn diagram showing malaria positive results by RDT, microscopy, qPCR and histopathology (B only) in peripheral blood (A), placental samples (B), placental blood (C), and cord blood (D) of parturient women in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 60)

Malaria positivity rate in combined peripheral blood of pregnant and parturient women

The overall malaria test positivity rate in the combined peripheral blood of pregnant and parturient women was 22.0% (141/640, 95% CI 18.9–25.14). The positivity rate of malaria RDT, microscopy and qPCR to detect Plasmodium infection in peripheral blood of pregnant and parturient women combined was 17.3% (111/640, 95% CI 14.5–20.5), 18.9% (121/640, 95% CI 15.9–22.2), and 25.7% (52/202, 95% CI 19.9–32.3), respectively. Based on the available results for all tests (n = 202), the concurrently positive and negative result rate among the three tests was 13.8% (n = 28) and 71.2% (n = 145), respectively, while the discordance rate was 15.0% (n = 29) (Fig. 4).

Fig. 4.

Venn diagram showing malaria positive results by RDT, microscopy, and qPCR in peripheral blood of pregnant women and parturient women in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 202)

Diagnostic test accuracy study among pregnant women

Performance of index tests using peripheral blood microscopy of pregnant women as reference

Considering peripheral blood film microscopy as reference, the sensitivity of RDT, and qPCR to detect MiP was 84.3% (95% CI 81.0–87.6), and 96.3% (95% CI 93.2–99.4), respectively, as well as the specificity of RDT, and qPCR to detect MiP was 97.6% (95% CI 96.2–99.0), and 92.2% (95% CI 87.7–96.6), respectively to detect Plasmodium infection in peripheral blood of pregnant women. Peripheral blood RDT and microscopy combined showed a very good overall performance scoring sensitivity of 84.3% (95% CI 81.0–87.6), specificity of 97.6% (95% CI 96.2–99.0), agreement of 0.835 (95% CI 0.768–0.901), and overall accuracy 0.974 (95% CI 0.949–0.998). (Supplement Table 1 and Supplement Fig. 2).

Performance of index tests using peripheral blood qPCR of pregnant women as reference

Considering peripheral blood qPCR as reference, the combination of RDT and microscopy showed a high performance with the sensitivity and specificity of 88.6% (95% CI 78.2–96.6), and 98.1% (95% CI 95.4–99.8), respectively, to detect MiP. It also showed an excellent diagnostic accuracy (AUC = 0.934 95% CI 0.870–0.997), and a very good agreement with the reference test (K = 0.884 95% CI 0.793–0.974). But, when these index tests were evaluated as a single, their performance was lower than their combination (sensitivity, specificity, AUC, and K of microscopy and RDT were 74.3%, 99.1%, 0.795, and 0.867, and 65.7%, 99.1%, 0.724, and 0.824, respectively), though not statistically significant (Table 2 and Fig. 5).

Table 2.

Diagnostic performance of index tests to detect Plasmodium infection among pregnant women using peripheral blood qPCR as reference test in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022-March 2023 (n = 142)

Index test methods	Peripheral blood qPCR of pregnant women as a reference test method (MiP = 142)
	TP	FP	FN	TN	Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	Accuracy (95% CI)	Kappa (95% CI)	AUC (95% CI)	p
1. RDT	23	1	12	106	65.7% (57.9–73.5)	99.1% (97.5–100.6)	95.8% (92.5–99.1)	89.8% (84.8–94.8)	90.8% (84.8–95.0)	0.724 (0.584–0.863)	0.824 (0.725–0.923)	< 0.0001
2. Microscopy	26	1	9	106	74.3% (67.1–81.4)	99.1% (97.5–100.6)	96.3% (93.2–99.4)	92.2% (87.7–96.6)	93.0% (87.5–96.6)	0.795 (0.673–0.916)	0.867 (0.778–0.956)	< 0.0001
3. Microscopy & RDT combined	31	2	4	105	88.6% (78.2–96.6)	98.1% (95.4–99.8)	93.9 (83.6–98.5)	96.3% (91.2–98.8)	95.8 (91.0–98.5)	0.884 (0.793–0.974)	0.934 (0.870–0.997)	< 0.0001

Fig. 5.

ROC curve showing the diagnostic performance of index tests to detect Plasmodium infection among pregnant women using peripheral blood qPCR as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 142)

Diagnostic test accuracy study among parturient women

Performance of index tests using peripheral blood microscopy of parturient women as reference

Considering peripheral blood microscopy as reference, peripheral blood RDT and microscopy combined scored the highest performance, with almost perfect sensitivity, specificity, PPV, NPV, and accuracy to detect Plasmodium infection among parturient women. Peripheral blood RDT and microscopy combined demonstrated a very good agreement with the reference test (K = 0.983 95% CI 0.951–1.014), and a very good accuracy (AUC = 0.989 95% CI 0.964–1.014). All index tests showed a poor agreement with the reference test, and failed the diagnostic accuracy test (AUC < 0.60 p ˃ 0.05) to detect Plasmodium infection in the cord blood, in which they scored the lowest sensitivity. All index tests were at least in a good agreement (K > 0.6) with the reference test, and demonstrated at least a good overall accuracy (AUC ≥ 0.7) to detect Plasmodium infection in peripheral and placental blood of the parturient women as well as met the minimum threshold for clinical utility (AUC ≥ 0.8) when their performance was evaluated using peripheral blood microscopy as a reference test (Supplement Table 2 and Supplement Fig. 3).

