Reliability of rapid diagnostic test for diagnosing peripheral and placental malaria in an area of unstable malaria transmission in Eastern Sudan

Malaria is a major public health problem in the tropics. Around 125 million pregnant women live in malaria-endemic areas, and 32 million of these are at risk of malaria in sub-Saharan Africa [1, 2]. Sequestration of malaria parasites in the placenta, where selection of pregnancy-associated Plasmodium falciparum erythrocyte membrane protein-1 (Pf EMP-1) variant surface antigen occurs in P. falciparum malaria in pregnancy, presents an enormous diagnostic challenge, especially in sub-Saharan African countries [3, 4].

There is a need for accurate and prompt diagnosis of malaria so as to achieve the desired level of disease control. This goal is of fundamental importance because prescription of any drug during pregnancy poses a risk to the unborn child [5]. The World Health Organization (WHO) now recommends a parasite-based diagnosis of malaria infection [6]. Microscopic examination using Giemsa-stained capillary blood slides for detection of malaria parasites remains the reference standard [6]. However, blood film microscopy is time-consuming and needs significant technical skills, good-quality reagents, and clean slides; thus, its accuracy is of highly variable quality in sub-Saharan African hospitals [7‐10]. Therefore, there is a need for the development of easier and faster diagnostic methods such as rapid diagnostic tests (RDTs). It has been shown that RDT is easy to use, are heat stable and have the ability to detect low parasitaemias [11]. Thus RDT is an ideal diagnostic tool for malaria diagnosis in settings that are resource constrained. While RDT have been extensively evaluated in malaria among the non-pregnant population, there are few published data on the performance of RDT for diagnosing malaria during pregnancy, especially in areas of unstable malaria transmission [12‐17].

Pregnant Sudanese women are at risk of malaria regardless of their age or parity and malaria is associated with adverse maternal and perinatal outcomes [18]. RDT has not yet been implemented as standard methods for diagnosing malaria during pregnancy in Sudan. Hence, this study was conducted to investigate the performance of RDT in the diagnosis of P. falciparum infections in febrile pregnant women and placental malaria. This study aimed to provide evidence-based data on the best diagnostic methods for malaria during pregnancy, based on our recent observation of the limitations of the different diagnostic methods for malaria during pregnancy in Sudan [19‐21].

This study was conducted at Gadarif Hospital in Eastern Sudan during September through December 2011. The area is characterized by unstable malaria transmission and P. falciparum is the sole malaria species present [22]. The cross-sectional study at the antenatal clinic that recruited febrile (temperature ≥ 37.5°C) pregnant women, compared the accuracy of RDT with microscopy; PCR was used as a reference standard for detecting peripheral parasitaemia in the participants. At delivery, a second cross-sectional study compared the accuracy of RDT and microscopy with placental histology (the reference standard), and with PCR performed to detect placental malaria.

After signing informed consent documents, those enrolled in both studies (i.e., the pregnant and newly delivered women) were screened and clinically examined for malaria. Information was collected on social demographics using a pre-tested questionnaire. Malaria screening was performed using microscopy, RDT and PCR during pregnancy, and placental histology at delivery. Pregnant women with a confirmed diagnosis of malaria were treated with artesunate sulfadoxine-pyrimethamine which is the first line treatment for P. falciparum malaria in Sudan [23]. The sample sizes were calculated based on 2-sided hypothesis tests using Epi Info with 80% power and a confidence interval of 95%.

The basic characteristics of the women enrolled in this study are shown in Table 1. The mean (SD) of the age, gestational age and temperature for the 156 febrile pregnant women was 27.0 (6.0) years, 19.0 (10.0) weeks and 38.2°C, respectively. Of these women, 17 (11.0%), 26 (16.7%) and 18 (11.5%) were P. falciparum-positive cases as detected by microscopy, RDT and PCR, respectively. The geometric mean of the parasite counts for the microscopy results was 8897.8 (1031.0) rings/μl.

