Rapid Dissemination of Plasmodium falciparum Drug Resistance Despite Strictly Controlled Antimalarial Use

BACKGROUND: Inadequate treatment practices with antimalarials are considered major contributors to Plasmodium falciparum resistance to chloroquine, pyrimethamine and sulfadoxine. The longitudinal survey conducted in Dielmo, a rural Senegalese community, offers a unique frame to explore the impact of strictly controlled and quantified antimalarial use for diagnosed malaria on drug resistance. METHODOLOGY/PRINCIPAL FINDINGS: We conducted on a yearly basis a retrospective survey over a ten-year period that included two successive treatment policies, namely quinine during 1990–1994, and chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) as first and second line treatments, respectively, during 1995–1999. Molecular beacon-based genotyping, gene sequencing and microsatellite analysis showed a low prevalence of Pfcrt and Pfdhfr-ts resistance alleles of Southeast Asian origin by the end of 1994 and their effective dissemination within one year of CQ and SP implementation. The Pfcrt resistant allele rose from 9% to 46% prevalence during the first year of CQ reintroduction, i.e., after a mean of 1.66 CQ treatment courses/person/year. The Pfdhfr-ts triple mutant rose from 0% to 20% by end 1996, after a mean of 0.35 SP treatment courses/person in a 16-month period. Both resistance alleles were observed at a younger age than all other alleles. Their spreading was associated with enhanced in vitro resistance and rapidly translated in an increased incidence of clinical malaria episodes during the early post-treatment period. CONCLUSION/SIGNIFICANCE: In such a highly endemic setting, selection of drug-resistant parasites took a single year after drug implementation, resulting in a rapid progression of the incidence of clinical malaria during the early post-treatment period. Controlled antimalarial use at the community level did not prevent dissemination of resistance haplotypes. This data pleads against reintroduction of CQ in places where resistant allele frequency has dropped to a very low level after CQ use has been discontinued, unless drastic measures are put in place to prevent selection and spreading of mutants during the post-treatment period

Characterization of chikungunya virus-like particles.

Chikungunya virus (CHIKV) is becoming a global concern due to the increasing number of outbreaks throughout the world and the absence of any CHIKV-specific vaccine or treatment. Virus-like particles (VLPs) are multistructured proteins that mimic the organization and conformation of native viruses but lack the viral genome. They are noninfectious and potentially safer vaccine candidates. Recent studies demonstrated that the yield of CHIKV VLPs varies depending on the strains, despite the 95% amino acid similarity of the strains. This might be due to the codon usage, since protein expression is differently controlled by different organisms. We optimized the region encoding CHIKV structural proteins, C-E3-E2-6k-E1, inserted it into a mammalian expression vector, and used the resulting construct to transfect 293 cells. We detected 50-kDa proteins corresponding to E1 and/or E2 in the cell lysate and the supernatant. Transmission electron microscopy revealed spherical particles with a 50- to 60-nm diameter in the supernatant that resembled the native CHIKV virions. The buoyant density of the VLPs was 1.23 g/mL, and the yield was 20 µg purified VLPs per 108 cells. The VLPs aggregated when mixed with convalescent sera from chikungunya patients, indicating that their antigenicity is similar to that of native CHIKV. Antibodies elicited with the VLPs were capable of detecting native CHIKV, demonstrating that the VLPs retain immunogenicity similar to that of the native virion. These results indicated that CHIKV VLPs are morphologically, antigenically, and immunologically similar to the native CHIKV, suggesting that they have potential for use in chikungunya vaccines

TEM analysis of purified VLPs expressed with the optimized codons.

<p>(A) Magnification at ×30,000, (B) ×150,000, including CHIKV virion (top, left corner).</p

The antigen-coated aggregation of the VLPs observed by immunoelectron microscopy.

<p>The antigen-coated aggregation of the VLPs was observed by TEM. The VLPs incubated with (A) serum from a patient (32097), and (B) with serum from a healthy individual.</p

Western blot assay of structural proteins expressed in 293T cells.

<p>We transfected 293T cells with an expression plasmid containing structural proteins derived from either natural or optimized codons. The supernatant was analyzed by western blot analysis using serum from a mouse immunized with formalin-inactivated CHIKV. Results for the following are shown: (1) the supernatants from 293T cells transfected with optimized structural protein genes, (2) natural structural protein genes, (3) plasmid vector, (4) no plasmid vector but lipofectamine, and (5) no transfection.</p

Time course of the formation of CHIKV VLPs.

<p>293T cells were transfected with the expression plasmid, and the supernatant was collected for 10 consecutive days. The supernatant (20 µL) was subjected to the VLP concentration as described in the Materials and Methods, and analyzed by western blotting using anti-CHIKV mouse serum. (A) Time course of the expression by the plasmid with the natural codons. (B) Results for the same experiment but using the plasmid with optimized codons. Lanes 1 through 10 are days 1 to 10 p.t. (C) CsCl equilibrium density gradient centrifugation of the VLPs purified from 293F cells. The proteins in each fraction were analyzed by western blotting.</p

Antigenicity and immunogenicity of VLPs.

