Adaptation of a Multi-drug Resistant Strain of Plasmodium falciparum from Peru to Aotus lemurinus griseimembra, A. nancymaae, and A. vociferans Monkeys

INTRODUCTION

The adaptation of different strains of Plasmodium falciparum to in vitro culture and New World monkeys has made possible the use of these infections for vaccine trials and drug studies as well as a number of basic biologic studies. Previously, we have reported the adaptation of isolates of P. falciparum from El Salvador, Panama, Haiti, West Africa, East Africa, Cambodia, Indochina, Malaysia, Vietnam, and Ghana.^1–11 These newer isolates are being adapted to provide a range of parasites for heterologous vaccine challenges and for the testing of candidate drug combinations. Of renewed interest has been the adaptation of strains from the Amazon basin to Aotus nancymaae and Aotus vociferans, 2 species of animals currently available for laboratory studies. Our goal has been 1) to obtain parasites representative of parasites currently circulating in endemic areas; 2) to passsage the parasites in as few animals as possible to determine the predictability of parasitemia; and 3) isolate and maintain the parasites that produce infective gametocytes. In recent years, fewer isolates of P. falciparum have been adapted to nonhuman primates, primarily because of the lack of Aotus lemurinus greiseimembra monkeys. For some reason, these animals are much more susceptible to primary adaptation of human isolates of malaria parasites than other species of Aotus monkeys. However, once a parasite is established in this host, it generally can then be passaged to another susceptible species of Aotus or other nonhuman primate species. A. l. greiseimembra monkeys have not been exported to the United States from Colombia for several decades, and laboratory-bred animals are rarely available in sufficient numbers for these studies.

Nevertheless, we report here the adaptation of a strain of multi-drug resistant P. falciparum originating in Peru to New World monkeys. The isolate designated as Peru 01–134 was received from the U.S. Naval Medical Research Center (NMRCD, Lima, Peru). The results of some of the studies conducted with this parasite in different New World monkeys are also reported.

MATERIALS AND METHODS

A. nancymaae and A. vociferans monkeys were wild-caught animals imported from Peru. A. l. griseimembra monkeys were laboratory-born animals. Upon arrival at the facility, all animals were quarantined for a 2-month conditioning period, weighed, and tested for tuberculosis. Parasitologic and serologic examination indicated that the animals were free of infection with malaria parasites before inoculation. All monkeys were splenectomized before exposure to infection. All surgeries were performed in an AAALAC (Association for the Assessment and Accreditation of Laboratory Animal Care, International, Inc.)-approved surgical suite appropriate for aseptic surgery. Protocols were reviewed and approved by the Centers for Disease Control and Prevention Institutional Animal Care and Use Committee, in accordance with procedures described in the 1986 U.S. Public Health Policy.

Aotus monkeys generally were housed doubly or in some cases singly to avoid injuries caused by fighting with cage mates. Space recommendations for laboratory animals were followed as set forth in the NIH Guide for the Care and Use of Laboratory Animals. All animals were fed a diet that has been proven to provide adequate nutrition and calories in captive Aotus monkeys used in malaria-related research. Feed was free of contaminants and freshly prepared. Daily observations of the animals’ behavior, appetite, stool, and condition were recorded. All were treated as medical conditions arose by an attending veterinarian.

Anopheles freeborni (F–1 strain originally from California), Anopheles gambiae (originally from The Gambia), Anopheles quadrimaculatus (originally from southeastern United States), and Anopheles stephensi (originally from Delhi, India) were laboratory-reared and maintained at the CDC/DPD insectaries. During periods when gametocytes were present in blood, mosquitoes were allowed to feed on tranquilized monkeys as previously described.¹² After feeding, mosquitoes were held in an incubator at 25°C until examined 1 week later for the presence of oocysts on their midguts.

Blood-stage parasitemia was monitored and quantified by the daily examination of thick- and thin-blood films by the method of Earle and Perez.¹³ Infections were terminated by treatment with various combinations of drugs as described. Drugs were administered by oral intubation.

DNA was purified by the Qiagen method from Peru 01–134 parasites collected from Aotus monkeys at various times in the adaptation process. The genes for dhfr, dhps, ert, and cytochrome b were amplified from the genome of Peru 01–134 using standard PCR methods. The amplified gene fragments were sequenced directly and in their entirety on both strands of DNA using an ABI 3130x sequencing system to determine the mutations that imparted resistance to particular drugs.

The parasites used for in vitro drug testing were cultured in RPMI 1640 medium (Gibco No. 31800) supplemented with 10% O⁺ human serum, 2,000 mg/L sodium bicarbonate, and 25 mM HEPES at pH 7.2.¹⁴ Additional glucose was added to a final concentration of 4 g/L.¹⁵ The parasites were cultured in 25 cm² tissue culture flasks at 10% hematocrit in O⁺ red blood cells (RBCs) under an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 37°C.

