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Malaria prophylaxis failure with doxycycline, central African republic, 2014.

To the Editor: Doxycycline is an effective antimalarial prophylactic drug when administered as a monotherapy 1 day before, daily during, and for 4 weeks after travel to an area where malaria is endemic (1). Doxycycline is currently a recommended chemoprophylactic regimen for travelers visiting areas where malaria is endemic and has a high prevalence of chloroquine or multidrug resistance (2). The World Health Organization also recommends doxycycline in combination with quinine or artesunate as the second-line treatment for uncomplicated Plasmodium falciparum malaria (3).

Prophylactic and clinical failures of doxycycline against P. falciparum have been associated with both inadequate doses (4) and poor patient compliance (5). However, resistance can also explain failures of prophylaxis. Cycline resistance in Plasmodium spp. has been documented as a consequence of selective drug pressure in a P berghei murine malaria model (6). The administration of increasing doses of minocycline to mice infected with 1 x 107 parasites for 86 successive passages over 600 days made it possible to obtain a resistant P. berghei strain with a median drug inhibitory concentration ([IC.sub.50]) of 600 mg/kg/d, which is 6-fold higher than that of the susceptible starting strain (100 mg/kg/d) (6). A Bayesian mixture modeling approach identified 3 different phenotypes (low, medium, and high doxycycline [IC.sub.50] phenotypic groups) among P. falciparum clinical isolates (7,8). Using 90 isolates from 14 countries, we demonstrated that increases in copy numbers of P. falciparum metabolite drug transporter gene (Pfmdt, PFE0825w) and P. falciparum GTPase TetQ gene (PfTetQ, PFL1710c) are associated with reduced susceptibility to doxycycline (9); this association was later confirmed (7). In addition, isolates with PfTetQ KYNNNN motif repeats are associated with in vitro reduced susceptibility to doxycycline and with a significantly higher probability of having an [IC.sub.50] above the doxycycline resistance threshold of 35 mM (9,10).

We report a case of documented malaria prophylactic failure with doxycycline in a 26-year-old soldier from France who was infected during a 6-week peacekeeping mission in the Central African Republic in 2014. According to his colleagues and the collective prophylaxis intake, the patient had been compliant with doxycycline prophylaxis. On admission to a hospital in Bangui, Central African Republic, the patient had fever (temperature 40[degrees]C), alteration of consciousness, and hypotension. The diagnosis of severe P. falciparum malaria was made on the basis of a rapid diagnostic test confirmed by a blood smear test (parasitemia 8% on day 0). Intravenous artesunate was immediately started, in accordance with World Health Organization recommendations (3). The patient's clinical condition worsened, and kidney failure developed. Twenty-four hours later (day 1), he was transported by airplane to Begin Military Teaching Hospital (Saint-Mande, France). On admission, he had cerebral edema and a P falciparum parasitemia level of 0.7%. The patient died 1 day later (day 2).

A blood sample obtained from the patient on day 1 in France showed a doxycycline concentration of 195 p.g/mL plasma. This concentration, which was determined by liquid chromatography coupled with tandem mass spectrometry, was compatible with a last doxycycline uptake 1 day before diagnosis (day -1). The finding of the expected doxycycline plasma concentration, together with assurances (colleague's statements and collective intake of doxycycline) that the patient had followed the drug regimen, was sufficient to suggest prophylaxis failure in a treatment-compliant patient.

The P. falciparum sample obtained from the patient on arrival in France was evaluated for in vitro susceptibility to doxycycline, but the evaluation was unsuccessful. The number of copies of PfTetQ and Pfmdt genes were evaluated relative to the single-copy P. falciparum b-tubulin gene (pffytubulin), as previously described (7,8). The sample was assayed in triplicate. The [2.sup.-[DELTA][DELTA]Ct] method (where C indicates cycle threshold) of relative quantification was used and adapted to estimate the number of copies of Pfmdt and PfTetQ by using the formula [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. Genomic DNA extracted from 3D7 P. falciparum, which has a single copy of each gene, was used for calibrator sample; Pffytubulin served as the control housekeeping gene. The experiment was assayed twice. The sample had 2 copies of PfTetQ and Pfmdt genes, which suggested decreased in vitro susceptibility of the sample to doxycycline (8,9). The genotyping of PfTetQ sequence polymorphisms was done by using conventional methods with the primers PfTetQ forward (5'-TCACGACAAATGTGCTAGATAC-3') and PfTetQ reverse (5'-ATCATCATTTGTGGTGGATAT-3'), as previously described (10). Two PfTetQ KYNNNN motif repeats were found in the sample; <3 KYNNNN motif repeats are predictive of in vitro P. falciparum-resistant parasites with an [IC.sub.50] of >35 mM (odds ratio 15) (10). The 2 copies of Pfmdt and the 2 KYNNNN motif repeats have been shown to be associated with parasites with in vitro resistance to doxycycline (9,10). The association of doxycycline resistance (prophylactic failure with statement of correct intake and the presence of an expected concentration) with increased Pfmdt copies and decreased PfTetQ KYNNNN motif repeats suggest that these molecular markers are predictive markers of doxycycline resistance that can be used for resistance surveillance.