Performance of index tests using placental blood microscopy of parturient women as reference

The overall sensitivity and specificity of RDT to detect malaria among parturient women were 87.5% (95% CI 82.6–92.3) and 95.3% (95% CI 92.2–98.3), respectively, using placental blood microscopy as reference. Peripheral blood microscopy and a combination of RDT and microscopy demonstrated an excellent diagnostic performance by scoring above 95.0% sensitivity and specificity, an excellent accuracy (AUC > 0.9), and a very good agreement with the reference test. Placental histopathology showed a moderate agreement and a very good diagnostic accuracy. All index tests showed a poor agreement with the reference test, and failed the diagnostic test accuracy evaluation (AUC < 0.60 p ˃ 0.05) to detect Plasmodium infection in cord blood by scoring the lowest sensitivity. (Supplement Table 3 and Supplement Fig. 4).

Table 3.

Diagnostic performance of index tests to detect Plasmodium infection among parturient women using peripheral blood qPCR as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022-March 2023 (n = 60)

Index test methods		Peripheral blood qPCR of parturient women as a reference test method (PM = 60)
		TP	FP	FN	TN	Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	Accuracy (95% CI)	Kappa (95% CI)	AUC (95% CI)	p
1. RDT	Peripheral blood	10	2	7	41	58.8% (46.3–71.3)	95.3% (90.0–100.7)	83.3% (73.9–92.2)	85.4% (76.5–94.3)	85.0% (73.4–92.9)	0.595 (0.361–0.828)	0.771 (0.618–0.924)	< 0.0001
	Placental blood	8	3	9	40	47.1% (34.4–59.7)	93.0% (86.6–99.4)	72.7% (61.4–84.0)	81.6% (71.8–91.4)	80.0% (67.6–89.2)	0.449 (0.192–0.705)	0.700 (0.537–0.864)	0.016
	Cord blood	1	0	16	43	5.9% (−0.07–11.8)	100.0% (100.0–100.0)	100.0% (100.0–100.0	72.9% (61.6–84.1)	73.3% (60.3–83.9)	0.082 (−0.0–0.235)	0.529 (0.362–0.696)	0.730
	Overall	11	2	6	40	64.7% (52.6–76.8)	93.0% (86.6–99.4)	78.6% (68.2–88.9)	87.0% (78.4–95.6)	85.0% (73.4–92.9)	0.610 (0.380–0.839)	0.789 (0.642–0.935)	< 0.0001
2. Microscopy	Peripheral blood	12	2	5	41	70.6% (59.0–82.1)	95.3% (90.0–100.7)	85.7% (76.8–94.5)	89.1% (77.2–94.8)	88.3% (77.4–95.1)	0.697 (0.489–0.904)	0.830 (0.693–0.967)	< 0.0001
	Placental blood	12	1	5	42	70.6% (59.0–82.1)	97.7% (93.9–101.5)	92.3% (85.5–99.0)	89.4% (81.5–97.1)	90.0% (79.4–96.2)	0.735 (0.539–0.931)	0.841 (0.705–0.977)	< 0.0001
	Cord blood	1	0	16	43	5.9% (−0.07–11.8)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	72.9% (61.6–84.1)	73.3% (60.3–83.9)	0.082 (−0.070–0.235)	0.529 (0.362–0.696)	0.730
	Overall	13	2	4	41	76.5% (65.7–87.2)	95.3% (90.0–100.7)	86.7% (78.0–95.2)	91.1% (83.9–98.3)	90.0% (79.4–96.2)	0.745 (0.552–0.937)	0.859 (0.733–0.986)	< 0.0001
3. Microscopy & RDT Combined	Peripheral blood	12	3	5	40	70.6% (59.0–82.1)	93.0% (86.6–99.4)	80.0% (69.9–90.1)	88.9% (80.9–96.8)	86.7% (75.4–94.1)	0.660 (0.444–0.875)	0.818 (0.680–0.956)	< 0.0001
	Placental blood	12	3	5	40	70.6% (59.0–82.1)	93.0% (86.6–99.4)	80.0% (69.9–90.1)	88.9% (80.9–96.8)	86.7% (75.4–94.1)	0.660 (0.444–0.875)	0.818 (0.680–0.956)	< 0.0001
	Cord blood	1	0	16	43	5.9% (−0.07–11.8)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	72.9% (61.6–84.1)	73.3% (60.3–83.9)	0.082 (−0.070–0.235)	0.529 (0.362–0.696)	0.730
4. qPCR	Placental blood	15	1	2	42	88.2% (80.1–96.4)	97.7% (93.9–101.5)	93.8% (87.6–99.8)	95.5% (90.2–100.7)	95.0% (86.1–98.9)	0.875 (0.737–1.012)	0.930 (0.836–1.024)	< 0.0001
	Cord blood	3	0	14	43	17.6% (8.0–27.3)	100.0% (100.0–100.0	100.0% (100.0–100.0	75.4% (64.5–86.3)	76.7% 63.9–86.6)	0.235 (0.009–0.460)	0.588 (0.417–0.759)	0.311
	Overall	17	2	0	41	100.0% (100.0–100.0	95.3% (90.0–100.7)	89.5% (81.1–97.2)	100.0% (100.0–100.0)	96.7% (88.4–99.6)	0.921 (0.813–1.028)	0.977 (0.939–1.014)	< 0.0001
5. Histopathology of placental biopsy		13	1	4	42	76.5% (65.7–87.2)	97.7% (93.8–101.5)	92.9% (86.3–99.3)	91.3% (84.1–98.4)	91.7% (81.6–97.2)	0.783 (0.602–0.963)	0.824 (0.693–0.956)	< 0.0001

Performance of index tests using peripheral blood qPCR of parturient women as reference

When peripheral blood qPCR was used as a reference test, placental blood qPCR with its highest sensitivity, and the largest AUC and Kappa score showed the best performance to detect Plasmodium infection among parturient women. On the other hand, cord blood RDT and microscopy, with their lowest sensitivity, smallest AUC, and Kappa score, showed the poorest performance to detect Plasmodium infection among parturient women. Placental histopathology demonstrated a good performance relative to other tests considering its high sensitivity (76.5% 95% CI 65.7–87.2), specificity (97.7% 95% CI 93.8–101.5), and AUC (0.824 95% CI 0.693–0.956) (Table 3 and Fig. 6).

