Comparative performance of microscopy, rapid diagnostic tests, and multiplex real-time PCR for detection of malaria parasites among pregnant women in northwest Ethiopia

Abstract

Background

Low malaria parasitaemia is a diagnostic challenge in pregnancy, leading to false negative microscopy and rapid diagnostic test (RDT) results. However, these submicroscopic or subpatent infections could cause adverse pregnancy outcomes. Thus, evaluating the diagnostic performance of microscopy, RDT, and multiplex qPCR in pregnancy is vital for informed decisions.

Methods

A total of 835 peripheral blood and 372 placental blood samples were collected from 835 pregnant women attending first antenatal care or admitted for delivery at selected health facilities in northwest Ethiopia between November 2021 and July 2022. In multiplex qPCR, all microscopy and/or RDT positive samples were extracted and amplified individually, whereas all samples negative by both RDT and microscopy were extracted after pooling ten samples together and tested for Plasmodium genus. The diagnostic performance of microscopy, RDT, and multiplex qPCR in pregnancy was compared and evaluated against each other.

Results

Using multiplex qPCR as a reference test, microscopy had a sensitivity of 73.8% (95% confidence interval (CI): 65.9–80.7) and 62.2% (95% CI: 46.5–76.2) to detect Plasmodium parasites in peripheral and placental blood samples, respectively, with a 100% (95% CI: 98.9–100) specificity in both samples. Similarly, the RDT had a sensitivity of 67.6% (95% CI: 59.3–75.1) and a specificity of 96.5% (95% CI: 94.9–97.8) for Plasmodium infection diagnosis in peripheral blood and a sensitivity of 62.2% (95% CI: 46.5–76.2) and a specificity of 98.8% (95% CI: 96.9–99.7) in placental blood samples. Considering microscopy as a reference test, multiplex qPCR showed a sensitivity of 100% (95% CI: 96.6–100) and a specificity of 94.8% (95% CI: 93.0–96.3) to diagnose Plasmodium infections in both peripheral and placental blood samples. Pooled multiplex qPCR detected 34 peripheral and 12 placental blood Plasmodium infections from microscopy and RDT negative samples. The pooled assay obviated about half of the reactions and its testing costs. Microscopy showed almost perfect agreement (κ = 0.823) with multiplex qPCR for detecting malaria parasites in pregnancy, whereas the RDT showed a substantial agreement (κ = 0.684).

Conclusion

Multiplex qPCR had a better performance for Plasmodium infection diagnosis in pregnancy compared to microscopy and RDT. Pooled multiplex qPCR could be a sensitive and resource-efficient strategy for epidemiological surveillance of Plasmodium infections in pregnancy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12936-025-05256-2.

Background

Malaria is considered the most important parasitic disease of humans, posing a risk to nearly half of the world’s population and causing its most serious and complex public health problems, mainly in tropical and subtropical countries [1]. Despite tremendous global control efforts and successes, malaria is still among the leading causes of death in many affected countries [2]. In 2022, there were an estimated 249 million malaria cases and 608,000 deaths in 85 countries [1]. Malaria is a significant health problem and a hindrance to socioeconomic development in Ethiopia. About three-quarters of the land in Ethiopia is suitable for malaria transmission, and more than half of the population is at risk of infection [3].

Ethiopia is co-endemic for both Plasmodium falciparum and Plasmodium vivax. Plasmodium falciparum accounts for about 65% of the malaria cases in the country, and Plasmodium vivax accounts for about 35%. Anopheles arabiensis is the main malaria vector in Ethiopia with a wide geographical distribution. Anopheles funestus, Anopheles pharoensis, and Anopheles nili are also malaria vectors in Ethiopia with limited distribution [4]. Recently, Anopheles stephensi is described as widely distributed in the country, particularly in the eastern part [5]. Despite a significant reduction in malaria morbidity and mortality in the last two decades, malaria showed a significant surge in recent years and remained a public health threat to the nation. In 2022, about five million malaria cases and ten thousand malaria-related deaths were reported in Ethiopia [1].

In endemic areas, pregnant women with altered immunological and physiological conditions are more susceptible to malaria than before pregnancy and other non-pregnant women [1, 6]. Plasmodium infections in pregnancy cause maternal anemia, intrauterine growth retardation, low birthweight, miscarriage, stillbirth, and sometimes maternal death [7–9]. In high malaria transmission areas, pregnant women are semi-immune to Plasmodium infections and often carry the infections with few or no symptoms (asymptomatic malaria) [10]. Asymptomatically infected women have low healthcare-seeking behaviour and low parasite densities, which would remain undiagnosed and untreated and be a potential reservoir for disease transmission as well as a cause of adverse pregnancy outcomes [10, 11].

Malaria control in Ethiopia is through the use of long-lasting insecticidal nets (LLINs), indoor residual spray (IRS), effective management of malaria cases and periodic surveillance. Pregnant women are prioritized and targeted during LLINs distribution, IRS campaigns and related social and behavioural change activities [12]. However, since malaria transmission intensity in Ethiopia is generally low to moderate, intermittent preventive treatment with sulfadoxine-pyrimethamine in pregnancy is not practiced [13].