Author affiliations: Institut de Recherche Biomedicale des Armees, Bretigny sur Orge, France (M. Madamet, N. Taudon, B. Pradines); Aix Marseille Universite, Marseille, France (M. Madamet, N. Taudon, B. Pradines); Centre National de Reference du Paludisme, Marseille (M. Madamet, N. Taudon, B. Pradines); Hopital d'Instruction des Armees Saint Anne, Toulon, France (T. Gaillard); Centre d'Epidemiologie et de Sante Publique des Armees, Marseille, France (G. Velut, R. Michel); Hopital d'Instruction des Armees Begin, Saint Mande, France (C. Ficko, S. Merat, P. Pasquier, C. Rapp); Hopital Saint-Louis, Paris, France (P. Houze); Hopital Bichat Claude Bernard, Paris (S. Houze); Institut pour la Recherche et le Developpement, Paris (S. Houze), Universite Paris Descartes, Paris (S. Houze); Centre National de Reference du Paludisme, Paris (S. Houze); Antenne Medicale de Fontevraud, Fontevraud, France (C. Bylicki); Ecole du Val-de-Grace, Paris (R. Michel, C. Rapp)


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(2.) Institut National de Veille Sanitaire. Recommandations sanitaires pour les voyageurs, 2014. Bulletin Epidemiologique Hebdomadaire. 2014;16 17:264-311.

(3.) World Health Organization. Guidelines for the treatment of malaria, 2nd ed. Geneva: The Organization; 2010.

(4.) Pang L, Limsomwong N, Singharaj P. Prophylactic treatment of vivax and falciparum malaria with low-dose doxycycline. J Infect Dis. 1988;158:1124-7.

(5.) Wallace MR, Sharp TW, Smoak B, Iriye C, Rozmajzl P, Thornton SA, et al. Malaria among United States troops in Somalia. Am J Med. 1996;100:49-55. S0002-9343(96)90011-X

(6.) Jacobs RL, Koontz LC. Plasmodium berghei: development of resistance to clindamycin and minocycline in mice. Exp Parasitol. 1976;40:116-23.

(7.) Briolant S, Baragatti M, Parola P, Simon F, Tall A, Sokhna C, et al. Multinormal in vitro distribution model suitable for the distribution of Plasmodium falciparum chemosusceptibility to doxycycline. Antimicrob Agents Chemother. 2009;53:688-95. 10.1128/AAC.00546-08

(8.) Gaillard T, Briolant S, Houze S, Baragatti M, Wurtz N, Hubert V, et al. PftetQ and pfmdt copy numbers as predictive molecular markers of decreased ex vivo doxycycline susceptibility in imported Plasmodium falciparum malaria. Malar J. 2013;12:414.

(9.) Briolant S, Wurtz N, Zettor A, Rogier C, Pradines B. Susceptibility of Plasmodium falciparum isolates to doxycycline is associated with pftetQ sequence polymorphisms and pftetQ and pfmdt copy numbers. J Infect Dis. 2010;201:153-9. 10.1086/648594

(10.) Achieng AO, Ingasia LA, Juma DW, Cheruiyot AC, Okudo CA, Yeda RA, et al. Reduced in vitro doxycycline susceptibility in Plasmodium falciparum field isolates from Kenya is associated with PftetQ KYNNNN sequence polymorphism. Antimicrob Agents Chemother. 2014;58:5894-9. AAC.02788-13

Address for correspondence: Bruno Pradines, Unite de parasitologie et d'entomologie, Institut de recherche biomedicale des Armees, BP 73, 91223 Bretigny sur Orge, France; email:
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Title Annotation:LETTERS
Author:Madamet, Marylin; Gaillard, Tiphaine; Velut, Guillaume; Ficko, Cecile; Houze, Pascal; Bylicki, Clair
Publication:Emerging Infectious Diseases
Article Type:Report
Date:Aug 1, 2015
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