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Reemergence of Chloroquine-SensitivePlasmodium falciparum Malaria after Cessationof Chloroquine Use in Malawi James G. Kublin,1,3,a,b Joseph F. Cortese,1,a Eric Mbindo Njunju,5 Rabia A. G. Mukadam,3 Jack J. Wirima,3
Peter N. Kazembe,4 Abdoulaye A. Djimde´,6 Bourema Kouriba,6 Terrie E. Taylor,2,3 and Christopher V. Plowe1
1Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore; 2Department of Internal Medicine,College of Osteopathic Medicine, East Lansing, Michigan; 3Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre,and 4Lilongwe Central Hospital, Lilongwe, Malawi; 5Tropical Diseases Research Centre, Ndola, Zambia; 6Malaria Research and Training Center, In 1993, Malawi became the first African country to replace chloroquine with sulfadoxine-pyrimethamine
nationwide in response to high rates of chloroquine-resistant falciparum malaria. To determine whether
withdrawal of chloroquine can lead to the reemergence of chloroquine sensitivity, the prevalence of the pfcrt
76T molecular marker for chloroquine-resistant Plasmodium falciparum malaria was retrospectively measured
in Blantyre, Malawi. The prevalence of the chloroquine-resistant pfcrt genotype decreased from 85% in 1992
to 13% in 2000. In 2001, chloroquine cleared 100% of 63 asymptomatic P. falciparum infections, no isolates
were resistant to chloroquine in vitro, and no infections with the chloroquine-resistant pfcrt genotype were
detected. A concerted national effort to withdraw chloroquine from use has been followed by a return of
chloroquine-sensitive falciparum malaria in Malawi. The reintroduction of chloroquine, ideally in combination
with another antimalarial drug, should be considered in areas where chloroquine resistance has declined and
safe and affordable alternatives remain unavailable.
In 1993, Malawi became the first sub-Saharan African (SP) to the antimalarial of first choice nationwide. The country to discontinue the routine use of chloroquine decision was based on chloroquine’s increasing failure to against Plasmodium falciparum malaria and to elevate produce adequate clinical and hematological recovery.
the antifolate combination sulfadoxine-pyrimethamine High-level parasitological resistance was observed in 180% of Malawian children treated with chloroquine in 1990 [1]. Since 1993, SP has been the only available Received 25 November 2002; accepted 27 January 2003; electronically published treatment for uncomplicated malaria in all government health facilities and is dispensed without prescription.
Presented in part: 51st annual meeting of the American Society of Tropical Medicine and Hygiene, Denver, 10–14 November 2002 (abstract 54).
Although chloroquine has remained available by pre- Informed consent was obtained from subjects or their parents or guardians, scription and through unauthorized private sources, a according to the human experimentation guidelines of the US Department of Health national information campaign was largely successful in and Human Services and/or those of the authors’ institutions.
Financial support: National Institute of Allergy and Infectious Diseases (grant convincing health practitioners and the public to accept SP as the treatment of choice for children with malaria.
a J.G.K. and J.F.C. contributed equally to this work.
Other African countries, including Kenya in 1999 [2], b Present affiliation: Merck Research Laboratories, West Point, Pennsylvania.
have instituted similar changes in national drug policy Reprints or correspondence: Dr. Christopher V. Plowe, Malaria Section, Center for Vaccine Development, University of Maryland at Baltimore, 685 W. Baltimore in the face of increasing chloroquine resistance.
St., HSF 480, Baltimore, MD ([email protected]).
Chloroquine resistance in P. falciparum is conferred The Journal of Infectious Diseases
2003; 187:1870–5
by mutations in the parasite pfcrt, which encodes a ᮊ 2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/18712-0006$15.00 putative transporter localized to the digestive vacuole 1870 • JID 2003:187 (15 June) • Kublin et al.