Fig. 6.

ROC curve showing the diagnostic performance of index tests to detect Plasmodium infection among parturient women using peripheral blood qPCR as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 60)

Performance of index tests using placental blood qPCR of parturient women as reference

When placental blood qPCR was used as a reference test to assess the diagnostic performance of index tests to detect Plasmodium infection among parturient women, cord blood RDT showed the lowest performance with its lowest sensitivity (6.3% 95% CI 0.13–12.3), and AUC (0.531 95% CI 0.361–0.702), whereas peripheral blood qPCR showed the highest performance with its highest sensitivity (93.7% 95% CI 87.6–99.8), specificity (95.5% 95% CI 90.2–100.7), and AUC (0.946 95% CI 0.868–1.024). Placental histopathology showed the best agreement with the reference test, and a very good overall accuracy. All index tests showed a poor agreement with the reference test, and failed the accuracy test (AUC < 0.60 p˃ 0.05) to detect Plasmodium infection in the cord blood, in which they scored the lowest sensitivity (Table 4 and Fig. 7).

Table 4.

Diagnostic performance of index tests to detect Plasmodium infection among parturient women using placental blood qPCR as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022-March 2023 (n = 60)

Index test methods		Placental blood qPCR of parturient women as a reference test method (PM = 60)
		TP	FP	FN	TN	Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	Accuracy (95% CI)	Kappa (95% CI)	AUC (95% CI)	p
1. RDT	Peripheral blood	11	1	5	43	68.8% (57.6–79.2)	97.7% (93.9–101.5)	91.7% (84.6–98.6)	89.6% (81.8–97.3)	90.0% (79.4–96.2)	0.722 (0.516–0.927)	0.832 (0.690–0.975)	< 0.0001
	Placental blood	9	2	7	42	56.3% (43.7–68.8)	95.5% (90.2–100.7)	81.8% (72.0–91.6)	85.7% (76.8–94.5)	85.0% (73.4–92.9)	0.574 (0.331–0.817)	0.759 (0.599–0.918)	0.002
	Cord blood	1	0	15	44	6.3% (0.13–12.3)	100.0% (100.0–100.0)	100.0% (100–100.0)	74.6% (63.5–85.6)	75.0% (62.1–85.3)	0.089 (−0.075–0.253)	0.531 (0.361–0.702)	0.720
	Overall	12	2	4	42	75.0% (64.0–85.9)	95.5% (90.2–100.7)	85.7% (76.8–94.5)	91.3% (84.1–98.4)	90.0% (79.4–96.2)	0.734 (0.534–0.934)	0.852 (0.719–0.985)	< 0.0001
2. Microscopy	Peripheral blood	13	1	3	43	81.3% (71.3–91.1)	97.7% (93.9–101.5)	92.9% (86.3–99.3)	93.5% (86.3–99.3)	93.3% (83.8–98.1)	0.822 (0.655–0.988)	0.895 (0.778–1.012)	< 0.0001
	Placental blood	13	0	3	44	81.3% (71.3–91.1)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	93.6% (87.4–99.8)	95.0% (86.1–98.9)	0.864 (0.715–1.013)	0.906 (0.791–1.022)	< 0.0001
	Cord blood	1	0	15	44	6.3% (0.13–12.3)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	74.6% (63.5–85.6)	75.0% (62.1–85.3)	0.089 (0.075–0.253)	0.531 (0.361–0.702)	0.720
	Overall	14	1	43	45	87.5% (79.3–95.8)	97.7% (93.9–101.5)	93.3% (87.0–99.6)	95.5% (90.2–100.7)	95.0% (86.1–98.9)	0.870 (0.727–1.013)	0.926 (0.827–1.025)	< 0.0001
3. Microscopy & RDT combined	Peripheral blood	13	2	3	42	81.3% (71.3–91.1)	95.5% (90.2–100.7)	86.7% (78.0–95.2)	93.3% (87.0–99.6)	91.7% (81.6–97.2)	0.783 (0.602–0.963)	0.884 (0.764–1.003)	< 0.0001
	Placental blood	13	2	15	42	81.3% (71.3–91.1)	95.5% (90.2–100.7)	86.7% (78.0–95.2)	93.3% (87.0–99.6)	91.7% (81.6–97.2)	0.783 (0.602–0.963)	0.884 (0.764–1.003)	< 0.0001
	Cord blood	1	0	15	44	6.3% (0.13–12.3)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	74.6% (63.5–85.6)	75.0% (62.1–85.3)	0.089 (−0.075–0.253)	0.531 (0.361–0.702)	0.720
4. qPCR	Peripheral blood	15	2	1	42	93.7% (87.6–99.8)	95.5% (90.2–100.7)	88.2% (80.1–96.4)	97.7% (93.9–101.5)	95.0% (86.1–98.9)	0.875 (0.737–1.012)	0.946 (0.868–1.024)	< 0.0001
	Cord blood	3	0	3	44	18.7% (8.8–28.6)	100.0% (100.0–100.0)	100.0% (100–100.0)	77.2% (66.6–87.8)	78.3% (65.8–87.9)	0.253 (0.014–0.492)	0.594 (0.419–0.769)	0.294
	Overall	16	3	0	41	100.0% (100.0–100.0)	93.2% (86.8–99.5)	84.2% (74.9–93.4)	100.0% (100.0–100)	95.0% (86.1–98.9)	0.879 (0.747–1.010)	0.966 (0.921–1.011)	< 0.0001
5. Histopathology of placental biopsy		14	0	2	42	87.5% (79.1–95.8)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	95.7 (90.5–100.8)	96.7 (88.4–99.6)	0.911(0.791–1.030)	0.892 (0.785–0.999)	< 0.0001

Fig. 7.

