Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance

The protozoan parasite Plasmodium which causes malaria is a vector-borne infectious disease, estimated to infect approximately 350-500 million and kill more than a million people world wide every year [1]. There are five species of the Plasmodium parasite that can infect humans: the most virulent form of the disease is caused by Plasmodium falciparum and Plasmodium knowlesi. Malaria caused by Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, are more chronic disease in humans. In the past three decades, with the globalization of economy and increasing immigration frequency, there has been a significant rise in the number of cases of imported malaria in non-endemic, developed countries, such as Canada [2]. Toronto is a large urban center in Canada with high rates of international travel and immigration and has recently been subjected to waves of imported outbreaks, such as SARS and swine H1N1 influenza [3].

Microscopic identification of malaria parasites relies on examination of Giemsa-stained peripheral blood smears. The sensitivity and specificity of this method are highly affected by the staining techniques used and the skill level of the microscopist [4‐9]. Although light microscopy is capable of defining parasite density, parasite stage, and speciation, this method is labour-intensive and requires well-trained experts at reference centres, not to mention delays in specimen transport to such centres. In order to make the diagnosis of malaria faster and simpler to perform, rapid tests have been developed based on antigen-capture immuno-chromatographic tests (ICTs) as a valuable adjunct to microscopy for the diagnosis of malaria [2]. The ICTs are useful for identifying P. falciparum infection, but cannot be used to specifically identify non-P. falciparum species infections such as P. vivax, P. ovale, and P. malariae[7].

In the past few decades, with the advent of technology in molecular biology, new molecular methods have been introduced, such as polymerase chain reaction (PCR) and real-time quantitative polymerase chain reaction (QPCR). These molecular methods can detect malaria parasites to the species level by targeting the parasite DNA with good analytical sensitivity and specificity in laboratory studies [2, 10‐13]. QPCR in particular has been a successful method for microbial detection from sterile sites with excellent sensitivity and has the ability to be quantitative. Many of the studies conducted to date seeking to validate malaria molecular diagnostics are limited to the research laboratory, but reports emphasizing the implementation of these techniques in clinical laboratory setup have not been adequately investigated in the past [14]. In the present study, validation of QPCR as compared to reference microscopy and two different ICTs was performed with particular attention to evaluating limits of detection, quality assurance, cost, staffing, and implementation in the clinical microbiology laboratory.

Although microscopy can be sensitive to a threshold of 5-50 parasites/μl depending on the expertise of the microscopist, the average microscopist is likely to achieve a sensitivity closer to 100 parasites/μl or higher [22, 23]. The possible explanation for the discordance in these samples could be the inability of microscopy to appreciate the degraded parasite, merozoites stage, sequestered parasites in the splenic vasculature, or the occult exo-erythrocytic schizogony stage. More likely, the LOD studies suggest that reference microscopy is unable to detect parasites when below a certain threshold. Interestingly, this study revealed that the limit of detection was significantly affected by the stage of malarial parasite. For example, the two P. malariae positive specimens "Pm1" and "Pm3" (see Table 4) had the same parasitaemia of 0.06%, but there was a marked difference of ten fold in the limit of detection. This may be due to the difference between the parasite stage proportions between the specimens. The specimen "Pm1" had higher proportion of parasite stages like growing trophozoites and mature trophozoites in comparison to "Pm3". Our QPCR appeared to be more sensitive in the detection of the malarial parasite in blood specimens than performed by our comparator institution in Alberta (see Additional file 1). This is likely because of the difference in the number of targets amplified in one tube. The QPCR screens two targets in one tube as opposed to four targets in Alberta. This suggests that the PCR reaction kinetics favours the lesser number of targets per reaction due to competition among the targets for amplification. Of note, this marginal difference did not affect result interpretation.

It has been reported that the sensitivity of ICT to detect malarial parasites decreases as the parasitaemia decreases [24]. False-negative results, particularly for P. falciparum with its more virulent natural history of infection, are of concern. For BINAXNOW four false-negative results occurred for P. falciparum infections, 13 for P. vivax, four for P. ovale, and one for P. malariae. For CARESTART, three false-negative results occured for P. falciparum infections, 10 for P. vivax, four for P. ovale, one for P. malariae and one for a P. ovale and P. malariae mixed infection. In most cases the false negative results were due to low parasitaemia, but in a very few cases the false negative results was even at high parasitaemia. This may be attributed to the prozone phenomenon (antibody excess) making antigen unavailable to be detected by ICT or a possible mutant gene resulting in altered HRP-2 antigen [24]. The sensitivity of CARESTART (85.2%) was slightly higher than BINAXNOW (83.67%), and both ICTs had similar specificities (99.26%). CARESTART sensitivity was higher because it was able to pick up non-falciparum species on more occasions than BINAXNOW, this suggests that Plasmodium lactate dehydrogenase (pLDH) used in CARESTART is a more sensitive marker than aldolase used in BINAXNOW for detecting non-falciparum species. In the majority of samples, both the ICTs have identified P. falciparum monoinfections (detected by microscopy and QPCR) as P. falciparum/mixed because both bands (P. falciparum and pan-malarial) were positive, thereby not allowing the technologist to rule out mixed infection on the basis of the ICT alone. The sensitivity of both ICTs for the detection of only P. ovale and P. malariae infections was 0% and 50% respectively.

