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Journal of Clinical Microbiology, May 2007, p. 1624-1627, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.02316-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Polymorphisms Influence Plasmodium ovale PCR Detection Accuracy{triangledown}

A. Calderaro,1* G. Piccolo,1 F. Perandin,2 C. Gorrini,1 S. Peruzzi,1 C. Zuelli,1,{dagger} L. Ricci,3 N. Manca,2 G. Dettori,1 C. Chezzi,1 and G. Snounou4

Department of Pathology and Laboratory Medicine, Section of Microbiology, University of Parma, Viale A. Gramsci, 14-43100 Parma, Italy,1 Department of Experimental and Applied Medicine, Institute of Microbiology, Spedali Civili, University of Brescia, Piazza Spedali Civili, 1-25123 Brescia, Italy,2 Arcispedale S. Maria Nuova, Centrale Operativa, Viale Risorgimento, 57-42100 Reggio Emilia, Italy,3 Parasitologie Comparée et Modèles Expérimentaux (Unité Scientifique Muséum 0307), Centre National de la Recherche Scientifique Institut Fédératif de Recherche 101, Muséum National d'Histoire Naturelle, CP52, 61 Rue Buffon 75231 Paris Cedex 05, France4

Received 15 November 2006/ Returned for modification 1 January 2007/ Accepted 3 March 2007


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ABSTRACT
 
Detection of Plasmodium ovale by use of a nested PCR assay with a novel Plasmodium ovale primer set was superior to detection of Plasmodium ovale by real-time PCR assays. Nested PCR was also better at detecting P. malariae. The detection of P. ovale in many patients first admitted >2 months following their return to Italy indicated that P. ovale relapses are common.


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TEXT
 
Two of the four Plasmodium parasites that infect humans, Plasmodium malariae and P. ovale, are relatively poorly investigated. Plasmodium ovale, the last of the malaria parasites of humans to be described (22), is mainly found in West Africa, is infrequently reported in Asia and Oceania, and is yet to be described in the Americas (3). Hypnozoites occur in P. ovale infections and lead to relapses, although these are considered infrequent. P. malariae, on the other hand, is globally distributed, although its prevalence is considered low. P. malariae infections do not reach high parasite levels but are associated with chronic nephropathy and can persist for decades, although dormant liver forms (hypnozoites) are not thought to occur. Sensitive molecular techniques that allow the sensitive detection and the accurate identification of Plasmodium parasites, on the basis of amplification of their small-subunit rRNA (ssrRNA) gene, revealed that the prevalences of P. malariae and P. ovale infections were higher than previously thought (9, 14, 18, 21, 25). Sequence variations were, however, detected in these genes in P. malariae and, in particular, in P. ovale, in which two types were found (4, 8, 28, 30). This led to the design of novel oligonucleotide primer combinations to increase the accuracy of P. ovale detection (5, 10, 19).

We wished to conduct a comparative study of some of the molecular diagnostic approaches for the detection of these two species and to test a new set of primers designed to recognize all types of P. ovale. To this end, we used a set of 200 blood samples, collected on admission from patients presenting to Parma University Hospital (189 patients) and the Arcispedale of Reggio Emilia (11 patients) with symptoms consistent with malaria between January 1999 and August 2005. The samples were processed as described previously (2). PCR analysis of a first set of 122 samples revealed that 62 were indeed positive for Plasmodium, and in 20 of these samples, non-P. falciparum species were found (13). The presence of P. ovale in a relatively high proportion of the samples (10/62) reflected the fact that many of the patients became infected following travel to a destination in West Africa. These observations made these samples suitable for the purposes of our study. We had noted in the course of the PCR analyses conducted with the samples described above and subsequent samples that for some patients, genus-specific primers revealed the presence of Plasmodium, but species identification could not be achieved with a set of species-specific primers (NP-1993 primers) that had been in use for the last 12 years (21). Some of these samples proved positive for P. ovale with a different set of primers (NP-2002 primers) designed to overcome the presence of polymorphisms in the ssrRNA gene of P. ovale that would affect detection efficiency (19). This provided an indication that some of the P. ovale strains in the samples collected were of the classic type, while others were of the variant type. In order to resolve this matter, primers rOVA3 and rOVA4, published previously and found to be specific for P. ovale (10), were used in the secondary reaction. The P. ovale samples described above were indeed found to be positive (amplified band of 434 bp); however, control DNAs purified from five patients infected with P. vivax alone were also positive (those from patients infected with P. malariae and P. falciparum proved negative). Sequence analysis confirmed that this primer set was not P. ovale specific under the conditions used (data not shown).