[3, 4]. One mutation, K76T, was perfectly associated with in .umaryland.edu/CVD/plowe.html. In brief, the dried thick smear vitro resistance in all progeny of a genetic cross between chlo- was detached from the slide with a new razor blade, and the roquine-sensitive and -resistant parental clones and among a debris were collected in a microcentrifuge tube. DNA was pre- set of geographically diverse parasite isolates [3]. Several field pared for PCR by a methanol-fixation/heat extraction method, studies summarized in a recent review have since confirmed as described elsewhere [7], for preparation of DNA from dried the absolute specificity of the pfcrt K76T to clinical chloroquine blood spots. Nested PCR and subsequent allele-specific restric- resistance [5]. Polymorphisms in pfmdr1, encoding the P. fal- tion analysis were performed to identify polymorphic codons of ciparum P glycoprotein homologue 1, modulate chloroquine interest. Infections presenting with mixed alleles in any assay resistance in mutant pfcrt-harboring parasites in vitro [6], al- were defined as polyclonal and were characterized as mutant for though their role in vivo has yet to be substantiated [7]. SP that assay. Direct DNA sequencing was performed at the Uni- resistance is conferred by mutations in 2 genes encoding en- versity of Maryland Baltimore Biopolymer Facility. The x2 test zymes in the parasite’s folate synthesis pathway, dihydrofolate for linear trend using Epi Info (version 6.04c; CDC) was used reductase (dhfr) and dihydropteroate synthase [8].
It is not known whether years of reliance on antimalarials In vitro chloroquine assays.
other than chloroquine can lead to the reemergence of chloro- collected in February 2001 at the Ndirande Health Centre from quine-sensitive P. falciparum and permit the reintroduction of consenting individuals presenting with uncomplicated malaria.
this safe and affordable drug. Using polymerase chain reaction Drug-sensitivity assays were performed essentially, as described (PCR)–based assays, we measured the prevalence of resistance- elsewhere [10], using the World Health Organization (WHO) conferring mutations in malaria-infected human blood samples microtest method, a field test that estimates drug susceptibility obtained from southern Malawi before and after the 1993 change by culturing fresh parasites for 1 48-h life cycle in the presence in drug policy. We demonstrate a progressive decline in the fre- of increasing drug concentrations and by determining parasite quency of the pfcrt K76T mutation over the decade after the survival by light microscopy. In brief, 0.1 mL of blood was proscription of chloroquine and a much lower prevalence of 76T collected in a sterile, 100-mL heparin-treated capillary tube and in Malawi than in neighboring Zambia, where chloroquine re- immediately was added to 0.9 mL of RPMI 1640 culture me- mained in use. We confirm these molecular findings by dem- dium (Life Technologies). Blood-medium mixture (50 mL) was onstrating chloroquine sensitivity in vitro and in vivo in con- added to each well of tissue culture plates predosed with chloro- temporary infections from this same region of Malawi.
quine diphosphate salt (Sigma) at 1, 2, 4, 8, 16, 32, and 64pmol/well. The plates were incubated at 37.5ЊC for 30 h, ac-cording to standard methodology. After incubation, Field’s- MATERIALS AND METHODS
stained thick blood films were prepared, and the number of Study sites and sample sources.
mature schizonts was counted per 200 asexual parasites. Ac- residents in southern Malawi with moderate-to-intense levels cording to the WHO protocol, chloroquine susceptibility was of seasonal P. falciparum transmission that peak during De- defined as complete schizont inhibition at р4 pmol of chloro- cember through March. Archived, anonymized microscope quine, and chloroquine resistance was defined as schizont for- slides with Field’s-stained thick smears were provided by the mation at у8 pmol of chloroquine. Isolates that formed schiz- Malaria Project, Blantyre, Malawi. These slides had been col- onts at 4 pmol of chloroquine but had complete schizont in- lected in the January–June malaria seasons in 1992–1996 from hibition at 8 pmol were considered to be intermediate.
children hospitalized for severe malaria. The 1998–2000 Ma- In vivo chloroquine efficacy.
lawian samples were collected at the Ndirande Health Centre vivo chloroquine efficacy studies showed unacceptably high in Blantyre from children with uncomplicated malaria, ac- rates of treatment failure, it was deemed unethical to assess cording to standard definitions [9], and were stored as dried chloroquine efficacy in persons with symptomatic malaria or blood spots on filter paper, as described elsewhere [7]. The in children, who are at higher risk of becoming symptomatic.
Zambian samples were collected in 1999 as filter papers and Therefore, we conducted a preliminary in vivo chloroquine blood smears from symptomatic children in the outpatient de- efficacy study in infected adults with no symptoms. Consenting partment of the Isoka district hospital in the northern province.
asymptomatic adults accompanying sick children to the Ndi- All available samples from 1992–1996 and randomly selected rande Health Centre in January–September 2001 were screened samples from the Malawi 1998–2000 and Zambia 1999 sample for occult P. falciparum infection by standard microscopy.
sets were analyzed. All samples were obtained before initiation Those with positive blood smears were treated by directly ob- served therapy with standard doses of two 300-mg tablets of Sample analysis.
chloroquine phosphate on the treatment day and on day 1 after tion-specific assays are detailed on the Internet: http://medschool treatment and one 300-mg tablet on day 2 after treatment.
