Molecular and Cellular Probes (1995) 9, 161-165

Species-specific 18S rRNA gene amplification for the detection of P. falciparum and P. vivax malaria parasites

Ashis Das, 1'2 Brian Holloway, 3 Wil l iam E. Collins,2 V. P. Shama, 4 Sushanta K. Ghosh, 4 Subrata Sinha, s* Seyed E. Hasnain, 1 Gursaran P. Talwar ~

and Altaf A. Lal 2

1National Institute of Immunology, Aruna Asaf A li Marg, New Delhi, 110067, India; 2Malaria Branch, Division of Parasitic Diseases, National Center for Infectious Diseases,

Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA; 3Biotechnology Core Facility, Scientific Resource Branch, NCID, CDC, Atlanta, GA, USA; 4Malaria Research Centre, Indian Council of Medical Research, Shamnath Marg, New Delhi,

India; SDepartment of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India

(Received 14 June 1994, Accepted 14 January 1995)

Based on the sequence diversity of the Plasmodium 18S ribosomal RNA (rRNA), we designed oligonucleotide primers for polymerase chain reaction (PCR) to yield different size fragments for P. falciparum and P. vivax. The primers for the PCR procedure were chosen such that the 5' primer was Plasmodium-conserved while the 3" primers were species-specific. Using primer cocktails and cloned plasmid DNAs containing the 18S rRNA genes or parasite genomic DNA as targets, we show that the PCR procedure yields 1-4-kb and 0.5-kb DNA fragments for P. falciparum and P. vivax, respectively. Limited field testing of this procedure demonstrated the utility of a ribosomal gene based species-specific malaria diagnosis.

KEYWORDS: diagnosis, malaria, polymerase chain reaction, ribosomal RNA.

INTRODUCTION

Four known Plasmodium species that cause malaria in humans are P. falciparum, responsible for most of the mortality; P. vivax, causing considerable mor- bidity; and the less prevalent P. malariae and P. ovale. Diagnosis of malaria is presently accomplished by the examination of Giemsa-stained blood smears. This method is both sensitive and specific with de- tections of less than 0.001% parasitemia. ~ The major disadvantage of microscopic diagnosis, however, is that it is time consuming, often subjective, and re- quires a skilled technician. For instance, to detect a 0-001% parasitemia or around 50 parasites per microlitre (assuming an erythrocyte COLInt of

* Author to whom correspondence should be addressed.

0890-8508/95/030161 + 06 $08.00/0

5x1061~1-~), a microscopist must routinely spend 30 min reading a thick blood smear. 2 These limitations of microscopy have encouraged researchers to de- velop alternate malaria diagnostic procedures.

A number of such systems have been proposed and are at different stages of development. Some of these assays are based o.n modified microscopic techniques, 3 while others use the parasite DNA coding sequences, encoded products, or antibodies as diag- nostic targets. We chose to exploit the use of the malaria parasite rRNA gene as the diagnostic target. 4 As in other eukaryotic systems, rRNA is the most abundant parasite nucleic acid representing nearly

161 © 1995 Academic Press Limited

162 Ashis

85-95% of the total cellular RNA. s The sequence characterization of the-plasmodial 18S rRNA genes has revealed considerable species-specific diver- sity. 6'7 Thesenaturally divergent sequences have been considered as interesting diagnostic targets. 6 Using oligonucleotide probes complementary to the P. fag ciparum 18S rRNA target, a diagnostic sensitivity of 0.0004% parasitemia was obtained. 8 In another study, nucleic acids (rRNA) equivalent to about ten parasites were detected by autoradiography. 9

The present study was conducted to develop a PCR-based malaria diagnosis procedure that would utilize the parasite ribosomal RNA genes as diagnostic targets. We show that sequen&e~diversity in the 18S ribosomal gene could be exploited to yield DNA fragments of different sizes in a species-specific man- ner in laboratory as well as field conditions.

0

MATERIALS AND METHODS

The 18S rRNA sequences from R falciparum,6P. vivax, 7 and human 1° were aligned to identify plas- modial genus and species-specific sequences. Com- plementary antisense oligonucleotides were synthesized for the amplification of malaria parasite 18S targets of defined size by PCR. A mixture of genus conserved and species-specific primers were used to amplify corresponding sequences present in cloned p[asmids or in genomic DNA. The 5" genus specific primers were AL286 (AGTGTGTATCAATCGAGT) and A1 (ATCAGCTTTTGATG'I-I'AGGGTATT). The P fal- ciparum specific primers were AL290 (CAG- TAATCAAATTAGGAT), AL291 (GCI-I-ATATTTGTATC- TTTGA) and' A291 (GC-I-I-ATAITIGTATCT-I-FGAGC). The P. vivax specific primers were AL289 (GGCTTGGAAGTCCTTGTT) and AL292 (-I-ICGCTT- -Iq-CATACTGT). The expected size of the amplified target was 1-2kb (AL290), 1-4kb (AL291, A291), 0-5 kb (AL289) and 1.3 kb (AL292). The plasmid tar- gets comprised of 18S rRNA genes cloned in Blue- script2 vector available in the laboratory of Altaf A. Lal at CDC, Atlanta. Genomic DNA was obtained from infected RBC (SDS and proteinase K treated) by the method of Sambrook etal. u PCR was carried out, as per protocol described in the GENEAMP kit (Perkin Elmer Cetus, USA), for 25 cycles using 100-200 ng of the isolated DNA (denaturation: 93°C, 1 rain; an- nealing: 42°C for AL 286 and 52°C for A1, 2 min; and extension 72°C, 3 rain), using the above primers. The amplified fragments were analysed on 0.8% aga- rose gel.