The in vitro response of Peru 01–134 to chloroquine phosphate (Sigma Chemical Co., St. Louis, MO), quinine hydrochloride (Sigma Chemical Co.), mefloquine hydrochloride (Hoffmann-La Roche Ltd., Basel, Switzerland), and artemisinin (Aldrich Chemical Co., St. Louis, MO) was assayed for growth by uptake of ³H-hypoxanthine (MP Biomedicals, Aurora, OH).¹⁶ W2, a chloroquine-resistant clone of Indochina III/CDC, and D6, a chloroquine-sensitive clone of Sierra Leone I/CDC served as controls.¹⁷ The two clones were obtained from the Walter Reed Institute of Medical Research (WRAIR, Silver Spring, MD). The tests were conducted in duplicate. Prior to testing, cultures were adjusted to 1% parasitemia. IC50 (50% inhibitory concentration) values and correlation coefficients r were determined using commercially available software, XLfit 4 (IDBS, Guildford, U.K.).

RESULTS

Parasites were obtained from a patient during a parasitologic survey. A cryopreserved blood sample collected on March 26, 1998, was established into in vitro culture on January 17, 2001. After 23 days of development in culture, parasitized erythrocytes were injected into splenectomized A. l. griseimembra monkey AI-2177 (Figure 1). The prepatent period was 28 days; the course of parasitemia was transient, and the maximum parasite count was 37,017/μL. Blood was passaged into splenectomized A. nancymaae monkey AI-1782. However, a maximum parasite count of only 10/μL developed. Blood was then passaged through 2 splenectomized A. l. griseimembra monkeys, AI-2172 and AI-2178 (Figure 1), which resulted in a complete adaptation to this host. A maximum parasite count of 372,000/μL was obtained in AI-2178, 17 days after inoculation (Figure 2). The count would likely have gone higher if it had not been controlled by treatment with chlorguanide (4 mg and 2 mg), quinine (50 mg), and chloroquine (15 mg × 2 days) during the first episode of parasitemia, and chloroquine (15 mg) plus mefloquine (20 mg) once during the second episode of recrudescent parasitemia. Gametocyte production was maintained for an extended period; mosquitoes were infected on 1 occasion.

Blood from AI-2178 was passaged to 2 splenectomized A. nancymaae monkeys, AI-1768 and AI-1770. The maximum parasite counts were 450 and 450/μL, respectively. When parasites from AI-1768 were passaged to A. nancymaae AI-1793, the maximum parasite count was 68,000/μL; this indicated that the parasite was adapting rapidly to develop in this species of monkey.

The initial passage to an A. vociferans monkey, AI-3004, was with parasites from A. nancymaae monkey AI-1770. The prepatent period was 45 days and the maximum parasite count was 2,340/μL. The second passage (Figure 1) to AI-2619 resulted in a maximum parasite count of 9,450/μL. It was during the next passage that the parasite began to become more adapted to splenectomized A. vociferans monkeys. AI-3003, AI-1785, and AI-3007 had maximum asexual parasite counts of 74,520, 156,000, and 25,020/μL, respectively. The next passage to AI-3006 resulted in an initial maximum parasite count of 76,000/μL followed by recrudescent peak parasite counts of 29,160 and 96,000/μL. Passage from this animal to T-1201 resulted in a maximum parasite count of 236,000/μL, indicating adaptation was essentially complete. However, in none of the splenectomized animals was an infection considered to be life-threatening.

A. vociferans monkey AI-3006 had a long-term chronic infection that lasted > 300 days (Figure 3). At day 133, it was reinoculated with frozen parasites taken earlier from its own infection. A marked increase in asexual parasite count occurred that was controlled with a combination of 8 mg of chlorquanide given over 3 days followed by 50 mg of quinine. After recrudescence, the infection was treated with a single dose of malarone (31.25 mg atovoquone + 12.5 mg proguanil). A recrudescence occurred that was confirmed after 49 days. The infection was retreated on 2 successive days with malarone (62.5 mg atovoquone + 25 mg proguanil). A recrudescence occurred 11 days later. The animal was then treated on 3 successive days with malarone (93.75 mg atovoquone + 37.5 mg proguanil), which appeared to have cured the infection.

After a series of passages through A. vociferans monkeys (Table 1), the parasite was introduced into A. nancymaae monkey T-0865, which had a maximum parasite count of 320,000/μL. The animal (Figure 4) was treated with malarone (62.5 mg atovoquone + 25 mg proguanil) given over 2 days on 4 different occasions. The infection was eventually treated with 20 mg of mefloquine, which appeared to have effected a cure.