Chloroquine-Sensitive Malaria in Malawi • JID 2003:187 (15 June) • 1871
Chloroquine efficacy was measured after slight modificationsof standard protocols, as described elsewhere [11], with activemicroscopic and clinical follow-up on days 3, 7, and 14 aftertreatment and passive surveillance by continuous availabilityof medical care throughout the follow-up period. Because studyparticipants were asymptomatic, parasitologic rather than ther-apeutic outcome definitions were used; efficacy results wererecorded as sensitive or as resistant at the RI, RII, or RIII levels[11]. An additional follow-up assessment was conducted onday 28 for study participants who could be located at this time.
DNA was recovered, and assays for pfcrt 76T were performedsuccessfully on 39/40 thick smears from 1992, 23/24 from 1993–1994, and 24/27 from 1995–1996. Similar results were obtainedfor the other assays (sample sizes are shown in figure 1). Success rates for 1998–2000 filter-paper samples were even higher, rang-ing from 93% to 100% (data not shown). The number of1998–1999 samples analyzed for dhfr mutations was higher,because a large set of samples had been analyzed previously for The chloroquine resistance mutation pfcrt 76T was detected in 30 (85%) of the 39 isolates obtained in 1992 (figure 1A),and its prevalence diminished progressively and significantly(P ! .000 ) 1 over the subsequent 8 years, from 11 (50%) of 22 isolates obtained in 1993–1994 to 10 (13%) of 75 isolates in2000. No infections with pfcrt 76T were found among 25 sam- ples collected in 2001 that were analyzed for both in vivo chlo-roquine resistance and the presence of this mutation. Thecurrent low prevalence of the pfcrt 76T in Malawi is in sharpcontrast to its frequency in neighboring Zambia, where chloro-quine has remained the first-line antimalarial drug. We analyzed Figure. 1.
Prevalence of drug-resistance mutations in Plasmodium isolates obtained in 1999 from the Isoka region of northern falciparum malaria infections in Malawi before and after chloroquine was Zambia, 80 km from the western border of Malawi, and found replaced with sulfadoxine-pyrimethamine (SP) in 1993. A, Chloroquine a 92% prevalence of the mutation (46/50 infections). These resistance–conferring 76T mutation in pfcrt; B, pyrimethamine resistance–conferring N51I and C59R mutations in dhfr; and C, possible chloroquine findings in Zambia are similar to those of a recent study con- resistance–modulating N86Y and D1256Y mutations in pfmdr1. Sample ducted in southern Mozambique, where a 91% prevalence of sizes are indicated beneath the years.
the mutant pfcrt was detected [12].
The institution of SP was followed by an increased prevalence of 2 mutations in the parasite dhfr that confer pyrimethamine some studies with chloroquine resistance [6, 15] and for which resistance and are common throughout East and Central Africa a role in antifolate resistance has not been described. Both [13]. In 1992, the dhfr N51I and C59R were present in only mutations exhibited a modest decrease in frequency over the 16 (6%) of 38 and 7 (18%) of 38 Malawi infections, respectively decade, with the decline in D1246Y, from one-half of the in- (figure 1B). By 1999, nearly all infections were mutant at these fections in 1993 to one-fourth in 2000, attaining statistical sig- codons: N51I, 96% (144/150); and C59R, 94% (217/231). The significant increase in dhfr mutations (P ! .0001) is consistent The 76T has invariably been found to be accompanied by with the increasing rates of antifolate resistance evident in Ma- sets of additional pfcrt mutations [3]. We analyzed a subset of the infections from 1993 (8 with 76K infections and 4 with We found less dramatic trends for 2 mutations in the pfmdr1 76T) and 1998–2000 (10 with 76K infections and 2 with 76T) (figure 1C), N86Y and D1246Y, which have been associated in for the resistance-associated mutations at pfcrt codons 74, 75, 1872 • JID 2003:187 (15 June) • Kublin et al.