ROC curve showing the diagnostic performance of index tests to detect Plasmodium infection among parturient women using placental blood qPCR as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 60)

Performance of index tests using placental histopathology of parturient women as reference

When placental histopathology was used as reference, placental blood qPCR showed the highest performance by scoring the highest sensitivity (100.0% 95% CI 100.0–100.0), and accuracy (AUC = 0.978 95% CI 0.943–1.014), whereas, cord blood RDT showed the lowest overall performance by scoring the lowest sensitivity (11.4% 95% CI 6.7–16.0), and accuracy (AUC = 0.536 95% CI 0.356–0.715). All index tests showed a poor agreement with the reference test, and failed the accuracy test (AUC < 0.60 p˃ 0.05) to detect Plasmodium infection in cord blood. The accuracy of microscopy and qPCR to detect Plasmodium infection in peripheral and placental blood of the parturient women was generally very good (AUC ≥ 0.8) (Table 5 and Fig. 8).

Table 5.

Diagnostic performance of index tests to detect Plasmodium infection among parturient women using placental biopsy histopathology as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022-March 2023 (n = 180)

Index test methods		Placental biopsy histopathology from parturient women as a reference test method (PM = 180)
		TP	FP	FN	TN	Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	Accuracy (95% CI)	Kappa (95% CI)	AUC (95% CI)	p
1. RDT	Peripheral blood	28	4	16	132	63.6% (56.6–70.6)	97.1% (94.6–99.5)	87.5% (82.6–92.3)	89.2% (84.6–93.7)	88.9% (84.6–93.7)	0.669 (0.535–0.802)	0.789 (0.629–0.949)	< 0.001
	Placental blood	25	4	19	132	56.8% (49.6–64.0)	97.1% (94.6–99.5)	86.2% (81.1–91.2)	87.4% (82.5–92.2)	87.2% (82.5–92.2)	0.609 (0.467–0.750)	0.753 (0.585–0.982)	0.003
	Cord blood	5	0	39	136	11.4% (6.7–16.0)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	77.7% (71.6–83.8)	78.3% (71.6–84.1)	0.162 (0.034–0.289)	0.536 (0.356–0.715)	0.740
	Overall	30	5	14	131	68.2% (61.4–74.9)	96.3% (93.5–99.1)	85.7% (80.6–90.8)	90.3% (86.0–94.6)	89.4% (84.1–93.5)	0.693 (0.565–0.820)	0.814 (0.664–0.963)	< 0.001
2. Microscopy	Peripheral blood	34	4	10	132	77.3% (71.1–83.4)	97.1% (94.6–99.5)	89.5% (84.3–93.5)	93.0% (89.2–96.7)	92.2% (87.3–95.7)	0.779 (0.670–0.889)	0.860 (0.726–0.994)	< 0.001
	Placental blood	30	2	14	134	68.2% (61.4–74.9)	98.5% (96.7–100.3)	93.8% (90.2–97.3)	90.5% (86.2–94.8)	91.1% (85.9–94.8)	0.735 (0.613–0.856)	0.918 (0.806–1.030)	< 0.001
	Cord blood	7	0	37	136	15.9% (10.5–21.2)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	78.6% (72.6–84.6)	79.4% (72.8–85.1)	0.222 (0.080–0.363)	0.536 (0.356–0.715)	0.697
	Overall	35	4	9	132	79.5% (73.6–85.4)	97.1% (94.6–99.5)	89.7% (85.3–94.2)	93.6% (90.0–97.2)	92.8% (87.9–96.1)	0.797 (0.691–0.903)	0.896 (0.781–1.011)	< 0.001
3. Microscopy & RDT combined	Peripheral blood	34	5	10	131	77.3% (71.1–83.4)	96.3% (93.5–99.1)	87.2% (82.3–92.0)	92.9% (89.1–96.6)	91.7% (86.6–95.2)	0.765 (0.653–0.876)	0.849 (0.714–0.985)	< 0.001
	Placental blood	31	4	13	132	70.5% (63.8–77.1)	91.7% (94.6–99.5)	88.6% (83.9–93.2)	91.0% (86.8–95.2)	90.6% (85.3–94.4)	0.725 (0.603–0.846)	0.896 (0.781–1.011)	< 0.001
	Cord blood	7	0	37	136	15.9% (10.5–21.2)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	78.6% (72.6–84.6)	79.4% (72.8–85.1)	0.222 (0.080–0.363)	0.536 (0.356–0.715)	0.740
4. qPCR	Peripheral blood	13	4	1	42	92.9% (86.3–99.3)	91.3% (84.1–98.4)	76.5% (65.7–87.2)	97.7% (93.9–101.5)	91.7% (81.6–97.2)	0.783 (0.602–0.963)	0.921 (0.829–1.013)	< 0.001
	Placental blood	14	2	0	44	100.0% (100.0–100.0)	95.7% (90.5–100.8)	87.5% (79.1–95.8)	100.0% (100.0–100.)	96.7% (88.4–99.6)	0.911 (0.791–1.030)	0.978 (0.943–1.014)	< 0.001
	Cord blood	3	0	11	46	21.4% (11.0–31.8)	100.0% (100.0–100.0)	100.0% (100.0–100.0)	80.7% (70.7–90.7)	81.7% (69.5–90.5)	0.295 (0.028–0.561)	0.607 (0.422–0.792)	0.328
	Overall	22	14	5	19	100.0% (100.0–100.0)	89.1% (81.2–97.0)	73.7% (62.5–84.8)	100.0% (100.0–100.)	91.7% (81.6–97.2)	0.793 (0.622–0.963)	0.946 (0.890–1.002)	< 0.001

Fig. 8.