Previous reports suggest that the expression levels of the pan-Plasmodium antigen (LDH and aldolase) in P. ovale and P. malariae may be minimal and this in combination with low parasitaemia in these infections accounts for relatively lower sensitivity for the detection of these malaria species [25, 26]. It is important to note that both the ICTs have the limitation that does not distinguish between the non-P. falciparum species (P. vivax, P. ovale, and P. malariae), nor can it reliably distinguish pure P. falciparum infections from mixed falciparum infections. However, the test was found to be simple to conduct, rapid, and easy to interpret, with excellent inter-reader agreement for BINAXNOW and CARESTART as K value = 0.872 and K value = 0.898 respectively, indicating good agreement between trained and non-trained readers. Discrepancies between readers occurred mainly when the test result was weakly positive (faint band), especially in cases of low parasitaemia. Rapid diagnostic assays like ICTs may be a useful tool when expert microscopy is not available or onsite field survey is to be conducted on large population. Considering the inherent limitation and advantages of all the assays evaluated in the present study and the evidence from previous reports, ICTs are a useful diagnostic tool and can be used as an adjunct to conventional microscopy in front-line clinical laboratories in areas where malaria is not endemic. These laboratories frequently lack sufficient specimen volume to maintain expertise in diagnostic microscopy. A rapid diagnostic test like ICT could provide a fast although still preliminary diagnosis, while results are confirmed from a reference laboratory. There were three false-positive BINAXNOW results and two false-positive CARESTART results in this study. Occasional false-positive results due to the presence of rheumatoid factor have previously been reported with diagnostic methods based on the detection of HRP II [27]. It is important to note that, due to the incidental false-negative results with ICTs, malaria infection cannot be ruled out on the basis of a negative ICT result. Reference-level microscopy remains essential especially with the capability for species identification, parasite quantitation, and capacity to rule out gametocyte only infections. However, due to its excellent LOD, QPCR at the reference level serves the essential purpose of confirming or refuting microscopic speciation in certain cases (discussed below). A limitation of QPCR and ICT was that a positive result was obtained in one blood specimen with pure P. falciparum gametocytes. This shows that the QPCR can be positive in convalescence or clinically cured infections due to persistent sexual-stage forms (gametocytes) implying that the QPCR is unable to distinguish gametocyte only infections from true infections.

The cost and the processing time for all the four techniques employed for the laboratory diagnosis of malaria were evaluated (Table 6). At the time of writing, when purchased in bulk, the BINAXNOW kit was priced at US$5.20 per sample, whereas the CARESTART™ Malaria was priced at US$1.20 per sample. Of note, no significant difference was observed in this study between either ICT in spite of the cost differential. The BINAXNOW test has the approval from regulatory bodies in North America, such as the Food and Drug Administration and Health Canada. Reference microscopy of thick and thin smears costs US$2.50 for reagents in our reference laboratory setting. The ABI 7900HT Fast Real-Time PCR System used here for QPCR had a per sample cost of US$14.80 for reagents. Of note, QPCR requires a one time capital equipment purchase of the thermocycler. For example, the FC2500 SmartCycler System (Cepheid, Sunnyvale. CA) costs approximately US$36,000 and is capable of 14 reactions per run which seems appropriate given the frequency of malaria requests and the "stat" nature of the test in a non-endemic setting. The cost analysis clearly showed that the CARESTART was the cheapest method. While ICTs do not require trained staff, reference parasitology laboratories continue to place emphasis on microscopy training. A challenge to staffing of QPCR is the lack of equivalent training in molecular techniques. This is in contrast to virology and bacteriology sections where cross-training in molecular techniques is now common place in the reference laboratory.

Taken together, QPCR should be implemented in all reference clinical laboratories and used in specific scenarios: (i) when a clinician does not agree with microscopic findings based on clinical pre-test probability of malaria; (ii) when two microscopists disagree on the findings particularly in cases of mixed infection, low parasitaemia, and sample degradation; (iii) when a microscopist does not visualize enough parasite stages to make a speciation call. Of note, the significance of a positive QPCR result following pharmacotherapy is still unclear as it could represent a fully treated infection with only DNA present and no live asexual stage organisms. As such, use of QPCR for test of cure is not encouraged until further studies are available. Due to the poor LOD with non-P. falciparum species and lack of advantage in diagnostic sensitivity when compared to reference microscopists for P. falciparum, the ICTs appear better suited to the community or hospital laboratory where experience with malaria smears may be limited and thus confer a diagnostic advantage.