François Peyron

Estimation of the avidity of immunoglobulin G for routine diagnosis of chronicToxoplasma gondii infection in pregnant women

Present serological methods differentiate poorly between acute and chronic toxoplasmosis in pregnant women, particularly when immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies toToxoplasma gondii are present simultaneously. In the present study, a simple test for discriminating between high-avidity antibodies, which are usually present in chronic infections, and low-avidity antibodies, typical of acute infection, was evaluated. Sera were evaluated forToxoplasma gondii antibodies using a commercial enzyme immunoassay, but a duplicate well was washed in 6M urea to disrupt lowavidity complexes. Results are expressed as the percentage of antibodies resisting elution by urea. Equivocal sera (n=493) containing both IgG and IgMToxoplasma gondii antibodies from 309 pregnant women whose status as chronically or acutely infected had been independently determined using standard methods were evaluated for antibody avidity. A value of >35% elution-resistant antibodies was always associated with chronic infection and could absolutely exclude a recent (<3 months) infectious incident. Values of <35% require repeat testing four weeks later to confirm the patients status, since a proportion of individuals with chronic toxoplasmosis maintain low-avidity antibodies over long periods. This inexpensive, simple method can provide reassurance to clearly chronically infected individuals and avoids the need for repeated testing in these cases.

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.

Comparative diagnostic performance of two commercial rapid tests for malaria in a non-endemic area

In the study reported here, the diagnostic performance of two new rapid tests for the diagnosis of malaria was evaluated in symptomatic patients in a non-endemic area. Of 557 consecutive patients, 109 (19.6%) had documented malaria. For the NOW ICT MALARIA P.f./P.v. (Binax, Portland, ME, USA) and OptiMAL IT (Diamed, Cressier, Switzerland) tests, respectively, sensitivity values were 96.3% and 79.8% (P-value, 0.0001), and specificity values were 98.8% and 98.4%. The NOW ICT test did not detect two of 80 Plasmodium falciparum infections, and it generated false-positive results for five patients. The OptiMAL IT test failed to detect ten of the P. falciparum infections, and it generated seven false-positive results. The results suggest that these rapid diagnostic tests for malaria may be useful, but they cannot replace microscopic examination of blood films.

Diagnosis of Congenital Toxoplasmosis by Using a Whole-Blood Gamma Interferon Release Assay

ABSTRACT Congenital toxoplasmosis in newborns is generally subclinical, but infected infants are at risk of developing ocular lesions. Diagnosis at birth relies mainly on serological tests. Cell-mediated immunity plays the major role in resistance to infection but is not routinely investigated for diagnostic purposes. Here, we describe a simple test based on the gamma interferon (IFN-γ) response after stimulation of whole blood by crude parasitic antigens. One milliliter of heparinized blood was centrifuged; plasma was kept for routine serological tests, and pellets were resuspended in culture medium. After 24 h of culture in the presence of crude Toxoplasma gondii antigen, the cells were centrifuged and the supernatant was assayed for IFN-γ. For 62 infants under 1 year of age born to mothers who were infected during pregnancy, the sensitivity and specificity of the test were 94% (with positive results for 16 of 17 infected infants) and 98% (with negative results for 44 of 45 uninfected infants), respectively. The false-negative result was for a treated baby who gave positive results after the withdrawal of treatment. The false positive was obtained for a 3-month-old baby. For a cohort of 124 congenitally infected patients between 1 and 30 years of age, the sensitivity of the assay was 100%. We present a simple test based on IFN-γ secretion to assess cell-mediated immunity in toxoplasmosis. As only 1 ml of blood is required to investigate humoral and cellular immunity, our assay is well adapted for the study of congenital toxoplasmosis in infants. Using purified antigens or recombinant peptides may improve the test performance.

Plasma levels of tumor necrosis factor during a longitudinal survey in an endemic area of malaria

The plasma levels of tumor necrosis factor were measured during a longitudinal survey of 84 subjects living in an endemic area of malaria. In most cases, the plasma tumor necrosis factor was found at its highest level during the malaria transmission peak and became normal again during the dry season. Children having suffered from malarial attack keep low tumor necrosis factor levels compared to adults and asymptomatic children. These results suggest that tumor necrosis factor could be associated with the development of resistance against malaria.

Cellular Immunity to Toxoplasma gondii in Congenitally Infected Newborns and Immunocompetent Infected Hosts

The aim of this study was to determine the frequency of anergy to Toxoplasma gondii in congenitally infected newborns and immunocompetent infected individuals. Specific anergy to Toxoplasma has been reported previously, especially in cases of congenital toxoplasmosis. Whole blood from 592 immunocompetent patients with suspected toxoplasmosis was cultured in the presence of soluble Toxoplasma antigen for 7 days. Activated T lymphocytes were detected by flow cytometry. In patients over 1 year of age, the percentage of soluble Toxoplasma antigen-stimulated T cells expressing the interleukin-2 receptor CD25 was higher in infected patients than in uninfected subjects (40.0±18.3% vs. 1.8±2.0%, P<0.0001). No differences were detected between seroconverters, i.e. those with recent rises in IgM and IgG antibodies, and those with acquired or congenital toxoplasmosis. Similar results were observed when congenitally infected (n=38) and uninfected (n=89) children under 1 year of age were compared (17.6±9.2% vs. 3.0±4.9%, P<0.0001). The sensitivity and specificity values of CD25 detection for diagnosis of congenital toxoplasmosis in infants were 95% and 89%, respectively, at a threshold value of 7% above control culture. The results show that specific cellular immunity is detectable in virtually all Toxoplasma-infected patients, including newborns. Detection of CD25 constitutes a simple, sensitive and specific test for diagnosis of congenital toxoplasmosis.

