Background
Malaria is a highly prevalent disease in tropical and subtropical regions, and nearly half of the world’s population is at risk of contracting it [
1]. The rapidly shrinking malaria map takes us a step closer to worldwide malaria eradication. Yet, great challenges remain. To achieve elimination and prevent resurgence, surveillance systems must adapt to the changing malaria epidemiology and be able to detect all possible malaria infections in a timely manner. Thus, the accurate identification of all malaria infections, including symptomatic and asymptomatic, has become a vital component of the control and elimination programmes [
2]. Asymptomatic malaria infection refers to malarial parasitaemia of any density in the absence of fever or other acute symptoms in individuals who have not received recent antimalarial treatments [
3]. Some asymptomatic infections have parasitaemia levels that are detectable by microscopy, whereas others can only be detected by molecular methods and are termed submicroscopic infections. At any given time, the vast majority of individuals with detectable malaria parasitaemia can be categorized as asymptomatic [
4], and they are regarded as important reservoirs sustaining malaria transmission [
5]. Therefore, low-cost, highly sensitive and specific screening tools would be very useful in the malaria elimination phase [
6].
Light microscopy (LM) is the cost-effective, gold standard for detecting symptomatic infections, but it has limitations for the diagnosis of malaria in asymptomatic individuals, especially in low-endemic settings [
6‐
13]. It has been reported that both microscopy and rapid diagnostic tests miss infections when parasite densities are low (<10 parasites/µL) [
14]. PCR is the most frequently used molecular method for detecting malaria. Several different target genes have been used, including the 18S ribosomal RNA gene (
18S rRNA) [
15‐
19],
tRNA [
20],
AMA1 [
21], and
cytochrome b [
22,
23], among which the 18S rRNA gene is the most commonly used [
11,
24‐
26]. PCR-based methods include nested PCR with DNA (nD-PCR) [
12,
24,
27‐
31], nested reverse transcriptase PCR (nRT-PCR) [
26,
30], quantitative RT-PCR [
27,
32‐
35], and more recently, capture and ligation probe-PCR (CLIP-PCR) [
36]. PCR can typically detect 5–10 parasites/µL, while nRT-PCR can detect as few as 22 parasites/mL [
26]. In addition, CLIP-PCR has been advocated for use in molecular epidemiological studies because of its higher throughput since samples can be pooled for analysis. It is necessary to compare the benefits of different methods, particularly for asymptomatic malaria.
Whereas most epidemiological surveillance has focused on the evaluation of parasite prevalence in representative populations, gametocyte carriage rarely has been assessed simultaneously. In this study, the prevalence of malaria infections was evaluated in 1005 healthy individuals in villages along the international border between China and Myanmar, where malaria elimination action plans are in place. The sensitivity and specificity of LM and three molecular diagnostic methods for detecting asymptomatic Plasmodium infections were compared. In addition, the relationship between parasite species detection using the Pv18s rRNA and gametocyte detection using the Pvs25 gene within individual samples was analysed.
Methods
Study area and sample collection
The study site is located in the northeastern Kachin State of Myanmar, along the China-Myanmar border (97.56°E and 24.75°N) [
37]. One thousand and five healthy individuals (344 males and 661 females, ages 1–82 years) living in seven villages near the Laiza township were recruited in May (530), July (235) and November (240) of 2015. Finger-prick blood samples (~100 µL) were collected on Whatman 3 M filter paper, air-dried, individually sealed in plastic bags, and stored at −20 °C until use. In addition, 100 µL of finger-prick blood were collected in EDTA tubes, kept on ice, and transferred to a nearby field laboratory on the same day for processing. The study protocol was approved by institutional review boards of the Pennsylvania State University and the local Bureau of Health in Kachin. All participants or legal guardians gave written informed consent before entering the study.
Malaria diagnosis by LM
Thick and thin blood films stained with Giemsa were prepared and read according to the World Health Organization standard operating procedure in basic malaria microscopy with an oil immersion lens (100×) by two microscopists who had at least five years of experience. Each slide was examined for at least 100 good fields by each microscopist. For positive slides, parasite density was quantified in 500 white blood cells (WBCs) on thick blood films assuming that 1 µL of blood contains 8000 WBCs [
38]. Thin films in the positive slides were further examined to identify the parasite species. For samples with discrepant results by the two microscopists, a third senior microscopist provided additional evaluation to reconcile the divergence.
