Chemosusceptibility analysis of Plasmodium falciparum imported from Comoros to Marseilles, France in 2001-2003

OBJECTIVES: The aim of this work was to study the chemosensitivity of Plasmodium falciparum strains isolated from patients presenting with malaria after having returned from Comoros Islands in 2002-2003, and hospitalized at the North University Hospital, in Marseilles, France. MATERIALS AND METHODS: In vitro drug susceptibility (for strains maintained in culture) and mutation-specific polymerase chain reaction (PCR) assays (for all strains) were performed. RESULTS: Out of 23 strains kept in culture, 50% were shown to be resistant in vitro to chloroquine, 50% were resistant to pyrimethamine, 40% to cycloguanil, 25% to atovaquone, and 7% to mefloquine. However all these strains were susceptible to quinine, halofantrine, and artemether. Moreover, 48 strains were tested by molecular methods. As a result, 69% were shown to have the Asp108 mutation in the dihydrofolate reductase gene (Pfdhfr), the basic mutation associated with antifolate resistance, and 54% had additional mutations Ile51 plus Arg59, associated with a high level of resistance. Furthermore, 90% of the 20 strains tested in 2003 were shown to have the point mutation Pfcrt76 in the P. falciparum chloroquine resistance transporter (Pfcrt) gene recently proposed as a molecular marker of chloroquine-resistance. CONCLUSION: Obtaining plasmodium strains from Comoros to be tested in Marseilles, where all laboratory facilities are available, is a unique opportunity to establish a surveillance of falciparum drug resistance in the Comoros islands.     

A network RER rooted on in vitro readout assays of Plasmodium falciparum sensitivity to chloroquine in the Indian Ocean Region

Chloroquine has been used as a first line drug to treat uncomplicated malaria cases during the last five decades in Madagascar and in the Comoros Union. The four plasmodial species known to infect humans occur on Madagascar Island. Chloroquine-resistant malaria cases, sometimes only suspected from presumptive malaria cases, have been reported in both countries. Thus, to redefine a strategy and a policy to cure malaria, there is a need to get relevant and updated data. In December 1999, the Madagascan Ministry of Health and the Institut Pasteur de Madagascar formed a network named RER for malaria resistance surveillance. In 2000 and 2001, 18 study sites (17 throughout Madagascar and 1 in Comoros) joined this network. Health-care workers were trained mainly for malaria diagnosis through the use of blood smear examination and for malaria case management. To alleviate the lack of competent medical teams within the health centres, and for technical and logistic reasons, as part of the network activities, it was decided to start with in vitro tests to assess the sensitivity of P.falciparum isolates to chloroquine by means of the isotopic method. Parasitized blood samples were collected from consenting patients. P.falciparum isolates were more predominant (989/1,036). Out of the 564 tests done, 432 (76.6%) could be assessed. Results demonstrated that 94.3% (381/404) of the Madagascan P.falciparum isolates were susceptible to chloroquine. In contrast, chloroquine-resistant isolates were prevalent in Comoros (8/28). The network set-up is presented. The usefulness of the in vivo approach and of the in vitro investigations (chemosusceptibility test and screening of mutations accounting for resistance to chloroquine) to monitor the emergence and the dissemination of chloroquine-resistant parasites is discussed.     

Imported malaria at the Marseilles Hôpital-Nord, France: a prospective study on 352 cases between 2001 and 2003

