A Non-Radioactive DAPI-based High-Throughput In Vitro Assay to Assess Plasmodium falciparum Responsiveness to Antimalarials��Increased Sensitivity of P. falciparum to Chloroquine in Senegal

The spread of Plasmodium falciparum drug resistance is outpacing new antimalarial development and compromising effective malaria treatment. Combination therapy is widely implemented to prolong the effectiveness of currently approved antimalarials. To maximize utility of available drugs, periodic monitoring of drug efficacy and gathering of accurate information regarding parasite-sensitivity changes are essential. We describe a high-throughput, non-radioactive, field-based assay to evaluate in vitro antimalarial drug sensitivity of P. falciparum isolates from 40 Senegalese patients. Compared with earlier years, we found a significant decrease in chloroquine in vitro and in genotypic resistances (> 50% and > 65%, respectively, in previous studies) with only 23% of isolates showing resistance. This is possibly caused by a withdrawal of chloroquine from Senegal in 2002. We also found a range of artemisinin responses. Prevalence of drug resistance is dynamic and varies by region. Therefore, the implementation of non-radioactive, robust, high-throughput antimalarial sensitivity assays is critical for defining region-specific prophylaxis and treatment guidelines.Plasmodium falciparum, which causes the most virulent human malaria, is responsible for approximately one million deaths annually. Malaria related morbidity and mortality in sub-Saharan Africa presents a significant obstacle for economic development in this part of the world.^1,2 Although a number of chemotherapeutic options are available for treating P. falciparum malaria, the rapid spread of drug resistance has marginalized the utility of many of these drugs. Chloroquine (CQ), quinine (QN), pyrimethamine, amodiaquine (AMQ), and artemisinin (ART) are among the most effective antimalarial agents. The latter two have been used in combination in many malaria endemic regions to thwart the emergence of drug resistance.³ In fact, the World Health Organization recommends the use of ART-based combination therapy (ACT) as a first-line regimen throughout much of Africa.⁴ However, monotherapy with ART and its derivatives has caused the emergence of parasites that show decreased sensitivity to these drugs, which is reflected in the higher IC₅₀ values of some P. falciparum clinical isolates.^5,6 Although clinical resistance to ART has not been adequately defined and reported, these decreased sensitivities may be the harbinger of clinical resistance. Periodic surveillance of drug efficacy through in vitro drug-sensitivity assays is essential for optimal selection of drug combinations, which will ensure successful administration of antimalarial treatment.High-throughput methods that enable analyses of drug responsiveness of clinical isolates from different countries where malaria is endemic are essential to define antimalarial treatment regimens in those regions. For example, ACT is routinely and widely used for managing most malaria infections, sulfadoxine-pyrimethamine is still used for intermittent preventative treatment in pregnancy, and QN remains an important option for parenteral use in severe disease.⁴ In addition, a wide variation in drug sensitivity may be present even within the same country,⁷ and thus, drug-sensitivity testing would be needed for each geographically distinct region. Moreover, such high-throughput methods will allow screening of lead compounds that could potentially be developed into novel antimalarials.Herein, we describe the field implementation of a non-radioactive, high-throughput assay to evaluate drug sensitivity on ex vivo isolates of P. falciparum collected from infected Senegalese patients. Previously, the only means of evaluating inhibition of parasite proliferation entailed microscopic examination of blood smears stained with modified Wright-Giemsa or use of the [³H]-hypoxanthine incorporation assay.⁸ The assays are either laborious or require the use of radioactive isotopes, whose safe disposal is difficult in a resource-limited setting. More recently, methods for quantifying parasite histidine-rich protein-2 (HRP-2) as well as non-radioactive fluorescence-based methods using SYBR green and 4–6-diamidino-2-phenylindole (DAPI) that measure parasite DNA have been developed.^9–11 We have adapted the DAPI-based method to evaluate drug sensitivity of ex vivo P. falciparum parasite cultures, thus directly assessing parasite samples collected from patients under field laboratory conditions in Senegal.We examined the drug-sensitivity profiles of P. falciparum parasites derived from Senegalese patients presenting during transmission season in the fall of 2007 to the Anti-Parasite Service in Thiès (SLAP), a city ~75 km southeast of Dakar, against a panel of antimalarial agents. In this region, malaria transmission is perennial and hypoendemic with an increase in cases during the end of the rainy season from September to December.¹² Approximately one-third of all outpatient consultations and 20–30% of mortality in healthcare facilities in Senegal can be attributed to infection with P. falciparum. CQ was recommended as the first-line malaria therapy in Senegal until 2002. However, in 2003, the Senegalese Ministry of Health altered the national treatment policy to include ACT using artesunate, a more soluble derivative of ART, plus AMQ as the first-line antimalarial regimen.Consenting outpatients with clinical signs suggestive of mild malaria and a positive Giemsa-stained thick film for P. falciparum were identified and enrolled from SLAP. Patients presenting with fever or those who had a history of fever and symptoms indicative of malaria were enrolled. The study protocol was approved by the Ethics Committee of the Ministry of Health in Senegal and the Human Subject Committee of the Harvard School of Public Health.Before the start of the transmission season, microtiter plates were pre-loaded with antimalarial drug dilutions (serial dilutions of CQ [0.02–400 nM], AMQ [0.02–20 μM], ART [3.12–35 nM], and QN [234.37–75 μM] and stored at −20°C until needed. Blood was collected from patients and transported on ice to the central laboratory. Only samples with 0.3% parasitemia or greater were used for the study. Inasmuch as this may introduce some bias, we were unable to use our method to obtain standard threshold 50% inhibitory concentration (IC₅₀) values for isolates with lower parasitemias. Packed erythrocytes were washed twice with RPMI (pH 7.4). The hematocrit of all samples was set to 2%, and samples with parasitemia greater than 1% were adjusted to 0.5–1.0% in complete medium using standard tissue-culture media.¹⁰ The cell suspension (180 μL per well) was dispensed into the thawed, pre-loaded, 96-well microtiter plates. The plates were incubated at 37°C in a gas environment of 5% CO₂, 1% O₂, and 94% N₂ for 72 hours. Smears prepared from zero drug wells were stained with Giemsa to ensure parasite growth at 72 hours. Samples that did not show any parasite growth in culture were eliminated from the subsequent analyses. At the end of the assay, the plates were frozen at −20°C. The microtiter plates were then thawed at room temperature and centrifuged, and media were aspirated with care to avoid disturbing the red cell pellet. The fluorochrome mixture was prepared as previously described¹⁰ and dispensed in each well at a final dilution of 1:100,000 of DAPI (5 mg/mL stock; Molecular Probes, Inc., Eugene, OR). The microtiter plates were then incubated in the dark for 30 minutes and centrifuged at 4,000 rpm for 10 minutes. Excess fluorochrome mixture was aspirated, and 30 μL of 1× phosphate buffered saline (PBS) was dispensed into each well. The microtiter plates were read using a Fluoroskan plate reader (ThermoFisher Scientific, Milford, MA) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. IC₅₀ values were calculated by non-linear regression analysis (GraphPad Prism, La Jolla, CA). Each antimalarial compound was evaluated in duplicate.Parasite resistance to CQ, QN, and AMQ was defined based on standard threshold IC₅₀ values, above which parasites are classified as resistant (13

