Background
Plasmodium falciparum is the principal cause of severe malaria while
Plasmodium vivax is increasingly recognized as capable of causing severe disease [
1‐
4]. According to the latest World Health Organization report of 2015, there were an estimated 214 million cases of malaria worldwide with 438,000 deaths. Ninety per cent of the deaths occurred in sub-Saharan Africa where pregnant women and children are significantly affected [
1].
Every year about 30 million African women in malaria-endemic areas become pregnant and are at risk of infection with malaria, and an estimated 75,000–200,000 infant deaths are reported due to malaria infection in pregnancy [
5,
6]. Malaria, particularly due to
P. falciparum, in pregnant women increases the risk of maternal death, miscarriage, stillbirth and neonatal death [
7,
8]. The impact of malaria during pregnancy may vary within a country depending on the intensity of malaria transmission. In areas of seasonal malaria transmission, pregnant women are three times more likely to suffer from severe malaria as compared to non-pregnant counterparts. In areas of stable malaria, adult pregnant women would have considerable acquired immunity and infection during pregnancy typically does not cause symptomatic malaria. The effect of malaria in pregnancy is primarily low birth weight and maternal anaemia [
9,
10].
The World Health Organization (WHO) recommends a three-pronged approach to reduce the burden of malaria in pregnancy: (1) provision and promotion of insecticide treated bet nets (ITN) or long-lasting insecticide- treated bed nets (LLINS); (2) administration of intermittent preventive treatment with sulfadoxine–pyrimethamine (IPTp-SP) after the first trimester of pregnancy in areas with stable malaria transmission; and (3) prompt diagnosis and appropriate treatment of malaria [
5,
11]. However, because of rising
P. falciparum resistance to SP in sub-Saharan regions, the use of rapid diagnostic tests to screen women for malaria at the first or each antenatal visit and then treat is likely more sustainable than IPTp without diagnosis [
12].
Early and accurate diagnosis with effective treatment is the best strategy to decrease malaria-related pregnancy complications and infant mortality. The current malaria diagnostic methods include Giemsa-based microscopy, rapid diagnostic tests (RDTs), polymerase chain reaction (PCR) and placental histology depending on the setting [
13]. The poor performance of routine malaria diagnostic techniques including RDTs and microscopy contribute to the burden of malaria in pregnant women [
14,
15]. This is in large part due to the sequestration of the parasite in the placenta making the parasitaemia lower than usual in the peripheral blood. Therefore, nPCR which targets the small subunit ribosomal RNA (ssrRNA) is the better alternative diagnostic technique due to its high sensitivity (as low as 0.1 parasite/µl of whole blood). However, nPCR it is not widely used for the diagnosis of malaria in resource-limited settings as it requires a very well-equipped laboratory, and the cost of diagnosis is more expensive [
16,
17].
Loop-mediated isothermal amplification (LAMP) is a simpler molecular procedure and better alternative for the field than nPCR [
18,
19]. LAMP has many operational advantages over PCR including: (1) the acceptability of a crude sample preparation; (2) minimal capital equipment requirements; (3) rapid time to obtain a result; (4) lower cost; and (5) technically less complex than PCR [
20].
Results
A total of 87 malaria suspected pregnant women with the mean (SD) age of 27.43 (
+5.2) years were enrolled into the study, of which 50.6% (45/87) had a previous malaria history (Table
1). The majority (52.9%) of patients were in the 25–30 age group. Second (41.4%) and third trimester (41.4%) pregnancies were more common than first trimester (17.2%). Close to three quarters (74.7%) of patients were multigravida. The overall malaria positivity rate among the study participants by nPCR was 11.5% (10/87). Ten malaria positive patients (11.5%) were detected by Giemsa microcopy with an overall median parasitaemia density of 3380/μl (parasitaemia range 400–24,760/μl). Five of the Giemsa microscopy positives were
P. falciparum (parasitaemia density minimum/maximum: 400/7840 per μl); three were
P. vivax (parasitaemia density minimum/maximum: 520/24,760 per μl), and two were mixed infections of
P. falciparum and
P. vivax (parasitaemia density minimum/maximum: 520/5360 per μl). LAMP identified 15 positive (17.2%) specimens and RDT 9 (10.3%) positives. A higher positivity rate of both single and mixed
Plasmodium species infection were reported by LAMP than other diagnostic techniques used in this study (Table
2).