OBJECTIVE: The authors had for aim to study epidemiological, clinical, and parasitological characteristics, as well as regimen received, of imported malaria cases hospitalised at the North University Hospital, in Marseilles, France. DESIGN: The patients presenting with imported malaria included in this study were hospitalised in the infectious and tropical diseases unit and in the pediatrics unit at the North University Hospital, from January 1, 2001 to December 31, 2003. Variables were prospectively collected and recorded. RESULTS: 352 patients including 240 adults and 112 children were included. Most of them (67% of the adults and 92% of the children) were contaminated during a trip to the Comoros Islands. Plasmodium falciparum was the most common species identified. 97.5% of adult and 98% of child patients back from Comoros did not take any chemoprophylaxis against malaria or took inadequate regimens. Halofantrin was the most commonly used drug for children to treat uncomplicated P. falciparum malaria. In adults, atovaquone-proguanil was used as a first line drug in the absence of vomiting, and a 3-day intravenous regimen of quinine-clindamycin in case of vomiting. CONCLUSION: The specificity of imported malaria in Marseilles is the high proportion of Comorian patients who go back home periodically to visit friends and relatives. A better education of the Comorian population in Marseilles, regarding malaria risks and prophylaxis, needs to be implemented.     

Point-of-care testing for malaria outbreak management

A rapid antigen assay for malaria was performed on blood samples collected during a simultaneous outbreak of falciparum malaria and vivax malaria on a remote island in the Indonesian archipelago. During the outbreak, a total of 89 patients (4.3% of the population) were diagnosed with malaria within a week. Microscopic examination revealed 78 malaria slide-positive cases, of whom 49 (62.8%) were identified as P. falciparum, 7 (9.0%) as P. vivax and 22 (28.2%) as mixed P. falciparum and P. vivax infections. The rapid malaria assay showed excellent correlation with expert-confirmed routine microscopy for P. falciparum and P. vivax monoinfections and mixed infections with a parasite density >50 parasites/microl. Several slide-negative blood samples collected from febrile patients with clinical malaria tested positive in the rapid test. The estimated sensitivity calculated for the rapid test (91.0%) was slightly higher than that of microscopy (87.6%). The result indicates that rapid antigen detection for malaria could be a useful alternative to microscopy to reduce the workload during emergency outbreak situations.     

A molecular surveillance system for global patterns of drug resistance in imported malaria

Analysis of imported malaria in travelers may represent a novel surveillance system for drug-resistant malaria. We analyzed consecutive falciparum malaria isolates from Canadian travelers from 1994 to 2000, for polymorphisms in pfcrt, dhfr, and dhps linked to chloroquine and pyrimethamine/sulfadoxine resistance. Forty percent of isolates possessed the K76 pfcrt allele, suggesting that many imported falciparum infections are still responsive to chloroquine. Travelers who had recently taken chloroquine had a significantly increased risk of harboring isolates with pfcrt resistance alleles (odds ratio = 4.47; p=0.03). The presence of two or more mutations in dhfr or dhps was found in 64.8% (95% confidence interval [CI] 54.6 to 73.9) and in 30.4% (95% CI 21.7 to 40.3) of isolates, respectively, and increased significantly over the course of the study. These molecular markers indicate that pyrimethamine/sulfadoxine resistance is increasing and is now too high to rely on this drug as a routine therapeutic agent to treat malaria in travelers.     

Lessons learnt from the six decades of chloroquine use (1945-2005) to control malaria in Madagascar

On the island of Madagascar, malaria was nearly eradicated in the highland areas and malaria transmission was significantly decreased in the coastal areas between the 1940s and 1960s. The success of the control programme was primarily achieved by chloroquine (CQ) use at the community level. CQ was administered to children weekly on a routine basis for malaria prevention in the period 1949-1971. Then, the Malagasy Government was unable to financially support the malaria control programme. The malarial situation worsened in the 1980s, partly due to the shortage of CQ. A malaria epidemic occurred. To deal with this epidemic, massive CQ use was urgently adopted. CQ has remained the first-line drug since 1945, but the prevalence of Plasmodium falciparum carrying the pfcrt mutation associated with CQ resistance remains low (<3%). However, late CQ treatment failure has been reported and the prevalence may be as high as 35% during 14-day follow-up since 1982. In an effort to eliminate malaria as a public health problem, a shift from CQ to artemisinin-based combination therapy has been advocated by a new policy since December 2005. A change of this kind is complex and the lessons learnt from the six decades of CQ use are of the utmost importance to achieve malaria control.     