ROC curve showing the diagnostic performance of index tests to detect Plasmodium infection among parturient women using placental histopathology as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 180)

Diagnostic test accuracy study among pregnant and parturient women combined

Performance of index tests considering peripheral blood microscopy of pregnant and parturient women as reference

When peripheral blood microscopy was used as reference, the combination of RDT and microscopy showed a very good performance with its highest sensitivity (100.0% 95% CI 100.0–100.0), and specificity (98.1% 95% CI 97.1–99.1) to detect Plasmodium infection in peripheral blood of pregnant and parturient women combined. It also showed a very good overall accuracy (AUC = 0.978 95% CI 0.959–0.997), and a very good agreement with the reference test (K = 0.951 95% CI 0.921–0.980) (Supplement Table 4 and Supplement Fig. 5).

Performance of index tests considering peripheral blood qPCR of pregnant and parturient women as reference

When peripheral blood qPCR of pregnant and parturient women combined was used as a reference test, the combination of RDT and microscopy showed a good performance with its highest sensitivity (82.7% 95% CI 77.5–87.9), and specificity (96.7% 95% CI 94.2–99.1) to detect Plasmodium infection in peripheral blood of the women. It also showed a good overall accuracy (AUC = 0.897 95% CI 0.834–0.960), and a very good agreement with the reference test (K = 0.814 95% CI 0.720–0.908) (Table 6 and Fig. 9).

Table 6.

Diagnostic performance of index tests to detect Plasmodium infection among pregnant and parturient women using peripheral blood qPCR as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022-March 2023 (n = 202)

Index test methods	Peripheral blood qPCR of pregnant and parturient women as a reference test method (MiP and PM = 202)
	TP	FP	FN	TN	Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	Accuracy (95% CI)	Kappa (95% CI)	AUC (95% CI)	p
1. RDT	33	3	19	147	63.5% (56.8–70.1)	98.0% (96.1–99.9)	91.7% (87.8–95.5)	88.6% (84.1–92.9)	89.1% (83.9–93.1)	0.683 (0.563–0.802)	0.807 (0.724–0.890)	< 0.001
2. Microscopy	38	3	14	147	73.1% (66.9–79.2)	98.0% (96.1–99.9)	92.7% (89.1–96.2)	91.3% (87.4–95.2)	91.6% (86.8–95.0)	0.764 (0.658–0.869)	0.855 (0.781–0.930)	< 0.001
3. Microscopy and RDT combined	43	5	9	145	82.7% (77.5–87.9)	96.7% (94.2–99.1)	89.6% (85.4–93.8)	94.2% (90.9–97.4)	93.1% (88.6–96.1)	0.814 (0.720–0.908)	0.897 (0.834–0.960)	< 0.001

Fig. 9.

ROC curve showing the diagnostic performance of index tests to detect Plasmodium infection among pregnant and parturient women using peripheral blood qPCR as reference in Majang Zone of Gambella Region, Southwest Ethiopia, November 2022–March 2023 (n = 202)

Factors affecting performance of malaria RDT and microscopy to detect Plasmodium infection among pregnant and parturient women using peripheral blood qPCR as reference

The sensitivity and specificity of RDT and microscopy were higher among participants who lack ITN, lack ANC contact and were symptomatic than their respective counterparts. However, these differences were not statistically significant (Supplement Fig. 6).

Discussion

This article presented the positivity rate and comparative performance of malaria diagnostic tests to detect Plasmodium infection in peripheral, placental, and cord blood, and placental biopsy among pregnant and parturient women in Majang Zone of Gambella Region, Southwest Ethiopia. It provided comprehensive data on the diagnostic performance of index tests to detect Plasmodium infection in these specimen types by alternatively using microscopy, qPCR, and histopathology as index and reference tests. Any malaria test using the umbilical cord blood specimen, and an RDT on any of the three blood specimen types, were considered only as an index test in this study. Positivity rate and diagnostic performance of malaria tests involved in this particular study are discussed as follows.

The overall malaria test positivity rate among pregnant and parturient women in Majang Zone was 21.1% and 28.9%, respectively. The positivity rate increased from malaria RDT to microscopy to PCR, although not statistically significant. Similarly, an increasing pattern of malaria positivity rate was observed from RDT to microscopy to qPCR, and from cord blood to placental blood to peripheral blood of parturient women. This pattern is conceivable considering the sensitivity of the diagnostic tests (from conventional RDT up to qPCR), the underlying flow pattern of Plasmodium infection (from peripheral up to cord blood), pattern of maternal immunological defense organization (weakened during pregnancy while trying to protect the fetus), and epidemiology of malaria in this particular study area (P. falciparum-dominated a high-transmission setting with relatively inadequate MiP mitigation and ANC service utilization) [4, 7–9, 65, 66]. The 21.1% overall malaria positivity rate among pregnant women recorded in the current study was concordant with the recently reported 21.3% positivity rate among pregnant women in Northwest Ethiopia [31]. The positivity rate of microscopy (18.0%) to detect Plasmodium infection in the peripheral blood of pregnant women in the current study was consistent with the 18.7% positivity rate of microscopy reported from a study conducted in the Democratic Republic of the Congo [67]. The positivity rate of malaria microscopy (21.2%), and qPCR (28.3%) to detect Plasmodium infection in peripheral blood of parturient women in the current study was nearly concordant to the reported positivity rate of microscopy (19.7%) and PCR (35.8%) from a study conducted in Colombia [68]. Likewise, the positivity rate of malaria qPCR to detect Plasmodium infection from placental blood recorded in the current study (26.7%) was consistent with the 27.4% positivity rate in placental blood reported from Colombia [68].

