Molecular method for the diagnosis of imported pediatric malaria

Médecine et maladies infectieuses 40 (2010) 115–118

Case report

Molecular method for the diagnosis of imported pediatric malaria Place des approches moléculaires dans le diagnostic du paludisme d’importation de l’enfant L. Delhaes Jeanne a,∗ , A. Berry b , E. Dutoit a , F. Leclerc c , J. Beaudou b , S. Leteurtre c , D. Camus a , F. Benoit-Vical b a

Laboratoire de parasitologie-mycologie EA3609, département de microbiologie de médecine, faculté de médecine, université de Lille 2, CHU de Lille, Lille, France b Service de parasitologie, hôpital de Rangueil, CHU de Toulouse, Toulouse, France c Réanimation pédiatrique, hôpital Jeanne-de-Flandre, faculté de médecine, université de Lille 2, CHU de Lille, Lille, France Received 5 July 2008; accepted 29 April 2009 Available online 23 July 2009

Abstract Malaria is a polymorphous disease; it can be life threatening especially for children. We report a case of imported malaria in a boy, illustrating the epidemiological and clinical aspects of severe pediatric malaria. In this case real-time PCR was used to quantify Plasmodium falciparum DNA levels, to monitor the evolution under treatment, and to determine genetic mutations involved in chloroquine resistance. The major epidemiological features of imported malaria, and the difficulty to diagnose childhood severe malaria are described. The contribution of molecular methods for the diagnosis of imported malaria is discussed. © 2009 Elsevier Masson SAS. All rights reserved. Keywords: Chloroquine-resistance; Plasmodium falciparum; Real-time PCR

Résumé Le paludisme est une maladie d’expression clinique polymorphe, pouvant mettre en jeu le pronostic vital, notamment chez l’enfant. Nous rapportons un cas de paludisme sévère chez un enfant pour lequel la PCR en temps réel a été mise à profit pour quantifier l’ADN parasitaire, suivre l’évolution sous traitement et rechercher les mutations génétiques impliquées dans la chloroquinorésistance. Nous rappelons les principales caractéristiques épidémiologiques du paludisme d’importation, les difficultés à diagnostiquer un accès palustre chez l’enfant sur le plan clinique et le risque de survenue d’un neuropaludisme. Nous discutons la place des méthodes moléculaires dans le diagnostic de paludisme d’importation. © 2009 Elsevier Masson SAS. Tous droits réservés. Mots clés : Chloroquino-résistance ; Plasmodium falciparum ; PCR en temps réel

1. Introduction Imported Plasmodium falciparum malaria is the most common cause of fatal infections in returning travellers [1]. Among these, children are particularly at risk from malaria since symptoms can develop rapidly and be severe [1–3]. The diagnosis of malaria may be difficult given the frequent febrile pediatric illnesses and their clinical features, which may differ from those in

adults, [1,3]. We report a case of imported malaria in a child that illustrates the epidemiological, clinical, and molecular aspects of severe pediatric malaria for which real-time PCR was used to determine clinical management. The prevention and management of such malaria are discussed, as well as the place of molecular methods for the diagnosis of malaria, treatment option, and follow-up. 2. Case report

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Corresponding author. EA3609, centre de biologie pathologie, département de parasitologie mycologie, Université de Lille 2, CHU de Lille, Lille, France E-mail address: [email protected] (L. Delhaes Jeanne). 0399-077X/$ – see front matter © 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.medmal.2009.04.011

A 6-year-old boy, living in the North of France with his family (his Guinean mother, his French father, and two other siblings),

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Fig. 1. Evolution of malaria: parasitemia. Évolution clinique : parasitémie.

returned from a four-week visit to Conakry-Guinea. During his stay in Africa, he was under prophylaxis therapy (chloroquine, 50 mg/day), but the antimalarial prophylaxis was stopped early, 10 days after leaving the endemic area. No repellent or mosquito net was used. Twelve days after leaving the endemic area, he presented with fever (40 ◦ C), cough, and vomiting which was first managed with ambulatory treatment (cefixim + josamycin) without further investigations. He became drowsy, confused, and was admitted to hospital seven days later. On admission, in our pediatric intensive care unit, he was no longer confused but presented with asthenia, a temperature of 37.3 ◦ C, and a heart beat rate of 139 bpm. The clinical examination was otherwise normal, except for a moderate hepato-splenomegaly. His creatinine and electrolyte levels, and his chest X rays were normal. P. falciparum trophozoite and parasitemia of 40% were found on blood smear examination (Fig. 1). Further investigations revealed: hypoglycemia (1.5 mmol/l for hypoglycemia, blood glucose < 2.2 mmol/l), anemia (hemoglobin, 6.4 g/dl, normal range from 7.75 to 9.3 g/dl), thrombocytopenia (platelet count 28 × 109 /l, normal range of 150–400 × 109 /l), high bilirubin concentration 53 ␮mol/l (normal < 17 ␮mol/l), metabolic acidosis (HCO3 = 15 mmol/l), and inflammatory syndrome (CRP = 134 mg/l, normal < 12 mg/l). He was treated

