Cytochrome b gene quantitative PCR for diagnosing Plasmodium falciparum infection in travelers.

A cytochrome b (cytb) gene quantitative PCR (qPCR) assay was developed to diagnose malaria in travelers. First, manual and automated DNA extractions were compared and automated DNA extraction of 400 µl of blood was found to be more efficient. Sensitivity was estimated using the WHO international standard for Plasmodium falciparum DNA and compared to that of a previously published qPCR targeting the 18S rRNA coding gene (18S qPCR). The limit of detection of the cytb qPCR assay was 20 DNA copies (i.e., 1 parasite equivalent) per 400 µl of extracted whole blood and was comparable for the two qPCR assays. Both qPCR assays were used on blood samples from 265 consecutive patients seen for suspicion of malaria. There were no microscopy-positive and qPCR-negative samples. Positive cytb qPCR results were observed for 51 samples, and all but 1 were also 18S qPCR positive. Eight (16%) of these 51 samples were negative by microscopic examination. The 8 cytb qPCR-positive and microscopy-negative samples were from African patients, 3 of whom had received antimalarial drugs. Three non-P. falciparum infections were correctly identified using an additional qPCR assay. The absence of PCR inhibitors was tested for by the use of an internal control of mouse DNA to allow reliable quantification of circulating DNA. The high analytical sensitivity of both qPCR assays combined with automated DNA extraction supports its use as a laboratory tool for diagnosis and parasitemia determination in emergencies. Whether to treat qPCR-positive and microscopy-negative patients remains to be determined.

Low prevalence of resistance to azoles in Aspergillus fumigatus in a French cohort of patients treated for haematological malignancies.

OBJECTIVES: An increase in invasive aspergillosis (IA) due to azole-resistant Aspergillus fumigatus isolates has been reported for 10 years. Our study aimed to estimate the prevalence of azole resistance in isolates prospectively collected in patients with haematological diseases. METHODS: One hundred and eighteen isolates were collected from 89 consecutive patients over 4 years. Fifty-one patients had proven or probable IA. Species identification was ascertained based on ß-tubulin gene sequencing. The MICs of azole drugs were determined using Etest(®), and the cyp51A gene and its promoter were sequenced to detect mutations. RESULTS: All isolates were identified as A. fumigatus and all of them but one had itraconazole and voriconazole MICs of ≤ 2 mg/L and posaconazole MICs of ≤ 0.25 mg/L. An isolate for which the itraconazole MIC was high (itraconazole MIC = 16 mg/L; voriconazole MIC = 0.38 mg/L; and posaconazole MIC = 0.25 mg/L) was recovered from a patient naive to azole treatment and had a new G432S substitution. To establish whether this mutation existed in other isolates, the 1426-2025 bp cyp51A locus was sequenced for all. G432S was not found. CONCLUSIONS: In A. fumigatus, the prevalence of azole resistance is currently low in the haematological population in the Paris area. Surveillance programmes for azole resistance to adapt antifungal treatments are warranted for clinical isolates of A. fumigatus.

Primary in vitro culture of porcine tracheal epithelial cells in an air-liquid interface as a model to study airway epithelium and Aspergillus fumigatus interactions.

Since the airway epithelium is the first tissue encountered by airborne fungal spores, specific models are needed to study this interaction. We developed such a model using primary porcine tracheal epithelial cells (PTEC) as a possible alternative to the use of primary human cells. PTEC were obtained from pigs and were cultivated in an air-liquid interface. Fluorescent brightener was employed to quantify the internalization of Aspergillus fumigatus conidia. Potential differences (Vt) and transepithelial resistances (Rt) after challenge with the mycotoxin, verruculogen, were studied. Primers for porcine inflammatory mediator genes IL-8, TNF-alpha, and GM-CSF were designed for a quantitative real-time PCR procedure to study cellular responses to challenges with A. fumigatus conidia. TEM showed the differentiation of ciliated cells and the PTEC ability to internalize conidia. The internalization rate was 21.9 ± 1.4% after 8 h of incubation. Verruculogen (10(-6) M) significantly increased Vt without having an effect on the Rt. Exposure of PTEC to live A. fumigatus conidia for 24 h induced a 10- to 40-fold increase in the mRNA levels of inflammatory mediator genes. PTEC behave similarly to human cells and are therefore a suitable alternative to human cells for studying interaction between airway epithelium and A. fumigatus.

Aerosolized liposomal amphotericin B: prediction of lung deposition, in vitro uptake and cytotoxicity.

To predict the efficacy and toxicity of pulmonary administration of liposomal amphotericin B (L-AMB) for the treatment or the prevention of pulmonary invasive aspergillosis, a multistage liquid impinger was used to estimate the concentrations of drug that could be attained in different lung compartments after nebulization. The highest concentration of amphotericin B was found in the alveolar compartment, where it was calculated that the concentration in the lung surfactant could reach 54 µM or more when 21.6 µmoles of drug as liposomes was nebulized. The uptake and toxicity of L-AMB were studied in vitro using the A549 human lung epithelial cell line. Uptake was time and concentration-dependent and reached intracellular concentrations exceeding the minimal inhibitory concentrations for most Aspergillus species. The toxicity of L-AMB toward these cells, estimated by the MTT reduction assay, was reduced compared with the conventional form, deoxycholate amphotericin B (D-AMB), with an IC(50) value of about 120 µM after 24 h of exposure for D-AMB, but only a 13% reduction in viability for 200 µM L-AMB at 24 h. These results indicate that aerosol therapy with nebulized L-AMB could be efficient but that doses need to be carefully controlled to avoid toxicity.

Genotyping of Candida albicans using length fragment and high-resolution melting analyses together with minisequencing of a polymorphic microsatellite locus.

Microsatellite length polymorphism (MLP) typing is a PCR-based method used for genotyping of the diploid yeast Candida albicans. However, MLP is subject to homoplasia which can hamper the accuracy of the results. We combined fragment length analysis, high-resolution DNA melting (HRM) analysis, and SNaPshot minisequencing after a single amplification of the CDC3 locus to study 95 epidemiologically independent C. albicans isolates. HRM analysis for a given electrophoretic group led to a maximum of three different curves due to the presence of a SNP upstream of the tandem repeat which could be characterized using the SNaPshot assay. The combination of the three methods had a discriminatory index of 0.88 in complete congruence with previous MLP typing (Mantel test R=0.99, P<10(-)(4)). HRM is a useful tool of adding resolving power to MLP genotyping in identifying SNPs.