Light microscopy using Giemsa-stained blood film is the gold standard for malaria parasite detection, identification, and confirmation at hospitals and health centers in Ethiopia. Rapid diagnostic tests (RDTs) are the malaria diagnostic tools at health posts in malaria-endemic rural areas [14]. However, microscopy and conventional RDTs have poor sensitivity for detection of Plasmodium species in low parasite infections, such as in asymptomatic semi-immune pregnant women living in endemic areas with low peripheral blood parasitaemia due to parasite sequestration in the placenta. Moreover, histidine-rich protein–II (HRP-II) based RDTs showed false negative results due to HRP-II gene deletion [15] and false positive results since antigens remain in circulation for about 28 days [16].

Real-time polymerase chain reaction (qPCR) represent a highly sensitive and specific method for malaria surveillance, providing information on parasite density and infecting species [17]. However, the application of PCR on malaria surveillance is limited due to the associated high cost, requirement of highly trained manpower, and advanced laboratory facilities [18]. Multiplex real-time (multiplex qPCR) has the advantage of simultaneous detection of multiple Plasmodium species in a single reaction. It was suggested that pooling samples prior to diagnostic testing by PCR for Plasmodium gene targets provides a cost-effective and resource-conserving alternative with better diagnostic certainty compared to microscopy and RDT, particularly among pregnant women in low-transmission settings [17, 19].

Thus, the current study aimed to compare the performance of multiplex qPCR and RDTs with microscopy and the performance of microscopy and RDTs with multiplex qPCR for the diagnosis of peripheral and placental Plasmodium infections among pregnant women in northwest Ethiopia.

Methods

Study design, setting and participants

A cross-sectional study was carried out among pregnant women at Bambluk Health Center, Jawi Health Center, and Jawi Primary Hospital in Jawi District, Amhara Regional State, northwest Ethiopia. Pregnant women who attended their first ANC contact (ANC1) or were admitted to the health facilities for delivery or spontaneous abortion between November 2021 and July 2022 were enrolled in the study. The district is among the regions with a high rate of malaria transmission [20]. Details of the study area and study participants were presented in the previous work [21].

Sample size determination and sampling technique

The sample size was determined using the Buderer’s formula [22]:

$$n=\frac{{Z^2}_{1-\alpha /2}\times S_N\times (1-S_N)}{L^2\times Prevalence}$$

where: Z_1−α/2 (standard normal deviate corresponding to the specified size of the critical region (α) = 1.96, S_N (anticipated sensitivity of microscopy compared to multiplex qPCR) = 67.9% [17], prevalence = 19.3% prevalence of malaria in the area [23], and L (absolute precision desired on either side of sensitivity) = 0.096. Accordingly, a minimum of 434 pregnant women were required for the study.

Socio-demographic and clinical data collection

Socio-demographic, clinical, obstetric, and malaria risk factors data were collected from consented pregnant women at ANC rooms and labour wards using pretested structured questionnaires and checklists [21]. The data was collected using Amharic or Awugni version questionnaires by data collectors who are native to these languages. The data collectors read and completed the Amharic or Awugni version of the questionnaire for the participants, regardless of their participants’ literacy level, to ensure consistency.

The questionnaire contained the women’s socio-demographic characteristics, malaria prevention practices, obstetric and clinical profiles, such as ANC follow-up, gravidity, parity, malaria symptoms, history of malaria during pregnancy, and usage of anti-malarial drugs. While the last menstrual cycle and the fundal height method were utilized to estimate gestational age in health centers, the Mindray DP-50 digital ultrasound machine was used in the hospital. Moreover, a digital thermometer was used to take the mother's axillary temperature.

Malaria diagnosis

Light microscopy, RDTs, and multiplex qPCR were used to diagnose Plasmodium infections from maternal capillary and placental blood. The collection of capillary blood and placental blood was detailed elsewhere [21].

Plasmodium infections were diagnosed using the Abbott Bioline™ Malaria Ag P.f/P.v test kit (05FK80) (Standard Diagnostics, Inc., Republic of Korea) RDT as per the manufacturer’s instructions. The RDT targets the Histidine Rich Protein-2 (HRP2) of P. falciparum and lactate dehydrogenase of P. vivax. For microscopical diagnosis of Plasmodium infections, thin and thick blood smears were prepared on a single slide, air-dried, fixed with absolute methanol, stained with 10% Giemsa, and air-dried in accordance with standard operating procedures [24]. Two trained and experienced laboratory technicians who were blind to the RDT data read the stained smears independently. In case of discordant results between the two readers, a third more experienced laboratory personnel crosschecked the slides and resolved the discrepancy. If malaria parasites (asexual and sexual stages) are not detected after examining at least 200 high-power microscope fields, a slide was classified as negative [24]. All positive slides and 10% of negative slides were re-checked by an expert microscopist who was blinded to the microscopy and RDT results.

