Development of insulated isothermal PCR for rapid on-site malaria detection

Malaria is a global health issue and presently endemic in 97 countries [1]. In 2013, an estimated 198 million cases of malaria occurred worldwide with 584,000 deaths. Africa’s endemic countries have most cases (80 %) and deaths (90 %).

The majority of malaria cases are reported from rural areas. Socio-economic factors related to poverty, low health consciousness and disease prevention, and poor infrastructure and transport contribute to a higher prevalence rate of malaria in rural areas compared to urban areas [2]. All these factors hinder early treatment of the disease, and prompt the development of diagnostic methods that are easily accessible and usable without delay or the need to travel or transport patient samples to laboratories, which can take hours or days to reach. Thus, this study aimed to develop a portable, user-friendly diagnostic method that can be hand-carried into endemic rural areas.

Insulated isothermal polymerase chain reaction (iiPCR) is established based on Rayleigh-Bénard convection method, which can amplify nucleic acids into significant amounts within 30 min in a simple heating device [3‐5]. It is a PCR assay whereby the copper ring attached to the bottom of a special polycarbonate capillary tube (R-tube™) is heated isothermally by the device and the PCR can occur when reagents travel through temperature gradient zones created by thermal convection in a tube [6]. Integration of fluorescent hydrolysis probe technology into iiPCR further upgrades its usefulness as detection results can be displayed directly on the device [5]. The device is now commercially available and is named POCKIT™ nucleic acid analyzer (GeneReach, Taichung City, Taiwan). The platform allows iiPCR or reverse transcription-iiPCR, and fluorescence signal detection and data interpretation upon completion of reaction within 1 h. One to eight reactions can be carried out concurrently in one run at the present setting and the device is a closed system where R-tubes™ are used and inserted into the device. The built-in algorithms in the device calculate the signal-to-noise (S/N) ratio, which is the fluorescence after/before reaction, and subsequently display them as ‘+’, ‘−’ or ‘?’ according to default thresholds [7]. The device is able to detect two fluorescence dyes, i.e., 6-FAM™ and VIC^® dyes at 520 and 550 nm, respectively.

Recently, several iiPCR assays were developed for detection of pathogens, including white spot syndrome virus [5, 6] and canine distemper virus [7]. The iiPCR assay for white spot syndrome virus has also been validated to have sensitivity and specificity comparable to those of nested PCR [8]. Since the device is small and portable, it is suitable for on-site pathogen detection or fieldwork purposes. Considering all the advantages, a malaria detection assay was developed based on iiPCR approach in POCKIT™.

This study demonstrated the possibility of using portable iiPCR device, POCKIT™, in the detection of Plasmodium spp. in clinical samples. Although it may not be as sensitive and quantitative as the qPCR method, it is useful and suitable for malaria detection in the field due to its small size (28 × 25 × 8.5 cm, W × D×H), light weight (2.1 kg), easy operation and direct interpretation of results. Given that this method allows for on-site diagnosis without needing to send samples to a laboratory, it may reduce shipping costs and shorten test turn-around time, which in turn improves disease management [7].

This newly developed malaria diagnostic method, which mainly focused on the detection of the five human Plasmodium spp, was the initial and ultimate aim of this study to apply the system in malarial endemic rural areas. It could also be utilized to detect Plasmodium spp. infecting monkeys, birds and rodents, which also made it deployable for field surveillance of animal malaria. Specificity test had shown that the assay did not detect organisms other than Plasmodium. However, since the assay conferred universal detection of Plasmodium spp, it can only serve as a preliminary screening of Plasmodium infections in humans or animals. Further confirmation of Plasmodium spp. may need to be carried out using another molecular approach. This assay will be ameliorated in the future by permitting it to detect and discern P. falciparum from non-P. falciparum species using both fluorescence channels (520 and 550 nm) equipped in POCKIT™ device.

