Françoise Faraut

Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity

ABSTRACT A real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to reach a sensitivity of 0.0125 parasites/ml of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the Leishmania DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different L. infantum and Leishmania donovani strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for Leishmania quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of 18 to 42, 0.7 to 42, and 0.12 to 22.5 parasites/ml for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to 188,700 parasites/ml, with a median of 837 parasites/ml), while in bone marrow, parasite load was more than 100 parasites per 106 nucleated human cells. After successful therapy, parasitemia levels remain lower than 1 parasite/ml. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of Leishmania DNA in the blood of 21% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.

Mucosal Leishmania infantum leishmaniasis: Specific pattern in a multicentre survey and historical cases

OBJECTIVE Leishmania infantum mucosally restricted leishmaniasis was rarely reported, so that diagnostic and treatment strategies remain debated. A long-term multicentric survey appeared thereby necessary. METHODS Cases were prospectively collected over 12 years in 3 academic hospitals of Southern France. Predisposing factors, clinical findings, diagnostic procedures, treatment and outcome were compared to medical literature. RESULTS Ten new cases and 40 historical reports were collected. Respectively 10/10 and 35/40 patients were adult males. Immunodeficiency was frequent (5/10 and 18/40). No previous cutaneous lesion was reported. Leishmaniasis affected mostly larynx (5/10 and 19/40), but also mouth (2/10 and 19/40) and nose (3/10 and 5/40). Lesions were highly polymorph. Mucosa histological examination provided respectively 1/10 and 2/40 false negative results, contrary to serum immunoblotting and PCR on mucosal biopsy. Although local response was always satisfactory even using topical treatment, subsequent visceral spreading was observed in 2/10 and 1/40 cases. CONCLUSION L. infantum mucosally restricted leishmaniasis exhibits a specific pattern, marked by tropism for adult males, high clinical and histological polymorphism. Immunoblot screening and PCR confirmation of suspected lesions are necessary because of direct examination occasional false negative results. The risk of visceral spreading sustains systemic therapy. SUMMARY Leishmania infantum mucosal leishmaniasis mostly affects adult males, half of them immunodeficient. Clinical and histological polymorphism makes the diagnosis difficult, stressing the need for immunoblot screening and mucosa PCR analysis of suspected cases. Possible visceralization sustains systemic therapy.

Identification of Leishmania at the species level with matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.

Timely Diagnosis of Disseminated Toxoplasmosis by Sputum Examination

ABSTRACT The diagnosis of disseminated toxoplasmosis in a 14-year-old allogeneic bone marrow recipient with graft-versus-host disease was determined by the detection of Toxoplasma gondii tachyzoites in sputum smears. Sputum analysis is a valuable alternative in the clinical assessment of pulmonary toxoplasmosis, especially when conventional invasive techniques are not practicable.

Heterogeneity of environments associated with transmission of visceral leishmaniasis in South-Eastern France and implication for control strategies.

Background Visceral leishmaniasis due to Leishmania infantum is currently spreading into new foci across Europe. Leishmania infantum transmission in the Old World was reported to be strongly associated with a few specific environments. Environmental changes due to global warming or human activity were therefore incriminated in the spread of the disease. However, comprehensive studies were lacking to reliably identify all the environments at risk and thereby optimize monitoring and control strategy. Methodology/Findings We exhaustively collected 328 cases of autochthonous visceral leishmaniasis from 1993 to 2009 in South-Eastern France. Leishmaniasis incidence decreased from 31 yearly cases between 1993 and 1997 to 12 yearly cases between 2005 and 2009 mostly because Leishmania/HIV coinfection were less frequent. No spread of human visceral leishmaniasis was observed in the studied region. Two major foci were identified, associated with opposite environments: whereas one involved semi-rural hillside environments partly made of mixed forests, the other involved urban and peri-urban areas in and around the region main town, Marseille. The two neighboring foci were related to differing environments despite similar vectors (P. perniciosus), canine reservoir, parasite (L. infantum zymodeme MON-1), and human host. Conclusions/Significance This unprecedented collection of cases highlighted the occurrence of protracted urban transmission of L. infantum in France, a worrisome finding as the disease is currently spreading in other areas around the Mediterranean. These results complete previous studies about more widespread canine leishmaniasis or human asymptomatic carriage. This first application of systematic geostatistical methods to European human visceral leishmaniasis demonstrated an unsuspected heterogeneity of environments associated with the transmission of the disease. These findings modify the current view of leishmaniasis epidemiology. They notably stress the need for locally defined control strategies and extensive monitoring including in urban environments.

Heterogeneity of molecular resistance patterns in antimony-resistant field isolates of Leishmania species from the western Mediterranean area.

ABSTRACT Antimonials remain the first-line treatment for the various manifestations of leishmaniasis in most areas where the disease is endemic, and increasing cases of therapeutic failure associated with parasite resistance have been reported. In this study, we assessed the molecular status of 47 clinical isolates of Leishmania causing visceral and cutaneous leishmaniasis from Algeria, Tunisia, and southern France. In total, we examined 14 genes that have been shown to exhibit significant variations in DNA amplification, mRNA levels, or protein expression with respect to resistance to antimonials. The gene status of each clinical isolate was assessed via qPCR and qRT-PCR. We then compared the molecular pattern against the phenotype determined via an in vitro sensitivity test of the clinical isolates against meglumine antimoniate, which is considered the reference technique. Our results demonstrate significant DNA amplification and/or RNA overexpression in 56% of the clinical isolates with the resistant phenotype. All clinical isolates that exhibited significant overexpression of at least 2 genes displayed a resistant phenotype. Among the 14 genes investigated, 10 genes displayed either significant amplification or overexpression in at least 1 clinical isolate; these genes are involved in several metabolic pathways. Moreover, various gene associations were observed depending on the clinical isolates, supporting the multifactorial nature of Leishmania resistance. Molecular resistance features were found in the 3 Leishmania species investigated (Leishmania infantum, Leishmania major, and Leishmania killicki). To our knowledge, this is the first report of the involvement of molecular resistance genes in field isolates of Leishmania major and Leishmania killicki with the resistance phenotype.