Malaria PCR Detection of Parasite Reservoir in the Field
Malaria PCR Detection of Parasite Reservoir in the Field
In the context of malaria elimination, the development of new diagnostic approach capable to detect low infection for mass screening in field settings is essential. In this study, a reliable and cost-effective molecular assay for malaria diagnostic was adapted for high throughput testing and transferred into a mobile laboratory.
The real-time PCR screening assay showed good amplification efficiency and was capable to detect Plasmodium 3D7 DNA as low as 10 ng/μl. When combined together with the Instagen DNA extraction, the detection limit using 5 μl of blood on dried blood spot was 2 parasites/μl. The melt curve analysis allowed the differentiation of falciparum from non-falciparum malaria positive samples (melt temperatures were consistently higher for falciparum amplicons compared to other species). The real-time PCR screening assay was also found more sensitive than the reference 18S rRNA nested PCR, by detecting more often very low parasitaemia. This increased sensitivity is likely related to the targeted gene and the method used to detect the PCR products. Indeed, in malaria parasites, the mitochondrial genome is presented at a higher copy number than 18S rRNA gene, and, while early ring-stage parasite typically have one mitochondrial organelle, mature gametocytes have many. Moreover, the melt curve analysis step following the real-time PCR allowed the differentiation of specific/unspecific late amplification curves (generally occurring at Ct > 35), and thus, enabled an increase in the number of amplification cycles up to 45.
Species identification of positive samples was highly comparable between the two PCR methods used, with 90% concordant results. The discordances observed in four/39 samples were due to a failure to amplify the species present at very low level in a mixed infection by the 18S RNA nested PCR in three cases, and by the real-time in one case. This finding suggests that the real-time PCR species assays have an improved ability to detect minor species in mixed infection. Indeed, plasmid mixing experiments showed that the presence of a highly concentrated species did not impact the detection of the minor species, even with a ratio of 1:100,000.
As Cambodia is a low transmission area, the adopted strategy was to first screen for malaria positive cases using a highly sensitive real-time PCR, followed by a species identification of those positive samples. This approach offers the advantages of reducing result turnaround times and PCR costs. A first round of amplification with genus-specific primers can be optionally added prior to performing the four real-time PCR species assays. Although the real-time species assays alone demonstrated good performances when tested on quality controls (similar to the real-time screening assay), the addition of an outer PCR was observed to be beneficial when working with clinical samples; likely because DNA extracted by Instagen method is not very stable and sensitive to freeze/thaw cycles, and so, low quantities of parasites DNA may start being degraded during the storage time between the "screening test" and the "species test". This additional step also offers the advantage of reducing the quantity of DNA templates needed to perform the analysis.
The next challenge after the validation of the methodology was to transfer it from the laboratory to the mobile laboratory. In less than one month, a total of 4,999 samples were screened for malaria parasites in the province of Ratanakiri in October-November 2012. Five four-person teams conducting sample collections and two laboratory technicians were permanently present in the field. The technicians typically performed four DNA extractions (96-well plate format) and four real-time PCR runs per working day. An average of 240 clinical samples and 40 quality controls were analysed per day. More than 97% of the results were delivered in less than 24 hours, allowing study participants identified as malaria positive and lacking any symptoms, to be offered a treatment free of charge in less than 48 hours.
Quality control performances demonstrated the real-time PCR screening to have a very good sensitivity and specificity (100% of both extraction and PCR positive controls being positive, even for the lowest positive control at 5 parasites/μl and 98.2% of negative controls being negative). In addition, a satisfactory low interassay coefficients of variation (CV <5%) proves its reproducibility.
As the goal was to develop a malaria diagnostic intended for large-scale malaria screening studies, the challenge was to keep the testing price at a minimum. The average cost for the real-time PCR screening, including DNA extraction, is estimated at 2.75 USD per sample, whereas the identification of the four Plasmodium species was performed at 3.75 USD per sample. In addition, the global cost of the mobile laboratory fully equipped for PCR was around 200,000 USD.
The main limitations of the PCR developed are related to the DNA extraction step and, mainly to the volume of blood analysed. Indeed, for low parasitaemia samples, it is obvious that the higher the volume of blood collected, the higher the probability to pick up parasites. When collecting 5 μl of blood, the absolute limit of detection that can be achieved is one parasite/5 μl blood (0.2 parasite/μl) if the entire DNA extract is analysed. In the protocol described here, only one/ten of the DNA extract is analysed, raising the theoretical detection limit to 2 parasites/μl blood. When the overall diagnostic method was assessed on a serial dilution of falciparum parasites, the last consistently positive dilution was actually 2 parasites/μl. Increasing the amount of blood tested during mass screening studies is technically challenging as blood samples are usually collected from finger prick, and high throughput testing is facilitated by 96-well plate format collection. Moreover, the use of a higher volume of blood would raise another issue: the high quantities of human DNA that will interfere with parasite DNA amplification by PCR. To date, no (or little) information is available on the real distribution of asymptomatic parasite carriers and the minimum parasitaemia in malaria parasite carriers, therefore, the proportion of infection being missed by a screening method with a detection threshold of 2 parasites/μl is impossible to predict.
Another limitation was related to the specificity of the real-time PCR species; indeed, samples highly positive for P. malariae could be misclassified as very weak positive for P. vivax. Thus, special attention must be paid if one sample simultaneously presents an early amplification curve for P. malariae (Ct <18) and a late amplification curve for P. vivax (Ct >35), even if this scenario appears to be rare. Finally, regarding the detection of P. knowlesi infection, the primers designed were found to be unspecific and were removed from the assay. As a consequence, a P. knowlesi infection would not be missed by the real-time PCR screening, but would be identified as a P. vivax infection by the real-time PCR species assays.
