Effects of Mefloquine Use on Plasmodium vivax Multidrug Resistance

Use of mefloquine against P. falciparum jeopardizes its future use against P. vivax.

codons 57, 58, 61, 117, and 173 of P. vivax DHFR (corresponding to codons 51, 59, 108, and 164 of P. falciparum DHFR) are involved in resistance to pyrimethamine, although P. vivax infections are not usually treated directly with SP (7).This resistance was confirmed by heterologous expression studies, invalidating the common idea that P. vivax was "intrinsically resistant" to pyrimethamine (8), which suggests that the high frequency of mixed P. falciparum/P.vivax infections that are not detected by microscopy (9)(10)(11) or relapses of P. vivax infection after P. falciparum infections probably exposes P. vivax parasites to antimalarial drugs used to treat falciparum malaria infections, especially those with a long half-life, and selects P. vivax genetic traits conferring antimalarial drug resistance.
The impact of antimalarial drugs, especially those with long half-lives (such as mefloquine), on the sympatric Plasmodium species is not clearly understood.In areas where P. falciparum and P. vivax are co-endemic, such as South America and Southeast Asia, mefloquine has been widely used (alone in monotherapy or in combination with artemisinin derivatives) to treat uncomplicated falciparum malaria (12).In both areas, emergence of P. falciparum parasites resistant to mefloquine has been demonstrated from therapeutic efficacy studies (treatment failure) or in vitro testing (increased IC 50 [half maximal inhibitory concentration]) and has been associated with the amplification of P. falciparum mdr-1 (Pfmdr-1) gene (13)(14)(15)(16).Recently, several studies performed on P. vivax samples collected in Southeast Asia (Thailand, Laos, Cambodia, and Myanmar) (17)(18)(19), South America (Brazil, Honduras) (20,21), and Africa (Mauritania) (22) have shown that mdr-1 amplification does occur in P. vivax.
In this context, and to confirm the impact of the mefloquine drug pressure on P. vivax parasite populations, we used a real-time PCR to assess the number of P. vivax mdr-1 (Pvmdr-1) gene copies to evaluate the worldwide distribution of Pvmdr-1 amplification in samples collected from travelers with vivax malaria returning to France and from residents in areas with different histories of mefloquine use (French Guiana, Cambodia, Madagascar, and Sudan).

DNA Extraction and PCR Detection of P. falciparum and P. vivax
We extracted parasite DNA from blood spots with Instagene Matrix (BioRad, Marnes-la-Coquette, France) or from whole blood samples using the phenol-chloroform method (23) or the QIAamp DNA Blood Mini Kit (QIA-GEN, Courtaboeuf, France), according to the manufacturer's instructions.Molecular detection and identification of Plasmodium parasites were performed by using real-time PCR as described by Chou et al. (24).

Statistical Analysis
Microsoft Excel 2010 (Microsoft, Redmond, WA, USA) and MedCalc software (v9.1.0.1, Mariakerke, Belgium) were used for data analysis.Categorical variables were compared by χ 2 test, and continuous variables were compared by using the 1-way analysis of variance or Mann-Whitney U test.We considered p values <0.05 as significant.

Ethical Approval
We obtained ethics clearance for the samples used in this study from National Ethics Committee in Cambodia (Ministry of Health), in Madagascar (Ministry of Health), in Sudan (Ministry of Health), and in France (National Reference Center for Malaria).All patients or their parents/ guardians provided informed written consent.

Amplification of Pvmdr-1 in Isolates from Travelers
The mean number of Pvmdr-1 copies was similar for travelers returning to France from South America or Asia and did not change by year of collection: 1997-2000 (1.42 and 1.24, respectively), 2001-2005 (1.17    4).