The sensitivity test of the iiPCR assay in this study showed that the lower detection limit was ≥100 copies of target DNA sequence, higher than that of qPCR assay which could go down to 1 copy number. Nevertheless, when screening the clinical samples with <100 copies of target DNA, some were found to be positively detected while the rest were either detected as ambiguous or negatively detected. This indicates that the assay could possibly detect samples with lower copy numbers. On the other hand, two samples with 212 and 213 copies of target DNA, which were anticipated to be positively detected, was interpreted as ambiguous. This suggests that iiPCR, which works on the basis of thermal convection, may not be as stable as qPCR, which employs direct heating from the block at desired temperatures. The results also revealed that the iiPCR method was not as quantitative as the qPCR method as the fluorescence ratios did not decline proportionally with the decreased copy numbers of target DNA. Furthermore, the observation that samples with high copy numbers did not consistently obtain high fluorescence ratio and vice versa, once again suggested that the iiPCR assay was qualitative. However, the assay is specific and sensitive enough to detect Plasmodium spp in the samples as long as the copy number of the target DNA is ≥ 100, therefore, a second attempt at diagnosis with higher DNA amounts might be needed if ambiguous results are acquired. If translating the copy number into parasite density, whereby a plasmodial genome carries four to eight copies of 18S SSU rRNA gene [12], the lower detection limit of this method would be 12.5–25 parasites/µl.

The evolution of iiPCR started with the development of Rayleigh-Bénard convection cell by Krishnan and team [3] to perform PCR in a steady convection flow, generated with top and bottom surfaces of the cavity maintained at 97 °C by hot plate and 61 °C by water cooled top plate, respectively. After that, Chou et al. [4] modified the method by using dry bath to heat the bottom of a capillary tube at a fixed temperature of 95 °C, followed by DNA amplification which is rendered by temperature gradient created when the tube is cooled by the surrounding air. But since fluctuations of ambient temperature, especially at temperatures >38 °C would affect the performance of the reaction, Chang and team [13] established the first insulated isothermal PCR (iiPCR) by adding thermal baffles which are able to stabilize the temperature gradient inside the reaction tube. Gel electrophoresis was used as the DNA product detection method until the fluorescent hydrolysis probe (Taqman™ probe) technology and optical detection module, which enables fluorescent signal detection, were integrated into the iiPCR system [5]. This further modified system was reported to be able to offer low-cost and rapid on-site pathogen detection, with a high sensitivity (100 %) and specificity (96.67 %) in the case of white spot syndrome virus detection [5]. Today, the system is commercially available with all-in-one functions allowing for iiPCR/reverse transcription, signal detection and data interpretation in one device.

Currently, the simplest way to diagnose malaria is using the rapid diagnostic test (RDT) and its use has increased over the past few years, especially in the developing countries. It is a test strip either in a plastic cassette enclosure or attached to cardboard that detects malaria antigen in a small amount of blood (5–15 µl) and results can be obtained in 5–20 min [14]. Among many commercial tests that have been developed and tested in various countries, only one product received FDA clearance in June 2007 [14‐20]. RDTs available on the market can detect P. falciparum alone, P. falciparum and P. vivax, as well P. falciparum from non-P. falciparum species [15‐20]. Overall, RDTs for P. falciparum might attain greater than 95 % sensitivity but parasite density needs to be ≥500/µl of blood [14]. Nevertheless, the product testing programme organized by WHO and other partners set an even lower parasite density of 200/µl (which is below the mean parasite density found in many populations with endemic malaria) as their evaluation standard of RDT in order to assure the product is reliable to identify clinical malaria in various settings [21, 22]. False-positive and false-negative results are common for RDT as it is affected by cross-reactivity with rheumatoid factor [23, 24] and the presence of inhibitor in blood [25], as well as temperature and humidity of endemic areas [14, 26].