However, the positivity rate of malaria RDT (17.2%), microscopy (18.0%), and qPCR (24.6%) to detect Plasmodium infection in peripheral blood of pregnant women in the current study was higher than the positivity rate of malaria RDT (9.6%), microscopy (11.4%), and PCR (18.7%) among pregnant women reported from Northwest Ethiopia [30]. This variation might be due to the inclusion of both symptomatic and asymptomatic pregnant women and the coverage of both high and low transmission seasons during data collection period in the current study than the other study that was conducted during dry season by including only asymptomatic pregnant women, both of which might reduce the positivity rate [8, 9, 30]. The positivity rate of malaria RDT, microscopy, and qPCR to detect Plasmodium infection in peripheral blood of pregnant women was much higher than the positivity rate of cRDT (2.4%), microscopy (2.7%), and nPCR (4.2%) among pregnant women reported from Colombia [17]. This disparity might be due to the restriction of the evaluation to P. falciparum only, and the low parasitaemia found in the study conducted in Colombia, together with geographical variation [8, 9, 17]. The positivity rate of malaria RDT, microscopy, and qPCR to detect Plasmodium infection among pregnant women in the current study was much higher than the positivity rate of RDT (4.3%) and microscopy (8.8%) among pregnant women reported from Nigeria [69]. Moreover, in the same study, the positivity rate of peripheral blood microscopy (2.4%) and RDT (3.4%) at delivery among those women was even lower than the corresponding positivity rate of microscopy (21.2%) and RDT (17.8%) in the current study [69]. The observed difference might be explained by the inclusion of pregnant and parturient women only with asymptomatic infection, which is often less detectable by microscopy and RDT [11, 66, 69]. The positivity rate of RDT, microscopy, and qPCR in peripheral and placental blood recorded in the current study was higher than that was reported from a study conducted among parturient women in Benin [13]. The use of a combo RDT and relatively larger sample size in the current study, as well as geographical variation, might explain the observed difference [9, 13]. The positivity rate of malaria RDT, microscopy, and qPCR to detect Plasmodium infection in placental blood of parturient women in the current study was higher than the positivity rate of RDT (5.4%), microscopy (6.9%), and PCR (8.3%) reported from Burkina Faso [70]. The discrepancy might be due to the involvement of parturient women who used intermittent preventive treatment during pregnancy (IPTp), which can bring-about a dose-dependent reduction of the risk of malaria during later weeks of gestation and at parturiency through parasite clearance and/or lowered risk of placental sequestration, thereby potentially reducing parasite detectability and test positivity rate in peripheral and placental blood in that study [16, 63, 66, 70]. On the other hand, the positivity rate of malaria RDT (17.2%) and microscopy (18.0%) in peripheral blood of pregnant women in the current study was much lower than the positivity rate of malaria RDT (77.9%), and microscopy (69.2%) among pregnant women reported from Cameroon [71]. The discrepancy might be due to the involvement of only symptomatic pregnant women and relatively lower sample size in that study [21, 66, 71]. The positivity rate of RDT, microscopy, and qPCR in peripheral blood of pregnant women found in the current study was lower than the positivity rate of RDT (58.2%), microscopy (59.9%), and PCR (61.1%) reported from a study conducted in Nigeria [72]. The inclusion of only symptomatic women in that study can potentially increase the positivity rate might explain the discrepancy [21, 66, 72]. Moreover, the positivity rate of peripheral blood RDT, microscopy, and qPCR to detect Plasmodium infection among pregnant women in the current study was lower than that was recorded among pregnant women at first ANC enrolment in Uganda (peripheral blood RDT = 38.2%, microscopy = 36.2%, and PCR = 44.0%) [16]. The disparity might be due to malaria endemicity variation, whereby that study was conducted in a holoendemic area where 95% of the detected infection was P. falciparum species [7, 16].

The diagnostic performance of malaria tests recorded in the current study was consistent with findings reported from studies conducted in Ethiopia and other parts of sub-Saharan Africa [9, 16, 30, 31, 68, 73, 74]. When peripheral blood microscopy of pregnant women was used as a reference test, the 84.3% sensitivity, and 97.6% specificity, and very good agreement of RDT (K = 0.835) with the reference test was consistent with findings from a study conducted in Jawi District, Northwest Ethiopia that reported 83.3% sensitivity, 99.3% specificity, and 0.893 agreement [30]. The 100% sensitivity and 93.6% specificity of placental blood qPCR using placental blood microscopy as reference test recorded in the current study was similar to the 100% sensitivity and 95.1% specificity of placental blood qPCR compared to microscopy to detect Plasmodium infection in placental blood of parturient women in Jawi District, Northwest Ethiopia [31]. Moreover, the 78.3% sensitivity and 97.3% specificity of placental blood RDT recorded in the current study were similar to the 82.1% sensitivity and 97.4% specificity of placental blood RDT compared to microscopy to detect Plasmodium infection in placental blood of parturient women in the same study [31]. Considering peripheral blood qPCR as reference, the 74.3% sensitivity and 99.1% specificity of peripheral blood microscopy recorded in the current study were similar to 69.7% sensitivity and 98.4% specificity of peripheral blood microscopy reported from a study conducted among pregnant women in Uganda [16]. The performance of peripheral blood microscopy (76.3% sensitivity, and 92.1% specificity) among parturient women reported from a study conducted in Uganda [24], and the 67.0% sensitivity of qPCR to detect PM reported from a study conducted in Ghana [74] were consistent with the results of these similar index tests in the current study using placental histopathology as reference. Furthermore, the diagnostic performance of malaria RDT (81.3% sensitivity, and 95.9% specificity), and qPCR (92.3% sensitivity, and 89.4% specificity) recorded in the current study to detect Plasmodium infection in peripheral blood using placental blood microscopy as reference was nearly similar to the findings of a SRMA that reported the pooled sensitivity of peripheral blood RDTs (81.0%), and PCR (94.0%), as well as the pooled specificity of peripheral blood RDTs (94.0%) [9].