Fig. 2. Yearly incidence of malaria cases in the Lille University Hospital, France. Incidence annuelle des cas de paludisme au CHU de Lille, France.

with intravenous quinine (8 mg/kg/d every 8 hours after a bolus of 17 mg/kg) associated to oxygen-therapy. The evolution was complicated firstly (12 hours after admission) by prostration associated with pyrexia (39–40 ◦ C), and secondly (12 hours later) hemolysis (hemoglobin = 5 g/dl) with tachycardia (200 bpm) that required erythrocyte transfusion. The efficiency of antimalarial treatment and outcome were assessed not only, by using clinical data and checking the patient’s parasitemia on blood smears, but also by quantifying P. falciparum DNA concentration and screening for parasite DNA mutations linked to chloroquine-resistance with real time PCR [4,5]. In addition to quantify P. falciparum DNA level, a set of three different real-time PCR was used to detect any non-falciparum infection. Pfcrt K76T mutant population of P. falciparum was successfully detected, using a locked-nucleic-acid oligomer in the clamped-probe assay [4,5]. Since LNA oligomer competes for binding with the detection probe, detection of the mutant variant by specific clamped-probe is enhanced, allowing to detect less than 5% of a mutant minority population [4]. On Day 5 after admission, the temperature remained below 37.5 ◦ C, the patient’s clinical state (especially neurological symptoms) was improving, and he was switched to oral route. Parasitemia was negative on blood smears, while parasite DNA was detected at very low concentrations (0.02–0.001%, Fig. 1). On Day 6, he was transferred from the intensive unit to a pediatric department with a stable partial recovery. The young patient then recovered fully and was discharged on Day 9. No other family members presented with malaria. 3. Discussion 3.1. Epidemiological and clinical aspects France is the European country the most affected by imported malaria, with about 5000 cases diagnosed per year [2,6]. Children account for approximately 20% of European imported malaria cases [1,2]. Our case illustrates the most specific aspects

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as well as the diagnostic difficulty that characterize imported pediatric malaria. The infection was acquired in Africa during a summer travel to visit family, one of the most common reasons to stay in an endemic area explaining our peak seasonal incidence (late summer/early autumn, Fig. 2) [1,3]. It was caused by P. falciparum, the most frequent parasite identified in imported malaria (> 80%). Chloroquine-prophylaxis was not completed and probably inadequate, since Guinea Conakry and Bissau) are chloroquine resistant malaria areas, promoting the emergence of a P. falciparum population mutated on Pfcrt gene, one of the molecular features of parasite chloroquine-resistance [7]. Malaria was not suspected initially, leading to a misdiagnosis and delayed diagnosis. Such a long interval between the onset of symptoms and the diagnosis is not unusual, since pediatric malaria may often be asymptomatic or present with misleading symptoms such as gastrointestinal features or a febrile illness [1,3]. Imported malaria in children is increasing and was recently considered as different from imported malaria in adults since there are different risk factors, clinical presentations, and a high risk of developing severe disease for children [1–3]. Children living in non-endemic countries and travelling during school holidays to visit family in their parents’ country of origin deserve special attention because when they present with malaria (a) they often have non-specific clinical features such as fever, vomiting, diarrhea, abdominal pain, asthenia, headache, confusion, and prostration [1,2], (b) they are particularly at risk to rapidly develop severe malaria for which prostration was suggested as a criterion of severity [1], and (c) they often take inadequate or do not take antimalarial prophylaxis nor any other preventive measures [1,2]. The severity of malaria was assessed in the present case report by using WHO criteria, even if they had not been validated in non-immune children. As severe malaria has a bad prognosis, it requires an early antimalarial treatment and close monitoring in an intensive care unit [1,3]. Intravenous quinine is the gold-standard treatment in Europe [1]. It was recently documented that laboratory values may vary [8], and that a rapid neurological deterioration can occur within 24–48 hours of quinine therapy initiation; this is what we observed in our case. More recently, treatment with a combination containing artemisinin was recommended, both for a better clinical efficiency and to limit the emergence of drug-resistant parasites [3]. Beside severe P. falciparum malaria, lethal human malaria due to the simian parasite Plasmodium knowlesi (morphologically close to Plasmodium malariae) was recently reported in Southeast Asia, requiring the same management as severe falciparum malaria [9]. 3.2. Place of new molecular approach Even if PCR-based assays (including nested PCR, multiplex PCR, and real-time PCR) are the most efficient technique for mixed infection detection and are more sensitive than conventional methods, detecting up to 0.05 parasite/␮L when thick drop examination or QBC® (quantitative buffy coat technique) methods detect 10 to 20 parasites/␮L [4,5,10], their use is still controversial. Microscopic blood smear examination