Malaria--Great Exuma, Bahamas, May-June 2006

Malaria in humans is caused by four distinct protozoan species of the genus Plasmodium (P. falciparum, P. vivax, P. ovale, and P. malariae). These parasites are transmitted by the bite of an infective female Anopheles mosquito. In the Caribbean region, malaria has been eliminated from all islands except Hispaniola, the island consisting of Haiti and the Dominican Republic. Elimination of malaria elsewhere resulted from a combination of integrated control measures, socioeconomic development, and close public health surveillance. However, even Caribbean islands where malaria is no longer endemic remain at constant risk for reintroduction of the disease because of their tropical climate, presence of competent malaria vectors, and proximity to other countries where malaria is endemic. This susceptibility was underscored by the recent outbreak of malaria on the island of Great Exuma in the Bahamas; during May-June 2006, a total of 19 malaria cases were identified. Four of the cases, in travelers from North America and Europe, are described in this report; such cases of imported malaria can signal the presence of a malaria problem in the country visited and thus assist local health authorities in their investigations. On September 19, after 3 months with no report of new cases, CDC rescinded its previous recommendation that U.S.-based travelers take preventive doses of the antimalarial drug chloroquine before, during, and after travel to Great Exuma.     

Drug-resistant malaria parasites introduced into Madagascar from Comoros Islands

To determine risk for drug-resistant malaria parasites entering Madagascar from Comoros Islands, we screened travelers. For the 141 Plasmodium falciparum isolates detected by real-time PCR, frequency of mutant alleles of genes associated with resistance to chloroquine and pyrimethamine was high. International-level antimalarial policy and a regional antimalarial forum are needed.     

Genetic diversity and population structure of Plasmodium falciparum over space and time in an African archipelago

The archipelago of São Tomé and Principe (STP), West Africa, has suffered the heavy burden of malaria since the 16th century. Until the last decade, when after a successful control program STP has become a low transmission country and one of the few nations with decreases of more than 90% in malaria admission and death rates.We carried out a longitudinal study to determine the genetic structure of STP parasite populations over time and space. Twelve microsatellite loci were genotyped in Plasmodium falciparum samples from two islands collected in 1997, 2000 and 2004. Analysis was performed on proportions of mixed genotype infections, allelic diversity, population differentiation, effective population size and bottleneck effects.We have found high levels of genetic diversity and minimal inter-population genetic differentiation typical of African continental regions with intense and stable malaria transmission.We detected significant differences between the years, with special emphasis for 1997 that showed the highest proportion of samples infected with P. falciparum and the highest mean number of haplotypes per isolate.This study establishes a comprehensive genetic data baseline of a pre-intervention scenario for future studies; taking into account the most recent and successful control intervention on the territory.     

Spleen rates in children: an old and new surveillance tool for malaria elimination initiatives in island settings

Spleen rates (SR) have been traditionally used to estimate the burden of malaria transmission. Results are presented from 51 surveys, which measured SR and parasite rates (PR) in 29,962 individuals in the archipelago of Vanuatu. Indices for spleen size computed with multivariate statistical tools outperformed the WHO average spleen index and showed that spleen sizes in a population can track shifts in malaria transmission. In general, a positive linear relationship between Plasmodium spp. PR and SR was found for the archipelago. In the context of malaria elimination and for the specific setting of this study we found that spleen examination is a useful tool in post-malaria elimination surveillance. Finally, results highlight the value of measuring spleen sizes to rapidly assess the impact of intervention packages aimed at malaria elimination or control.     

An Overview of the Malaria Situation in Zanzibar

Zanzibar is composed of two neighbouring islands with about a 1.1 million inhabitants in 2600 square kilometers. Both islands are located off the northeastern coast of the Tanzanian mainland. Zanzibar is a typical malaria endemic area. About a third of all blood samples currently examined for malaria in Zanzibar are positive to Plasmodium falciparum. Previous attempts failed to significantly improve the malaria situation in Zanzibar due to administrative problems, antimalarial drug and DDT resistance. We visited Zanzibar in order to evaluate the magnitude of the malaria problem and the medical logistics. This paper reviews the results of the visit, past and present attempts to eliminate malaria and our conclusions regarding Zanzibar as a model site for attempts to eradicate malaria.     