Assuming a total white blood cell count of 8000/µl, the density of both sexual and asexual stages of P. falciparum, P. vivax, and mixed P. falciparum and P. vivax infections was estimated on thick film against 200 leucocytes. After that, the parasite density was categorized as low (below 1000 parasites per microlitre of blood), intermediate (1000–4999 parasites per microlitre of blood), and high (≥ 5000 parasites per microlitre of blood) [25].

Plasmodium species detection using multiplex real-time PCR

Dried blood spots were collected by applying capillary and placental blood samples on Whatman filter papers, air-dried, sealed in a ziploc with desiccants, and transported by cold chain to the Aklilu Lemma Institute of Pathobiology and kept at − 20 °C until tested. The multiplex qPCR assay was carried out at the Ethiopian Public Health Institute National Parasitology Laboratory.

Dry blood spots (DBSs) were collected from 420 ANC1-attending women, 372 delivering women, and 43 aborting women. Accordingly, 835 peripheral blood DBSs and 372 placental blood DBSs were collected. One hundred thirty-seven peripheral blood DBSs and 47 placental blood DBSs were from women who had microscopic and/or RDT-positive results.

DNA extraction and multiplex qPCR assays

The Geneius™ Micro gDNA Extraction Kit (Geneaid Biotech Ltd., Taiwan) was used to extract the genomic DNA (gDNA). In short, 3-mm-diameter circles of DBSs were punched out and processed following the manufacturer’s instructions in 1.5-ml Eppendorf tubes. After that, the DNA was eluted with 100 µl volume of elution buffer and kept at − 20 °C until tested. DBS samples from microscopy and/or RDT-positive women were extracted and analysed individually. Whereas, DBSs from microscopy and RDT-negative women were detected using pooled DBS sample extraction and analysis with slight modifications as described by Zhou et al. [19, 21] (Fig. 1).

Fig. 1.

Flow chart showing dry blood spot pooling and analysis procedure

Briefly, ten 3-mm-diameter DBS circles were placed together in 2 ml Eppendorf tubes. To guarantee adequate lysis, these DBSs were treated overnight with lyse buffer and proteinase K solution. Subsequently, DNA extraction was carried out in accordance with standard protocols. Multiplex qPCR was used to analyse the extracted DNA for the presence of Plasmodium genus. Pools of samples with genus qPCR negative results were considered negative. For positively tested genus-specific qPCR pools, individual DBSs were punched out and extracted in accordance with the standard technique, tested for genus-specific qPCR, and then for species-specific qPCR.

The Applied Biosystems’ QuantStudio 5 Real-Time PCR system was utilized for DNA amplification and detection, employing the TaqMan fluorescence assay with a final volume of 10 μl in two cycles. As stated in previous works [21, 26], pan-Plasmodium-specific small unit of ribosomal RNA (18S rRNA) and P. falciparum-specific var gene acidic terminal sequence primers (varATS) primers were used to test all samples in the first run, while P. falciparum (varATS) and P. vivax-specific small unit of ribosomal RNA primers (P.v18S rRNA) primers were multiplexed during the second run. The primers and probes used for Plasmodium species detection were described previously [21].

The following thermal cycling conditions were used to carry out the PCR amplifications: 95 °C for 1 min, followed by 45 rounds of 95 °C for 15 s and 57 °C for 45 s for the first PCR run; and 95 °C for 1 min, followed by 45 rounds of 95 °C for 15 s and 53 °C for 45 s for the second run. The 3D7 DNA standard was run as a positive control and nuclease-free water as a negative control in each of the tests. The Ct values of 25.0 to 30.0 for the positive control and Ct values < 30.0 for HsRNaseP to all samples taken as qualified PCR runs. Samples with Ct values between 12 and 40 and sigmoidal shape amplification curve were considered positive [21].

Statistical analysis

Data was analysed using the Statistical Package for Social Sciences (SPSS) version 25 statistical software (IBM Corp., New York, USA). The Cochran’s Q test was used to compare the proportion of women diagnosed positive for malaria by light microscopy, RDT, and qPCR techniques in each of the peripheral and placental blood specimens. The Kappa (κ) measure of agreement was used to assess the consistency between the tests. The κ value was interpreted as slight agreement (κ = 0.01–0.20), fair agreement (κ = 0.21–0.40), moderate agreement (κ = 0.41–0.60), substantial agreement (κ = 0.61–0.80), and almost perfect agreement (0.81–1.00) [27]. The MedCalc statistical software was used to determine the sensitivity, specificity, positive and negative predictive value of light microscopy, RDT, and qPCR for malaria diagnosis. Moreover, the Mann–Whitney U test and Kruskal-Walis H test were used to compare the median values of malaria parasite density. A p < 0.05 was used to indicate statistical significance.