Nevertheless, the diagnostic performance of malaria tests demonstrated in the current study was inconsistent with findings reported from various studies conducted in Ethiopia, Colombia, Indonesia, and many countries in sub-Saharan Africa [13, 17, 21, 33, 63, 69–73, 75]. Using peripheral blood PCR as reference, the respective 67.5% and 74.3% sensitivities of peripheral blood RDT and microscopy recorded among pregnant women in the current study were higher than the respective 50.0% and 60.0% sensitivities of RDT and microscopy reported from Jawi District in Northwest Ethiopia [30]. Besides, these index tests in the current study were found to be in better agreement (K of RDT = 0.724 and K of microscopy = 0.795) with the reference test than that of the Jawi District study (K of RDT = 0.62 and K of microscopy = 0.71) [30]. Variation in malaria Epidemiology between the two study settings and the exclusion of pregnant women with malaria symptoms from the Jawi District study conducted during the dry season might have contributed to the observed discrepancy [9, 30]. The sensitivity of microscopy (74.3%) and RDT (65.7%) to detect Plasmodium infection in peripheral blood of pregnant women using peripheral blood qPCR as reference test in the current study was higher than the sensitivity of microscopy (59.0%), and RDT (53.8%) reported from a study conducted in Colombia [17], and the sensitivity of RDT and microscopy (both below 50%) reported from a study conducted in Indonesia using peripheral blood PCR as reference [75]. This disparity might be attributed to the lower prevalence and parasitaemia of the Plasmodium infection recorded in those studies than the current study [9, 17, 30, 75–77]. The sensitivity of peripheral blood RDT and microscopy of both pregnant and parturient women found in the current study using qPCR as reference was much higher than that was reported (ranged from 1.6 to 30.3%) from a study conducted in Nigeria [69]. The observed difference could be justified by the involvement of asymptomatic pregnant women with lower parasite density combined with the disproportionately lower prevalence of malaria compared to the reference test in that study. In fact, sensitivity and specificity metrics are affected by the prevalence of the disease under study [33, 69]. The sensitivity of malaria RDT in the current study was higher than that was reported from a study conducted in Benin, where the cRDTs demonstrated a lower sensitivity (44.2%) than the 65.7% found in the current study using PCR as reference [13]. The possible cause for this discrepancy might be due to the lower prevalence of MiP and the use of a single specie targeting (P. falciparum) type of RDT in that study unlike to the current study conducted in P. falciparum and P. vivax co-endemic area using a combo RDT targeting more than one species found a higher prevalence of MiP [8, 13]. The sensitivity of microscopy to detect Plasmodium infection in peripheral blood of parturient women found in the current study, considering peripheral blood qPCR as reference, was much higher (97.6%) than that was recorded (37.2%) in Mozambique [21]. Likewise, the same study reported a lower sensitivity of histopathology to detect PM (41.8% considering only active infections) than the 87.5% sensitivity recorded in the current study using qPCR as reference [21]. The presence of high parasitaemia in the current study than the mentioned study might justify the observed inconsistency. It is noteworthy that a higher parasitaemia increases the overall diagnostic performance of tests both as an index and reference [8, 9, 21]. When peripheral blood qPCR was considered as reference, a relatively higher sensitivity (74.3%) of microscopy to detect Plasmodium infection in peripheral blood was recorded in the current study than the 54.9% sensitivity of microscopy reported from a study conducted by following pregnant women up to delivery in Colombia [68]. The variation could be explained by variation in study design and higher prevalence of P. falciparum (which has a higher density that can be relatively easily detected than P. vivax) in the current study area than in Colombia [9, 68]. On the other hand, the sensitivity of malaria RDT to detect Plasmodium infection in the peripheral blood of pregnant women recorded in the current study was slightly lower than the 91.6% sensitivity recorded in Cameroon using peripheral blood microscopy as reference [71]. The discrepancy might be due to the involvement of only symptomatic pregnant women and the recorded high prevalence of MiP (microscopy = 69.2%, and RDT = 77.9%) in that study, and thus potentially increase the sensitivity of diagnostic tests [14, 71, 78]. The diagnostic performance of malaria microscopy compared to placental histopathology to detect Plasmodium infection in peripheral blood of parturient women in the current study was slightly lower than the 80.7% sensitivity and 100% specificity reported from a study conducted in Cameroon [73]. The observed discrepancy might be due to the use of placental impression smear, rather than placental biopsy histopathology, as reference that might not have similar accuracy as histopathology [8, 10].

