Stéphane Picot

A systematic review and meta-analysis of evidence for correlation between molecular markers of parasite resistance and treatment outcome in falciparum malaria

BackgroundAn assessment of the correlation between anti-malarial treatment outcome and molecular markers would improve the early detection and monitoring of drug resistance by Plasmodium falciparum. The purpose of this systematic review was to determine the risk of treatment failure associated with specific polymorphisms in the parasite genome or gene copy number.MethodsClinical studies of non-severe malaria reporting on target genetic markers (SNPs for pfmdr1, pfcrt, dhfr, dhps, gene copy number for pfmdr1) providing complete information on inclusion criteria, outcome, follow up and genotyping, were included. Three investigators independently extracted data from articles. Results were stratified by gene, codon, drug and duration of follow-up. For each study and aggregate data the random effect odds ratio (OR) with 95%CIs was estimated and presented as Forest plots. An OR with a lower 95th confidence interval > 1 was considered consistent with a failure being associated to a given gene mutation.Results92 studies were eligible among the selection from computerized search, with information on pfcrt (25/159 studies), pfmdr1 (29/236 studies), dhfr (18/373 studies), dhps (20/195 studies). The risk of therapeutic failure after chloroquine was increased by the presence of pfcrt K76T (Day 28, OR = 7.2 [95%CI: 4.5–11.5]), pfmdr1 N86Y was associated with both chloroquine (Day 28, OR = 1.8 [95%CI: 1.3–2.4]) and amodiaquine failures (OR = 5.4 [95%CI: 2.6–11.3, p < 0.001]). For sulphadoxine-pyrimethamine the dhfr single (S108N) (Day 28, OR = 3.5 [95%CI: 1.9–6.3]) and triple mutants (S108N, N51I, C59R) (Day 28, OR = 3.1 [95%CI: 2.0–4.9]) and dhfr-dhps quintuple mutants (Day 28, OR = 5.2 [95%CI: 3.2–8.8]) also increased the risk of treatment failure. Increased pfmdr1 copy number was correlated with treatment failure following mefloquine (OR = 8.6 [95%CI: 3.3–22.9]).ConclusionWhen applying the selection procedure for comparative analysis, few studies fulfilled all inclusion criteria compared to the large number of papers identified, but heterogeneity was limited. Genetic molecular markers were related to an increased risk of therapeutic failure. Guidelines are discussed and a checklist for further studies is proposed.

Contribution of the (1→3)-β-d-Glucan Assay for Diagnosis of Invasive Fungal Infections

ABSTRACT Diagnosis of invasive fungal infection (IFI) remains a challenge. A retrospective study was performed on 279 patients at three French university hospitals to evaluate the performance of the (1→3)-β-d-glucan assay (BG assay; Fungitell; Associates of Cape Cod, Inc.) for the diagnosis of IFI. The results of one serum per subject were analyzed for 117 patients who had probable or proven IFI according to the European Organization for Research and Treatment of Cancer criteria (70 invasive pulmonary aspergilloses [IPA], 27 fungal bloodstream infections, and 20 Pneumocystis jiroveci pneumonias), 40 blood donors, and 122 patients who were hospitalized in hematology wards or intensive care units and were at risk for IFI but in whom IFI had not been diagnosed. For the overall IFI diagnosis, the BG assay had 77.8% sensitivity and specificities of 92.5 and 70.5% for blood donors and patients at risk, respectively. The assay was positive in 48 patients with IPA (68%), in 23 with bloodstream infections (85.2%), and in all who had P. jiroveci pneumonias (100%), and the false-positive rate varied depending on the controls used. It allowed a higher rate of detection among IPA patients compared to the galactomannan enzyme-linked immunosorbent assay (ELISA) (48 versus 39 patients, respectively) and among candidemia patients compared to the mannan ELISA (20 versus 11 patients, respectively). This assay therefore appears to be useful in the diagnosis of IFI, particularly for serum analysis of pneumocystosis pneumonia patients, but further studies are needed to evaluate false-positive rates and its future role in IFI diagnosis.

Apoptosis related to chloroquine sensitivity of the human malaria parasite Plasmodium falciparum.

As chemoresistance of Plasmodium falciparum to chloroquine has arisen, new ways of combating the infection are needed. Similarities exist between the multidrug resistance of mammalian cells and chloroquine resistance of P. falciparum, based on the occurrence of internucleosomal deoxyribonucleic acid (DNA) breakdown and the ability of some anticancer drugs and chloroquine to induce apoptosis. Using chloroquine, oligonucleosomal DNA fragmentation was observed with a sensitive strain of P. falciparum, but not with a resistant one. This suggests that apoptosis may be involved in the action of chloroquine on the parasite.

Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

ABSTRACT In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC50s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC50 values when phenol red was included in the medium. The IC50s and the IC90s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC50 and IC90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.