220, 271, and 371 and found that the full array of mutations in selection of drug-sensitive phenotypes [17, 18]. Incomplete always cosegregated with 76T, whereas none of the infections withdrawal of the drug, cross-resistance to agents still in use, that were exclusively 76K were mutant at these codons (data and compensatory mutations that reduce the burden of resis- not shown). Thus the pfcrt in Malawi appears to have main- tance without jeopardizing the resistance itself [19] may allow tained the integrity of its fully mutant and wild alleles, and the for resistant organisms to persist, despite restricted availability reemerging 76T pfcrt are not mutants that simply reverted to of an established antimicrobial agent. In P. falciparum malaria K76, which alone can confer the in vitro chloroquine-sensitive in Malawi, the in vivo fitness burden of harboring the mutant phenotype [3]. Variation was detected at pfcrt codons 326 and pfcrt appears to be substantial in the absence of selective drug 356, which are polymorphic but not associated with resistance pressure, and with the mechanisms of antifolate resistance dis- [7], among both the sensitive and the resistant infections (data tinct from those of the aminoquinolines, there appears to have not shown). This heterogeneity at the pfcrt locus, along with been a strong selection of chloroquine-sensitive parasites after our finding that 55% of pfcrt 76T-harboring infections over the the nationwide replacement of chloroquine with SP.
time course were mutant at dhfr codons 51 and/or 59 (data The slower decline in prevalence of mutations in pfmdr1 sug- not shown), are indicative of the polyclonal nature of malaria gests that these mutations may be less deleterious to parasite infections in this setting and the high recombination rates of fitness than are pfcrt mutations. Although genetic transformation P. falciparum in Africa [16]. Malaria harboring the sensitive experiments have found that pfmdr1 mutations can modulate pfcrt appears to have arisen from low levels in the indigenous the level of chloroquine resistance once it has been conferred by parasite populations and/or to have emigrated from bordering mutations in pfcrt, mutations in pfmdr1 are by themselves in- countries where chloroquine pressure persists. But we can ex- sufficient to confer chloroquine resistance [20]. Recent studies clude as an explanation for the diminished prevalence of the have reported that pfmdr1 mutations, in addition to pfcrt mu- mutant pfcrt a selective clonal replacement of antifolate-sensi- tations, are no more strongly associated with chloroquine treat- tive/chloroquine-resistant parasites by antifolate-resistant para- ment failure than pfcrt mutations alone [7] and that pfmdr1 sites that coincidentally harbor a sensitive pfcrt. mutations do not add to the predictive value of pfcrt mutations Of the 11 isolates successfully assayed for in vitro drug suscep- for chloroquine treatment failure [21]. Even if pfmdr1 mutations tibility, 9 (82%) were determined to be fully chloroquine-sensitive, do contribute to treatment failure, they require pfcrt mutations whereas 2 isolates were of intermediate chloroquine susceptibility.
to exert an effect on the response to chloroquine. Therefore, No isolates exhibited in vitro chloroquine resistance.
persistence of pfmdr1 mutations in a population with a very low Seventy-two adults were enrolled in the study of in vivo prevalence of pfcrt mutations would not be expected to reduce chloroquine resistance. As expected, initial parasitemias were chloroquine efficacy in that population.
low, with a geometric mean of 600 asexual forms/mm3 and a The increase in mutations in dhfr is consistent with what is range of 50–12,250 asexual forms/mm3. Nine adults were lost known about SP efficacy in this area during the 1990s. Before to follow-up before day 3, precluding any determination of SP was introduced in Malawi as the first-line drug, efficacy outcome. Each of 63 remaining asymptomatic P. falciparum rates as high as 100% were reported [22], and parasitological infections in adults was successfully cleared within 3 days after cure rates of 90.5% were found in southern Malawi 1 year after chloroquine treatment without development of fever or other the introduction of SP [23]. Our own studies from the site of symptoms of malaria. Fifty-five adults completed the 14-day the current study have found that, by 2000, SP’s parasitological follow-up, and 42 were followed-up for 28 days. Five partici- cure rate had decreased to !70% and its clinical efficacy to pants were lost to follow-up after treatment day 3, and 3 were ∼80% (authors’ unpublished data), corresponding to the in- lost to follow-up after day 7. No recurrent infections were creasing prevalence of dhfr mutations.
detected during the follow-up period; thus, 100% had sensitive Standard oral doses of chloroquine cleared asymptomatic P. falciparum infections in adults with no exceptions, and therewas no evidence of recurrent infection among the 53 subjects DISCUSSION
monitored for at least 14 days, thus meeting the criteria forsensitive parsitological outcome in high transmission areas.