Table 1
Demographic and clinical data of the study particpants
Age |
18–24 | 3 (13.0) | 20 (87.0) | 1.05 (0.789) |
25–30 | 5 (10.9) | 41 (89.1) |
31–35 | 1 (7.1) | 13 (92.9) |
36+ | 1 (25.0) | 3 (75.0) |
Previous malaria history |
Yes | 5 (11.4) | 39 (88.6) | 0.001 (0.969) |
No | 5 (11.6) | 38 (88.4) |
Trimester |
First | 2 (13.3) | 13 (86.7) | 0.61 (0.74) |
Second | 5 (13.9) | 31 (86.1) |
Third | 3 (8.3) | 33 (91.7) |
Gravid |
Primigravidae | 2 (9.1) | 20 (90.9) | 0.167 (0.683) |
Multigravidae | 8 (12.3) | 57 (87.7) |
Table 2
Malaria positivity rate by diagnostic techniques among the study participants
Pf | 5 | 3 | 5 | 7 |
Pv | 3 | 3 | 5 | 2 |
Pf + Pv | 2 | 3 | 5 | 1 |
Total positives | 10 | 9 | 15 | 10 |
Total negatives | 77 | 78 | 72 | 77 |
Using nPCR as the gold standard, LAMP had the highest sensitivity (100%; 95% CI 100) compared to microscopy (90%; 95% CI 66.3–113.7) and RDT (70%; 95% CI 33.8–106.2). Microscopy had the greatest specificity (98.7%; 95% CI 96.5–101.9) compared to RDT (97.4%; 95% CI 92.9–101.9) and LAMP (93.5%; 95% CI 86.5–100.5). There were five discordant results between nPCR and LAMP where five of them were negative by nPCR but positive by LAMP. There was also one study participant with a parasitaemia load of 520/μl negative only by nPCR. In this study, LAMP showed better sensitivity but lower specificity than microscopy and RDTs using nPCR as the gold standard (Table
3). Discordant results are itemized in Table
4. RDTs had the quickest turnaround time at 23 min compared to LAMP (60 min), microscopy (60 min), and nPCR (130 min) (Table
5). Both RDT and LAMP can be performed at the point of care (community-based testing) without the need for laboratory facilities.
Table 3
Performance characteristics of Microscopy, RDTs and LAMP compared to nPCR for the diagnosis of malaria among study participants
Microscopy | 90 (66.3–113.7) | 98.7 (96.5–101.9) | 90 | 98.7 | 0.887 |
RDTs | 70 (33.8–106.2) | 97.4 (92.9–101.9) | 77.8 | 96.2 | 0.705 |
LAMP | 100 (100) | 93.5 (86.5–100.5) | 66.7 | 100 | 0.768 |
Table 4
Discordant analysis for microscopy, RDT, LAMP and nPCR for malaria diagnosis in malaria suspected pregnant women
Pf
| 2 | 1 | 1 | 1 | 1 | |
Pv (pan) | 3 | | | | | |
Pf, Pv (pan) | | | | | | |
Negatives | | | | | | 71 |
Table 5
Total turnaround time required for the diagnosis of malaria with diagnostic tools among study participants at Kola Diba health center
RDTs | 2 | 21 | 23 |
Microscope | 40 | 20 | 60 |
LAMP | 5 | 55 | 60 |
nPCR | 60 | 250 | 310 |
Discussion
Pregnant women have an increased susceptibility to infection by
Plasmodia spp. Parasites sequestered in the placenta are sometimes not detectable in peripheral blood smears by Giemsa microscopy [
14]. Infection can result in maternal anaemia, prematurity and intrauterine growth retardation (IUGR) and infant low birth weight (LBW) [
6]. A study from Cameroon revealed that 20.9% of pregnant women who had placental malaria were negative by peripheral blood smear [
27].
Malaria remains a leading cause of morbidity and mortality especially among pregnant women and children in the developing world [
1]. In the current study, the rate of malaria positivity was lower than a similar study conducted in Cameroon which had 21.9% positivity by microscopy [
27]; Ghana, 19, 34, and 53% positivity by microscopy, RDTs and PCR, respectively [
28]; Nigeria, 27 and 30% positivity by RDTs and microscopy, respectively [
29]; Mozambique, 18.7, 15.4 and 44.8% positivity by RDTs, microscopy and quantitative PCR, respectively [
14]. The discrepancy in positivity may be due to the seasonality of transmission levels in Ethiopia. The rate of malaria may vary depending on the district, intensity of malaria transmission, season, density of parasitaemia, immunity level acquired, administration of malaria chemoprophylaxis, and diagnostic methods used [
27,
30‐
33]. Although the current study area was reported to be endemic to malaria, its transmission was low during the dry season. Of note, malaria transmission has decreased over time in this region presumably due to the introduction of WHO-endorsed control strategies except the deployment of intermittent preventive therapy [
21].