The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the microscopic analyses were 94.4%, 100%, 100% and 99.3%, respectively. The corresponding values for RDT were 83.3%, 92.0%, 57.7% and 97.7%, where PCR was used as the gold standard, Table 2, Figure 1 and Figure 2.

The sensitivity, specificity, PPV and NPV for the RDT kit were 17.4%, 81.7%, 29.6% and 69.6%, respectively. The corresponding values for PCR were 6.5%, 82.7%, 14.3% and 66.7%, where histology was used as the gold standard, Table 3, Figure 2 and Figure 3.

Likewise, the sensitivity, specificity, PPV and NPV for the RDT kit were low (14.3%, 81.4%, 11.1% and 85.43%, respectively), where PCR was used as the gold standard, Table 4.

The main findings of the current study were that the RDT kit had moderate sensitivity (83.3%) and acceptable specificity (92.0%) for the diagnosis of peripheral P. falciparum among febrile pregnant women, but low sensitivity (17.4%) and specificity (81.7%) for diagnosing placental malaria when compared with PCR as the gold standard. The performance of the same brand of kit (SD Bioline P.f / P.v) was recently compared with PCR in eastern Sudan among febrile non-pregnant patients where it was found to have a sensitivity of 69% and specificity of 84% [28]. Recently low sensitivity (31.8%) but full specificity (100%) was reported for RDT kits used during pregnancy in Uganda, where PCR was used as the gold standard [13]. The performance of the RDT kit for malaria diagnosis used in the current study is in agreement with the findings of Schachterle et al., who showed that RDT kits had high false positive and negative rates in a region of malaria hypoendemicity in Tanzania [29]. However, the results of the later study were based on microscopy data without PCR correction. Furthermore, Mayor et al., [16] have recently shown that among the 122 women that were PCR-positive for P. falciparum (as judged by peripheral and/or placental blood sampling) 87 (71.3%) and 74 (60.7%) were not considered positive by peripheral microscopy and the HRP2 RDT, respectively.

Nevertheless, it has been observed that RDT had high sensitivity (96.8%) and specificity (73.5%) for the diagnosis of P. falciparum malaria among febrile pregnant women in a hyper-endemic region in Uganda, when compared with microscopy as the gold standard [15]. However, it was observed that the RDT had a modest level of accuracy (80.9% sensitivity, 87.5% specificity) for detecting placental malaria using peripheral blood at time of delivery, in the later study [15]. Recently, the prevalence of placental infection, as determined by microscopy and RDT, was 5.1% and 5.0%, respectively, with highly significant agreement (82.9%); however discordances were observed between the two methods at low level parasitaemias [12]. Previous studies conducted at the time of birth have shown that RDT detecting P. falciparum HRP2 are more sensitive than blood smears, and appear to be reliable predictors of adverse outcomes of malaria in pregnancy [30‐32].

Although the manufacturer’s (Bio Standard Diagnostics, Gurgaon, Korea) instructions were strictly followed, the poor performance this RDT kit in the current study is disappointing, and perhaps somewhat difficult to explain. Furthermore, the RDT used in the current study was very sensitive and specific when evaluated by WHO/ FIND [11]. High sensitivity is needed to provide confidence to the practicing physician that the RDT is unlikely to miss a malaria infection in pregnancy. However, the PPV was very low (14.3%); this could be due to false positive results, possibly attributable to the persistent nature of HRP-2 antigenaemia that has been documented already in previous studies [33, 34]. It should be mentioned that HRP2 based RDT positivity among pregnant women can persist for up to 28 days after antimalarial drug treatment, especially among women with low gravidity and those with a higher parasite density at enrolment [14]. It seems to be still there is a great challenge in diagnosing malaria and its treatment adverse effects and associated anemia [35‐37].

The Sudanese National Malaria Control Programme recommends the use of RDT in those settings where no expert microscopy is available, and maintains microscopic examination in those places where microscopy is of an adequate level. This RDT strategy was investigated earlier in Sudan for the home management of malaria using artemisinin-based combination therapy [38]. Therefore, based on the findings of the current study, it appears likely that implementation of malaria RDT in Sudan in settings where microscopic expertise is available should not be recommended.