Of the four human malaria species, three (Plasmodium vivax, P. falciparum, and P. malariae) have been reported in Sri Lanka. Currently, only P. vivax and P. falciparum are prevalent. The transmission of P. malariae was interrupted after eradication efforts in the mid-to-late 1960s, except for a sporadic case reported in 1984.1 Plasmodium ovale, which was traditionally prevalent only in Africa is now emerging in the Asian and Southseast Asian regions and has been reported in India, Indonesia, Laos, Myanmar, Thailand, Vietnam and Cambodia.2–6 We report here an indigenous case of P. ovale infection in Sri Lanka.
A 37-year-old man from Ilukhena in North Western Province came to the Base Hospital in Kuliyapitiya, Sri Lanka, on November 27, 2003 with fever, chills, and rigors. He had a history of two attacks of P. vivax malaria in 1996 and three attacks of P. vivax malaria in 2003, of which the last attack occurred two weeks before this episode. He was treated with chloroquine and primaquine. All previous attacks had been confirmed by microscopy. He gave no history of travel outside Sri Lanka or blood transfusions. During this visit for treatment, P. vivax infection with asexual stages was suspected on microscopy and blood was sampled on filter paper for analysis of molecular markers of drug resistance. Because the previous attack occurred two weeks before this episode, he was treated as a case of treatment-failure with 600 mg of quinine, every 8 hours for 10 days and 15 mg of primaquine for 5 days. Sequential blood smear examinations were performed on days 0, 2, 3, 7, 14, 21, and 28. Parasitemia disappeared by day 3. No treatment failure was reported during the four-week follow-up.
DNA extraction was carried out on a day 0 blood sample on filter paper using Instagene Matrix® resin (Bio-Rad, Marnes la Coquette, France). The sample was subjected to a real-time polymerase chain reaction (PCR) assay as previously described7 for detection of the four human Plasmodium species. Specific primer pairs have been designed for the Plasmodium small subunit ribosomal RNA gene. Real-time PCR technology was performed with fluorescent SYBR Green I. Analysis of the melting temperatures confirmed the identity of the PCR product with P. ovale control DNA. The experiment was repeated twice and the melting curves matched. DNA sequencing was not successful because of the small amount of DNA on the filter paper.
We report a case with P. ovale infection in Sri Lanka. This patient had no history of travel overseas or receipt of a transfusion of blood or any blood products, which makes this case a likely indigenous acquisition through a mosquito bite. This raises the question whether P. ovale parasites were prevalent in this region or whether the parasite had been imported recently from Africa as a result of human movement between the continents. If the infection was already prevalent in the area, the only possible explanation for it not being reported earlier is that previous infections were misdiagnosed as P. vivax on microscopy, as would have happened in this case unless a PCR analysis was performed.
On routine microscopy, it is difficult to distinguish morphologically between P. vivax and P. ovale parasites because the characteristic oval-shaped erythrocytes are not obvious on thick blood smears that are routinely used to detect malaria infections by the national malaria control program. There is also no difference in clinical presentation between infections with P. vivax and P. ovale, with both parasites resulting in pathognomonic chills and rigors every 48 hours. Plasmodium ovale is also sensitive to chloroquine, the drug of first choice for treatment of malaria in Sri Lanka. This finding would eliminate the possibility of repeat visits to health centers for more rigorous scrutiny.
Newer and more reliable diagnostic techniques currently available that are capable of detecting rare species, as was the case in this particular instance, may detect species that were always present but not detected previously by microscopy. There have been several recent reports of P. ovale infections in areas of low transmission of malaria in southern and southeast Asia.2–6 These parasites may have been introduced into Sri Lanka more recently by increased travel of tourists to Africa and by persons involved in commercial enterprises such as the gem trade.