Chloroquine blood concentrations and molecular markers of chloroquine-resistant Plasmodium falciparum in febrile children in northern Ghana

Plasmodium falciparum malaria is a predominant reason for health care utilization among children in sub-Saharan Africa. Despite the spread of resistance, chloroquine (CQ) is the most commonly used antimalarial. Little is known about the pattern of CQ use and resistance to the drug prior to attendance at a health care facility, and its impact on clinical presentation in children attending health care facilities in endemic regions. In a cross-sectional study among 840 febrile children presenting at a primary health care facility in northern Ghana from September to December 2000, CQ blood levels were measured by enzyme-linked immunosorbent assay and parasite isolates were genotyped for the CQ resistance markers pfcrt T76 and pfmdr1 Y86. Plasmodium falciparum was present in 95% by polymerase chain reaction and CQ was detected in 64% of the children. Concentrations of CQ in blood ranged from 31 to 3897 nmol/L (median 198 nmol/L). The pfcrt T76 and pfmdr1 Y86 resistance markers were detected in 84% and 57% of the isolates, respectively, and were selected by CQ. A significant trend for higher frequencies of the resistance markers with increasing CQ concentrations was observed. In this typical primary health care setting in sub-Saharan Africa, CQ use prior to attendance at a health care facility and CQ-resistant P. falciparum are frequent. As CQ selects resistant P. falciparum genotypes, CQ should be omitted as a first-line drug even in primary health care facilities when self-treatment with CQ is common.     

输入性抗性恶性疟综合防治与监测方法研究

目的　研究恶性疟病例,为进一步开展恶性疟防治工作提供依据。方法　对1989~2003年1 140名外出海南做工回归人员进行疟疾患病情况调查。结果　疟疾患病率为54. 04%,疟原虫阳性率为14. 39%,其中恶性疟原虫阳性率为12. 19%,占阳性总数的84. 76%。观察16例恶性疟原虫无性体带虫者和18例输入性恶性疟病人,对氯喹均呈现抗药性。经过15年来对疫点和疫区全民抗复发治疗、外出回归人员药物根治,以及连续3年“DDT”室内滞留喷洒和6年溴(氯)氰菊脂浸泡蚊帐灭蚊,控制了输入性恶性疟传播蔓延。结论　加强健康教育,支持流动人口自我保护,是控制输入性恶性疟的关键。要继续加强易感人群,特别是流动人群及媒介的监测。发现问题,及时妥善处理。     

The malaria and typhoid fever burden in the slums of Kolkata, India: data from a prospective community-based study

Recent research has indicated that the malaria burden in Asia may have been vastly underestimated. We conducted a prospective community-based study in an impoverished urban site in Kolkata, India, to estimate the burden of malaria and typhoid fever and to identify risk factors for these diseases. In a population of 60452 people, 3605 fever episodes were detected over a 12-month period. The blood films of 93 febrile patients contained Plasmodium (90 P. vivax, 2 P. falciparum and 1 P. malariae). Blood cultures from 95 patients grew Salmonella enterica serotype Typhi. Malaria patients were found to be significantly older (mean age 29 years) compared with patients with typhoid fever (15 years; P<0.001) but had similar clinical features on presentation. Having a household member with malaria, illiteracy, low household income and living in a structure not built of bricks were associated with an increased risk for malaria. Having a household member with typhoid fever and poor hygiene were associated with typhoid fever. A geographic analysis of the spatial distribution of malaria and typhoid fever cases detected high-risk neighbourhoods for each disease. Focal interventions to minimise human-vector contact and improved personal hygiene and targeted vaccination campaigns could help to prevent malaria and typhoid fever in this site.     