Table 1

Median IC₅₀ values of field isolates from Senegal for a panel of antimalarials

Characteristics of potential living kidney donors in Ivory Coast: a survey prior to a project of kidney transplantation in French Black Africa

The source of living kidney donors in the general population remains underused. The present study aims at assessing the prevalence and the characteristics of potential living kidney donors in Ivory Coast in a view of a project of kidney transplantation in French Black Africa. A survey was undertaken in Abidjan from 30 June to 7 July 2006. Nine hundred (and) sixty-two subjects living in the capital and aged between 19 and 64 years old were randomly chosen using data from the 1998 population census. Subjects were asked their age, gender, nationality, marital status, information on kidney graft and renal failure, and their willingness to donate kidney to a relative or friend for transplantation purpose. Seventy per cent of the population study appeared favourable to kidney donation. Potentials living kidney donors have displayed following characteristics: age inferior to 26 years old [OR=2.08, P<0.02, 95%CI: 1.10-3.92]; Ivorians national [OR=2.72, P<0.002, 95% CI 1.42-5.21]; having heard of kidney transplantation (OR=1.89, P<0.047, 95% CI 1-3.54]); the death of a relative or friend from renal failure [OR=1.82, P<0.002, 95% CI 1.25 2.67]. Being married adversely affect kidney donation [OR=0.52, P<0.0002, 95% CI 0.34-0.79]. Potentials living kidney donors are in great number in Ivory Coast, who had specific characteristics.     

