Development of a highly sensitive genus-specific qRT-PCR assay for detection and 1

quantitation of Plasmodium by amplifying RNA and DNA of the 18S rRNA genes 2

3

Edwin Kamau1*

, LaDonna S. Tolbert2, Luke Kortepeter

1, Michael Pratt

1, Nancy Nyakoe

3, 4

Linda Muringo3, Bernard Ogutu

3, John N. Waitumbi

3 and Christian F. Ockenhouse

1. 5

6

1Division of Malaria Vaccine Development, United States Military Malaria Vaccine Program, 7

Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America. 8

2Division of Entomology, Molecular Diagnostics, Walter Reed Army Institute of Research, 9

Silver Spring, Maryland, United States of America. 10

3Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya 11

12

Running title: Total Nucleic Acids PCR Assay for Malaria Diagnosis 13

Key words: Plasmodium, Malaria Diagnosis, real-time PCR, Microscopy, Malaria Elimination 14

Abbreviations: Real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (qRT-15

PCR), Limit of Detection (LOD), nucleic acids (NA), Malaria Research and Reference Reagent 16

Resource Center (MR4), American Type Culture Collection (ATCC), reverse transcriptase (RT) 17

*Corresponding author 18

Division of Malaria Vaccine Development, Center for Molecular Diagnostics and Genomic 19

Studies, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, 20

Maryland, United States of America. 21

22

Telephone number: (301) 319 7572 23

E-mail: Edwin.kamau@us.army.mil 24

Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00276-11 JCM Accepts, published online ahead of print on 8 June 2011

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Abstract 25

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A highly sensitive genus-specific quantitative reverse transcriptase real-time PCR (qRT-PCR) 27

assay for detection of Plasmodium has been developed. The assay amplifies total nucleic acids 28

(RNA and DNA) of the 18S rRNA genes with a limit of detection of 0.002 parasites/L using 29

cultured synchronized ring stage 3D7 parasites. Parasite densities as low as 0.000362 30

parasites/L were detected when analyzing clinical samples. Analysis of clinical samples 31

showed that detection of 18S rRNA genes from total nucleic acids increased the analytical 32

sensitivity of the assay by more than a log-fold compared to DNA only. When clinical samples 33

with no parasites present by microscopy were analyzed by qRT-PCR, 90% (117 of 130) were 34

positive for presence of Plasmodium nucleic acids. Quantification of clinical samples by qRT-35

PCR using total nucleic acid versus DNA was compared to microscopy. There was a 36

significantly greater correlation of parasite density to microscopy when DNA alone was used 37

compared to total nucleic acid. We conclude that analysis of total nucleic acids by qRT-PCR is a 38

suitable assay in detection of low parasite levels in patients with early-stage malaria and/or 39

submicroscopic infections and could greatly benefit malaria diagnosis, intervention trials, 40

malaria control and elimination efforts. 41

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Introduction 48

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Malaria remains one of the most devastating infectious diseases in the world. After many years 50

of neglect, various philanthropies such as Roll Back Malaria (http://www.rbm.who.int) and 51

Gates Foundation have now committed to its eventual eradication (7). The success of these new 52

initiatives hinges in part with the use of effective diagnostic and surveillance methods (11). 53

However, despite the revolutionary gains from molecular approaches in diagnosis of malaria, 54

microscopy remains the gold standard for malaria diagnosis, clinical trials efficacy evaluation 55

and epidemiological surveys not withstanding it shortcomings (15, 16). 56

In expert hands, microscopy has a detection limit of 10 to 50 parasites/L (9, 12), but the 57

average microscopist has a detection limit of about 100 parasites/L, thereby limiting the use of 58

microscopy in cases of low parasite burden (2). Studies have shown that even at submicroscopic 59

infections, mosquitoes do get infected and can potentially transmit malaria (13, 19). Therefore, 60

as we move to the era of malaria control and elimination, highly sensitive methods with high 61

throughput capabilities will be critical in parasite detection and surveillance. Such methods will 62

be important in quantifying the extent of submicroscopic infections and giving a better insight 63

into the dynamics of malaria transmission. 64

Molecular techniques for detection of specific Plasmodium nucleic-acid sequence have 65

enabled measurement of infections that are a log-fold lower than microscopy or antigen detection 66

tests (18, 20). For example, polymerase chain reaction (PCR) assays allow explicit identification 67

of malarial species and can be easily adapted for high throughput application. Real-time PCR 68

