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Journal of Clinical Microbiology, December 2006, p. 4547-4549, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01611-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Assessing the Genetic Diversity of the Aldolase Genes of Plasmodium falciparum and Plasmodium vivax and Its Potential Effect on Performance of Aldolase-Detecting Rapid Diagnostic Tests{triangledown}

Nelson Lee,1,2 Joanne Baker,2 David Bell,3 James McCarthy,1 and Qin Cheng2*

The Australian Centre for International and Tropical Health, a Joint Program of the Queensland Institute of Medical Research and the School of Population Health, University of Queensland, Brisbane, Australia,1 Department of Drug Resistance and Diagnostics, Australian Army Malaria Institute, Brisbane, Australia,2 World Health Organization—Regional Office for the Western Pacific, Manila, Philippines3

Received 3 August 2006/ Returned for modification 7 September 2006/ Accepted 22 September 2006


   ABSTRACT
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Malaria-specific rapid diagnostic tests (RDTs) targeting aldolase show highly variable sensitivities. We assessed diversity in Plasmodium falciparum and P. vivax aldolases by sequencing the coding genes from parasites of various origins. The results show that aldolases are highly conserved, indicating that antigenic diversity is not a cause of variable RDT sensitivity.


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Malaria-specific rapid diagnostic tests (RDTs) are lateral-flow immunochromatographic tests that detect specific antigens produced by malaria parasites. Malaria RDTs offer the potential to improve the accuracy of malaria diagnosis and case management, particularly when microscopic diagnosis is not available or is unreliable.

Many branded RDT products are now commercially available; some tests detect Plasmodium falciparum only, while others detect P. falciparum plus one or more of the three other species that commonly cause human malaria. The major target antigens in RDTs specific for P. falciparum are histidine-rich protein 2 (PfHRP2) and Plasmodium lactate dehydrogenase, while pan-specific Plasmodium lactate dehydrogenase and aldolase are targeted to detect the other three Plasmodium species.

Recently, we reported extensive diversity in PfHRP2 in isolates collected globally and demonstrated that this diversity affected the lower detection limits of two PfHRP2-detecting RDTs (2). We have also shown that the epitopes of anti-PfHRP2 monoclonal antibodies vary significantly in composition and frequency among different parasite isolates, resulting in antibodies that recognize different isolates at different strengths (11). These findings highlighted the potential effect of parasite genetic diversity on the performance of malaria RDTs and the need to investigate the degree of genetic diversity in antigens that are targeted by antibodies used in non-HRP2-detecting RDTs. Since a number of published studies have shown poor sensitivities of aldolase-detecting RDTs (4, 7, 9, 12, 14), genetic diversity is a plausible explanation.

Aldolase is a key enzyme in the glycolysis pathway in malaria parasites. Unlike higher vertebrates, with three tissue-specific aldolase isoenzymes (13), P. falciparum and P. vivax possess only one aldolase isoenzyme (6, 10), similar to Trypanosoma brucei (5) and Giardia lamblia (8). The P. falciparum and P. vivax aldolases are both 369 amino acids long, and their nucleotide and amino acid sequences are relatively conserved (6). However, genetic variation within P. falciparum and P. vivax aldolases has not been examined systematically.

To determine the degree of diversity and the potential effect of diversity on the performance of aldolase-detecting RDTs, we examined and compared the P. falciparum and P. vivax aldolase gene sequences for 36 P. falciparum lines and 18 P. vivax isolates originating from geographically different areas. The origins of the P. falciparum lines (2) and the collection and origin of P. vivax samples (1) were described previously and are summarized in Table 1.


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  		TABLE 1. Origins of P. falciparum and P. vivax isolates and SNPs in aldolases

 
Genomic DNA was extracted from guanidine hydrochloride-preserved blood as previously described (3) and from frozen packed cells by using a QIAamp DNA mini kit (QIAGEN, Germany) following the manufacturer's instructions. Full-length P. falciparum and P. vivax aldolase genes were amplified by PCRs using gene-specific primers. PCRs were carried out in 50-µl reaction mixtures containing 7.5 ng of each primer, 2.5 mM MgCl2, 1.25 units of Amplitaq Gold DNA polymerase (PE Applied Biosystems), a 200 µM concentration of each deoxynucleoside triphosphate (Promega, Madison, Wis.), and buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3). For P. falciparum aldolase, the primers used were 5'-TGCACTGAATATATGAATGCC-3' and 5'-GACATATTTCTTTTCATATCCTG-3', while for P. vivax aldolase, the primers were 5'-ATGGCCACTGGATCCG-3' and 5'-ACGTACTTCTTTTCGTAAAGGG-3'. PCR cycling conditions consisted of a 94°C denaturation step for 10 min followed by 40 cycles of amplification (94°C for 50 seconds, 50 seconds at 50°C for P. falciparum or 55°C for P. vivax, and 70°C for 1 min). PCR products were visualized following agarose gel electrophoresis, purified using a QIAquick PCR purification kit (QIAGEN, Valencia, CA), and then sequenced in an ABI PRISM 370 sequencer (Applied Biosystems, Inc., Foster City, CA).

