Background
Mass screening and treatment (MSAT) for malaria is defined as testing of an entire population in a wide geographical area followed by treating only positive individuals, regardless of whether they have malaria symptoms. Such an approach provides important information on the epidemiology of malaria, which can be useful for further disease containment efforts [
1], and is more likely to be accepted by the target population than mass drug administration [
2]. Moreover, mass prophylactic treatment with primaquine to control
Plasmodium vivax outbreaks could be dangerous because of a high rate of glucose-6-phosphate dehydrogenase deficiency (up to14.8%) in the population of the target district [
3]. Although recently the MSAT strategy has received renewed attention in the context of malaria elimination, the World Health Organization (WHO) did not recommend MSAT as an intervention strategy to interrupt malaria transmission, as there is a lack of a highly sensitive and high throughput diagnostic tool that is both cost effective and field-friendly to operate [
2,
4].
To overcome this challenge, a high-throughput molecular assay on a 96-well plate platform called Capture and ligation probe-PCR (CLIP-PCR) has been developed [
5], enabling direct malaria RNA detection without nucleic acid purification and reverse transcription, with a limit of detection of 0.01 malaria parasite/µL blood or around 0.33 parasite/µL if using 3 mm dried blood spot (DBS) [
5,
8]. This meets the target detection limit recommended by the WHO (2 parasites/µL or lower) for PCR-based malaria screening in low-transmission settings [
6], while making malaria screening easier and less expensive than microscopy and standard nested PCR [
7,
8]. In this method, 18 S rRNA targets in blood samples are released through cell lysis and captured by a series of specific, tailed probes onto the wells of a 96-well capture plate through sandwich hybridization. After removal of extra probes and impurities, bound tailed probes are ligated to form single stranded templates for subsequent qPCR amplification and detection [
5]. Recently, an improved version of CLIP-PCR, the multi-section CLIP-PCR (mCLIP-PCR), was developed enabling
Plasmodium genus- and species-identification with a much-shortened assay time [
9].
Kachin Special Region II (KSR2), a poor mountainous area located in northern Myanmar, shares a land border of 214.6 km with Yingjiang County of Yunnan Province, China. Since the regional conflict in KSR2 in 2012, a large number of internally displaced persons immigrated and resettled along the China-Myanmar border, posing a risk to the achievements of joint cross-border malaria prevention and control activities between China (Yunnan Province) and Myanmar [
10‐
14]. In KSR2 of Myanmar, the incidence of malaria increased from 2.1% to 2012 to 5.1% in 2016 [
15]. Besides, an outbreak of
P. vivax was observed in Laiza City and the surrounding areas in 2016 [
16], where the number of malaria cases increased sharply by 2.3 times from 940 in 2015 to 2080 in 2016 [
16]. The number of malaria cases imported from Myanmar to Yingjiang County rapidly increased from 35 in 2012 [
17] to 185 in 2016 [
16], jeopardizing the goal of malaria elimination in Yingjiang County by 2020 [
18‐
20].
In this study, the authors evaluated the feasibility of using CLIP-PCR in MSAT to determine the malaria prevalence in the Laiza district of Myanmar. Understanding the level of malaria prevalence in Laiza district can provide a basis for controlling the P. vivax outbreak and reducing the cross-border malaria importation into Yunnan, China.
Discussion
For decades since the invention of PCR, most molecular diagnostic innovations focused exclusively on amplification and detection to improve the sensitivity and specificity, but few focused on preanalytical process to reduce the complexity of molecular assays, which is the real impediment to their widespread applications. CLIP-PCR is conceptually similar to ELISA, with the capture probe and detection probe being analogous to the capture antibody and detection antibody. Subsequent PCR amplification is a signal amplification (rather than target amplification) analogous to the enzymatic cascade for signal detection in ELISA. Furthermore, the ligation step in CLIP-PCR affords two important benefits ELISA cannot have: (1) significantly better specificity and reduced background, as individual probes will not generate signal; and (2) ability to significantly avoid contamination, as it is only the ligated probes that are being detected. The reduced complexity of CLIP-PCR makes it possible to achieve high-throughput and sensitive molecular screening.
Contamination, high cost associated with tedious procedures, and low throughput are some of the challenges in PCR-based detection. Therefore, the WHO requires that a laboratory performing PCR analyses with diagnostic purposes be divided into at least three physically separated compartments for reagent preparation, sample preparation, and amplification and product detection [
22,
23]. In this study, CLIP-PCR was successfully conducted in one room with only a biosafety cabinet, a refrigerator, a desktop shaking incubator (Shaker) and a qPCR machine (Fig.
2), suggesting that it is feasible to accomplish CLIP-PCR-based diagnostics regardless of laboratory space compartmentalization, provided that standard measures to prevent contamination are observed. For example, standard cares were taken to prevent cross-contamination at the sampling stage (e.g., by use of trifold sampling cards, and flaming of puncher head after each punch). In CLIP-PCR, most of the target RNA is captured to the bottom of the well, not in the solution, making it less prone to aerosol-related cross-contamination. Any unbound, cross-contaminating target RNA is unable to be a source of signal as, most importantly, what is amplified is not the target RNA but instead the ligated bound probes, which remain bound at the bottom. The amplification template does not form until after the ligation step ligated the bound probes right before amplification. These features make CLIP-PCR less prone to cross-contamination and suitable for operation in a multi-well plate format even at a room other than dedicated PCR clean-room. This study also demonstrated that a CLIP-PCR laboratory setup bypassing nucleic acid extraction would help solve the challenges of timely PCR diagnosis and prompt treatment [
2,
24,
25], with a solution for MSAT for malaria: sampling on day 0, PCR screening on day 1, and treatment on day 2. In this study the CLIP-PCR testing capacity was 538 persons/day and the screening cost was only US$0.92 /person. Although it has been reported that CLIP-PCR detection can be conducted by pooling up to 36 samples/well [
5] and that the cost per sample decreases even more with the increase in the number of pooled samples [
7], the matrix pooling strategy for high-throughput screening requires concentration from the laboratory staff, and the possibility of a sample mishandling increases with the sample pooling number. Therefore, this study decided on a 10 × 10 matrix pooling strategy, with a double-checking mechanism intrinsic to the matrix pooling approach to minimize possible error (i.e. each positive sample should also have its raw-pool and column-pool both positive. If not, something is wrong). The screening capacity was limited to < 600 persons/day per qPCR machine, based on the sample collection speed and processing capacity of the shaker and PCR machine.