Results

Characteristics of pregnant women

The characteristics of the delivery room admitted parturient women, Plasmodium infections, and associated risk factors were previously reported [21]. The aim of this work was to comparatively evaluate the performance of microscopy, rapid diagnostic tests, and multiplex qPCR in the diagnosis of Plasmodium infections among pregnant women in Jawi District, northwest Ethiopia, and to make useful recommendations that can contribute to alleviating the diagnostic challenges of malaria in pregnancy.

In the current study, 835 pregnant women were included. Among them, 420 (50.3%) were recruited at their first ANC contact, and 415 (49.7%) were pregnant women admitted to the delivery rooms for delivery or spontaneous abortion. The mean ± SD age of the women was 25.29 years ± 5.38 years, with a minimum age of 16 years and a maximum of 47 years. Among the women, 79.5% were adults aged 20–34 years, 60.8% were multigravid, and 50.5% were in their third trimester of pregnancy. Similarly, 45.7% were illiterate, 94% were married, 52% were rural residents, and 47.9% were engaged in farming. Only 4.7% of the women had fever at presentation or within 24 h of presentation (Table 1).

Table 1.

Characteristics of study participant pregnant women in Jawi District northwest Ethiopia, 2022 (N = 835)

Characteristics	n (%)
Age category in years
< 20	95 (11.4)
20–34	664 (79.5)
≥ 35	76 (9.1)
Residence
Urban	401 (48.0)
Rural	434 (52.0)
Marital status
Single	50 (6.0)
Married	785 (94.0)
Educational status
Illiterate	382 (45.7)
Primary education	258 (30.9)
Secondary education	113 (13.5)
College and above	82 (9.8)
Occupation
House wife	254 (30.4)
Farmer	400 (47.9)
Trader	106 (12.7)
Civil servant	56 (6.7)
Others	19 (2.3)
Graviditya
Primigravidae	256(30.7)
Multigravidae	508 (60.8)
Grand multigravidae	71 (8.5)
Gestational age
First trimester	95 (11.4)
Second trimester	318 (38.1)
Third trimester	422 (50.5)
Fever at presentation
Yes	39 (4.7)
No	796 (95.3)

^aPrimigravidae refers a woman who is pregnant for the first time, multigravidae a woman who is pregnant for the second time and above, whereas grand multigravidae refers a woman who had six and above pregnancies

Prevalence of Plasmodium infections in pregnancy

Among the total 835 pregnant women, 178 women (21.3%) were infected with Plasmodium species. Plasmodium infection was 13.1% by microscopy, 15.1% by RDT, and 18.4% by qPCR (p = 0.001) (Table 2). About 24.5% of ANC1 attending women and 18.1% of parturient women had malaria (p = 0.028). Among delivering women, Plasmodium infection was 12.9% (48/372) in peripheral blood and 13.4% (50/372) in placental blood specimens (p = 0.824). Among microscopic malaria cases, the median (lower quartile–upper quartile) malaria parasite density was 4920 (1560–12600) parasites/mm³ in peripheral blood and 1700 (260–5410) parasites/mm³ in placental blood. The positivity rate of malaria significantly increased among the adolescent and primigravid women by microscopy, RDT, and multiplex qPCR.

Table 2.

Prevalence of Plasmodium infections by different malaria diagnostic methods and maternal characteristics, northwest Ethiopia (N = 835)

Characteristics	Maternal malaria positivity			Totala
	Microscopy	RDT	Multiplex qPCR
	n (%)	n (%)	n (%)	n (%)
Plasmodium infection	109 (13.1)	126 (15.1)	154 (18.4)	178 (21.3)
Age in years
< 20	29 (30.5)	34 (35.8)	34 (35.8)	41 (43.2)
≥ 20	80 (10.8)	92 (12.4)	92 (12.4)	137 (18.5)
p-valuec	< 0.001	< 0.001	< 0.001	< 0.001
Gravidity
Primigravid	58 (22.5)	63 (24.4)	75 (29.1)	82 (31.8)
Multigravidb	51 (8.8)	63 (10.9)	79 (13.7)	96 (16.6)
p-valuec	< 0.001	< 0.001	< 0.001	< 0.001
Gestational age
First trimester	10 (10.5)	13 (13.7)	14 (14.7)	17 (17.9)
Second trimester	52 (16.4)	56 (17.6)	74 (23.3)	81 (25.5)
Third trimester	47 (11.1)	57 (13.5)	66 (15.6)	80 (19.0)
p-valuec	0.084	0.280	0.018	0.069

^aOverall malaria positivity was determined if a woman had a positive RDT and/or detection of malaria parasites using light microscopy and/or qPCR

^bMultigravidae refers to a woman who is pregnant for the second time and above

^cBold values indicate statistically significant result

Plasmodium species infection positivity rate by microscopy, RDT, and multiplex qPCR