Our findings suggest that chloroquine-sensitive P. falciparum Some loss to follow-up was expected in this asymptomatic parasites have reemerged and are now predominant at a site urban adult population, and we cannot rule out the possibility in Africa where chloroquine was withdrawn in 1993, whereas of recurrent infections among subjects who were lost to follow- high levels of chloroquine resistance have persisted unabated up between 3 and 14 days after treatment. However, the com- in nearby areas where the drug has remained in use. The with- plete lack of recurrent infections in the 76% of subjects who drawal of antimicrobial drug pressure does not always com- were followed for у14 days is strong evidence of chloroquine’s promise the fitness of resistant microorganisms and result ability to clear P. falciparum infections in this setting.
Chloroquine-Sensitive Malaria in Malawi • JID 2003:187 (15 June) • 1873
It is possible that some of these semi-immune adults would other drugs that will eventually fail. Many African countries have spontaneously cleared their malaria infections even with- are delaying decisions to change antimalarial therapies, despite out drug treatment, but it was felt to be unethical to include high rates of chloroquine resistance that are associated with a placebo or nontreatment arm in this first in vivo assessment increasing malaria-attributable disease and death, fearing a slip- of chloroquine in Malawi since the drug was withdrawn. On pery slope of increasingly expensive antimalarial drugs with the basis of the preliminary evidence of chloroquine efficacy limited useful therapeutic lives. The preliminary in vivo data presented here, a controlled trial of chloroquine alone or in reported here justify the conduct of controlled trials of chlo- combination with other drugs may now be ethically justifiable roquine efficacy in areas where chloroquine use has been sub- stantially reduced for a period of years. If these trials confirm Nine of 11 fresh P. falciparum isolates were sensitive to chlo- a return of chloroquine’s clinical efficacy, governments can con- roquine in vitro, consistent with a recent report of a high prev- sider withdrawing chloroquine and switching to other drugs alence of in vitro chloroquine-sensitive P. falciparum in central on an interim basis, knowing that they may be able to later Malawi [24]. The finding that 2 infections yielded microtest val- reintroduce chloroquine, which is unparalleled in its safety and ues in the intermediate range is most likely explained by inac- low cost. However, it must be emphasized that Malawi made curacies of the in vitro test, or by mixed resistant and sensitive a concerted and sustained effort to withdraw chloroquine from parasites infections. Microtest assays are well known to be sub- use and that less rigorous efforts to reduce chloroquine drug ject to variation and artifact, and, although they are useful as pressure may not result in the same reemergence of chloroquine general measures of in vitro resistance in field surveys, they lack the reliability to definitively characterize individual infections Chloroquine’s useful therapeutic life span in Malawi is al- [25–27]. P. falciparum infections in settings like Malawi also most certain to be short-lived if it is reintroduced as mono- are frequently polyclonal, so that discrepant results could arise therapy. There is growing consensus that combination chemo- if in vitro survival of a minor number of resistant parasites is therapy should be the rule in malaria treatment, to deter the detected among a predominantly sensitive population. Such par- development of drug resistance, and that the artemesinin de- asites would have had to be too few to be detected by the mo- rivatives, because of their rapid reduction of parasite biomass lecular assays, which may not detect subpopulations comprising and continued efficacy against multidrug resistant P. falciparum,should constitute a component of most such regimens [34].
!5%–10% of an infection. A more careful evaluation of the chloroquine in vitro response using labeled nutrient uptake as- As SP failure rates increase, the reintroduction of chloroquinein Malawi in combination with artesunate or with other short- says and assessing the verapamil-reversal phenotype associated acting drugs, such as chlorproguanil-dapsone, should be con- with chloroquine resistance would have been more definitive but sidered and would likely extend the useful life span of each of was not possible on these fresh isolates collected under field the drugs. This study reinforces the need for rational anti- malarial drug policies at both the national and regional levels Earlier reports from China [28] and Gabon [29] showed less and demonstrates the utility of surveillance using molecular dramatic declines in measures of chloroquine resistance after assays to monitor the effects of these policies on drug resistance.
reductions in chloroquine use. However, regions of Columbiaand Venezuela, where chloroquine use is reportedly minimized,continue to sustain mutant pfcrt 76T–harboring parasites [30], Acknowledgments
which suggests that the trends observed in Malawi may not bereplicated in settings with different epidemiological and trans- We thank Standwell Nkhoma, Jean-Claude Akpa, and Love- mission characteristics. Nonetheless, in malaria-endemic coun- more Kaetano, for technical assistance, and Rosemary Musonda, tries with semi-immune host populations, such as Malawi, even a partial resumption of chloroquine sensitivity may positivelyimpact public health. In Mali, up to two-thirds of infectionscarrying the mutant pfcrt are cured by chloroquine treatment, References
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