One factor in the worldwide increase of imported malaria cases has been the presence of armed forces and United Nations (UN) peacekeeping missions in malaria-endemic areas and their subsequent return to their countries of origin [1]. As expected, the majority of the cases are caused by Plasmodium falciparum or P. vivax. However, P.ovale infection has been established as a relevant cause of imported malaria in these groups, resulting in challenges for accurate diagnosis and treatment [2].

P. ovale is mainly distributed in sub-Saharian Africa and the islands of western Pacific with prevalence between 3-10% depending on the region, population and type of diagnostic tests used [3,4]. Recently, Sutherland et al. demonstrated there are two non-recombining sympatric species of P. ovale, named P. ovale curtisi (classic type) and P. ovale (variant type) [5]. In Latin America, no autochthonous P. ovale cases have occurred and there have been just a few reports of imported P. ovale infections [6-8].

Guatemala is a Central American country, with approximately 80% of its territory considered a malaria transmission setting [9]. P. vivax and P. falciparum are the etiologic agents of malaria, with the last case of P. malariae reported in 1971 and no P. ovale cases ever documented in Guatemala [10]. In the last decade, and largely based on the scale up of vector-control strategies, a significant reduction in the number of malaria cases in the country had been observed, from 39,703 cases in 2005 [11] to 3,292 cases in 2013 (Guatemalan Malaria Control Program, unpublished data). The same trend has been observed in all Mesoamerican countries and the Española Islands (Dominican Republic and Haiti) (Malaria Control Programs, unpublished data). Thus, malaria programs in Guatemala and Mesoamerica are now aiming towards malaria elimination rather than only prevention and control. However, imported cases pose a threat to this goal [12].

Since 2004, Guatemala has deployed military personnel to contribute to UN peacekeeping missions. In 2010, an epidemiological investigation detected 12 cases of imported P. falciparum malaria in 150 Guatemalan soldiers returning from the Democratic Republic of the Congo (DRC) [13,14]. Since this event, molecular screenings to detect P. vivax /P. falciparum malaria carriers are conducted annually among returning contingents from the DRC as part of a program established between the Guatemalan National Reference Laboratory, Universidad del Valle de Guatemala and Military Healthcare and Social Services. Here we present the first P. ovale case detected in this population. To the best of our knowledge this is the first report of molecularly-confirmed imported P. ovale infection in Central America. The challenges that this case presented for an accurate diagnosis and the need to incorporate more sensitive methods will be discussed.

A 38-year-old male Guatemalan soldier presented to the facilities of the Military Hospital on June 29, 2012, to confirm suspected P. vivax malaria. Three months previously he had returned from a 9-month deployment in Africa, as part of a Guatemalan Army operation to aid the UN Organization Stabilization Mission in DRC.

The soldier reported that during his time in DRC he stayed in the towns of Dungu, Bangadi and Bunia. He travelled for a short period of time to Entebbe and Kampala (Uganda) and Mombasa (Kenya) as part of his rest and recuperation time. During the entire mission, he used an untreated bed net and received mefloquine prophylaxis (250 mg base/week). He reported high compliance with the drug, although he admitted to skipping some doses during his period in Uganda and Kenya. Upon his return to Guatemala, he and all deployed personnel were screened by malaria microscopy and P.vivax/P.falciparum PCR. Microscopic and molecular results were negative for the entire contingent.

Two months after his return to Guatemala, the patient travelled to Puerto de San José, Escuintla (a coastal zone in the southern Guatemala) and then returned to his residence in Jalpatagua, Jutiapa (eastern region). Malaria is endemic in both of these regions. He reported fatigue and fever, and malaria was clinically diagnosed by a private physician without any laboratory confirmation. He was given chloroquine diphosphate injections (no available dosage data) because the standard treatment in P. falciparum/P. vivax (the two endemic malaria parasites of the country) includes chloroquine [15].

The patient reported an improvement and returned to military duties. Three weeks later, he again presented with fever and fatigue. Malaria microscopy conducted at the Jutiapa Malaria Vector Control Program erroneously diagnosed a P. vivax infection. The patient was transferred to Guatemala City to be evaluated in the Military Medical Center.

Venous and capillary blood was taken for laboratory testing. A thin blood film identified P. vivax, although concerns about the morphology of some of the observed parasites were raised by the hospital microbiologist. A complete blood count at admission revealed thrombocytopenia (71,000 platelets/μl) and anemia (haemoglobin 12 g/dl). A rapid dengue diagnostic assay showed a current or recent infection. Other laboratory tests results were within reference values.

Based on the travel history of the patient and the doubts arisen as to its species identity, molecular testing was conducted to confirm the Plasmodium species. A P. ovale mono-infection was identified on both the blood film and whole blood of the patient using a Plasmodium genus-specific nested PCR (Figure 1) and a SYBR Green-based quantitative real-time PCR [16,17]. The blood film was re-examined to identify the morphological features of this parasite (Figure 2). Sequence analysis was conducted to discriminate between the classic or variant P. ovale. Basic local alignment search tool (BLAST) analysis revealed 95% similarity with P. ovale ssrRNA gene. The strain was classified as the classic type, P. ovale curtisi, based on its nucleotide polymorphisms [5]. The Genetics and Immunology Laboratory, Malaria Branch at Centers for Disease Control (Atlanta, US) confirmed these findings.Figure 1

Molecular detection of Plasmodium ovale in blood samples from patient by the amplification of 18S gene. Nested PCR with Plasmodium genus-conserved primers was followed by P. ovale specific primers. PCR products were separated by electrophoresis through a 2.0% agarose gel stained with ethidium bromide and flanked by a 50 bp DNA ladder (Novagen) as a size marker. Lane MM: Molecular marker 50 bp. Lane TBF: Patient’s thin blood film. LaneTBF-2: Patient’s thin blood film (duplicate). Lane WB: Patient’s whole blood. Lane Pf : Plasmodium falciparum control. Lane Pv : Plasmodium vivax control. Lane Pm : Plasmodium malariae control. Lane Po: Plasmodium ovale positive control. Lane C-: Negative control.

Imported malaria cases could lead to the introduction of drug resistant strains in Central America, as well as atypical malaria species, especially in areas where competent hosts could harbor these parasites and transmit them [3]. An integrated and timely malaria surveillance system is warranted in order to rapidly detect and control the emergence of these imported infections in the country. In the era of malaria elimination, improved guidelines for the diagnosis, treatment and control of imported malaria cases are crucial in Mesoamerica to mitigate the risk posed by such novel cases of imported infections.

Written informed consent was obtained from the patient for publication of this Case Report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.