Avian Gyrovirus 2 DNA in Fowl from Live Poultry Markets and in Healthy Humans, China

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 µg/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 (pfβtubulin), as previously described (7,8). The sample was assayed in triplicate. The 2 –ΔΔCt method (where C t indicates cycle threshold) of relative quantification was used and adapted to estimate the number of copies of Pfmdt and PfTetQ by using the formula DDC t = (C t (PfTetQ or Pfmdt) –C t (Pfβtubulin)) Sample –(C t (PfTetQ or Pfmdt) –C t (Pfβtubulin)) Calibrator. Genomic DNA extracted from 3D7 P. falciparum, which has a single copy of each gene, was used for calibrator sample; Pfβtubulin 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 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 doxy-cycline 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. Prophylactic treatment of vivax and falciparum malaria with …