Generally, malaria RDT showed the lowest positivity rate and the poorest diagnostic performance than microscopy, qPCR, and histopathology in detecting Plasmodium infection among pregnant and parturient women. The recorded diagnostic accuracy of the malaria RDT to detect Plasmodium infection in the current study setting was below the WHO-recommended minimum standard. This limited accuracy of the RDT might hamper the productivity and impact of interventions introduced to eliminate malaria. This limited accuracy of the RDT might be due to the deletion of hrp2 genes that code for the target protein in the diagnosis. However, since the current study did not assess the status of hrp2 genes among the Plasmodium parasites found in the current study setting, it is difficult to correctly identify the reasons for the limited accuracy of the RDT, warranting further study. Since microscopy showed better accuracy and very good agreement with reference tests in peripheral and placental blood than RDT, it might continue serving as gold standard and reference test for clinical and research utility, respectively. The very good performance of microscopy to detect Plasmodium infection in peripheral and placental blood, combined with its technical ease and low logistic demand relative to histopathology and qPCR, highlights its potential suitability for MiP screening during ANC contact and delivery. This is particularly important to control MiP and predict the potential burden of PM that has and/or would have been prevented by integrating anti-malarial intervention with ANC service. Most importantly, the improved performance of RDT combined with microscopy underscores the possibility to overcome their separate limitation and leverage their combined strength for increased case detection that can optimize efforts to mitigate MiP, in particular, and eliminate malaria, in general. In the current study, qPCR showed slightly lower overall performance than placental histopathology to detect PM. This implies the potential advantage and relevance of histopathology to assess, differentiate, and indicate the association of PM (active and past infection) with adverse pregnancy outcomes [18]. Taken together, the results of this study highlighted the clinical and public health importance of microscopy and qPCR to detect MiP from peripheral, placental, and cord blood, and histopathology from placental biopsy, as well as the limited overall performance of malaria RDT.

Strengths and limitations

Evaluating diagnostic performance of malaria tests in a multidimensional approach (i.e., by alternatively considering tests as index and reference on different specimen types collected from pregnant women during ANC visit, and parturient women after child delivery) helps to generate fine-tuned comprehensive data that can assist evidence-based decision-making and designing a targeted intervention. The involvement of many specimen types and diagnostic tests, particularly histopathology and qPCR, as well as ROC analysis, shows the overall robustness of findings from this study. Conversely, the use of cross-sectional design and small sample size, particularly for qPCR owing to resource constraints, might reduce the overall generalizability of this finding.

Conclusion and recommendation

Generally, qPCR showed the best accuracy to detect Plasmodium infection in peripheral, placental, and cord blood. Histopathology demonstrated the best accuracy to detect PM, implicating its robustness to characterize the burden of PM for research and policy purposes. Malaria RDT showed the lowest accuracy to detect Plasmodium infection in peripheral, placental, and cord blood, warranting for more sensitive test. Performance of microscopy and RDT combination was better than the separate performance of these tests, reflecting the potential to complement the suboptimal accuracy of each other. This has a particular implication for its potential relevance to mitigate the burden of MiP in low-resource-high-transmission settings where PCR, histopathology, and IPTp are not readily used.

Malaria qPCR could be used as reference to evaluate the diagnostic performance of a new and/or an existing malaria diagnostic test to detect Plasmodium infection in peripheral, placental, and cord blood. Histopathology could serve as a potential tool for research and policy purposes to characterize the burden of PM and monitor its mitigation efforts. Since the evaluated RDT showed suboptimal performance in detecting Plasmodium infection among pregnant and parturient women, more sensitive tools such as hsRDTs and molecular tools are needed. Microscopy and RDT combined test strategy can be a good option for stakeholders, particularly dedicated to mitigating MiP and improving the overall fetomaternal health. This strategy could also be indicated when early detection and appropriate MiP-case management are very critical in some mandatory conditions (such as during early gestation among primigravida in high-transmission settings, and settings approaching malaria elimination). Further studies involving a longitudinal design and larger sample size aimed to evaluate the diagnostic performance of malaria tests in detecting PM are needed.

Abbreviations

AMiP

Asymptomatic malaria in pregnancy

ANC

Antenatal care

CM

Congenital malaria

cRDT

Conventional rapid diagnostic test

DNA

Deoxyribonucleic acid

EDHS

Ethiopian Demographic Health Survey

HC

Health centre

hsRDT

Highly sensitive rapid diagnostic test

GM

Gestational malaria

HRP

Histidine-rich protein

IPTp

Intermittent preventive treatment in pregnancy

iRBCs

Infected red blood cells

IRS

Indoor residual spraying

ITN

Insecticide-treated net

JUIH

Jimma University Institute of Health

LAMP

Loop mediated isothermal amplification

LBW

Low birth weight

MiP

Malaria in pregnancy

MIS

Malaria Indicator Survey

NBFFPE

Neutral buffered formalin fixed paraffin embedded

NPV

Negative predictive value

PM

Placental malaria

PPV

Positive predictive value

qPCR

Quantitative polymerase chain reaction

RDT

Rapid diagnostic test

SPSS

Statistical package for social science

SRMA

Systematic review and meta-analysis

TIDRC

Tropical and infectious diseases research centre

uRDT

Ultra sensitive rapid diagnostic test

Author contributions

AA designed the study, participated in the data collection, conducted laboratory tests, performed data analysis and interpretation of the result, and drafted the manuscript. DY, AZ and JB contributed significantly by designing the study, supervising the overall work; critically reviewing the manuscript and providing final approval for publication. All authors read and approved the final manuscript.

Funding

This study received financial support from Jimma University and Arba Minch University.

Availability of data and materials

The data used for this research can be accessed from the corresponding author upon a reasonable request.

Declarations

Ethics approval and consent to participate

This study was conducted with the approval of the relevant Ethical Committee Review as well as prior written informed consent for each participant.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.