Reverse transcription quantitative PCR to detect low density malaria infections

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. Here we evaluated a real-time quantitative reverse transcription PCR (qRT-PCR) method that significantly 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 qRT-PCR. Plasmodium spp. qRT-PCR confirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specific qRT-PCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both qRT-PCR results increased the sensitivity to 100% and specificity was 95.1%. These results show that malaria detection in areas of low transmission and LDMI, can benefit from the increased sensitivity of qRT-PCR especially where sample volume is limited.

2 blood limits the widespread tractability of this method. Here we evaluated a real-time quantitative reverse 24 transcription PCR (qRT-PCR) method that significantly reduces the required sample volume compared to uPCR.

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To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were 26 compared using uPCR and qRT-PCR. Plasmodium spp. qRT-PCR confirmed 18 of 21 uPCR Plasmodium 27 falciparum positives, while P. falciparum specific qRT-PCR confirmed 17 of the 21 uPCR P. falciparum positives.

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Combining both qRT-PCR results increased the sensitivity to 100% and specificity was 95.1%. These results

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show that malaria detection in areas of low transmission and LDMI, can benefit from the increased sensitivity 30 of qRT-PCR especially where sample volume is limited. As malaria burden reduces globally, the international community is working toward its elimination. Successful 34 targeted malaria elimination strategies will require increased surveillance and highly sensitive tests capable of 35 detecting asymptomatic and low density malaria infection (LDMI). These infections are often well below 200 36 parasites per milliliter and are an important disease reservoir capable of transmitting malaria that must be 37 detected and eliminated (1, 2). Light microscopy and antigen based rapid diagnostic tests (RDTs) are the most 38 common tests used in malaria prevalence surveys, and usually assess 5 μL of whole blood per test, a volume 39 which precludes reliable detection of LDMI. Ultrasensitive RDTs improve detection sensitivity of patients with a 40 parasitaemia between 200 parasites/mL and 10,000 parasites/mL (3), but are still limited by their low input 41 volume. Molecular methods using the polymerase chain reaction (PCR) remain the only common and reliable 42 method to detect LDMI. This technique's sensitivity is due to its ability to detect single, specific DNA molecules 43 and use concentrated DNA from a large sample. Widespread use of PCR based assays, namely real-time 44 quantitative PCR (qPCR) and reverse-transcription qPCR (qRT-PCR), have revealed a new landscape of malaria 45 prevalence particularly in low transmission areas (4, 5).

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The targeted malaria elimination project (TME) on the Eastern Myanmar border used a high blood volume 47 ultrasensitive qPCR (uPCR) to consistently detect parasitaemia down to 22 parasites per mL (6), and revealed a 48 high proportion of LDMI (7). Major features of uPCR are its 7 copies of gene target, the high volume of blood 49 tested and the ability to accurately quantify low density parasitaemia. Although increasing the blood volume of 50 3 a PCR leads to higher sensitivity (6), the collection of large numbers of high volume samples have their own 51 specific limitations. These can include, the ethics approval required for venous blood draw, sample logistics, 52 increased nucleic acid extraction cost and increased sample processing time. Another way to increase the 53 sensitivity of PCR is to increase the number of specific nucleic acid targets per parasite by targeting specific 54 RNA and DNA using qRT-PCR. As previously reported by Kamau et al. (8), the primer set used in uPCR can be 55 made more sensitive by incorporating reverse transcription prior to qPCR, enabling detection of the 7 genes 56 encoding Plasmodium 18S ribosomal nucleic acid (rRNA) and its rRNA transcripts.

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In this study, we compare the sensitivity and specificity of high sample volume, low target copy number 58 ultrasensitive qPCRs, with reduced sample volume, high target copy number qRT-PCR for the detection and 59 quantification of Plasmodium spp. and P. falciparum.

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LDMI detection is essential for effective targeted elimination programs, necessitating the careful selection of a 140 detection assay that is appropriate to the setting and study requirements. Sample volume, storage conditions 141 and transit time are important factors, as well as the required assay sensitivity, specificity and its cost. RNA is 142 generally less stable than DNA, and accurate RNA quantification often requires normalization due to variable 143 transcription rates. It is for these reasons that DNA based qPCR was chosen for the original study as this 144 approach enabled accurate quantification of LDMI in a setting where samples from remote locations would 145 likely experience delays.

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The targeted malaria elimination (TME) project on the Eastern Myanmar border used conserved regions of the 147 18S rRNA genes as the target for qPCR. This high sample volume uPCR assay was modified from the qRT-PCR 148 published by Kamau et al. 2011 for their detection of low density malaria infections (LDMI). After uPCR 7 detected a high prevalence of LDMI in the region (9), and with continued surveillance in mind, we wanted a 150 lower sample volume assay with similar LDMI detection sensitivity. As Kamau et al. has shown, using this 151 primer set as a qRT-PCR significantly increases the sensitivity of the assay. The increased sensitivity of qRT-PCR 152 is due to the increased number of targets per parasite (compared to uPCR). These amplification targets include 153 the 18S rRNA genes, and the structural RNA of each ribosome. Plasmodium genus specific uPCR amplifies type 154 A and S 18S ribosomal RNA genes distributed on chromosomes 1, 5, 7, 11 and 13. A positive qPCR reaction 155 requires at least one of these genes to be included in the assay. Alternatively, qRT-PCR can detect these gene  Alternatively, LDMI detection relying on DNA, will have a significant reduction in sensitivity if only a fraction of 171 the DNA template is tested. This is exemplified by the lowest concentration standard used for quantification in 172 uPCR. This standard theoretically contains 0.032 parasites per PCR reaction, at this concentration there is a 1 in 173 5 chance for the reaction to be positive (7 target genes per parasite x 0.032 = 0.2 copies per reaction). These 174 factors need to be considered when setting up a qPCR standard curve for LDMI quantification. A reliable 175 standard curve for qPCR requires at least one copy of its target at the lowest concentration. In conclusion, the 176 success of any LDMI detection protocol relies on the careful consideration of the following factors: sample 177 8 volume, elution volume, template volume per assay reaction, primer set target and assay type (uPCR or qRT-178 PCR). Our experience of the different types of assay suggest that for a LDMI program requiring highly sensitive, 179 accurate quantification and where venous blood collection is possible, uPCR is recommended. In an 180 environment where blood volume is limited (i.e. finger prick sampling) and quantification accuracy of 181 parasitaemia is less important, qRT-PCR is a suitable alternative capable of detecting a single parasite in a given 182 sample volume.  Myanmar. Parasitaemia by uPCR ranged from 17 to 9.91x10 6 parasites per mL whole blood. One-way 241 ANOVA with multiple comparisons revealed no significance between quantification results. 242 uPCR, Plasmodium spp. RT-qPCR and P. falciparum RT-qPCR including total positives, total negatives, false positives, false negatives, sensitivity and specificity using Plasmodium spp. uPCR results as gold standard.