Antimalarial drug resistance

Drug resistant malaria is mostly due to Plasmodium falciparum, the highly prevalent species in tropical Africa, Amazon, and Southeast Asia. P. falciparum is responsible for severe involvement of fever or anemia causing more than a million deaths per year. Rationale for treatment is becoming weak as multiple drug resistance against well-tolerated drugs develops. P. falciparum drug resistant malaria originates from chromosomal mutations. Analyses using molecular, genetic and biochemical approaches showed that: 1) impaired uptake of chloroquine by the parasite vacuole is a common characteristic of resistant strains, this phenotype correlates with pfmdr1 and pfcrt gene mutations; 2) one S108N to four (N51I, C59R, I164L) point mutations of dihydrofolate reductase, the enzyme target of antifolinics (pyrimethamine and proguanil), give moderate to high level of resistance to these drugs; 3) resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (A437G, K540E), their enzyme target, impairing their capacity to potentiate antifolinic drugs; 4) resistance to atovaquone plus proguanil involves one single mutation on atovaquone target, cytochrome b (Y268S, C or N); 5) resistance to mefloquine is thought to be linked to the over expression of pfmdr1, a pump expelling toxic waste from eukaryotic cells. P. falciparum resistance levels may differ according to places and time, depending on malaria transmission and drug pressure. Coupling in vivo to in vitro tests, and using molecular tests is essential for the surveillance of replacement drugs. Low cost biochemical tools are urgently needed for a prospective monitoring of resistance.     

武汉市116例境外输入性恶性疟临床治疗效果分析

目的分析和评价境外输入性恶性疟患者临床特点及青蒿素类药物治疗效果,促进合理规范用药,提高临床治愈率。方法收集武汉市省级疟疾定点医院收治的116例输入性恶性疟病例,对患者临床症状及体征、青蒿素药物使用情况及用药效果等进行回顾性分析。结果输入性恶性疟临床表现复杂多样且并发症较多,主要为发热、纳差、贫血、黄疸等;使用青蒿素治疗后症状均减轻或消失,无明显药物不良反应,退热中位数为2d,疟原虫转阴中位数为3d,临床症状缓解中位数为3d;109例普通型恶性疟采用青蒿素复方口服或青蒿琥酯针剂联合青蒿素复方口服治疗,其中30例口服青蒿素复方治疗1~4d, 1例复燃;79例青蒿琥酯针剂联合青蒿素复方口服治疗4~6d, 4例出现复燃。7例重症病例采用青蒿琥酯针剂联合口服多种青蒿素复方长疗程大剂量后均治愈,总治愈率95.69%(111/116)。结论患者以口服和注射青蒿素杀灭疟原虫治疗恶性疟安全有效,治愈率高,但部分患者停用青蒿素类药物后出现疟原虫复燃,为遏制患者重症和抗药风险,应"规范、全程、足量"使用青蒿素类药品。     

Chloroquine resistant Plasmodium falciparum in indigenous residents of Cameroon

Thirty-nine percent (36 of 92) of children in Limbe, Cameroon, treated with chloroquine (10 mg/kg body weight on days 1 and 2, and 5 mg/kg on day 3) for falciparum malaria failed to respond within 7 d of treatment. Twenty-two of these children with chloroquine-resistant malaria were successfully treated with Fansidar [one-half tablet (250 mg sulfadoxine and 25 mg pyrimethamine) per 10 kg body weight], while the other 14 children were cured with mefloquine (25 mg/kg body weight). In vitro, a combination of verapamil at 1.0 x 10(-6) M with chloroquine or desethylchloroquine reversed resistance to the antimalarial drug and its primary metabolite in each of the 2 isolates successfully adapted and maintained in continuous culture. Similar combinations had no effect on susceptibilities of a sensitive reference clone, D6, used as control. Both chloroquine-resistant isolates from Cameroon were significantly more susceptible to mefloquine and halofantrine in vitro than the chloroquine-sensitive reference clone. Clinical observation, and in vitro confirmation, of chloroquine-resistant falciparum malaria in these indigenous children from Cameroon, and the current socio-economic condition in West Africa, underscore the need for pragmatic health management policies for efficient use of alternative antimalarial drugs in controlling drug resistant Plasmodium falciparum in the region. This observation of reversal of chloroquine resistance in isolates of P. falciparum obtained from West Africa, and a previous report on clones obtained from south-east Asia and South America, suggest that the mechanism(s) of resistance to chloroquine may be identical in resistant parasites from the 3 continents.     