Of late, loop-mediated isothermal amplification (LAMP), developed by Notomi et al. [27], emerged as another, similar isothermal DNA amplification method and has gained popularity as many researchers have reported it to be rapid, accurate, cost-effective, and suitable for on-site surveillance and diagnosis. The set-up of LAMP is relatively simpler and cheaper than POCKIT-iiPCR as no specific device, only water/dry bath, and no specific tubes, only normal microcentrifuge tubes, are required to perform the amplification. LAMP is also claimed to be able to amplify a few copies of DNA to a vast amount of end product, in which turbidity is observable with the naked eye within an hour. The reagents used are also much cheaper than those used for POCKIT-iiPCR. Although multiplexing cannot be done for LAMP method as compared to POCKIT-iiPCR, which permits duplexing, LAMP can be performed in high-throughput even with single-plexing in separate tubes because it only uses water/dry bath for heating and thus the capacity of the water/dry bath is much more dependable. Its resistance to inhibitory substances present in biological samples allows simpler sample preparation [28], such as heating of blood samples [29], which in turn saves time and cost in the sample processing steps required for PCR and POCKIT-iiPCR. This method has been widely developed for malaria detection as it can be performed in resource-limited settings, and a LAMP kit for malaria detection, namely Loopamp™ MALARIA Pan/Pf detection Kit, has now been brought to the market [30, 31]. LAMP method developed by Han and team has achieved the detection of up to four species of Plasmodium, with detection limit of ten copies of the target 18S rRNA genes for P. malariae and P. ovale, and 100 copies for P. falciparum and P. vivax [32].

However, the tedious part of the LAMP method development is the primer design, as four or six primers should be included in a reaction. The method developed by Han and team involved a total of 30 primers, six primers in each primer set for genus Plasmodium and four Plasmodium species [32]. Although it has been said that the recognition of six or eight distinct regions on target sequence by the primers may increase the specificity of the method, it will also increase the likelihood of primer-primer interactions, as well as primer design constraints. The major shortcoming of LAMP is false-positive results resulting from carry-over contamination of amplicons and non-specific amplification of non-target sequence [33], which are ruinous for a diagnostic method. The elimination of post-amplification procedures and the coupling of sequence-specific Taqman™ probes, as well as the use of uracil-DNA glycosylase (UDG) contained master mix, in POCKIT-iiPCR can preclude both issues. Multiplexing in a reaction is not applicable with LAMP method because Bst polymerase, which lacks 5′-3′ exonuclease activity, does not support the use of hydrolysis probes, although Tanner and his group developed a multiplexed real-time LAMP which allowed detection of one to four targets simultaneously [34]; the fluorescent signal detection is another problem to be solved. On the other hand, with the all-in-one function of POCKIT, fluorescence signal emitted by FAM™- and VIC^®-labelled probes during iiPCR can be detected directly by the built-in detector in the device.

The POCKIT-iiPCR assay developed in this study provides another option besides RDT and LAMP in the diagnosis of malaria. Despite the slightly higher cost (approximately US$2 per sample) and longer result generation time, the high specificity and low detection limit are some of the advantages of POCKIT-iiPCR assay over RDT (US$0.15–1.50 per sample) [14]. A POCKIT nucleic acid analyser costs approximately US$7000, which might be worth investing in a point-of-care PCR platform to be used in the field. POCKIT-iiPCR does not require separate equipment to perform PCR and detection, and most importantly, it does not have carry-over contamination and false-positive result issues, as occurs in the LAMP method. The design of primers and probe is relatively simpler than the LAMP method, although the cost is unfortunately higher than that of LAMP, as probe is utilized and final volume of a reaction is larger (50 µl compared to 25 µl in LAMP). The use of probes expands the potential of POCKIT-iiPCR to perform multiplexed detection in a reaction, which in turn can save time and reagents for a diagnosis. Electricity is required to operate the device but a normal generator is sufficient enough as the device’s power consumption is low. DNA extraction step cannot be omitted in this method at the moment, however direct loading of blood samples will be attempted in future by modifying the master mix in order to withstand inhibitors present in the blood yet allowing iiPCR and fluorescent signal emission to occur. Another limitation of this study was the unavailability of plasmodial cultures to evaluate the assay sensitivity. Overall, the iiPCR assay can be refined by enabling it to detect P. falciparum and other species or distinguish it from non-P. falciparum species.