Plasmodium falciparum was detected among 18.2% of women, which accounted for 80.9% (144/178) of the total Plasmodium infections in pregnancy. Plasmodium vivax was detected among 3.1% of pregnant women which accounted for 14.6% of Plasmodium infections. A significantly increased number of P. falciparum cases were detected by multiplex qPCR compared to microscopy and RDT both in peripheral blood (119 vs 88; 119 vs 96, respectively, p = 0.001) and placental blood specimens (38 vs 24; 38 vs 26, respectively, p = 0.001). Multiplex qPCR detected more P. vivax cases compared to microscopy and RDT in peripheral blood (20 vs 14; 20 vs 18, respectively, p = 0.097) but not in placental blood specimens (4 vs 2; 4 vs 5, respectively, p = 0.097). Detection of mixed infections (P. falciparum and P. vivax) among the three methods showed little variation both in peripheral and placental blood specimens (Table 3). Multiplex qPCR and RDT showed a substantial agreement in detecting P. falciparum both in peripheral blood (κ = 0.7) and placental blood (κ = 0.693) (Additional file 1).

Table 3.

Prevalence of Plasmodium infections by microscopy, RDT, and multiplex qPCR among pregnant women, northwest Ethiopia (N = 835)

Plasmodium species	Diagnostic methods			p-valuea	Overallc
	Microscopy	RDT	Multiplex qPCR
	n (%)	n (%)	n (%)		n (%)
Peripheral blood Plasmodium infection
Plasmodium falciparum	88 (10.5)	96 (11.5)	119 (14.3)	0.001	134 (16)
Plasmodium vivax	14 (1.7)	18 (2.2)	20 (2.4)	0.097	25 (3)
Mixed	5 (0.6)	6(0.72)	6 (0.72)	0.779	8 (1)
Total peripheral Plasmodium infection	107(12.8)	120 (14.4)	145 (17.4)	< 0.001	167(20.0)
Placental Plasmodium infection (N = 372)b
Plasmodium falciparum	24(6.5)	26 (7)	38 (10.2)	0.001	41(11.0)
Plasmodium vivax	2(0.5)	5 (1.3)	4 (1.1)	0.097	6 (1.6)
Mixed	2(0.5)	1 (0.3)	3 (0.8)	0.223	3 (0.8)
Total placental Plasmodium infection	28 (7.5)	32 (8.6)	45 (12.1)	< 0.001	50 (13.4)
Total maternal Plasmodium infectiond
Plasmodium falciparum	90 (10.8)	101 (12.1)	128 (15.3)	0.001	144(18.2)
Plasmodium vivax	14 (1.7)	19 (2.3)	20 (2.4)	0.092	26 ((3.1)
Mixed	5 (0.6)	6(0.7)	6 (0.7)	0.779	8(1)
Total maternal Plasmodium infectiond	109 (13.1)	126 (15.1)	154 (18.4)	< 0.001	178(21.3)

Bold values indicate statistically significant result

^aThe Cochran Q test was used to test statistical significance

^bPlacental Plasmodium infection was diagnosed among 372 delivered women

^cOverall malaria positivity was determined if a woman had a positive RDT and/or detection of malaria parasites using light microscopy and/or qPCR

^dTotal maternal malaria was determined if a woman had Plasmodium infection in peripheral and/or placental blood specimen

Malaria diagnostic performance of microscopy, RDT, and multiplex qPCR

Considering microscopy as a reference diagnostic method, multiplex qPCR detected all Plasmodium parasites from peripheral and placental blood specimens, which tested positive by microscopy. Additionally, 38 peripheral blood and 17 placental blood samples that were diagnosed as negative by microscopy were tested positive by multiplex qPCR. Real-time PCR had a sensitivity of 100% (95% CI 96.6–100) in peripheral blood and 100% (95% CI 87.7–100) in placental blood specimens, whereas a specificity of 94.8% (95% CI 93.0–96.3) in peripheral blood and 95.1% (95% CI 92.2–97.1) in placental blood compared to microscopy. Microscopy and qPCR showed almost perfect agreement in peripheral blood (κ = 0.823) and a substantial agreement in placental blood in detecting Plasmodium parasites (κ = 0.743) (Table 4). Similarly, microscopy and qPCR showed almost perfect agreement in peripheral blood (κ = 0.814) and a substantial agreement in placental blood in detecting P. falciparum parasites (κ = 0.761) (Additional file 1).

Table 4.

Diagnostic performance of RDT and multiplex qPCR in diagnosing Plasmodium infections in pregnancy in comparison with microscopy, northwest Ethiopia

Diagnostic method	Microscopy			Sensitivity, % [95% CI]	Specificity, % [95% CI]	PPV, % [95% CI]	NPV, % (95% CI)	Kappa value
	Positive	Negative	Total
Peripheral blood
Multiplex qPCR
Positive	107	38	145	100 [96.6–100]	94.8 [93.0–96.3]	73.8 [67.4–79.3]	100 [99.5–100]	0.823
Negative	0	690	690
Total	107	728	835
RDT
Positive	94	28	122	87.9 [80.1–93.4]	96.2 [94.5–97.4]	77.1 [69.9–82.9]	98.2 [97.0–98.9]	0.793
Negative	13	700	713
Total	107	728	835
Placental blood
Multiplex qPCR
Positive	28	17	45	100 [87.7–100]	95.1 [92.2–97.1]	62.2 [50.9–72.4]	100 [98.9–100]	0.743
Negative	0	327	327
Total	28	344	372
RDT
Positive	23	9	32	82.1 [63.1–93.4]	97.4 [95.1–98.8]	71.9 [56.7–83.3]	98.5 [96.8–99.3]	0.746
Negative	5	335	340
Total	28	344	372

