Reduced In Vitro Doxycycline Susceptibility in Plasmodium falciparum Field Isolates from Kenya Is Associated with PfTetQ KYNNNN Sequence Polymorphism

Abstract

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC 50 s) of >35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC 50 s of >35,000 nM relative to those with IC 50 s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC 50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC 50 of 9,912 nM (P ‫؍‬ 0.0245). Isolates with 1 copy of PftetQ had a median IC 50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC 50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC 50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC 50 of 3,352 nM (P ‫؍‬ 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.

Delayed Onset of Plasmodium falciparum Malaria after Doxycycline Prophylaxis in a Soldier Returning from the Central African Republic

“…In addition, isolates with Ͻ3 PfTetQ KYNNNN motif repeats are associated with in vitro reduced susceptibility to doxycycline and a significantly higher probability of having a half-maximal inhibitory concentration (IC 50 ) above the doxycycline resistance threshold of 35 M (odds ratio of 15) (6,8).…”

“…The number of copies of the pftetQ and pfmdt genes was evaluated relative to the single-copy gene pf␤tubulin as previously described (6,7). The genotyping of pftetQ sequence polymorphisms was performed by a conventional method as previously described (5,8). The sample had only one copy of the pftetQ and pfmdt genes; one PfTetQ KYNNNN motif repeat was also present but was not associated with an IC 50 of Ͼ35 M as previously shown (8).…”

“…The genotyping of pftetQ sequence polymorphisms was performed by a conventional method as previously described (5,8). The sample had only one copy of the pftetQ and pfmdt genes; one PfTetQ KYNNNN motif repeat was also present but was not associated with an IC 50 of Ͼ35 M as previously shown (8). In this case, the single copy of pftetQ and pfmdt suggested in vitro susceptibility of P. falciparum to doxycycline, and the single PfTetQ KYNNNN motif repeat was not predictive of in vitro resistance to doxycycline.…”

Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand

“…Two recent studies have demonstrated that there was an association between the pfmdt and pftetQ copy numbers and the level of doxycycline susceptibility in Africa (3,4). In addition, in Kenya, Achieng et al showed that isolates with one copy of pfmdt had a median IC 50 lower than those with two or more pfmdt copies (5 (3,4,17). Two isolates possessed two copies of pftetQ and one copy of pfmdt.…”

“…In addition, isolates with PfTetQ KYNNNN motif repeats have been associated with reduced in vitro susceptibility to doxycycline and with a significantly greater probability of a 50% inhibitory concentration (IC 50 ) greater than the doxycycline resistance threshold of 35 M (3,5).…”