Field testing of the procedures was carried out on 15 clinical samples collected in Orissa State, India. Finger prick or intravenous heparinized blood was

Das et a/.

Table 1. Ampl i f icat ion of c loned 18S r ibosomal DNA.

Lane Target PCR pr imer Expected Observed code/Species size (kb) size'(kb) specificity with specific

target

2 P.f. 289/P.v. 0.521 - - 3 P.f. 290/P. f. 1.2 1.2 4 P.f. 291/P.f. 1-4 1.4 5 P.f. 292/P.v. 1.3 --* 6 P.v. 289/P.v. 0.521 0.521"1" 7 P.v. 290/P.f. 1.2 - - 8 P.v. 291/P.f. 1-4 - - 9 P.v. 292/P.v. 1.3 1.3

10 Rv. 289/P.v. 0.521 0.521 11 P.v. 290/P.f. 1.2 - - 12 P.v. 291/P.f. 1-4 - - 13 . . . . 14 P.v. 292/P.v. 1.3 1.3

* Band of expected size was not observed but one corresponding to 0-5 kb was obtained. tThe band intensity could not be captured on the photograph. DNA molecular size marker was loaded in lane 1.

collected, thin and thick blood smears were prepared for microscopic examination. Genomic DNA from the blood samples was extracted by previously described procedures, u About 100ILl blood obtained from a fingerprick yielded ,-,200 ng DNA.

RESULTS

The strategy for primer design was such that the 5' amplifying primer (Table 1) was common for both the Plasmodium species while the 3' amplifying primer was species-specific yielding amplified frag- ments of different sizes. Experiments were initially conducted with cloned 18S ribosomal genes as targets using 100 ng of plasmid DNA for each amplification reaction. Results (Fig. 1, Table 1) demonstrate that except in the case of one P. vivax-specific primer, AL292, all primers yielded DNA amplification prod- ucts of tFie size expected for the respective parasite species. AL292 in addition to generating a 1.3-kb amplification product for P. vivax, also produced a fragment of ~500 bp with P. falciparum target (Fig. 1, lane 5). The apparent sequence homology between the 3' end of AL292, and a stretch of the P. falciparum small subunit rRNA gene at nucleotide positions 753 to 760 may have allowed priming leading to the observed amplification product. However, the other P. vivax-specific primer AL289 did not amplify P. falciparum targets. Non-specific amplification of P. vivax 18S plasmid DNA with P. falciparum specific

Detection of P. falciparum and P. vivax malaria parasites 163

T a r g e t D N A

P r i m e r c o d e

S p e c i f i c i t y

liiiilililIBil IIIIIIIIIIIIII mmm mm ImI

" I Q I

I I

Q I

Q

Q

[ ' 0 ~

I ' 0 - -

1 2 3 4 5 6 7 8 9 1 0 11 12 1 9 14 1 5

Fig. 1. Amplification of cloned plasmid DNA. 100 ng of cloned 18S ribosomal gene target was amplified. The 5' primer used in all reactions was AL286. The target, primer code and designed specificities of the 3' primers are indicated in the figure. DNA molecular size markers are indicated in lane 1 and 15. Results are summarized in Table 1.

oligonucleotide AL291 was also observed. In sub- sequent field studies, the primers AL292 and AL291 were therefore not used.

Subsequent experiments were conducted on P. fal- ciparum and P. vivax genomic DNA targets using primers AL286 (Plasmodial), AL291/AL290 (P. fal- ciparum), and AL 292/AL289 (P. vivax). Genomic DNAs (100 ng) isolated from R falciparum cultures and P. vivax-infected monkey erythrocytes were used. As expected, the 3' primers AL291 and AL290 yielded fragments of 1.4 kb and 1.2 kb for R falciparum gen- omic DNA, while the 3' primers, AL292 and AL289, gave 1.3-kb and 520-bp fragments with P. vivax genomic DNA (Fig. 2).