Similarly, except for 13 peripheral blood and five placental blood samples, all microscopy-positive samples for Plasmodium infections were also positive by the RDT. The RDT detected 28 peripheral blood and nine placental blood samples as positive for Plasmodium infections, which were diagnosed negative by microscopy. The RDT had a sensitivity of 87.9% (95% CI 80.1–93.4) in peripheral blood and 82.1% (95% CI 63.1–93.4) in placental blood specimens in detecting Plasmodium parasites compared to microscopy. Similarly, it had a specificity of 96.2% (95% CI 94.5–97.4) in peripheral blood and 97.4% (95% CI 95.1–98.8) in placental blood to rule out Plasmodium infections compared to microscopy. Microscopy and the RDT had a substantial agreement in detecting Plasmodium infections in general (κ = 0.793) and P. falciparum infections in particular (κ = 0.792) in peripheral blood. Similarly, the two methods showed a substantial agreement in detecting Plasmodium infections (κ = 0.746) as well as P. falciparum infections (κ = 0.786) in placental blood specimens (κ = 0.746) (Additional file 1).

Considering multiplex qPCR as a reference diagnostic method, microscopy detected only 107 peripheral blood and 28 placental blood Plasmodium infections from a total of 145 peripheral and 45 placental blood Plasmodium infections, which were tested positive by qPCR. On the other hand, microscopy identified all qPCR-negative peripheral and placental blood samples as negative. Microscopy had a sensitivity of 73.8% (95% CI 65.9–80.7) for detecting Plasmodium infections in peripheral and 62.2% (95% CI 46.5–76.2) in placental blood samples, whereas a specificity of 100% (95% CI 99.5–100) to rule out Plasmodium infections both in peripheral and placental blood specimens compared to multiplex qPCR. Multiplex qPCR and microscopy showed almost perfect agreement in peripheral blood (κ = 0.823) and a substantial agreement in placental blood in detecting Plasmodium parasites (κ = 0.743) (Table 5).

Table 5.

Performance of RDT and microscopy in diagnosing malaria in pregnancy in comparison with multiplex qPCR

Diagnostic method	Multiplex qPCR			Sensitivity, % [95% CI]	Specificity, % [95% CI]	PPV, % [95% CI]	NPV, % (95% CI)	Kappa value
	Positive	Negative	Total
Peripheral blood
Microscopy
Positive	107	0	107	73.8 [65.9–80.7]	100 [99.5–100]	100 [96.6–100]	94.8 [93.3–96.0]	0.823
Negative	38	690	728
Total	145	690	835
RDT
Positive	98	24	122	67.6 [59.3–75.1]	96.5 [94.9–97.8]	80.3 [73.1–86.0]	93.4 [91.8–94.7]	0.684
Negative	47	666	713
Total	145	690	835
Placental blood
Microscopy
Positive	28	0	28	62.2 [46.5–76.2]	100 [98.9–100]	100 [87.7–100]	95.1 [93.0–96.6]	0.743
Negative	17	327	344
Total	45	327	372
RDT
Positive	28	4	32	62.2 [46.5–76.2]	98.8 [96.9–99.7]	87.5 [72.0–95.0]	95 [93.0–96.5]	0.697
Negative	17	323	340
Total	45	327	372

Similarly, the RDT identified only 98 peripheral blood and 28 placental blood Plasmodium infections as positive from 145 peripheral and 45 placental blood Plasmodium infections, which were tested positive by multiplex qPCR. Moreover, 24 peripheral blood and four placental blood Plasmodium infections that had negative results by qPCR got a positive diagnosis by the RDT. The RDT showed 67.6% (95% CI 59.3–75.1) sensitivity in detecting Plasmodium parasites in peripheral blood and 62.2% (95% CI 46.5–76.2) in placental blood samples compared to multiplex qPCR. It also had a specificity of 96.5% (95% CI 94.9–97.8) in peripheral blood and 98.8% (95% CI 96.9–99.7) in placental blood to rule out Plasmodium infections compared to multiplex qPCR. Multiplex qPCR and RDT had a substantial agreement in both peripheral blood (κ = 0.684) and placental blood specimens in detecting Plasmodium parasites (κ = 0.697) (Table 5). Using multiplex qPCR as a reference test, microscopy had better sensitivity and specificity than the RDT for Plasmodium parasite detection both in peripheral and placental blood samples.