Comparison of the DNA sequences of the P. falciparum aldolase genes from 36 different strains originating from eight different regions showed a high level of conservation, with a synonymous single nucleotide polymorphism (SNP) at nucleotide 174 (A to G) observed in only two isolates. Both parasites with this SNP, PH1 and Palawan130, originated from the Philippines. The remaining 34 sequences were identical to those of strains FCBR (GenBank accession no. M2881) and 3D7 (PlasmoDB accession no. NP_702314) but different from that of K1 (GenBank accession no. AAA29473) at nucleotide 512, resulting in an amino acid change (I to N). Notably, however, when the 34 sequences were aligned with another P. falciparum aldolase sequence (GenBank accession no. AF179421 [FCC1]), greater sequence divergence was observed, with 13 distinct SNPs, five of which were synonymous and eight of which resulted in an amino acid change (data not shown). There are two possible explanations for this difference. First, the differences may be due to a sequencing error in the FCC1 aldolase sequence. Alternatively, FCC1 has a rare form of aldolase that is different from those in other parasite lines. Since the aldolase from the same parasite line was sequenced by a different laboratory more recently and reported to be identical to other published aldolases (15), it is most likely that the divergent sequence deposited in GenBank contains sequence errors.

The aldolase genes from 18 P. vivax isolates from five different regions were sequenced and compared. A single synonymous SNP at base pair 615, from A to G, was observed in 3 of the 16 isolates. Two of these isolates (J54 and J55) were from China, while isolate X061 was from the Philippines. Alignment with the previously published P. vivax WDK sequence (6) (GenBank accession no. AF247063) showed a SNP at bp 1,066 resulting in an amino acid change of P to A.

In contrast to PfHRP2, both the P. falciparum and P. vivax aldolase isoenzymes appear to be highly conserved among the parasite lines and isolates we examined, which originated from geographically distinct areas. No insertions/deletions or nonsynonymous changes that are likely to affect the binding efficiency of anti-aldolase antibodies were observed in the aldolases of 36 P. falciparum and 18 P. vivax isolates. Although we cannot completely rule out the possibility that some strains of P. falciparum or P. vivax may possess a form of aldolase that has profound amino acid changes, our data suggest that it is highly unlikely. The conservation of the sequence potentially makes this enzyme a consistent target for detection by malaria-specific rapid diagnostic tests detecting P. falciparum and P. vivax infections in different regions. The overall limited sequence variation observed in both P. falciparum and P. vivax aldolases suggests that antigen variation is an unlikely cause of variation in the sensitivity of aldolase-detecting RDTs for either species. Reports of low sensitivities of aldolase-detecting RDTs are therefore more likely to be due to low antigen concentrations, to a single/small number of epitopes per parasite, or to other factors affecting RDT quality.

Nucleotide sequence accession numbers. The novel nucleotide sequences of the P. falciparum and P. vivax aldolases reported in this paper have been deposited in the GenBank database under accession numbers DQ874471 to DQ874473.


   ACKNOWLEDGMENTS
 
We thank Dennis Kyle (Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Md.) for providing some of the P. falciparum lines and Le Ngoc Anh (Military Institute of Hygiene and Epidemiology, Department of Military Medicine, Vietnam), Gao Qi (Jiangsu Institute for Parasitic Diseases, Wuxi, China), Jason Maguire (U.S. Naval Medical Research Unit 2, Jakarta, Indonesia), colleagues at the University of the Philippines and Research Institute for Tropical Medicine, Manila, Philippines, and volunteer health workers and staff for collection of P. vivax-infected blood samples. We are also grateful to John Barnwell for constructive suggestions. We thank the Australian Red Cross Blood Service (Brisbane) for providing human erythrocytes and serum used for in vitro cultivation of P. falciparum.

P. vivax isolates from Vietnam were obtained as part of the Vietnam Australia Defense Malaria Project (VADMP), a defense cooperation between the Vietnam People's Army and the Australian Defense Force. The VADMP is sponsored by the International Policy Division, Department of Defense, Australia. This project was partially funded by AusAID through the World Health Organization—Regional Office for the Western Pacific, Manila, Philippines.

The opinions expressed herein are those of the authors and do not necessarily reflect those of the Defence Health Service or any extant policy of Department of Defence, Australia, or World Health Organization.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Drug Resistance and Diagnostics, Australian Army Malaria Institute, Brisbane, Australia. Phone: 61-7-3332 4834. Fax: 71-7-3332 4800. E-mail: qin.cheng{at}defence.gov.au. Back

{triangledown} Published ahead of print on 4 October 2006. Back


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Journal of Clinical Microbiology, December 2006, p. 4547-4549, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01611-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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