A. nancymaae monkey T-1106 was also infected. This animal had a maximum parasite count of 96,000/μL and a recrudescence peak of 4,140/μL 46 days after inoculation; the animal died on day 87.

As shown in Figure 2 of the infection in A. l. griseimembra monkey AI-2178, treatment with 30 mg chloroquine and quinine temporarily cleared the primary parasitemia, but recrudescence occurred. The animal was then treated with a combination of 15 mg of chloroquine and 20 mg of mefloquine, which resulted in clearance for only a single day. The animal eventually cleared parasites on its own. However, other animals were cured by treatment with 20 mg of mefloquine. Thus, it appeared advisable to retreat the animals again with 20 mg of mefloquine 2 weeks after the first treatment to ensure cure.

Mutations in the dhfr, dhps, crt, and cytochrome b genes were analyzed from parasite isolations from Aotus AI-2171 (the first monkey passage) on March 20, 2001. Subsequent samplings for analyses were from AI-2178 (June 6, 2002), AI-3006 (December 26, 2002), AI-3006 (July 15, 2003), AI-2003 (August 12, 2003), T-865 (May 18, 2004), and T-1422 (April 8, 2004). The mutations found in the targeted genes were identical whether analysis was conducted on DNA from the first animal passage or collected much later post-drug treatment.

The dhfr gene had nonsynonymous mutations at codons 51, 108, and 164 that confer drug resistance, producing amino changes that correspond to N51I, S108N, and I164L. The Bolivian repeat sequence was also present. The dhps gene had mutations in codons 437, 540, and 581. The gene encoding the CQ-resistance transporter, crt, had the canonical CQ resistance-conferring mutation IA codon that changes K to T. The Peru 01–134 crt mutations were typical for South American isolates having the sequence SVMNT. The sequence of the cytochrome b gene was the same as the wild type.

Mosquitoes were fed on 8 different monkeys. Only A. l. griseimembra and A. vociferans were shown to produce infectious gametocytes; highest-level infections were obtained from A. vociferans monkeys AI-3003 and AI-2619. Of 2,253 An. freeborni dissected and examined, 279 were positive for oocysts (12.4%); 17.5% of An. stephensi were positive, 6.1% of An. gambiae were positive, and 15.7% of An. quadrimaculatus were positive. However, An. freeborni were more often fed on days when infection was less likely to occur. In general, this parasite readily produced gametocytes infective to standard laboratory vectors.

Drug sensitivity testing showed that the in vitro responses of the Peru 01–134 isolate of P. falciparum were comparable to those of the W2 clone for chloroquine and quinine; the IC50 values (Table 2) indicate that Peru 01–134 is resistant to both these antimalarials. Additionally, Peru 01–134 appears to be slightly more sensitive to mefloquine than does W2 but shows a similar degree of sensitivity to artimisinin.

DISCUSSION

To have available more current isolates of P. falciparum for in vivo testing of antimalarial drugs and candidate vaccines, a drug-resistant parasite from Peru was adapted to develop in currently available Aotus spp. monkeys. In vivo, the parasite was mildly resistant (R–1) to chloroquine by having recrudescent parasites after an initial clearance. The Peru 01–134 also exhibited some level of tolerance to treatment with malarone because the amount of malarone given was ≃20% greater than the standard total curative pediatric dosage (50 mg/kg proguanil). Giving upward of 94 mg of atovaquone and 37.5 mg of proguanil was needed to cure infection. In addition, it appeared to be less responsive to mefloquine, but this will require further testing to be sure. The R-1 level of resistance to CQ is not surprising given the crt gene of the Peru 01–134 isolate has the K-T marker for CQ resistance. However, there was also a possible low level of resistance to MQ. This will require more in vivo and in vitro testing to confirm as well as determining mutations and copy number for mdr-1 of Peru 01–134. The demonstrated resistance to malarone (atovaquone + proguanil) as well as chlorguanide is predicted by the presence of 3 mutations in dhfr and 3 others in dhps, but most relevant is the I164L mutation in dhfr that confers resistance to proguanil and chlorguanide. In splenectomized monkeys, gametocytemia was maintained and standard laboratory vectors were infected.

Continued adaptation of new isolates from different endemic areas is hampered by the lack of access to A. l. griseimembra monkeys. Limited numbers are needed for the initial adaptation before subsequent passage to the available A. nancymaae and A. vociferans animals. Current laboratory-bred stocks are not adequate for the needs. However, continued isolation of emerging drug-resistant P. falciparum parasite lines is important for drug screening and development as well as vaccine evaluation.