From 698 peripheral blood samples that were diagnosed negative for Plasmodium infections by both microscopy and RDT, pooled multiplex qPCR detected Plasmodium infections in 34 (4.9%) samples. Similarly, pooled multiplex qPCR detected Plasmodium infections in 12 (3.7%) placental blood samples from a total of 325 placental blood samples, which were diagnosed negative by both microscopy and RDT. The pooling strategy obviated about half of the extraction and genus qPCR reactions, thus halving the costs of consumable supplies, reagents, and processing time.

Discussion

The WHO recommended quality-assured microscopy and RDTs as appropriate tools for routine clinical malaria surveillance in malaria-endemic settings [28]. However, malaria in semi-immune pregnant women remains largely asymptomatic and often below the detection level of microscopy and conventional RDT due to sequestration of malaria parasites in the placenta, producing false negative results in peripheral blood samples [29]. These sub-microscopic infections might cause adverse pregnancy outcomes and would be a hub of malaria transmission in the community [11]. Thus, evaluating the diagnostic performance of the different malaria diagnostic methods in pregnancy is vital for informed decisions.

In the current study, 21.3% of the pregnant women were infected with Plasmodium species, and more than 80% of the infections were due to P. falciparum. Moreover, multiplex qPCR showed the highest positivity rate for detection of Plasmodium infections compared to microscopy and the RDT. This was comparable with a recent report in the study area, which reported 19.3% asymptomatic malaria among pregnant women, most of which were due to P. falciparum parasites, and the highest positivity rate was by real-time PCR [23]. The highest positivity rate of malaria by multiplex qPCR compared to microscopy and RDTs was due to the lower limits of detection by qPCR, which is as low as 0.1 parasites/µL) [30] compared to microscopy and RDT, which have limits of detection of about 50 parasites/µl of blood and 100–200 parasites/µl of blood, respectively [31].

The multiplex qPCR showed excellent sensitivity for the detection of Plasmodium parasites in peripheral and placenta blood samples compared to microscopy. The assay detected 100% of microscopically positive Plasmodium infections and about 80% of RDT-positive Plasmodium infections. The positivity rate of Plasmodium infections in pregnancy by the three methods significantly increased among the adolescent and primigravid women. A study in Ethiopia reported that multiplex qPCR had 100% sensitivity for detection of microscopically positive Plasmodium infections and 97% of RDT-positive Plasmodium infections from malaria-suspected febrile outpatients [26]. The increased susceptibility of adolescent and primigravid women compared to the adults and multigravid ones could be due to the fact that acquired anti-malarial immunity development in pregnancy is dependent on level of previous malaria exposure [32].

Pooled qPCR testing was a sensitive method for detection of submicroscopic and subpatent Plasmodium infections, reducing processing time, reagent, and supply cost [17, 19]. In the current study, the pooled multiplex qPCR assay detected Plasmodium infections from 34 peripheral blood and 12 placental blood samples from a total of 698 peripheral blood and 325 placental blood samples that were diagnosed negative by both microscopy and RDT. The pooled multiplex qPCR assay halved the cost of testing. Similarly, Tylor et al. reported detection of Plasmodium parasites from 35 samples from a total of 1092 microscopically negative peripheral blood samples, which were pooled in four samples and tested. The study also reported that about 50% of the reactions were obviated, and the cost of testing is halved [17].

This study revealed that multiplex qPCR showed lower specificities for ruling out Plasmodium infection in both peripheral blood (94.8%) and placental blood samples (95.1%) compared to microscopy and RDT. The finding was in agreement with Belachew et al. [26]. This might indicate that microscopy and RDT are producing false negative results of Plasmodium infection in pregnancy, which might be associated with their lower limit of detection. This could be due to the poor sensitivity of microscopy for detection of Plasmodium species in low parasite infections, such as in asymptomatic, semi-immune pregnant women living in endemic areas with low peripheral blood parasitaemia associated with placental sequestration of P. falciparum [31].

Microscopy and multiplex qPCR demonstrated substantial agreement in detecting Plasmodium parasites in peripheral blood and a relatively perfect agreement in placental blood. However, compared to multiplex qPCR, microscopy missed 31 P. falciparum, six P. vivax, and one mixed infection, misidentifying two mixed infections as P. falciparum, one P. falciparum case as P. vivax, and one mixed infection as P. vivax in peripheral blood. Similarly, microscopy missed 14 P. falciparum cases, two P. vivax cases, and one mixed infection that were diagnosed positive by qPCR in placental blood specimen. Tilahun et al. reported about 60% sensitivity of microscopy for detection of asymptomatic Plasmodium infections in pregnancy compared to multiplex qPCR [23]. Similarly, Belachew et al. reported only 58% sensitivity of microscopy, missing a significant number of Plasmodium infections that were diagnosed positive by multiplex qPCR among symptomatic patients in Ethiopia [26]. These submicroscopic Plasmodium infections are indicated to cause maternal anemia and adverse birth outcomes such as low birthweight and premature birth [11].