Efficacy of artesunate plus chloroquine for the treatment of uncomplicated malaria in children in Burkina Faso: a double-blind, randomized, controlled trial

Chloroquine (CQ)-resistant Plasmodium falciparum is compromising malaria control in Africa. Combining artesunate (AS) with standard antimalarial drugs increases cure rates and may delay drug resistance. We compared the safety and efficacy of CQ alone and CQ combined with AS (CQ-AS) for treating uncomplicated P. falciparum malaria in Burkina Faso between August 1999 and August 2000. Chloroquine (25 mg/kg over 3 d) combined with AS or placebo (4 mg/kg/d for 3 d) was administered to 300 children aged 6 to 59 months in a randomized, double-blind study. Follow-up extended over 28 d. No adverse drug reactions were recorded. By day 14, parasites were cleared in 120/147 (81.6%) CQ AS-treated children compared with 53/143 (37.1%) CQ-treated children (odds ratio [OR] = 7.55, 95% CI 4.27-13.43, P < 0.001). Corresponding rates for day 28 were 71/145 (49.0%) vs. 27/142 (19.0%) (OR= 4.09, 95% CI 2.33-7.21, P < 0.001). Children who received CQ-AS had significantly faster parasite and fever clearance. Despite the beneficial effects of adding AS, the high failure rate at day 28 of CQ-AS precludes its use as the first-line regimen for treating CQ-resistant P. falciparum in Burkina Faso.     

Recrudescence of imported falciparum malaria after quinine therapy: potential drug interaction with phenytoin

Quinine remains a reliable treatment for falciparum malaria in most parts of the world. We report recrudescence of imported Plasmodium falciparum malaria following quinine treatment in the context of concurrent phenytoin use. Supported by a subtherapeutic quinine level, we hypothesise that a drug interaction with phenytoin may compromise the efficacy of quinine in the treatment of malaria.     

Therapeutic response of multidrug-resistant Plasmodium falciparum and P. vivax to chloroquine and sulfadoxine-pyrimethamine in southern Papua, Indonesia

To determine the level of antimalarial drug resistance in southern Papua, Indonesia, we assessed the therapeutic efficacy of chloroquine plus sulfadoxine-pyrimethamine (CQ+SP) for Plasmodium falciparum infections as well as CQ monotherapy for P. vivax infections. Patients with P. falciparum failing therapy were re-treated with unsupervised quinine+/-doxycycline therapy and those with P. vivax with either unsupervised quinine+/-doxycycline or amodiaquine. In total, 143 patients were enrolled in the study (103 treated with CQ+SP and 40 with CQ). Early treatment failures occurred in four patients (4%) with P. falciparum and six patients (15%) with P. vivax. The failure rate by Day 28 for P. vivax was 65% (95% CI 49-81). After PCR correction for re-infections, the Day 42 recrudescence rate for P. falciparum infections was 48% (95% CI 31-65). Re-treatment with unsupervised quinine+/-doxycycline resulted in further recurrence of malaria in 48% (95% CI 31-65) of P. falciparum infections and 70% (95% CI 37-100) of P. vivax infections. Eleven patients with recurrent P. vivax were re-treated with amodiaquine; there were no early or late treatment failures. In southern Papua, a high prevalence of drug resistance of P. falciparum and P. vivax exists both to first- and second-line therapies. Preliminary data indicate that amodiaquine retains superior efficacy compared with CQ for CQ-resistant P. vivax.