Activation of minority-variant Plasmodium vivax hypnozoites following artesunate + amodiaquine treatment in a 23-year old man with relapsing malaria in Antananarivo, Madagascar
© Andrianaranjaka et al.; licensee BioMed Central Ltd. 2013
Received: 4 February 2013
Accepted: 27 May 2013
Published: 31 May 2013
In endemic areas, Plasmodium vivax relapses are difficult to distinguish from new infections. Genotyping of patients who experience relapse after returning to a malaria-free area can be used to explore the nature of hypnozoite activation and relapse. This paper describes a person who developed P. vivax malaria for the first time after travelling to Boriziny in the malaria endemic coastal area of Madagascar, then suffered two P. vivax relapses 11 weeks and 21 weeks later despite remaining in Antananarivo in the malaria-free central highlands area. He was treated with the combination artesunate + amodiaquine according to the national malaria policy in Madagascar. Genotyping by PCR-RFLP at pvmsp-3α as well as pvmsp1 heteroduplex tracking assay (HTA) showed the same dominant genotype at each relapse. Multiple recurring minority variants were also detected at each relapse, highlighting the propensity for multiple hypnozoite clones to activate simultaneously to cause relapse.
In Madagascar, the combination of artesunate + amodiaquine (ASAQ) is recommended since December 2005 as the first-line treatment for uncomplicated and non-severe malaria, regardless of the parasite species involved. One of the important differences between Plasmodium falciparum and Plasmodium vivax is the formation of hypnozoites that can cause relapses after a course of treatment . Such relapses have been described as clonal in origin in individuals returning to non endemic areas , but more recent evidence suggests that multiple low-frequency variants commonly arise during relapse [3, 4]. In this report is described a case where sequential P. vivax malaria relapses in a patient living in Antananarivo, a malaria-free urban area, following ASAQ treatment comprised multiple minority-variant parasites, suggesting simultaneous reactivation of multiple hypnozoite clones.
Following the first malaria episode on November 23, 2010, the patient remained in Antananarivo and did not travel back to Boriziny. However, on February 9, 2011 (D78), he developed fever and chills again and returned to the malaria unit at IPM. Plasmodium vivax monoinfection was confirmed by microscopy with a parasitaemia of 3,208 parasites/μl. Blood sample was collected on filter paper. The patient was again treated with ASAQ as described. The medical staff at IPM wanted to add primaquine to the treatment but none was available in Madagascar. After one week, the patient reported that symptoms had completely resolved.
Again, the patient remained in Antananarivo, but on April 22, 2011 (D150 with reference to the first attack in November 2010), he experienced similar symptoms as previously reported and returned to the the malaria unit at IPM. Microscopy was performed and P. vivax monoinfection was confirmed again with a parasitaemia of 63 parasites/μl. A blood sample was collected on filter paper, and the patient was treated with ASAQ for a third time. After a week, the patient reported that symptoms had completely resolved. Since November 2011, the subject has been living in France and has reported no further symptoms. Last contact was made with the subject on January 16, 2013.
Plasmodium vivax genotyping
Parasite DNA extracted from filter paper blood samples was analysed to compare the genotypic profile of the P. vivax isolates collected from the patient during each of the three malaria episodes. Using an Instagen kit (Bio-Rad Laboratories Headquarters, France), DNA was extracted from the blood spots. Nested-PCR was used to confirm P. vivax monoinfection in the three samples from D0, D78 and D150 . DNA from P. vivax collected from Maevatanana – a study site distant from Boriziny, was used as positive control. Human DNA from a blood donor from the HJRA University Hospital in Antananarivo was used as negative control. The lack of DNA contamination was checked using a blank (without DNA) in each run.
The case reported herein involves P. vivax malaria imported from the coastal area to the central highland area in Madagascar. Repetitive ASAQ treatment administered according to the national malaria treatment policy was effective in treating the acute illness in the patient but relapses occurred twice. Parasitaemia was lower at each subsequent attack. Pvmsp-3α genotyping indicated that the same major strain was present during the three consecutive attacks. However, such PCR-based genotyping can miss other genotypes also present in low numbers. The HTA analysis suggests that multiple other clones were involved in the relapse.
Plasmodium vivax malaria is characterized by relapses after resolution of the primary infection, derived from activation of dormant hypnozoites in the liver . The efficacy of ASAQ has been proven for treating uncomplicated P. falciparum malaria  and P. vivax malaria (Randrianarivelojosia, personal communication) in Madagascar. Regarding the case reported in this paper, P. vivax malaria occurring more than 70 days post-treatment in a patient living in the central highland does not indicate recrudescence due to ASAQ treatment failure; rather, it is indicative of typical relapse. Plasmodium vivax is the second most prevalent species of human malaria parasite behind P. falciparum in Madagascar, but it occurs in less than 5 %; of biologically confirmed malaria cases [12, 13]. Thus, it is logical to give ACT universally for all Plasmodium species whether P. falciparum or P. vivax. However, P. vivax-specific treatment of hypnozoites in the form of primaquine for radical cure is not the policy in Madagascar. A subset of patients will suffer relapse as in this gentleman. From a public health perspective, it would be important eventually to take into account the use of primaquine to eradicate dormant P. vivax hypnozites and prevent these relapses.
To some degree, the level of genetic complexity seen in malaria infections is associated with transmission intensity. Even though P. falciparum dominates malaria transmission in Madagascar, the detection of multiple minority-variants of P. vivax in the reported case suggests that genetic diversity of P. vivax parasites exists to the extent that polyclonal P. vivax infections are not rare. None of the minority variants detected evolved into the predominant strain at relapse. However, the detection of these minority variants allowed a more nuanced appreciation of the mechanisms of hypnozoite activation within a polyclonal infection. Simultaneous hypnozoite activation and relapse of multiple clones promotes genetic diversity in the parasite population as whole, and such relapse may be an important mechanism for maintaining high P. vivax genetic diversity even in areas of relatively low transmission. Furthermore, the ability to detect multiple recurring strains of the same type can lend further evidence that a recurrent malaria episode indeed represents relapse rather than new infection.
In line with any activities part of the national network for malaria drug resistance surveillance (ethical clearance no. 013/04-SANPF/CAB on January, 21, 2004), written informed consent was obtained from the patient for blood and information collection, for follow up, and for publication of this report.
The laboratory works were financially supported by the Institut Pasteur de Madagascar mainly through the Project NSA Project MDG-910-G19-M; and by the University of North Carolina, USA from the National Institutes of Health [grant number AI089819 to JJJ]. JTL was supported by a National Institutes of Health Infectious Disease Pathogenesis Research Training Grant [grant number 5T32AI0715132] and the North Carolina Clinical and Translational Science Award [grant number UL1RR025747]. We are grateful to Elisabeth Ravaoarisoa, Seheno Razanatsiorimalala and Rogelin Raherinjafy for their technical support.
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