In the current study, age group, trimester and parity were not statistically associated with malaria infection. Based on nPCR results, seventy percent (7/10) of malaria infected pregnant women were multigravida. In contrast, a study from Gabon revealed that primigravida and young pregnant women were associated with increased malaria susceptibility. Although not statistically significant, based on microscopy results, primigravida women demonstrated a higher median parasite density (5200 parasites/μl) than multigravida (1560 parasites/μl) [
34]. This is similar to a report from Nigeria, where primigravida women demonstrated higher parasitaemia than multigravida women [
35].
Polymerase chain reaction (PCR) in its various formats has emerged as the most sensitive method able to detect low levels of parasites in the blood especially in this setting [
36,
37]. However, PCR requires high capital investment costs, service agreements, reagent supplies, and trained staff in molecular technologies with robust quality assurance programmes. LAMP while being molecular in nature permits crude, easy-to-perform DNA extraction and visual detection even possible in the field. Previous studies from Ethiopia and Thailand revealed that a rapid and user friendly LAMP had comparable performance to nPCR for the diagnosis of malaria in the general population [
19,
24]. The current study also indicated that LAMP had better sensitivity (100%) than RDTs (70%) and Giemsa microscopy (90%) for the diagnosis of malaria in pregnant women. Increased sensitivity is essential to the malaria eradication campaign especially in populations such as pregnant women where low levels of infection go undetected.
The current study showed lower specificity of LAMP (93.5%) than RDTs and Giemsa microscopy. This result was in line with a study conducted in Bangladesh where LAMP revealed lower specificity (58.3%) than microscopy and RDT (both 100%) when compared with nPCR [
18]. In the current study, there were five discordant results between LAMP and nPCR in which five samples positive by LAMP ended up negative by nPCR. This result was similar to a study conducted in Thailand where four LAMP positive results were negative by nPCR [
38]. These authors suggested the need for a more sensitive PCR technique to accurately evaluate the performance of LAMP. Indeed, the role of nPCR as the
bona fide gold standard could be questioned in this study as it relied on filter paper samples which may be subject to DNA degradation in transport to Canada from Ethiopia. DNA degradation on filter paper may ultimately explain the lower kappa value between nPCR and LAMP versus nPCR and microscopy. In support of this supposition, one specimen was positive by all three methods except nPCR. Thus, additional LAMP positive cases may be true positives and warrant treatment. However, others have reported that genomic DNA is stable on filter papers over time making this less likely a contributing factor to the discordance observed [
39]. All discordant specimens were repeated by nPCR and/or LAMP to confirm the discrepancy. It is also possible that LAMP resulted in false positive amplification due to contamination as observed previously in this setting [
24]. To limit this confusion, future studies should attempt LAMP and reference PCR on the same specimen at the field site to remove the confounder of filter paper versus fresh blood.
In the current study, two samples which were negative by RDTs ended up positive by LAMP, Giemsa microscopy and nPCR. For accurate diagnosis of malaria with RDTs, at least 100–500 parasites/μl of whole blood are required in peripheral blood. This was supported by a study from Tanzania which revealed that lower density of malaria parasitaemia is highly associated with the negative result of RDTs [
32]. In the present study, malaria parasitaemia level in these two samples was 400 and 640 parasites/μl. However, the performance of RDTs could be also be affected by incorrectly reading faint positive or invalid results as negative [
40]. There was one sample which was positive for
P. falciparum only by RDTs. Histidine rich protein-2 (HRP-2) antigen could persist for a long time even after effective treatment, giving false positive RDT test results in the absence of active
P. falciparum infection [
41].
Authors’ contributions
SG and DRP conceived the study. BT, SG, WL and DRP designed the proposal. BT collected and analysed the data, and carried out LAMP analysis. SG and WL supervised BT while collecting the data and LAMP analysis. ANM performed the nPCR analysis. All authors read and approved final manuscript.