Atypical Chikungunya virus infections: clinical manifestations, mortality and risk factors for severe disease during the 2005-2006 outbreak on Réunion

In April 2005, an outbreak of Chikungunya fever occurred on the island of Réunion in the Indian Ocean. During winter 2005, six patients developed meningoencephalitis and acute hepatitis due to Chikungunya virus. Our objectives were to determine the incidence and mortality of atypical Chikungunya viral infections and to identify risk factors for severe disease. A hospital-based surveillance system was established to collect data on atypical Chikungunya cases. Case reports, medical records and laboratory results were reviewed and analysed. We defined an atypical case as one in which a patient with laboratory-confirmed Chikungunya virus infection developed symptoms other than fever and arthralgia. We defined a severe atypical case as one which required maintenance of at least one vital function. We recorded 610 atypical cases of Chikungunya fever: 222 were severe cases, 65 affected patients died. Five hundred and forty-six cases had underlying medical conditions (of which 226 suffered from cardiovascular, 147 from neurological and 150 from respiratory disorders). Clinical features that had never been associated with Chikungunya fever were recorded, such as bullous dermatosis, pneumonia, and diabetes mellitus. Hypertension, and underlying respiratory or cardiological conditions were independent risk factors for disease severity. The overall mortality rate was 10.6% and it increased with age. This is the first time that severe cases and deaths due to Chikungunya fever have been documented. The information presented in this article may assist clinicians in identifying the disease, selecting the treatment strategy, and anticipating the course of illness.     

Inflammatory demyelinating diseases of the central nervous system in Niger

Background

In sub-Saharan Africa (SSA), few studies have been reported on inflammatory demyelinating diseases of the central nervous system (CNS). Neuromyelitis optica spectrum disorders (NMOSD) seems to be the most frequent inflammatory demyelinating disease of CNS in sub-Saharan Africans or people of sub-Saharan African descent.

蒿甲醚与双氢青蒿素治疗恶性疟患儿疗效对比观察

为对今后临床选择抗恶性疟药物提供可能的依据,本文比较蒿甲醚及双氢青蒿素治疗恶性疟的疗效,156例2～13岁恶性疟患儿分为双氢青蒿素组76例及蒿甲醚组80例,两组均口服给药,用药5d,剂量各异,治疗期间禁用糖皮质激素及解热镇痛药,以用药后疟原虫无性体在体内消失及复发做疗效观察指标。结果,两组在24h平均原虫下降率、平均原虫转化时间、平均退热时间上比较差异均无统计学意义。     

Safety of the yellow fever vaccine during the September 2001 mass vaccination campaign in Abidjan, Ivory Coast

In 2001, a vaccination campaign against yellow fever was carried out in Abidjan, Cote d'Ivoire. During the campaign and 4 weeks after an active surveillance system for adverse events following immunization (AEFI) was set up. More then 2.6 million doses were administered and 87 AEFI were notified. Eight suspected YF cases were reported after vaccination and considered as AEFI. However, none had IgM for YF and all recovered without sequels. This surveillance system provided reassuring data about the safety of the YF vaccine and proved that it is feasible to set up an active surveillance system during a mass campaign.     

Malaria imported into Réunion Island: is there a risk of re-emergence of the disease?

After a long period of endemicity until the 1950s, the World Health Organization considered autochthonous malaria eliminated from Réunion in 1979. To prevent secondary transmission and re-emergence of autochthonous malaria, permanent epidemiologic and entomological surveillance and vector control measures are conducted.The objective of this study is to report sociodemographic characteristics of imported malaria patients and incidence rates from 2003-2008 using mandatory notification with the aim of identifying risk groups and destinations.During this period, 684 imported malaria cases were reported. Median age of patients was 34.4 years and 22.1% were children ≤ 15 years. Men represented 67.7% of cases and 59.1% of patients reported having taken chemoprophylaxis based on chloroquine alone. Incidence of malaria was considerably different by country destination. For Comoros, incidence was stable and high during the period accounting for 1481 cases per 100 000 travels in 2008. The rate was lower for travels to Madagascar, South Africa and Mayotte and decreased over the period to 37, 19 and 3 per 100 000 respectively, by 2008.To avoid re-emergence of malaria on the island and to protect themselves, travelers should reduce their risks of acquisition and importation of parasites by using adequate preventive measures. A special preventive program and social mobilisation should be a priority, essentially for the Comorian community in Réunion.