Discussion
In the context of malaria elimination, the development of new diagnostic approach capable to detect low infection for mass screening in field settings is essential. In this study, a reliable and cost-effective molecular assay for malaria diagnostic was adapted for high throughput testing and transferred into a mobile laboratory.
The real-time PCR screening assay showed good amplification efficiency and was capable to detect Plasmodium 3D7 DNA as low as 10 ng/μl. When combined together with the Instagen DNA extraction, the detection limit using 5 μl of blood on dried blood spot was 2 parasites/μl. The melt curve analysis allowed the differentiation of falciparum from non-falciparum malaria positive samples (melt temperatures were consistently higher for falciparum amplicons compared to other species). The real-time PCR screening assay was also found more sensitive than the reference 18S rRNA nested PCR, by detecting more often very low parasitaemia. This increased sensitivity is likely related to the targeted gene and the method used to detect the PCR products. Indeed, in malaria parasites, the mitochondrial genome is presented at a higher copy number than 18S rRNA gene, and, while early ring-stage parasite typically have one mitochondrial organelle, mature gametocytes have many. Moreover, the melt curve analysis step following the real-time PCR allowed the differentiation of specific/unspecific late amplification curves (generally occurring at Ct > 35), and thus, enabled an increase in the number of amplification cycles up to 45.
Species identification of positive samples was highly comparable between the two PCR methods used, with 90% concordant results. The discordances observed in four/39 samples were due to a failure to amplify the species present at very low level in a mixed infection by the 18S RNA nested PCR in three cases, and by the real-time in one case. This finding suggests that the real-time PCR species assays have an improved ability to detect minor species in mixed infection. Indeed, plasmid mixing experiments showed that the presence of a highly concentrated species did not impact the detection of the minor species, even with a ratio of 1:100,000.
As Cambodia is a low transmission area, the adopted strategy was to first screen for malaria positive cases using a highly sensitive real-time PCR, followed by a species identification of those positive samples. This approach offers the advantages of reducing result turnaround times and PCR costs. A first round of amplification with genus-specific primers can be optionally added prior to performing the four real-time PCR species assays. Although the real-time species assays alone demonstrated good performances when tested on quality controls (similar to the real-time screening assay), the addition of an outer PCR was observed to be beneficial when working with clinical samples; likely because DNA extracted by Instagen method is not very stable and sensitive to freeze/thaw cycles, and so, low quantities of parasites DNA may start being degraded during the storage time between the "screening test" and the "species test". This additional step also offers the advantage of reducing the quantity of DNA templates needed to perform the analysis.
The next challenge after the validation of the methodology was to transfer it from the laboratory to the mobile laboratory. In less than one month, a total of 4,999 samples were screened for malaria parasites in the province of Ratanakiri in October-November 2012. Five four-person teams conducting sample collections and two laboratory technicians were permanently present in the field. The technicians typically performed four DNA extractions (96-well plate format) and four real-time PCR runs per working day. An average of 240 clinical samples and 40 quality controls were analysed per day. More than 97% of the results were delivered in less than 24 hours, allowing study participants identified as malaria positive and lacking any symptoms, to be offered a treatment free of charge in less than 48 hours.
Quality control performances demonstrated the real-time PCR screening to have a very good sensitivity and specificity (100% of both extraction and PCR positive controls being positive, even for the lowest positive control at 5 parasites/μl and 98.2% of negative controls being negative). In addition, a satisfactory low interassay coefficients of variation (CV <5%) proves its reproducibility.
As the goal was to develop a malaria diagnostic intended for large-scale malaria screening studies, the challenge was to keep the testing price at a minimum. The average cost for the real-time PCR screening, including DNA extraction, is estimated at 2.75 USD per sample, whereas the identification of the four Plasmodium species was performed at 3.75 USD per sample. In addition, the global cost of the mobile laboratory fully equipped for PCR was around 200,000 USD.
The main limitations of the PCR developed are related to the DNA extraction step and, mainly to the volume of blood analysed. Indeed, for low parasitaemia samples, it is obvious that the higher the volume of blood collected, the higher the probability to pick up parasites. When collecting 5 μl of blood, the absolute limit of detection that can be achieved is one parasite/5 μl blood (0.2 parasite/μl) if the entire DNA extract is analysed. In the protocol described here, only one/ten of the DNA extract is analysed, raising the theoretical detection limit to 2 parasites/μl blood. When the overall diagnostic method was assessed on a serial dilution of falciparum parasites, the last consistently positive dilution was actually 2 parasites/μl. Increasing the amount of blood tested during mass screening studies is technically challenging as blood samples are usually collected from finger prick, and high throughput testing is facilitated by 96-well plate format collection. Moreover, the use of a higher volume of blood would raise another issue: the high quantities of human DNA that will interfere with parasite DNA amplification by PCR. To date, no (or little) information is available on the real distribution of asymptomatic parasite carriers and the minimum parasitaemia in malaria parasite carriers, therefore, the proportion of infection being missed by a screening method with a detection threshold of 2 parasites/μl is impossible to predict.
Another limitation was related to the specificity of the real-time PCR species; indeed, samples highly positive for P. malariae could be misclassified as very weak positive for P. vivax. Thus, special attention must be paid if one sample simultaneously presents an early amplification curve for P. malariae (Ct <18) and a late amplification curve for P. vivax (Ct >35), even if this scenario appears to be rare. Finally, regarding the detection of P. knowlesi infection, the primers designed were found to be unspecific and were removed from the assay. As a consequence, a P. knowlesi infection would not be missed by the real-time PCR screening, but would be identified as a P. vivax infection by the real-time PCR species assays.
