DRUG-RESISTANT MALARIA IN BANGLADESH: AN IN VITRO ASSESSMENT

H. NOEDL 0 1 M. A. FAIZ 0 1 E. B. YUNUS 0 1 M. R. RAHMAN 0 1 M. A. HOSSAIN 0 1 R. SAMAD 0 1 R. S. MILLER 0 1 L. W. PANG 0 1 C. WONGSRICHANALAI 0 1 0 U.S. Army Medical Component, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Department of Specific Prophylaxis and Tropical Medicine, Institute of Pathophysiology, University of Vienna , Vienna , Austria; Department of Medicine, Chittagong Medical College , Chittagong , Bangladesh 1 Authors' addresses: H. Noedl, Department of Specific Prophylaxis and Tropical Medicine, Institute of Pathophysiology, University of Vienna , Kinderspitalgasse 15, A-1095, Vienna, Austria. MA Faiz, EB Yunus, MR Rahman, MA Hossain, MA Samad , Department of Medicine, Chittagong Medical College , Chittagong, Bangladesh. RS Miller, LW Pang, C Wongsrichanalai , U. S. Army Medical Compo- nent , Armed Forces Research Institute of Medical Sciences , 315/6 Rajvithi Road, Bangkok 10400 , Thailand Forty-four Plasmodium falciparum isolates from Bangladesh and 22 from western Thailand were successfully tested for their drug susceptibility. High degrees of resistance were observed against chloroquine with geometric mean IC50s of 114.25 and 120.5 nM, respectively, for Bangladesh and western Thailand. Most isolates from both sites were sensitive to quinine, and all were sensitive to artesunate. Many isolates were considered in vitro resistant to mefloquine, but the geometric mean IC50 for the Thai isolates (98.79 nM) was 1.6 times (P 0.002) higher than that of isolates from Bangladesh (60.3 nM). The high prevalence of in vitro mefloquine resistance in Bangladesh suggests that close surveillance is necessary to delay widespread multidrug resistant problems in the area. - Multidrug resistant malaria is a public health threat in Southeast Asia, and its potential spread is imminent. However, drug sensitivity reports from countries surrounding Thailand are sparse. To some extent, active malaria control efforts in countries like Thailand have prevented this problem from escalating in recent years. In Bangladesh, however, drug resistance has increased the burden of the countrys malaria control program. Up to 70,000 laboratory-confirmed and 900,000 clinical cases of malaria, with more than 500 deaths per year, were accounted for in Bangladesh in the late 1990s,1 but these numbers may represent a gross underestimate of the disease burden because of shortcomings in surveillance and information systems. Approximately 88% of the 125 million people in the country are at malaria risk.2 The aim of this study is to characterize the dimension of in vitro antimalarial drug resistance in Bangladesh and to define baseline data for future assessments of drug susceptibility. MATERIALS AND METHODS Plasmodium falciparum isolates were collected in 1999 at the outpatient department of Ramu Health Complex (Coxs Bazar district), Chittagong, Bangladesh, and the malaria clinic in Maesod, western Thailand. The samples were cryopreserved, culture-adapted, and tested for their drug susceptibility at the laboratory of the Armed Forces Research Institute of Medical Sciences in Bangkok using a [3H]hypoxanthine uptake assay.4,5 The Thaisamples, whcih represent isolates from a region with an exceptionally high prevalence of multidrug resistance, were gathered to serve as a comparative reference for the magnitude of drug resistance in Bangladesh. Forty-four specimens from Bangladesh (out of 72 collected) and 22 from Thailand (out of 26) were successfully culture-adapted and tested. The differences in the success rate were primarily attributable to logistic problems in storing and transporting the specimens from Bangladesh. Febrile patients (> 100F) or those with a history of fever within the past 48 hours with microscopically confirmed P. falciparum monoinfections (parasite density 5,000 to 200,000 asexual forms per L blood) were enrolled. Pregnant and lactating females, patients with severe malaria, those with a history of pretreatment, as well as young children were excluded. The samples were obtained before any treatments. Patients subsequently received mefloquine (Lariam 15 mg/ kg single dose) for Bangladesh and artesunate-mefloquine combination (mefloquine 750 mg stat followed by 500 mg six hours later, plus artesunate 300 mg once per day for two days) for Thailand. Informed consent was obtained from all adult volunteers or from parents or legal guardians of minors. The study protocols were approved by the ethical review boards of Chittagong Medical College and the Thai Ministry of Public Health. Inhibitory concentrations (ICs) were estimated by nonlinear regression analysis.4 For graphic display, the data were adapted to a log-probit model.6 Potency ratios (PR IC50A/ IC50B) were calculated as a measure of the different activity of antimalarial drugs at the two study sites. Geometric mean ICs were compared by Students t test. Most of the P. falciparum isolates from Bangladesh tested (37/44; 84%) were found to be resistant to chloroquine (i.e., IC99 > 200 nM). The corresponding percentage was even higher in Thailand (95%, 21/22). Table 1 shows the geometric mean IC50 and IC90 values. With potency ratios (PR) of 1.05 (P > 0.05), at both IC50 and IC90, chloroquine in vitro sensitivity levels for Thailand and Bangladesh were not significantly different. Only 13 isolates from Bangladesh (30%) and five from Thailand (23%) were found to be resistant to quinine (i.e., IC99 > 2000 nM). The isolates from the Thailand-Myanmar border gave a higher geometric mean IC50 value; with a PR of 1.31, the difference was statistically significant at the IC50 level (P 0.027). The most notable difference was found for mefloquine. Twenty-seven of 44 isolates from Bangladesh (61%) and 18 of 22 from Thailand (82%) were in vitro resistant to mefloquine (i.e., IC99 > 200 nM). The geometric mean IC50 for the Thai isolates, however, was 1.6 times higher than that for the Bangladeshisiolates (PR 1.64; P 0.002). At the IC90 level, a similar relationship was found (PR 1.31; P 0.024). All Bangladeshi patients were clinically cured based on 28-day follow-up. Artesunate-mefloquine combination is the current first-line regimen for the study area in Thailand and, according to the Thai Malaria Control Program, the regimen continues to be fully effective in that population. DRUG-RESISTANT MALARIA IN BANGLADESH No in vitro resistance threshold has been defined for artemisinin compounds. However, the artesunate ICs for both Bangladesh and Thailand were of low levels and did not show any significant differences [IC50 (PR 1.13; P > 0.05), IC90 (PR 1.14; P > 0.05)]. Highly significant correlations were found between artesunate and mefloquine at EC50 (r 0.651; P < 0.001) level. A similarly close relationship also was found between artesunate and quinine (r 0.681; P < 0.001) as well as between mefloquine and quinine (r 0.545; P < 0.001). Our data suggest a moderate level of in vitro antimalarial drug (...truncated)