Real-time PCR indicated that this was not an infection with any of the other human malaria parasites. Melting curves analysis provides definitive information when compared with control DNA, as previously demonstrated.7 However, we cannot exclude the possibility that this case had a zoonotic P. simiovale infection because the DNA primers used have not been evaluated against the DNA of this closely related simian parasite. The small subunit ribosomal 18S RNA gene of P. simiovale was described recently, and a phylogenetic analysis based on the gene encoding the cytochrome b protein suggests that the Sri Lankan primate malaria parasites form a separated group with the human P. ovale parasites.8,9 Unfortunately, sequencing the 18S ribosomal RNA gene was not successful, probably because of the small amount of DNA available. Cloning of these amplicons should have been performed. All DNA extracted was used in two experiments, which excluded testing for P. simiovale cytochrome b-specific primers. The morphology of P. simiovale is similar to that of P. ovale, which makes microscopic distinction unlikely. Infection with P. simiovale is prevalent in Macaca sinica monkeys in Sri Lanka,10 Brazil, and Papua New Guinea.11
The incidence of malaria in Sri Lanka has been decreasing since 2000. In 2003, 10,510 malaria cases were reported, of which 9,237 were P. vivax. In 2006, this number decreased to 586. Strategies used for malaria control focus on early detection and treatment of cases and selective vector control by indoor residual spraying and use of insecticide treated nets. With relatively good malaria control, there is a selective advantage for malarial species developing hypnozoites that emerge during times of low incidence. Furthermore, when transmission intensities are extremely low, as at the present time, imported malarias and zoonotic infections could pose a relatively greater threat to malaria control than at other times.
This case of infection with P. ovale was detected during a therapeutic efficacy trial of chloroquine for treatment of P. vivax infections in Sri Lanka. This incidental finding of a case of P. ovale infection has implications for malaria control in this country and highlights the need to rigorously monitor malaria incidence, as well as prevalent Plasmodium species, with newer and more reliable diagnostics.
Address correspondence to Ananda R. Wickremasinghe, Department of Public Health, Faculty of Medicine, University of Kelaniya, PO Box 06, Thalagolla Road, Ragama, Sri Lanka. E-mail:
Authors’ addresses: Renu D. Wickremasinghe and Rushika S. Wijesinghe, Department of Parasitology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka, E-mails:
Acknowledgments: We thank the staff of the Anti Malaria Campaign, Kurunegala, the staff of the Medical Officer of Health Office, and the staff of the Department of Parasitology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Sri Lanka for their support during this study.
Financial support: This study was supported by the Global Malaria Program of the World Health Organization
Disclaimer: Kamini N. Mendis and Pascal Ringwald are staff members of the World Health Organization. These authors are responsible for the views expressed in this publication, which do not necessarily represent the decisions, policy, or views of the World Health Organization
Ministry of Health, 1985. Administrative Report of the Anti-Malaria Campaign. Colombo, Sri Lanka: Ministry of Health.
Incardona S, Chy S, Chiv L, Nhem S, Sem R, Hewitt S, Doung S, Mercereau-Puijalon O, Fandeur T, 2005. Large sequence heterogeneity of the small subunit ribosomal RNA gene of Plasmodium ovale in Cambodia. Am J Trop Med Hyg 72 :719–724.
Win TT, Lin K, Mizuno S, Zhou M, Liu Q, Ferreira MU, Tantular IS, Kojima S, Ishii A, Kawamoto F, 2002. Wide distribution of Plasmodium ovale in Myanmar. Trop Med Int Health 7 :231–239.
Toma H, Kobayashi J, Vannachone B, Arakawa T, Sato Y, Nambanya S, Manivong K, Inthakone S, 1999. Plasmodium ovale infections detected by PCR assay in Lao PDR. Southeast Asian J Trop Med Public Health 30 :620–622.
Kawamoto F, Liu Q, Ferreira MU, Tantular IS, 1999. How prevalent are Plasmodium ovale and P. malariae in east Asia? Parasitol Today 15 :422–426.
de Monbrison F, Angei C, Staal A, Kaiser K, Picot S, 2003. Simultaneous identification of four human Plasmodium species and quantification of Plasmodium DNA load by real time polymerase chain reaction. Trans R Soc Trop Med Hyg 97 :1–4.
McCutchan TF, Rathore D, Li J, 2004. Compensatory evolution in the human malaria parasite Plasmodium ovale. Genetics 166 :637–640.
Escalante AA, Freeland DE, Collins WE, Lal AA, 1998. The evolution of primate malaria parasites based on the gene encoding cytochrome b from the linear mitochondrial genome. Proc Natl Acad Sci U S A 95 :8124–8129.
Dissanaike AS, Nelson P, Garnham PCC, 1965. Plasmodium simiovale sp. nov., a new simian malaria parasite from Ceylon. Ceylon J Med Sci 14 :27–32.