This paper describes the development of a multiplex PCR assay for the detection and differentiation of malaria parasites combined with NALFIA to enable fast and easy readout of the results. This assay has been shown to be sensitive and specific and the readout with NALFIA is straightforward.
The assay was evaluated in three different phases. First, the analytical sensitivity and specificity was assessed and found to be very good, although the detection of P. vivax was slightly less sensitive than the detection of P. falciparum which is likely caused by the lower parasite densities present in a P. vivax sample.
Next, the robustness, reproducibility and repeatability of the assay was tested in the ring trial. This phase is often not performed in diagnostic test development and evaluation but it can give extremely valuable results. The current trial has shown, for example, that although in general the reproducibility of the assay was acceptable, there were differences in the results. Laboratory 3 failed to correctly identify some of the samples. Although the overall agreement between the laboratories was good, it was striking that laboratory 3 identified more samples as negative or a mono-infection, pointing to a reduced sensitivity of the assay in this laboratory. The exact mechanism behind this reduced sensitivity in this laboratory was not further investigated and can be the result of a variety of factors altough all reagents, samples and materials were provided to the laboratory and were the same as the other laboratories received. It should be noted that this was the only laboratory that performed the amplification on a Biorad-CFX Real-Time PCR machine, whereas the other laboratories used conventional PCR machines. The Real-Time machinery has a variable ramping time which can be installed manually whereas the other conventional PCRs have fixed ramping times and are therefore comparable in this respect. It was however not investigated whether or not the Real-Time PCR machine was the cause of the slightly poorer results but it cannot be excluded. This issue would not have would not have surfaced if a ring trial had not been performed. In all three laboratories and in the field evaluation, use of the NALFIA as a readout system was received enthusiastically by performers and was carried out without any particular difficulties.
In the final field evaluation, the sensitivity, specificity and predictive value of the assay was studied in a moderate transmission area for P. falciparum and P. vivax parasites. In this study the results of PCR-NALFIA were compared to both microscopy and RDT. Although microscopy is often considered to be the gold standard, it is not sensitive enough compared to PCR or other molecular diagnostics and may consequently obscure the results of the specificity of the assay under investigation . RDTs are even less sensitive but were used in this study to compare the performance of the PCR-NALFIA with another assay that is used on a day-to-day basis . Because of these limitations the discordant results were also analysed by a sensitive nested PCR. This study showed that PCR-NALFIA performs very well for the detection of P. falciparum and is as sensitive and specific as microscopy and PCR. The assay was also very sensitive compared to RDT and was able to pick up all samples over 50 parasites/ul that were found positive in microscopy. For P. vivax, the microscopy performed in this study was more sensitive although some samples that were found negative with microscopy were found positive in PCR and confirmed positive with the nested PCR as well. The microscopy performed extremely well in this study and it is therefore essential to mention that the protocol used for slide reading is different from the one that is used as standard in routine care, and which is usually less sensitive.
While it was not observed in the present study, a possible concern might be the risk of contamination when a PCR sample is transferred to the NALFIA device. Altough this could partly be circumvented by performing the PCR set up and amplification in one room and do the read-out in another, a closed system in which no leakage to the environment is possible should be the next step in the development. Another current disadvantage is that this assay relies on PCR technology for amplification, which demands electricity. This does not have to be a problem if the assay is going to be performed in a setting where constant supply of electricity can be guaranteed. However, if one desires to use this assay in more remote settings in which no constant supply of electricity is available, alternative platforms that are less dependent on electricity, such as LAMP  or NASBA , could be considered. Nevertheless, although these assays are generally considered to be sensitive and specific they should be subjected to a thorough analysis on their robustness, reproducibility, repeatability and user-friendliness before implementation can be considered.
The current NALFIA strips were produced by a manufacturer specialized in the production of lateral flow devices and because of the large amount of strips produced the price for the NALFIA strips is around €0,50 cent. The final costs will depend on the quanitity of devices produced. This will also depend on the shelflife of the NALFIA devices. This has not been extensively studied in this study but the devices used were performing well up to a period of 6 months after dispatching at room temperature.
Even though all reagents and materials were provided by the developers of the assay and the technicians that performed the assay, in both the ring trial and laboratory evaluation, had previous experience in molecular techniques, no training on the current protocol was given. This shows PCR-NALFIA is a user friendly procedure that can be performed with no or little extra training.
The assay in its current format, or slightly modified with more sensitive detection of P. vivax, could be an excellent tool for epidemiological studies on prevalence or distribution of parasites. In addition, it could be used as a screening tool at regional level for malaria control programmes especially in countries with declining transmission . Molecular tools have been shown to be especially valuable in areas where there is moderate to little transmission  and PCR-NALFIA may be a straightforward method to implement.