Comparison of the available P. ovale ssrRNA sequences (as found in the GenBank database in June 2006) confirmed that they fell into two distinct types (i.e., a classic type and a variant type), and these were aligned with primer sequences used in the 14 different P. ovale-specific PCR protocols (1, 5-7, 10-13, 15-17, 21, 23, 24, 27, 29) that we could identify from the literature. It was clear that some were suitable for the detection of both P. ovale ssrRNA types, whereas others were not (Table 1). Primers rOVA1 and rOVA2 span regions that differ between both types. Thus, oligonucleotide primers corresponding to the variant sequence were designed (primers rOVA1v and rOVA2v) and tested in the secondary P. ovale-specific reaction (with NP-2005 primers). By using a subset of 107 samples, detection of P. ovale (with both classic and variant ssrRNA types represented) and P. malariae was found to be more efficient when a nested PCR protocol was used than when two distinct real-time PCR (Rt-PCR) protocols were used (Table 2). The higher degree of accuracy of P. ovale detection by use of the new set of oligonucleotides (NP-2005 primers) compared to the accuracy of the previous protocols (with the NP-1993 and NP-2002 primers) was confirmed in a retrospective analysis of all samples found to be positive for this species (Table 3). The sensitivity of nested PCR amplification was consistently found to be on the order to 5 to 50 parasites genomes per aliquot analyzed (data not shown).


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  		TABLE 1. Oligonucleotides used for specific PCR-based detection of P. ovale


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  		TABLE 2. Detection of P. malariae and P. ovale by different PCR-based protocols


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  		TABLE 3. Characteristics of P. ovale samples and efficacy of detection by different PCR-based protocols

The detection of Plasmodium parasites based on species-specific sequences in the ssrRNA gene (26) could be achieved with a high sensitivity through a nested PCR approach (21) and provided a significant improvement over microscopic examination of blood smears. The efficacy and the accuracy of detection rely on a lack of variation in the target sequences to which the amplification primers hybridize. To date, evidence of polymorphisms in the ssrRNA genes was obtained for P. ovale and P. malariae (4, 8, 28, 30), mainly isolates collected in Southeast Asia, but not for P. falciparum and P. vivax. This might reflect a different and possibly more ancient evolutionary pathway for P. malariae and P. ovale. From a practical point of view, polymorphisms in P. ovale gene regions coincident with the sequences of the primers used in the PCR protocols can adversely affect the efficacy of detection of the variant types observed (Table 1). Thus, caution must be exercised when P. ovale- or P. malariae-specific oligonucleotides are selected from the literature, whether these were designed before or after the polymorphisms came to light (1, 13). Careful assessment of primer species specificity must be carried out experimentally, since minor variations in amplification conditions (which could, for example, result from the use of different thermocyclers or enzymes from different suppliers) might lead to a decrease in the specificity. This might explain why the rOVA3/rOVA4 combination designed to be specific for P. ovale (10) proved to be equally effective for the amplification of P. vivax genomic DNA. Taken together, these observations strongly advocate the development of standardized protocols for the detection of Plasmodium parasites and that these protocols be validated by reference laboratories before their adoption as an adjunct to routine diagnosis.

From a biological point of view, two aspects of the data on P. ovale are of interest. Parasites with the variant sequences are not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired the P. ovale infection in West Africa. For nine of the patients, the period between the date of return from the country of endemicity and the time of presentation at the hospital in Italy was known. Only two of these patients presented within a month of their return, whereas for all the others latency extended between 2 months and 2 years (Table 3). For these patients, the correct intake of chemoprophylaxis would not account for the suppression of the parasitemia for more than 2 months after their return to Italy. The most likely explanation is that the infections on admission in Italy were initiated from P. ovale hypnozoites, implying that relapses by this species might be more frequent than was previously thought.


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ACKNOWLEDGMENTS
 
This study was supported by Ministry of University and Scientific Research grant FIL (Parma, Italy) and Ministry of University and Scientific Research grant FIL (Brescia, Italy).


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FOOTNOTES
 
* Corresponding author. Mailing address: Faculty of Medicine and Surgery, University of Parma, Department of Pathology and Laboratory Medicine, Section of Microbiology, Viale A. Gramsci, 14-43100 Parma, Italy. Phone: 39 0521 988885. Fax: 39 0521 993620. E-mail: adriana.calderaro{at}unipr.it Back

{triangledown} Published ahead of print on 14 March 2007. Back

{dagger} Present address: Department of Laboratory Diagnostic, Unit of Immunohematology and Transfusion, University Hospital of Parma, Viale A. Gramsci, 14-43100 Parma, Italy. Back


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Journal of Clinical Microbiology, May 2007, p. 1624-1627, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.02316-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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