Reverse transcription PCR to detect low density malaria infections.

Peter Christensen,Francois Nosten,Zbynek Bozdech,Laurent Renia,Wanitda Watthanaworawit,Clare Ling,Benoit Malleret

doi:10.12688/wellcomeopenres.16564.1

Abstract

Background: Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Methods: Here we evaluated a real-time reverse transcription PCR (RT-PCR) method that reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and RT-PCR. Results: Plasmodium spp. RT-PCR confirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specific RT-PCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both RT-PCR results increased the sensitivity to 100% and specificity was 95.1%. Conclusion: Malaria detection in areas of low transmission and LDMI can benefit from the increased sensitivity of ribosomal RNA detection by RT-PCR, especially where sample volume is limited. Isolation of high quality RNA also allows for downstream analysis of malaria transcripts.

Highlights

What is the target? What is the comparison with methods used as the gold standard of polymerase chain reaction (PCR)?
Light microscopy and antigen based rapid diagnostic tests (RDTs) are the most common tests used in malaria prevalence surveys, and usually assess 5 μL of whole blood per test, a volume which precludes reliable detection of low density malaria infections (LDMI)
reverse transcription PCR (RT-PCR) was 85.7% and 96.8% respectively when compared to ultrasensitive quantitative PCR (uPCR) (Table 113)

Summary

Introduction

What is the target? What is the comparison with methods used as the gold standard of PCR?. Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Successful targeted malaria elimination strategies will require increased surveillance and highly sensitive tests capable of detecting asymptomatic and low density malaria infection (LDMI). These infections are often well below 200 parasites per milliliter and are an important disease reservoir capable of transmitting malaria that must be detected and eliminated[1,2]. Widespread use of PCR based assays, namely real-time quantitative PCR (qPCR) and reverse-transcription PCR (RT-PCR), have revealed a new landscape of malaria prevalence in low transmission areas[4,5]

Methods

Results

Discussion

Conclusion