Plasmodium vivax Resistance to Chloroquine in Madagascar: Clinical Efficacy and Polymorphisms in pvmdr1 and pvcrt-o Genes

ABSTRACT No data were available concerning Plasmodium vivax resistance to chloroquine (CQ) in Madagascar. We investigated the therapeutic efficacy of CQ in P. vivax malaria, the prevalence of mutations in the pvcrt-o and pvmdr1 genes before treatment, and the association between mutant parasites and the clinical response of the patients to CQ treatment. Clinical isolates were collected at six sentinel sites located in the three epidemiological strata for malaria throughout Madagascar in 2006. Patients were enrolled, treated, and followed up according to the WHO 2001 guidelines for P. vivax infections. Sequencing was used to analyze polymorphisms of the pvcrt-o (exons 1 to 6) and pvmdr1 genes. The treatment failure rate, after adjustment for genotyping, was estimated at 5.1% for the 105 patients included, ranging from zero in the South to 14.8% in the foothills of the Central Highlands. All samples were wild type for pvcrt-o but mutant for the pvmdr1 gene. Ten nonsynonymous mutations were found in the pvmdr1 gene, including five new mutations, four of which were present at low frequencies (1.3% to 7.5%) while the S513R mutation was present at a much higher frequency (96.3%). The other five mutations, including Y976F, had been described before and had frequencies of 97.8% to 100%. Our findings suggest that CQ-resistant P. vivax isolates are present in Madagascar, particularly in the foothills of the Central Highlands. The 976Y pvmdr1 mutation was found not to be useful for monitoring CQ resistance. Further efforts are required to develop suitable tools for monitoring drug resistance in P. vivax malaria.

Recombinant Human Erythropoietin Prevents the Death of Mice during Cerebral Malaria

Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction

The incidence of imported malaria cases in travellers returning from endemic areas has considerably increased over the last few years. The microscopical examination of stained blood films is the gold standard method to confirm clinical suspicion of malaria but diagnosis is difficult in the case of mixed infections, low-grade parasitaemia, or forms altered by uncompleted treatment. We have developed a real-time polymerase chain reaction (PCR) for the simultaneous identification of the 4 human Plasmodium spp. and quantification of Plasmodium DNA in human blood. The rapid turnaround and reduction in the risk of PCR product carryover are major advantages compared with conventional PCR. In combination with conventional tests, this method could be a powerful tool for the diagnosis of malaria infections among travellers from endemic areas and during the follow-up of patients in reference centres involved in travel and tropical medicine. Quantitative real-time PCR could also be used for the follow-up of patients during drug resistance studies managed by national malaria programmes, the testing of new drugs, and vaccine trials.

Identification of the Plasmodium vivax mdr-Like Gene (pvmdr1) and Analysis of Single-Nucleotide Polymorphisms among Isolates from Different Areas of Endemicity

Because of the lack of methods for continuous in vitro culture of Plasmodium vivax, little is known about drug-resistance mechanisms in this malaria-causing parasite. Therefore, identification of all the genes potentially involved in drug resistance and of molecular markers related to drug resistance would provide a framework for studying the incidence and spread of drug-resistant P. vivax strains. We have identified the P. vivax orthologue of the pfmdr1 gene (pvmdr1), which was shown to have a role in the drug resistance of Plasmodium falciparum. Comparison of the alignments of both nucleotide and amino acid sequences of pvmdr1 with those of other Plasmodium multidrug-resistance genes revealed an open-reading frame of 4392 base pairs encoding a deduced protein of 1464 amino acids. Nucleotide polymorphisms at 2 codons of the pvmdr1 gene--Y976F and F1076L--were found in 14 of 23 P. vivax isolates from different areas of endemicity, including Thailand, Indonesia, Turkey, Azerbaijan, and French Guyana.

Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.

Reduction of Invasive Aspergillosis Incidence among Immunocompromised Patients after Control of Environmental Exposure

BACKGROUND The objective of the study was to assess the impact of the relocation of an adult hematological intensive care unit on invasive aspergillosis (IA) incidence. METHODS A quasi-experimental study, including a control group and an intervention group that both underwent pretest and posttest evaluations, was conducted in the 3 adult hematological intensive care units (each composed of 14 single rooms) in a university hospital from 14 April 2005 through 1 February 2006. One of these units was relocated from the main building to an adjoining modular construction. In this unit, 4 rooms were equipped with laminar airflow before relocation; all rooms were equipped with positive pressure isolation after relocation. The 2 other units (control group), each containing 8 rooms with laminar airflow, did not undergo environmental modification. The diagnostic criteria for IA were based on the criteria of the European Organization for Research and Treatment of Cancer. RESULTS In total, 356 hospitalized patients were included. Of the 21 cases of IA, 18 were nosocomial, and 3 were of undetermined origin. In the relocated unit, the incidence of IA decreased from 13.2% (9 patients) before relocation to 1.6% (1 patient) after relocation (P=.018). Eight of the 9 patients with IA before relocation stayed in rooms without specific air treatment. The rate of IA did not change in the control group. Patient characteristics were similar in each unit before and after relocation. CONCLUSION We detected a straightforward association between environmental modification and decreased IA incidence, which emphasizes the use of an environmental strategy, including high-efficiency air filtration, in the prevention of IA.