Compared with “ordinary” PCR with pooling [
26,
27], which is another efficient and valid means for large scale malaria screening, CLIP-PCR with pooling has the following important advantages: (1) It does not involve nucleic acid extraction, therefore is more amenable to high throughput processing, and is much less concerned with sample contamination or nucleic acid degradation during- or post-extraction; It is also much less expensive than using automated DNA/RNA extraction devices. (2) The CLIP-PCR assay can test pooled DBS samples without losing sensitivity [
5], as all the background RNAs from the negative samples are washed away without interfering the subsequent amplification. In contrast, in standard PCR with pooling, either the extraction or the amplification efficiency may be adversely affected in pooled samples, when interfering background nucleic acids in negative samples were added. (3) CLIP-PCR is less prone to cross contamination than ordinary PCR. CLIP-PCR does, however, require a qPCR machine, which can be too costly for laboratories in resource-limited countries. A similar strategy using LAMP [
28] may reduce the overall setup cost.
The performance of CLIP-PCR, nested PCR, and quantitative reverse transcription-PCR (qRT-PCR) have been evaluated by Zhao et al. in testing of 1,005 individual samples collected from Laiza City and surrounding areas in 2015 [
29]. The author claimed significantly lower sensitivity of CLIP-PCR than the other molecular methods [
29], in contrast to similar field comparisons from the same reported area [
7‐
9]. One important flaw in Zhao et al. [
29] as a comparison study was that the CLIP-PCR was evaluated using only dried blood spot (DBS) samples with unknown quality, while nested PCR, qRT-PCR were all evaluated with fresh blood sample only [
29]. No attempt was made to evaluate the nested PCR, qRT-PCR using the same DBS samples, while when CLIP-PCR was evaluated using standard blood samples the same high sensitivity of 0.01 parasites/µL as the other molecular methods was observed by Zhao et al. [
29]. Therefore, the conclusion of Zhao et al. [
29] remains questionable. In a recent screening of 4,580 asymptomatic DBS samples, a side-by-side comparison of CLIP-PCR genus assay with standard qPCR on a subset of 100 asymptomatic DBS samples showed 100% agreement between the two methods, with high correlation (R
2 = 0.81) between Cq values of the two methods [
9].
As both CLIP-PCR and mCLIP-PCR were originally validated with standard qPCR using dried blood spots [
5,
9], and as most standard PCR setups require high-level laboratory conditions and are not suitable for high-throughput screening [
24], this study no longer verified CLIP-PCR results using other PCR protocols in the field laboratory. Instead, quality control with no-template controls, kit positive controls (cultured
P. falciparum 3D7 lysates), as well as 9 RDT-positive samples identified on day 0 in the same population, was used. The RDT positives included 8 confirmed positive
P. vivax and 1 confirmed positive
P. falciparum by microscopy on day 1. Blind screening with CLIP-PCR indicated 100% positivity, and mCLIP-PCR identified the expected species.
As shown in Table
3, according to PCR results malaria prevalence was the highest in Sha-it Yang Village (19.10%), which can be attributed to its location. Sha-it Yang Village is far from Laiza City (Fig.
1) in a remote valley close to the forest, where an ethnic army camp was set up, indicating a high risk of malaria transmission in conditions where medical service was not easily accessible [
30]. The study findings also showed that malaria was mainly detected in Laiza City and nearby areas, with the highest prevalence in the city, whereas no cases were detected in distant areas. One reason for such distribution of cases could be that Laiza City is situated in a hot, low-altitude, densely populated area, where malaria transmission is perennial with a seasonal peak, whereas in the distant high-altitude areas (except for Sha-it Yang Village), the transmission is interrupted in the cold season [
16]. Another reason is that this study started at the end of April 2017 – the early stage of the malaria epidemic peak in Laiza City, which experienced the
P. vivax outbreak in 2016 [
16]; thus, there could be a large number of asymptomatic carriers and newly infected individuals in Laiza City during the study period. This study showed that the total PCR-based parasite prevalence rate in the analyzed population was 1.36% and that the
P. falciparum/
P. vivax ratio was 0.09, which are indicative of a very low transmission area according to the WHO categorization [
31]. The proportion of subpatent infection was 64.7%, which was similar to the 67% rate of submicroscopic
P. vivax infection reported by Moreira et al. [
32] and Cheng et al. [
33], indicating a very large potentially infectious parasite reservoir in the study area.
This study has some limitations. Only 64.9% of the target population was covered by the screening. Although the sample collection team informed the target community one day in advance, some residents remained unwilling to volunteer, whereas others could not come in time, especially those who often work in the forest and are therefore at a high risk. This may have caused sampling bias. These results indicate that MSAT needs efficient logistics support. Furthermore, the PCR laboratory was established on the Chinese side of the border because of safety concerns, accommodation of researchers, and overall better conditions than in the Myanmar area under study. Although the laboratory was only 200 m away from some of the collection sites, it was not located in the field per se. Lastly, the rate of submicroscopic infection was calculated based on RDT results not on microscopy analysis, the actual number may be smaller.
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