The utility of the PCR procedures on field derived parasite material was investigated. Field samples were collected from Rourkela district in Orissa State, India. Finger-prick or heparinized intravenous blood was collected from individuals who presented with symp- toms of malaria. Simultaneously, thin and thick smears were made for microscopy. The genomic DNA was extracted ~ from these blood samples, and 100-200 ng was used for amplification. In order to avoid non- specific amplification observed earlier with plasmid targets (Fig. 1), the 5' genus conserved primer was redesigned and designated as A1, while the 3' primer (AL291) for P. falciparum was redesigned and desig- nated as A291. These modifications in the primer design facilitated annealing at higher temperatures

"rerget D N A P f P f Pv Pv

P r i m e r c o d e 2 9 1 2 9 0 2 9 2 2 8 9

S p e c i f i c i t y P f P f Pv Pv

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I

I I

I 1 2 3 4 5 6

Fig. 2. Amplification of parasite genomic DNA. 100 ng of parasite genomic DNA was used per reaction. The generic 5' primer used was AL286. The target, primer code and designed specificities of the 3' primers are indicated in the figure. DNA molecular size markers are shown in lane 1.

164 Ashis Das et al.

I P'"'°' no. 11121 s I" I" I' 17 I" I I o11111 11 1.11.1

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Fig. 3. Amplification of genomic DNA extracted from field samples. A primer cocktail of A1 (generic 5' primer). A291 (P. falciparum-specific 3' primer) and AL289 (P. vivax-specific 3' primer) was used. The annealing temperature was 52°C '. The amplification products were resolved on a 0.9% agarose gel. The lanes show the result of amplifications using genomic DNA extracted from patient samples. The lane numbers correspond to the patient numbers.

(52°C as compared to 42°C when primers AL286 and AL291 are used) to minimize the chances of false priming. The results (Fig. 3) show that of the 15 samples used in this study, 10 were diagnosed by microscopy as P. falciparum-positive (samples 1-5 and 9-13) and all of these showed the expected ,fragment size of approximately 1.4 kb for P. fal- ciparum. The remaining five, which were determined by microscopy to be positive for P. vivax (samples 6-8, 14 and 15), yielded bands of ~500bp in size as expected for this species. An interesting observation was the presence of P. vivax-specific bands in sample number 3 and 9 (lanes 3, 9), which were designated positive for P. falciparum only by microscopy. This suggested the presence of mixed infections in these samples. Non-infected blood did not generate any signal after PCR (data not shown).

D I S C U S S I O N

We demonstrate a very good concordance of micro- scopy with the amplification of parasite 18S ribosomal DNA. For wider field applicability, however, various issues still remain to be addressed. The system has yet to be analysed with regard to the sensitivity of the procedure, but the fact that the PCR procedure worked well with field samples collected in a low- transmission season suggests that the concerns of sensitivity might not be a hindrance for the use of this procedure in field. We used the method of Sam- brook et al. u to extract DNA from blood samples. The processing of the parasitized blood for the PCR,

however, needs to be simplified for the procedure to be amenable to field conditions. Several groups are working towards developing such simple procedure. In a recently published study, ~-' 20 I-d of fresh blood {with anticoagulant EDTA) was applied to the center of the glass-fibre filter disks, followed by drying and washing with water and normal saline. A piece of the filter disk containing parasite material yielded a desired DNA fragment upon amplification. We have found this procedure useful in our preliminary ex- periments with field material (data not shown). We have also found that a mixture P. falciparum-infected blood and detergent solution yields 18S ribosomal gene fragment when used as a source of DNA for amplification (Ollivera et al., personal commun- ication). Further work on these lines wil l provide the required technologic back-up for the development of a field-usable nucleic-acid-based diagnostic assay.

After this study was completed, we became aware of a complementary investigation ~3 on the use of ribosomal RNA for malaria parasite detection. The only difference between our study and that reported by Snounou and co-workers ~3 is in the design of primer. Unlike the choice of different 5' primers for each malaria parasite species, we have used common Plasmodium-specific 5' primer and different species- specific 3' primers to minimize false priming. None- theless, taken together, the results of these two studies conducted in two different malaria-endemic regions show that a PCR based assay is both sensitive and specific as compared to routine microscopy. With rapid advances in the non-radioactive diagnostic tech- niques it wil l not be long before simple nucleic acid

Detection of P. falciparum and P. vivax malaria parasites 165

ribosomal RNA-based malaria diagnostic procedures wil l be available for clinical use and epidemiologic studies including those of mixed infection.

ACKNOWLEDGEMENTS

We would like to thank the staff of the CDC Core Bio- technology Facility for synthesis of oligonucleotides. This work was supported (in part) by the United States Agency for International Development (USAID) PASA BST-0453- P-HC-2086-07 and an USAID/NIAID/CDC intra-agency agreement Y-02-A1-20004-01. The cooperation of Dr Rajpal Yadav in field studies is gratefully acknowledged.

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