Discussion
Developed in the 1970s at the US Department of Defense's Walter Reed Army Institute of Research as a synthetic analog of quinine (25), mefloquine was introduced in 1983 in Thailand to replace chloroquine as first-line treatment for falciparum malaria (26).Since then, mefloquine alone or in combination with artesunate has been widely used, especially in Southeast Asia (including Cambodia) and South America (including French Guiana), where it was introduced for second-line treatment and for chemoprophylaxis in 1990 (27).In contrast, mefloquine has not been used extensively in Africa and has not been introduced in Madagascar.Mefloquine has been available for malaria chemoprophylaxis since 1985 in Europe and since 1990 in the United States and has been used by >35 million travelers from France for this indication Pfmdr-1 gene amplification has been described as the major mechanism of P. falciparum mefloquine resistance associated with treatment failure or in vitro resistance (13)(14)(15)(16).Previous studies, including ours, confirm that mdr-1 amplification does occur in P. vivax (17)(18)(19)(20)(21)(22).In addition, the epidemiologic data in our current study show that in regions where mefloquine has never been used, such as in Madagascar and Sudan, amplification in Pvmdr-1 is rare (1% and 2% of total isolates, respectively), whereas in areas with current or past intense use of mefloquine, such as in French Guiana and Cambodia, Pvmdr-1 amplification is frequent and detected in 59% and 33% of isolates, respectively, and with a mean of 2 and 1.3 copies, respectively.Both the number of copies and prevalence of Pvmdr-1 of isolates with multiple mdr-1 copies we observed here are much higher than reported in other studies.For instance, Imwong et al. reported Pvmdr-1 amplification in 6/66, 2/50, and 1/49 isolates from Thailand, Laos, and Myanmar, respectively (17); Jovel et al. observed Pvmdr-1 amplification in 1/37 in Honduras (20); and Lin et al. recently reported 39% and only 4% prevalence in P. vivax isolates from Thailand and Cambodia, respectively (18).A reason for the discrepancy between observations from Lin et al. in Cambodia and our observations could be because their isolates were collected during 2006-2007 and our samples were collected in 2010, indicating an increase in Pvmdr-1 amplification over 3 years.Another reason is the location of collection.Indeed, drug resistance and drug pressure markedly differ across Cambodia.Lin et al. studied isolates from southern Cambodia (Kampot Province), whereas we studied isolates from areas in which drugs were highly resistant in western (Pailin Province) and southeastern Cambodia (Kratie Province), where multidrug resistance of P falciparum is emerging (30) and drug pressure (including artesunate-mefloquine combination) has been intense in recent years.To our knowledge, the previous maximum number of Pvmdr-1 copies detected was 3 (18,19); in this study.however, we observed up to 5 copies in isolates from South America and 4 copies in isolates from Southeast Asia.This difference is likely to be due to our real-time PCR approach using 6 standards of mixed plasmids, which enabled detection of P. vivax isolates with mdr-1 amplification with up to 6 copies.This observation also could indicate an ongoing selection of mdr-1-amplified parasites.Although, our data need to be confirmed and supported by in vivo data from mefloquine-treated patients or in vitro experiments showing a direct relationship between mefloquine pressure and P. vivax mdr-1 amplification, our findings advocate for an integrated drug policy whereby all sympatric malaria species are considered regarding treatment efficacy but also drug pressure and selection of resistance.
We were able to assess the number of Pvmdr-1 copies in isolates collected from travelers returning to France.These data must be analyzed with caution because information about the location of infection might be erroneous, particularly given the fact that relapses from hypnozoites can occur several months after primary infection.We cannot exclude that a patient declaring having returned from Africa was previously infected during a trip to a different location because we did not include this information in the questionnaire administered at recruitment.Nevertheless, we found significant differences between travelers from France and residents from a given geographic origin, especially in isolates from Africa, where most (99%) isolates from residents displayed no amplification, whereas most (57%) isolates from travelers from France had Pvmdr-1 amplification.We assume this indicates within-host selection by mefloquine prophylaxis, which has been and continues to be widely used among travelers from France who go to malaria-endemic countries.Such pressure does not exist in residents, who usually do not take any prophylaxis.These data are of concern because they suggest that selection of Pvmdr-1 amplification is a more rapid process than previously thought, reminiscent to atovaquone resistance.Although selection in travelers from France returning to nonendemic areas bears no transmission risk, chemoprophylaxis and intermittent preventive treatments in malaria-endemic areas might contribute to the emergence of resistant parasites.This possibility certainly warrants further investigation.
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Figure 2 .
Figure 2. Comparative distribution of numbers of Plasmodium falciparum and P. vivax mdr-1 copies in isolates collected from residents of Madagascar and Cambodia.Gray dot, median; dark bars, interquartile range (25%-75%).

Table 1 .
Variation in number of copies of Plasmodium vivax mdr-1 gene in 607 isolates from residents of and travelers from France to malaria-endemic areas, South America, Asia, andAfrica, 1997Africa,  -2009

Table 2 .
Variation in number of copies of Plasmodium vivax mdr-1 gene among isolates from residents of malaria-endemic continents and from travelers from France to those areas, 1997-2010

Table 3 .
Emerging Infectious Diseases • www.cdc.gov/eid• Vol. 20, No. 10, October 2014 1641 Variation in number of copies of Plasmodium vivax mdr-1 gene in isolates from residents of selected Asian and African countries

Table 4 .
Variation in number of copies of Plasmodium vivax mdr-1 gene among isolates from persons from France who reported having traveled during the previous month to malaria-endemic continents, 1997-2010