(qPCR) especially has improved the application of PCR because the assay is fast, has very low 69

risk of contamination, is highly sensitive, specific and quantitative. These qualities are not only 70

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ideal for diagnosis and clinical trials efficacy evaluation, but will increasingly become needed for 71

use in epidemiological surveys and to evaluate the success of malaria control and elimination 72

campaigns. Superiority of PCR as a diagnostic tool for detection of Plasmodium over 73

microscopy has been extensively described. For example in a recent systematic review of 74

surveys of endemic populations in which P. falciparum prevalence was measured by both 75

microscopy and PCR-based technique, the prevalence of infection measured by microscopy was 76

shown to be on average, 50.8% of that measured by PCR (17). 77

Most PCR assays target DNA of the Plasmodium multicopy 18S ribosomal RNA (rRNA) 78

genes, which, due to their high copy number and mosaic of conserved and variable regions, 79

provide an ideal molecular target for malaria genus and species identification and quantification. 80

However, even slight genetic variation within 18S rRNA gene sequences of the same species has 81

been problematic. The Plasmodium genome lacks the long tandemly repeated arrays of rRNA 82

genes found in other eukaryotes. Instead, it contains several, single 18S-5.8S-28S rRNA units 83

distributed on different chromosomes with sequence encoded by rRNA gene in one unit differing 84

from the sequence of the corresponding rRNA in the other units (8). Additionally, the expression 85

of each rRNA unit is developmentally regulated, with different sets of rRNAs being expressed at 86

different stages of the parasite life cycle (10, 21). As such, it is important to consider all these 87

factors when designing a nucleic-acid sequence based assay which targets 18S genes. 88

In this study, we describe development of a highly sensitive genus-specific quantitative 89

reverse transcriptase real-time PCR (qRT-PCR) assay for detection of Plasmodium and shown 90

that amplification of total nucleic acids (RNA and DNA) of the 18S rRNA genes significantly 91

increases analytical sensitivity of the assay. We also compared quantification of clinical samples 92

in detection of Plasmodium by qRT-PCR and microscopy. 93

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Material and Methods 94

Samples. 95

Samples used in this study were obtained from a Phase IIb pediatric clinical trial conducted 96

between March 2005 and April 2006 at the KEMRI/Walter Reed Project, Kombewa Clinic in the 97

Kombewa Division of Kisumu District, Nyanza Province, Western Kenya. The study was 98

approved by the Ethical Review Committee of the Kenya Medical Research Institute, Nairobi, 99

Kenya and Walter Reed Army Institute of Research Institute Review Board, Maryland, United 100

States. 101

Details about this study have been described elsewhere (23). Briefly, EDTA-treated 102

blood samples were collected from study participants at enrolment (Day 0) and one month after 103

administration of the third and final vaccination. In addition, blood was also drawn during 104

unscheduled clinical visits from children who were sick and suspected to have malaria. For 105

assessment of malaria, a peripheral blood smear was obtained from subjects who presented to the 106

Walter Reed Projects Kombewa Clinic with fever or a history of fever within 48 h, or an illness 107

that the attending doctor suspected might be due to malaria infection. After Giemsa staining, thin 108

and thick blood smear slides from each sample were independently examined by three expert 109

microscopists for detection of Plasmodium and count where applicable. All malaria 110

microscopists were fully trained and were required to pass