In this study, the RDT missed 38 P. falciparum cases and misidentified two cases in peripheral blood and missed 16 P. falciparum cases and misidentified one case as P. vivax in placental blood from a total of 119 P. falciparum cases in peripheral blood and 38 P. falciparum cases in placental blood. The RDT had a sensitivity of 67.6% in peripheral blood and 62.2% in placental blood compared to multiplex qPCR. From the study area, Tilahun et al. reported about 50% false negative results of RDT among asymptomatic pregnant women compared to real-time PCR [23]. This could be attributed to poor transport and storage conditions of the RDTs as well as parasitic factors, such as the prozone effect because of antigen saturation caused by high parasitaemia, low parasite density, and P. falciparum HRP-II/III gene deletion, which is reported high in Ethiopia [15, 33].

Considering multiplex qPCR as a reference standard, microscopy had increased sensitivity (73.8%) and specificity (100%) for detection of malaria parasites in peripheral blood compared to the RDT, which had a sensitivity of 67.6% and specificity of 96.5%. The finding was in line with the finding of Tilahun et al. [23] from the study area that reported increased sensitivity and specificity of microscopy compared to HRP2-based conventional RDTs for diagnosis of malaria in pregnancy. On the other hand, Kyabayinze et al. reported lower sensitivity and specificity of microscopy compared to HRP2 detecting conventional RDTs among pregnant women in Burkina Faso and Uganda [34]. Similarly, a study in Kinshasa, the Democratic Republic of Congo, revealed a significantly higher sensitivity and specificity of HRP2 detecting RTD compared to microscopy. This could be due to the fact that the malaria transmission in the current study area is relatively lower compared to the study sites of Kyabayinze et al. and Kinshasa, which were all described to have high malaria transmissions and about 40% malaria prevalence in pregnancy, which could lead to high parasite densities and thus higher RDT positivity [35, 36]. Moreover, the high proportion of P. falciparum HRP-II/III gene deletion reported in Ethiopia could result in false negativity of HRP2-based RDTs in the current study [15].

Further, 24 peripheral blood and four placental blood samples that had negative results by microscopy and/or qPCR got a positive diagnosis by the RDT. This could result from transcriptional error while reporting, persistence of the malaria antigens in the circulation, such as HRP-II, despite elimination of malaria parasites by the use of anti-malarials, or cross-reactivity with other protozoan parasite antigens [37].

Overall, the current study estimated the prevalence of Plasmodium infection in pregnancy using different diagnostic methods and assessed the comparative performances of microscopy, RDT, and multiplex qPCR for Plasmodium infection diagnosis in pregnancy, which is highly required for evidence-based decisions. However, the findings of the current study should be interpreted with its limitations. The current study did not assess detection limits of the diagnostic methods since Plasmodium parasite density was determined by microscopy. Moreover, placental histology examination was not carried out due to resource and expertise limitations.

Conclusion

This study showed that the prevalence of Plasmodium infections among pregnant women was 23.1%, with a significantly increased positivity rate by multiplex qPCR compared to microscopy and RDT. Microscopy and RDT missed a significant amount of Plasmodium infections in pregnancy, which could cause adverse pregnancy outcomes and be a source of malaria transmission in the community. Pooled multiplex qPCR obviated about half of the reactions and its testing costs; thus, it could be a resource-efficient strategy for epidemiological surveillance of malaria in pregnancy.

Abbreviations

ANC

Antenatal care

CI

Confidence interval

COR

Crude odds ratio

Ct

Cycle threshold

DBSs

Dry blood spots

gDNA

Genomic deoxyribonucleic acid

HRP-II

Histidine rich protein–II

mm

Millimeter

µL

Microliter

OR

Odds ratio

qPCR

Quantitative polymerase chain reaction

RDT

Rapid diagnostic tests

SD

Standard deviation

Author contributions

Z.E., A.A., S.D., A.G. and B.E. conceptualize and designed the study protocol; Z.E., A.A, S.D. and B.E. Conducted the field work; Z.E. analysed the data, interpret the data and wrote the draft manuscript; Z.E., M.B. and A.Ab, performed the laboratory works; All authors reviewed and approved the manuscript.

Funding

This study was financially supported by the School of Graduate Studies, Addis Ababa University and as part of the thematic research funded by the Office of Vice President for Research and Technology Transfer (VPRTT) of Addis Ababa University. The funder had no role in study design, data collection, and analysis, the decision to publish, or preparation of the manuscript.

Availability of data and materials

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

This study was ethically approved by the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University (reference number: ALIPB IRB/60/2013/21). Before commencement of the study, permission to conduct the study was obtained from the Amhara Public Health Institute, the Awi Zone Health Office, the Jawi District Health Office, and the management of each health facility. Written informed consent was obtained from pregnant women after briefing clearly about the purpose and benefits of the study. They were also informed that they have the full right to withdraw from the study at any time and at any stage they want. All personal identifiers were removed, and only codes were used to maintain the confidentiality of the participants’ data. Pregnant women who were infected with Plasmodium parasites were treated according to the national treatment guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.