Imported cutaneous leishmaniasis in a short-term traveler returning from Central Mali – The role of PCR

Leishmaniasis is a parasitic infection caused by the obligate intracellular protazoa leishmania. The most commonly encountered form is cutaneous leishmaniasis (CL), which generally manifests as a chronic, painless ulcer. Recent increases in the incidence of CL worldwide due in large part to increased immigration and international travel, combined often with the lack of familiarity with the disease in non-endemic settings, pose the continued problems of delayed diagnosis and inappropriate treatment. A case is described of imported cutaneous leishmaniasis occurring in a 48 year-old male who presented with multiple painless, progressively ulcerating lesions after returning from a one week trip to Bandiagara, Mali, West Africa. After four months of misdiagnoses and ineffective treatments, he was referred to a tropical disease specialist where the diagnosis was made with a skin biopsy followed by a tissue impression smear, culture and PCR. Appropriate treatment was initiated and the lesions resolved with minimal scarring. The goals of this case report are threefold: first, to stress the importance of associating chronic ulcers in a traveler with potential cutaneous leishmaniasis; second, to emphasize the clinical utility of PCR for the diagnosis; and third, to discuss the clinical approach to treatment.

Flow cytometric application of the Sabin and Feldman dye test in the diagnosis of toxoplasmosis.

Although time-consuming and requiring live parasites, the Sabin and Feldman dye test (DT) is still considered the gold standard among the serological tests for toxoplasmosis diagnosis. The present study was initiated to compare detection of dead parasites using optical microscopy with flow cytometry and a fluorescent nonvital dye, propidium iodide. After incubation with sera (N = 150) and a complement source, tachyzoites were washed, then stained using a fluorescein-conjugated Toxoplasma-specific antiserum. Dead tachyzoites were detected by flow cytometry after addition of propidium iodide. Intra- and inter-assay reproducibilities of percentages of dead parasites varied between 7 and 14%, and 8 and 21%, respectively. When comparing flow cytometry with the classical DT, no discrepancy was noted for positive (N = 118) and negative sera (N = 32). Correlation was good (r = 0.85) for positive sera. In conclusion, when easily available, flow cytometry is a very sensitive, specific and time-sparing method to detect specific antibodies to Toxoplasma gondii.

Mefloquine adverse effects with atypical facial lesions in an overweight patient

BACKGROUNDnThe recommended dosage of mefloquine to treat Plasmodium falciparum infection is 25xa0mg/kg, with no recommendation for dosage exceeding 1500xa0mg. We describe an original case of adverse reaction to mefloquine in an overweight patient.nnnMETHODnCase report.nnnRESULTSnA 32-year-old woman weighing 139xa0kg presented with uncomplicated P. falciparum infection after returning from Cameroon. She received 3250xa0mg of mefloquine (i.e. 23xa0mg/kg) administered in four doses. On day 2, she developed neuropsychiatric disorders and facial lesions. Nasal mucocutaneous vesicles and bullae, depressive mood, mild thrombocytopenia and hepatic cytolysis were evidenced. Parasitemia was negative. Recovery was complete on day 17. High mefloquine serum levels were measured (8.030xa0mg/L on day 3, 6.880xa0mg/L on day 8, and 3.370xa0mg/L on day 17).nnnCONCLUSIONSnThe causal relationship between mefloquine and the occurrence of these adverse effects is probable. However, as no viral or bacteriological investigations were performed, the drug responsibility remains uncertain. Mefloquine-induced bullous and facial lesions reversible upon drug withdrawal have already been described. The associated neuropsychiatric symptoms were strongly suggestive of mefloquine adverse effects, as such events are more frequently observed in cases of overdosage. Our case emphasizes the difficulties of dosage adaptation in overweight patients.

Détection des marqueurs moléculaires de la résistance de Plasmodium falciparum par PCR en temps réel

Resume Le paludisme est une infection severe qui touche les personnes residant en zone d’endemie palustre et les voyageurs. Son impact medical est du a l’augmentation du reservoir vectoriel et du reservoir humain, aux difficultes des programmes de lutte et a l’existence de resistance aux antipaludiques. La resistance a la chloroquine, medicament utilise en premiere intention dans le traitement des acces palustres simples en zone d’endemie, est liee a la presence de mutations ponctuelles sur les genes pfcrt (plasmodium falciparum chloroquino resistance transporter) et pfmdr1 (plasmodium falciparum multidrug resistance 1). La disparite des populations (patients residant en zone d’endemie ou voyageurs non immuns se deplacant en zone endemique), des zones geographiques etudiees et de methodes employees pour detecter ces mutations rend difficile l’analyse des resultats obtenus. Le but de notre travail etait de developper une methode alternative permettant de detecter cinqxa0mutations ponctuelles sur le gene pfmdr1 et troisxa0mutations ponctuelles sur le gene pfcrt.