Comparison of three molecular methods for the detection and speciation of Plasmodium vivax and Plasmodium falciparum

In 2005 more than three billion people were at risk of malaria infection [1]. In addition to Plasmodium falciparum there are four other malaria species known to infect humans; Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi [2].

Since P. falciparum infections are potentially fatal, quick and accurate diagnosis is vital for the effective treatment of this parasitic disease with artemisinin combination therapy (ACT) [3]. In Thailand, chloroquine remains the first line treatment of P. vivax since it is a safe and cost effective anti-malarial and its long half-life protects the patient from the first relapse after treatment. The current "gold standard" for Plasmodium spp. speciation is microscopic examination of blood smears [4‐8]. Microscopy provides a cost effective, rapid diagnostic tool which can easily be applied in the field. However, even the best malaria microscopists are proned to misdiagnosis, especially in cases of mixed infection and when only tiny rings are present [9]. In 2002, the United Kingdom National External Quality Assessment Scheme for Parasitology cross-checked the results of species identification by microscopists from 262 laboratories and found that the accuracy varied from 64% to 95% [6].

PCR offers an alternative to microscopy which has shown in many cases to have superior sensitivity and specificity [10‐12]. There are many techniques which utilise PCR to speciate malaria parasites [9, 13, 14], though these techniques are rarely evaluated side by side.

The aim of this study was to compare three molecular techniques for the detection and speciation of P. falciparum and P. vivax from dried blood spots collected from malaria patients in western Thailand. The results from the three molecular speciation techniques; nested PCR [9], multiplex PCR [13] and real-time PCR [14] were compared to microscopy.

Of the 136 patients presenting to the clinic with malaria symptoms, only 65.4% (89/136) had a Plasmodium spp. infection detected by microscopy. According to microscopic examination 24.3% (33/136) of specimens were positive for P. falciparum and 38.2% (52/136) for P. vivax. In 2.9% (4/136) of specimens a mixture of both species were detected (Table 2).

In 91 (67%) specimens, all three PCR techniques detected Plasmodium spp., two more than by microscopy. In positive specimens the speciation results for the PCR methods concurred in all but 4 (4.4%) cases. All conflicting speciation results were present in samples with a mixed infection result from at least one speciation method. In three of these cases Multiplex PCR did not detect P. vivax and in one case the nested PCR sample indicated a mixed infection when the consensus was a P. vivax single infection (Table 2).

The sensitivity and specificity results for the four speciation techniques differed dependent upon whether microscopy or molecular consensus was used as the gold standard. Using microscopy as gold standard, the sensitivity of all three molecular techniques for the overall detection of Plasmodium spp. was 96.6% (86/86+3) and the specificity was 89.4% (42/42+5) (Table 3B). However, when molecular consensus was used as the gold standard the sensitivity and specificity of microscopy was 94.5% (86/86+5) and 93.3% (42/42+3) respectively (Table 3A).

Predictably the specificity of all molecular techniques increased when the molecular consensus as gold standard for both P. falciparum and P. vivax, sensitivity, while sensitivity and specificity of microscopy drops.

The following costs are based on Australian prices and equipment requirements based on the set up at the Menzies School of Health Research (Table 4). Microscopy is the cheapest method in terms of both set up and execution at 0.27 USD per sample with an initial set up cost of approximately 3770 USD for a microscope. The next cheapest speciation technique is multiplex PCR at 6.28 USD per sample followed by nested and real-time PCR at 7.84 and 8.76 USD respectively. The set up costs for any PCR method are high, multiplex and nested require the same equipment which cost 36,028 USD, the major items of value being the gel documentation system and thermal cycler. Real-time PCR is cheaper to set up costing approximately 31,031 USD, the bulk of funds going towards a real-time thermal cycler.

Microscopy has the shortest amount of 'hands on' time for a single sample compared to all molecular techniques (Table 4). From DNA extraction to obtaining a result, nested PCR was the longest molecular technique to perform at approximately 11 hours and 30 minutes, real-time PCR was the shortest at four hours and 30 minutes while multiplex PCR took 10 hours.

Although this data shows that 9.6% (13/136) of microscopic diagnosis was probably incorrect, microscopy is clearly the only cost effective method for the rapid diagnosis of malaria in a field setting. In contrast, all three PCR methodologies investigated were sensitive, specific and are capable of detecting very low parasitaemia. Although economies of scale can be applied to PCR methods to reduce the time and cost involved in processing each sample, the capital costs and infrastructure needed to run and maintain PCR methods are not practical in most field settings where even intermittent electrical supply is a luxury.

In malaria research and reference laboratories the issue is not whether PCR should be the accepted gold standard, but which method to adopt. This study faced the same problem faced by others when comparing novel malaria detection assays; the traditional gold standard clearly lacks the sensitivity and specificity of the newer assays. The data from this study and data throughout much of the literature, in particular the paper by Coleman et al [15] demonstrates that the use of microscopy as gold standard devalues the effectiveness of novel malaria speciation techniques. To counter this a 'molecular consensus gold standard', based on the results of the three PCR assays was developed. Consensus was reached when two or more of the three molecular techniques were alike. Although this is not a perfect solution, it provided an objective way to assess each assay used in this study.

The results for each of the three molecular techniques agreed with the molecular consensus except in four cases. In three of these cases the multiplex failed to detect P. vivax in mixed infections and in one case the nested PCR sample gave a mixed infection false positive where the consensus was P. vivax single infection. The most likely reason behind the failure of the multiplex to detect P. vivax in the mixed infections is that the two PCR products were not visualized as separate bands after electrophoresis since the band sizes for P. falciparum and P. vivax differ by only 24-bp. These bands could have been separated with increased agarose gel density and/or a longer electrophoresis step. Conducting the multiplex amplifications in separate tubes for each species primer, paired with the reverse genus primer (Table 1) will also improve P. vivax detection in mixed species samples, but increase its cost almost fourfold. It should also be noted that the multiplex method uses twice as much template as the nested technique.

The 100% sensitivity and specificity of real-time PCR methodology supports its status as the best PCR methodology of the three tested. The rapid melting curve output provides an unambiguous result, without the need for hazardous and time consuming gel electrophoresis. While real-time PCR was the most expensive in regards to consumables, it was the least time consuming of the three PCR assays. Savings in labour costs and an increased sample throughput should offset the increased costs of running the real-time assay.

The scope of this assay is limited by the absence of P. malariae and P. ovale in the isolates tested. Future comparative studies should incorporate samples from geographical regions endemic for these species, noting that the nested PCR primers used in this study would be inappropriate for the detection of certain P. ovale strains in Southeast Asia [16]. As this study only used samples from dried filter paper spots that limit the volume of blood tested, future work should also investigate sample collection methods that allow for a greater potential DNA yield. Such a method would have to be useful in an environment lacking a cold chain. It is hoped that the molecular consensus approach used in this study will provide a more objective method to assess novel malaria diagnostic kits and PCR assays, avoiding the pitfalls of using an anachronistic gold standard.

Although rapid cost effective microscopy remains the most appropriate method for clinical diagnosis in a field setting, it lacks the sensitivity and specificity to be considered as a gold standard. Using three published molecular methods, a molecular consensus was established, providing an alternative gold standard to assess Plasmodium spp. detection and speciation methods. The data from this study suggests that the real-time PCR methodology was the most sensitive and specific method to detect P. falciparum and P. vivax from clinical blood spots dried on filter paper. The real-time PCR melting curve method is the recommended method for use in malaria reference and research laboratories