Nucleic acid extraction and cDNA synthesis
Total RNA and genomic DNA were extracted from peripheral blood samples with Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, 0.1 mL of blood sample in a 1.5 mL tube was mixed with 1 mL of Trizol and incubated at room temperature for 5 min. Two hundred µL of chloroform was added and mixed vigorously by hand for 15 s. Phase separation was done by centrifugation at 12,000×g for 15 min at 4 °C. The aqueous phase was transferred to a fresh 1.5 mL tube, where 10 µg of the carrier GlycoBlue Coprecipitant (Invitrogen) and 0.5 mL of 100% isopropanol were added to precipitate total RNA. The RNA pellet was washed with 1 mL of 75% ethanol twice, air-dried, dissolved in 30 µL RNase-free water, and stored at −70 °C. Genomic DNA was isolated from the interphase and phenol phase following the protocol for DNA isolation. Genomic DNA was dissolved in 30 µL of 8 mM HEPES buffer at pH 7.0–8.0 and stored at −20 °C. One microgram of each total RNA sample was directly used as the template for nested-PCR with primers for the Plasmodium 18S rRNA gene to verify that isolated total RNA had no genomic DNA contamination.
cDNA was synthesized from 1 µg of each RNA sample (~1/6 to 1/4 of total RNA) using the Takara RNA PCR kit (AMV) version 3.0 (Takara, Japan) in a total volume of 20 µL consisting of 1 µg RNA, 4 µL 5× reverse transcriptase buffer, 2 µL dNTP mix (10 mM each),1 µL primer of Random 9mer mixed with oligo dT-adaptor primer, 20 U (0.5 µL) RNase inhibitor, 10 U (2 µL) AMV reverse transcriptase, and RNase-free water to 20 µL.
PCR detection targeting the 18S rRNA
Nested PCR with genomic DNA (nD-PCR) or cDNA (nRT-PCR)
Modified nested PCR (nD-PCR) was performed as previously described based on the 18S rRNA gene [
12,
15,
31,
39‐
41].
Plasmodium genus- and species-specific primers for
P. falciparum,
P. vivax,
Plasmodium malariae, and
Plasmodium ovale, and expected sizes of PCR fragments and PCR reaction conditions are shown in Additional file
1. Primary PCR reactions were performed in 25 µL containing 14 µL distilled H
2O, 1.0 µL each of rPLU5 and rPLU1 primers (10 µmol/L), 2.5 µL 10 × buffer, 2 µL dNTP mixture (2.5 mM), 0.5 µL rTaq (2.5 U), and 4 µL of genomic DNA. Nested PCR was performed used 2 µL of the primary PCR product as a template and species-specific primers for the four human malaria species in separate reaction tubes. PCR products were separated in 1.2% agarose gels. For PCR assessment, one positive control (from a symptomatic
P. vivax case) and one negative control (sterile water) were used in a blind test for analysis. nRT-PCR was performed similarly as for nD-PCR using 1 µL cDNA template in the primary PCR. Three clinical
P. vivax samples with an average density of 3000, 3800 and 4400 parasites/µL blood, respectively, were used to define the limits of detection (LOD) for nD-PCR and nRT-PCR. The average parasite density of each sample was determined by two microscopists, who counted parasites per 500 WBCs in thick smears assuming 8000 WBCs/µL blood. All three samples were first diluted to the same parasite density using the same whole blood from a healthy person, and then subjected to fourfold serial dilutions (2000–0.488 parasites/µL) and threefold serial dilutions (5.6–0.0026 parasites/µL) for nD-PCR and nRT-PCR, respectively. For each dilution and nucleic acid extraction, nD-PCR or nRT-PCR was performed in triplicates, and the lowest parasite density at which all three PCR replicates were positive was considered the LOD.
Capture and ligation probe-PCR (CLIP-PCR)
For CLIP-PCR, a 3-mm circle of dried blood spot on 3 M Whatman filter paper was punched out and lysed with 100 µL lysis mixture (Diacurate, Paris, France), 191 µL water, 3 µL mixed capture and detection probe for the
Plasmodium genus, and 6 µL proteinase K (50 g/L) at 56 °C for 30 min with vigorous shaking. Pooled sample spots were lysed in the same manner. Lysates were then transferred at 100 µL per well to a 96-well capture plate (Diacurate). After incubation at 55 °C for 3 h, each well was washed three times with 150 µL wash buffer and incubated with 50 µL ligation mix at 37 °C for 30 min. The plate was then washed again and used for qPCR with 25 µL/well of PCR mixture containing 1× SYBR
® Premix Ex (Takara) and 100 nmol/L primers. Amplification and detection were performed on an ABI 7500 apparatus (Applied Biosystems, Foster City, CA, USA) under the following conditions: 30 s at 95 °C, 45 cycles of 5 s at 95 °C, and 20 s at 60 °C. The melting curve was prepared from 65 °C to 90 °C using a default setting. The standard curve was made by threefold serial dilutions (8–0.004 parasites/µL) of a lysate of cultured
P. falciparum 3D7 strain diluted with a parasite-negative whole blood lysate. For CLIP-PCR, the sample was considered positive if the fluorescent signal increased within 29 cycles and the melting curve was the same as that of the positive control [
36].