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remains the “gold standard” for malaria detection and speciation. Despite some drawbacks (such as interpretation of results that requires considerable expertise, especially at low parasite levels), microscopy of Giemsa-stained thick drop and smears by a skilled microscopist still is a cheap, and rapid conventional method for the diagnosis of malaria in endemic or non-endemic regions. Given their sensibility, molecular methods should be considered as second-line methods especially useful to rule out imported malaria in a febrile patient when the PCR-result is negative, confirming the absence of parasites on blood film microscopic observation. Real-time PCR can detect and quantify very low parasitemia, and is a valuable tool to monitor malaria treatment. In the reported case, real-time PCR quantified the parasitemia ranging from 50 to 0.001%, and showed a good correlation between microscopic and PCR quantification of parasitemia (Fig. 1). In addition to monitoring the therapy response, PCR assays that combine published molecular mutation detection method with a wild-type-specific LNA containing oligomer to suppress wild-type PCR amplification [4], can detect minority Pfcrt K76T mutant populations, which were correlated with in vitro chloroquine resistance. The strain in our case exhibited a K76T mutation on Pfcrt gene that could partly explain the failure of chloroquine-prophylaxis. The LNA-based clamped-probe assay for the diagnosis of minority resistant populations could detect such minority populations more rapidly, thus, improving the control of emerging resistant clones with a fast adaptation of therapy. More recently, molecular methods were used to identify Plasmodium knowlesi malaria in humans, a new lethal Plasmodium species that can easily be misdiagnosed for Plasmodium malariae [9]. 4. Conclusion A considerable number of children are still diagnosed with malaria in France, and in Europe. Improving the appropriate chemoprophylaxis should decrease the incidence of imported malaria. The diagnosis and treatment should also be improved. Imported malaria must be suspected in all travelers returning from malaria endemic areas, especially in febrile travelers. For these patients, molecular malaria diagnosis could be useful to confirm or to rule out malaria. Molecular methods are primarily used to help with the clinical management, especially to rapidly detect antimalarial resistance, to diagnose cases with low parasitemia (such as in partially-treated cases where the blood smear might initially be negative but the PCR would be sensitive enough to detect very low parasite DNA levels), and to specifically identify the Plasmodium species, especially to identify P. falciparum or P. knowlesi responsible for severe malaria. Patients must be hospitalized for at least 48 hours, in order to initiate efficient antimalarial treatment associated with a range of supportive measures. Given the parasite’s chemoresistance, there is a crucial need for careful surveillance of drug efficiency, which should include molecular methods. Real-time PCR could help to improve the initial diagnosis, to follow treatment response, and to quickly prove an early relapse.

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5. Conflicts of interests The authors declare that there is no conflict of interests. Specific funding did not support this study. It was accepted for a poster presentation in the Xth EMOP congress (Paris, August 2008). References [1] Castéla F, Legros F, Lagardère B. Imported malaria in children in France. Arch Pediatr 2003;10:758–65. [2] Ladhani S, Aibara RJ, Riordan FA, Shingadia D. Imported malaria in children: a review of clinical studies. Lancet Infect Dis 2007;7:349–57. [3] Petrognani R, Peytel E, Ponchel C, Carpentier J-P, Saïssy J-M. Severe imported malaria in adults. Med Mal Infect 2006;36:492–8. [4] Senescau A, Berry A, Benoit-Vical F, Landt O, Fabre R, Lelievre J, et al. Use of a locked-nucleic-acid oligomer in the clamped-probe assay for detection of a minority Pfcrt K76T mutant population of Plasmodium falciparum. J Clin Microbiol 2005;43:3304–8.

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