<p>(A) Anti-CHIKV IgG responses. Microplates were coated with purified VLPs (5 ng/well) and used to detect CHIKV-specific antibodies. IgG antibodies in the 1∶50 diluted serum from one rabbit (Rb1-post) and the sera from three mice (mouse #1, #2 and #3) immunized with inactivated CHIKV, and three sera from convalescent chikungunya patients (# 32095, 32097 and 32221) were examined. Preimmune (Rb1-pre) and PBS were included as controls. (B) Microneutralizaton assays to examine the neutralizing activity of anti-VLP sera. Serial twofold dilutions of the preimmune and postimmune sera were mixed with an equal volume of 100 TCID₅₀ of the ROSS strain and used to inoculate 10⁴ Vero cells. Cell viability was measured at 48 h after incubation by using a WST1 assay according to the manufacturer's instructions. The serum dilution at 50% cell viability indicated by a broken line was defined as the neutralizing antibody titer. pre Rb; rabbit preimmune serum, Rb 4w; rabbit serum from 4 wks postimmunization, Rb 5w; rabbit serum from 5 wks postimmunization, Gp 5w; guinea pigs serum from 5 wks postimmunization, vc; virus control, cc; cell control.</p

Temporal distribution of CQ and SP drug pressure and drug resistance in Dielmo in 1990–9.

<div><p>The drug pressure is expressed as No of treatments/person/year (first graph) and as overall No of treatment courses administered per year (second graph). Panels A and B refer to CQ and SP, respectively. The prevalence of the <i>Pfcrt</i> mutant alleles was calculated from molecular beacon studies (N = 324) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000139#pone-0000139-g002" target="_blank">Figure 2</a>), while the prevalence of the <i>Pfdhfr-ts</i> triple mutant was calculated from the full gene sequences available (N = 202) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000139#pone-0000139-g003" target="_blank">Figure 3</a>). <i>In vitro</i> susceptibility assays were carried out in 1990–4 during the rainy season (N = 26) and from 1995 onwards for the last 2–3 months of the year, namely from 7/11/1995–26/12/1995 (N = 46) ; 6/01/1996–3/12/1996 (N = 59); 27/10/97–15/121997 (N = 26) ; 10/01/1998–15/11/1998 (N = 54) and 29/09/1999–08/11/1999 (N = 25). The proportion of interpretable CQ and pyrimethamine susceptibility tests was 68–81% and 72–81%, respectively, depending on the year. The prevalence of resistance is expressed as the percent of interpretable assays presenting a CI₅₀ for CQ >100 nM or a CI₅₀ for pyrimethamine>2000 nM. The occurrence of a second clinical malaria episode within 7, 14, 21 and 28 days of treatment was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000139#s2" target="_blank">Materials and Methods</a>. The bars correspond to the 95% confidence interval. The years before implementation of CQ and SP (1990–4) are grouped together.</p> <p> A. CQ pressure, <i>Pfcrt</i> 76T resistance mutation, CQ <i>in vitro</i> resistance and prevalence of clinical attacks following a CQ treatment</p> <p> B. SP pressure, <i>Pfdhfr-ts</i> triple mutant, pyrimethamine <i>in vitro</i> resistance and prevalence of clinical attacks following a SP treatment</p> <p>Colour codes: 1990–4: grey; 1995: purple; 1996: yellow; 1997: light green; 1998: light blue; 1999: orange.</p></div

Temporal variation of the multiplicity of infection in Dielmo (A), frequency of Pfcrt codon 76 and <i>Pfdhfr-ts</i> codon 108 genotypes (B) and frequency of infections with only mutant type detected (C).

<div><p>The number of isolates typed at each locus is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000139#pone-0000139-t001" target="_blank">Table 1</a>. (A). Multiplicity of infection is depicted separately for each locus. For <i>Pfmsp1</i> block2, the figures derive from nested PCR analysis using family-specific primers and allele identification based on allelic family assignment and size polymorphism. For <i>Pfcrt</i> and <i>Pfdfhr-ts</i>, it is based on K76T and S108N genotypes determined by molecular beacons, respectively. Symbols used: (Red triangles): <i>Pfcrt</i> codon 76 genotype; (green squares): <i>Pfdhr-ts</i> codon 108 genotype, (blue open circles) <i>Pfmsp1</i> block2.</p> <p>B) Allelic frequency of resistance genotypes, calculated as percentage of mutant genotype within the total number of alleles detected for each locus. Symbols used as in A.</p> <p>C) Percentage of isolates containing only the mutant type. Symbols used as in A.</p></div

Frequency distribution of <i>Pfcrt</i> intron 4 microsatellite types by codons 72–76 and 220 haplotype.

<p>247 isolates were typed (19, 23, 22, 33, 28, 22, 29, 17, 39 and 15 isolates in 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998 and 1999, respectively) for the intron 4 microsatellite by gene sequencing (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000139#s2" target="_blank">Materials and Methods</a>). There were 31 CVIETS haplotypes and 216 wild type haplotypes. The haplotype codes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000139#pone.0000139.s002" target="_blank">Table S2</a>.</p