RT-PCR detection targeting the Pvs25 gene
Two RT-PCR methods were used to detect
P. vivax gametocytes in samples. For detection of
P. vivax gametocytes in all 1005 samples, the 645 bp full-length
Pvs25 gene was amplified using
Pvs25-specific primers under specified reaction conditions (Additional file
1: Table S1) [
42]. The PCR reaction contained 2 µL of 10× KOD-Plus-Neo buffer, 2 µL of 2 mM dNTPs, 0.8 µL of 25 mM MgSO
4, 0.5 µL of 10 µM of pvs25_fw and pvs25_rev primers, 0.5 U of KOD Plus-Neo DNA polymerase (Toyobo, Osaka, Japan), and 1.0 µL cDNA in a final volume of 20 µL. PCR products were separated on 1.2% agarose gels. Three
P. vivax gametocyte-positive samples with average gametocyte densities of 240, 208, and 320 gametocytes/µL blood, respectively, were used to define the LOD of the Pvs25 RT-PCR. Similarly, threefold serial dilutions of the three
P. vivax gametocyte-positive samples (all diluted to 5.6–0.0026 gametocytes/µL) were used for RT-PCR, and the lowest gametocyte density at which all three PCR replicates were positive was considered the LOD.
The subset of samples that were positive for
P. vivax parasites was further analysed by the TaqMan probe-based quantitative RT-PCR (qRT-PCR) targeting the
Pvs25 transcript [
43]. Sequences of primers as well as the FAM-BHQ1-labeled probes for
Pvs25 (GenBank Accession No. XM_001608410), expected size of the PCR fragment and PCR conditions are shown in Additional file
1. The PCR reaction consisted of 10 µL of 10× primer Extaq buffer, 0.2 µL of 50× Rox Reference Dye II, 0.8 µL of pvs25_probe, 0.4 µL of 10 µM of pvs25_fw and pvs25_rev primers, 1.0 µL cDNA, and sterile water to 20 µL (Takara). A 115 bp fragment of
Pvs25 was amplified with primers pvs25_fw and pvs25_rev from the cDNA of a symptomatic
P. vivax gametocyte-positive case, cloned into the pMD-18T vector (Takara), sequenced, and used as the positive control plasmid. Tenfold dilutions of plasmid DNA (3 × 10
9 to 3 × 10
−1 copies/µL) were made in triplicates to calculate a standard curve. The amplification efficiency (E) was calculated using the slopes of the standard curves (E = 10
(−1/Slope) − 1). The LOD was measured using the threefold serial dilutions of the same three
P. vivax gametocyte-positive samples (all diluted to 9–0.001 gametocytes/µL). qRT-PCR was carried out on an ABI 7500 apparatus (Applied Biosystems, Foster City, CA, USA) and analysed with 7500 Fast Software v2.3. To identify gametocyte-positive samples, the C
t values of standard curves obtained from assay-specific plasmids were routinely included in each 96-well plate.
Statistical analysis
Pair-wise comparison among the proportions of positive detections for the different methods was made by the McNemar’s exact test. Sensitivity and specificity were calculated from the numbers of true/false positives and negatives when each of the methods was considered to be the reference method by statistical analysis software SPSS and data combined using Microsoft Excel 2010 for Windows.
Discussion
Asymptomatic infections of malaria play an important role in sustaining transmission and present an obstacle to malaria elimination in low-endemicity regions [
5]. Accurate knowledge of malaria epidemiology and transmission dynamics of the parasites in pre-elimination regions is critical for implementing effective and targeted control measures such as detect-and-treat and mass drug administration strategies. Low-cost, highly sensitive and specific screening tools for malaria are required for this purpose [
44]. Molecular detection methods, though not suitable for field operations on a large scale, are normally applied in order to obtain representative assessment of the malaria situation in a subpopulation of an area. With the ease and speed of detection, PCR is a commonly used molecular tool and the detection limit is generally 50–100 times lower than those of LM and RDT [
24,
45‐
47]. CLIP-PCR recently has been developed with a claimed level of sensitivity as low as 0.01 parasites/µL and a much increased throughput that might be suitable for active screening of malaria parasites in low-transmission settings [
36]. This study compared the sensitivity and specificity of both DNA- and RNA-based methods for detecting
Plasmodium infections during cross-sectional surveys in an area of the Greater Mekong Subregion, which aims to eliminate malaria by 2030. This study identified that the two RNA-based detection methods, one for detecting
Plasmodium 18S rRNA in asexual stages and the other for detecting the
Pvs25 in gametocytes, were the most sensitive and detected a major proportion of the infections as submicroscopic.
WHO recommend LM as the ‘gold standard’ for symptomatic malaria, but its performance for detecting asymptomatic infections, especially under low endemic settings, is generally poor. In this survey, LM only detected 1.19% of the study population carrying asymptomatic
P. vivax infections, which is consistent with prior reports of a threshold for LM of around 10 parasites/µL for a research setting [
48] and 50–100 parasites/µL for outside a research setting [
49]. Because of low parasite densities with the asymptomatic infections, LM is time-consuming and has much lower sensitivity than molecular methods. Thus, it is not favored for screening for asymptomatic infections in low-endemic settings like the present one.
Species-specific nD-PCR is a frequently used method in molecular epidemiological studies since parasite DNA can easily be preserved on filter papers, and cheap DNA-binding agents such as Chelex can be used for DNA extraction [
50]. This method, in our hands, had a parasite detection limit of less than 2 parasites/µL, similar to an earlier report [
6], and the number of infections detected was more than double that detected using LM. In comparison, nRT-PCR, based on the detection of asexual stage rRNA with a LOD of 0.01 parasites/µL blood, detected 18.61% of the study population as
Plasmodium carriers. The presence of ~3500 18S rRNA transcripts in a single asexual parasite circulating in peripheral blood largely explains the superior sensitivity of nRT-PCR [
26,
51]. Furthermore, this study extracted RNA directly from freshly collected blood samples, which may have improved the efficiency of RNA extraction.
This study specifically assessed the detection efficiency of the recently developed CLIP-PCR. Though this method had a LOD of as low as 0.01 parasites/µL of
P. falciparum, it performed only slightly better than LM and detected 1.89% of the population carrying
Plasmodium infections. First, the increased throughput means that significant pooling of the samples was used. With 500 tests in a 96-well plate, CLIP-PCR incurs significant pooling of the samples and dilution of the targets [
36]. Second, this method used parasite RNA preserved on filter papers without the addition of any stabilizers, thus target degradation may also have accounted for the lower detection sensitivity. Third, the LOD was determined for
P. falciparum, which might be different for
P. vivax. Furthermore, the inferior performance of CLIP-PCR may be due to lower number of parasites used. For nD-PCR, RT-PCR, and nRT-PCR, the nucleic acids were extracted from 100 µL of whole blood. For nD-PCR, the amount of DNA used per reaction (4/30 µL of total DNA) corresponded to ~13 µL of whole blood, while the amount of RNA used for RT-PCR and nRT-PCR corresponded to ~1 µL of whole blood. In comparison, the CLIP-PCR used a 3-mm punch of dried filter paper, which is likely equivalent to <10 µL of whole blood. Nevertheless, CLIP-PCR demands further testing and improvement if future uses in molecular epidemiological studies in low endemic settings are considered. However, the lack of transparency on the design, and unavailable information about the sequences of the capture or detection probes and the kit components (of the assay lysis mixture, wash buffers, or ligation mix), hinder wide applications of this method [
52].
With the predominant status of
P. vivax infections in the study area, the presence of gametocytes also was evaluated with two RT-PCR methods targeting the
Pvs25 transcripts. RT-PCR for
Pvs25 detected gametocyte carriage in 6% (61/1005) of the study population, further increasing the
P. vivax infection rate from 18% to 20%. Analysis of the
P. vivax-positive samples from other methods by qRT-PCR targeting
Pvs25 transcripts revealed 115 of them as gametocyte-positive. Interestingly, 101 of the 182
P. vivax-positive samples detected by nRT-PCR targeting the 18S rRNA were gametocyte-positive, whereas 28 samples were only
Pvs25 positive. Negativity by nRT-PCR in a gametocyte-positive sample could be explained by the presence of significantly higher numbers of gametocytes (
Pvs25 transcripts) than the asexual forms [
43], which may have attributed to host conditions (including pH, drug, immunity, anaemia) that stimulate gametocyte formation and decrease asexual parasites [
10,
53]. Nevertheless, the relatively high rates of gametocyte carriage suggest that a large proportion of the asymptomatic and submicroscopic infections may serve as important reservoirs of continued malaria transmission in this area of low endemicity.
This study identified the nRT-PCR method targeting the 18S rRNA as an extremely sensitive, robust, and scalable procedure for molecular surveillance. A considerable overlap of detected infections with Pvs25-based method further indicates the validity of this method. The sensitivity (LOD of 10 parasites/mL) is similar to the high-volume qPCR method (>20 parasites/mL) that uses venous blood [
25], which is logistically difficult to conduct in large epidemiological studies. The availability of improved methods for conserving nucleic acids before processing will guarantee detection of malaria prevalence even in remote regions [
19,
25,
26].
Authors’ contributions
YHZ, YZ, and YL performed field work, lab experiments, and data analysis. QW, PL, ZZ, FL and YJL participated in data analysis. YHZ wrote the first draft of the manuscript. YC, QF, and LC conceived the study and participated in the design of the study and revision of the manuscript. All authors read and approved the final manuscript.