Linking Pneumocystis jiroveci sulfamethoxazole resistance to the alleles of the DHPS gene using functional complementation in Saccharomyces cerevisiae.

Curative and prophylactic therapy for Pneumocystis jiroveci pneumonia relies mainly on cotrimoxazole, an association of trimethoprim and sulfamethoxazole (SMX). SMX inhibits the folic acid pathway through competition with para-aminobenzoic acid (pABA), one of the two substrates of the dihydropteroate synthase (DHPS), a key enzyme in de novo folic acid synthesis. The most frequent non-synonymous single nucleotide polymorphisms (SNPs) in P. jiroveci DHPS are seen at positions 165 and 171, the combination leading to four possible different genetic alleles. A number of reports correlate prophylaxis failure and mutation in the P. jiroveci DHPS but, because of the impossibility of reliably cultivating P. jiroveci, the link between DHPS mutation(s) and SMX susceptibility is not definitively proven. To circumvent this limitation, the yeast Saccharomyces cerevisiae was used as a model. The introduction of the P. jiroveci DHPS gene, with or without point mutations, directly amplified from a clinical specimen and cloned in a centromeric plasmid into a DHPS-deleted yeast strain, allowed a fully effective complementation. However, in the presence of SMX at concentrations >250 mg/L, yeasts complemented with the double mutated allele showed a lower susceptibility compared with strains complemented with either a single mutated allele or wild-type alleles. These results confirm the need for prospective study of pneumocystosis, including systematic determination of the DHPS genotype, to clarify further the impact of mutations on clinical outcome. Additionally, the S. cerevisiae model proves to be useful for the study of still uninvestigated biological properties of P. jiroveci.

Molecular identification of Cryptococcus neoformans serotypes.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections primarily in immunocompromised hosts. Based on the genetic characteristics and serologic properties of capsular polysaccharides, three varieties and five serotypes have been defined: C. neoformans var. neoformans (serotype D), C. neoformans var. grubii (serotype A), hybrid serotype AD, and C. neoformans var. gattii (serotypes B and C). Epidemiologic features, such as geographic distribution and ecologic niche, and clinical characteristics have been shown to be associated with serotypes. At the present time, serotyping is based on agglutination tests with either commercial or "homemade" antisera or on immunofluorescence assays using a monoclonal antibody directed against the capsule polysaccharide. In this paper, we describe two molecular methods (PCR-restriction enzyme analysis and length polymorphism analysis) for C. neoformans serotype identification. Both are based on the sequence characteristics of a fragment of the CAP59 gene required for capsule biosynthesis. Testing of 72 C. neoformans strains including representatives of the five serotypes demonstrated the reliability of these methods.

Pneumocystis jirovecii dihydropteroate synthase genotypes in French patients with pneumocystosis: a 1998-2001 prospective study.

Dihydropteroate synthase gene (DHPS) mutations at codons 55 and 57 have been associated with sulfa/sulfone resistance in Pneumocystis jirovecii strains from patients who previously received prophylaxis. To evaluate the prevalence of these mutations, a portion of P. jirovecii DHPS gene was analysed using PCR combined with restriction fragment length polymorphism (RFLP) analysis in 92 bronchoalveolar fluid samples collected between January 1998 and September 2001 from French patients with pulmonary pneumocystosis (PCP). Seventy-six samples contained the wild-type DHPS genotype (82.6%) and 16 contained a mutant genotype (17.4%). Twelve out of the 16 isolates with a mutant DHPS genotype corresponded to patients who had never received sulfa or sulfone prophylaxis, suggesting that DHPS mutants may be acquired de novo. There was no significant difference in favourable or adverse outcome in PCP caused by the wild or mutant DHPS genotypes (P = 0.34).

Development of an RT-PCR on the heat shock protein 70 gene for viability detection of Pneumocystis carinii f. sp. hominis in patients with pneumocystosis and in air sample.

To test the viability of Pneumocystis carinii f. sp. hominis, an RT-PCR assay that employs specific primers from the Heat Shock Protein 70 gene was developed. Using this method, the viability of P.c. hominis in bronchoalveolar lavage fluids from patients developing PCP and in the environment of PCP patients was established.

[Experimental Schistosomiases. I. Study of S. mansoni fecondity as regards to its adaptation to different Biomphalaria glabrata strains (author's transl)]. / Schistosomose expérimentale. I. Etude de la fécondité de Schistosoma mansoni en fonction de son adaptation a la souche de Biomphalaria glabrata

We compared the infestation of different strains of B. glabrata from Brasil (Recife). Guadeloupe, Martinique and Porto-Rico with 6 to 8 miracidia of S. mansoni (from Recife. We noted the four following points: 1. The planorbid snails from Martinique and Guadeloupe had a low resistance to infestation. 2. The guadeloupean snails showed the lesser rate of positivity and the lower medium amount of emitted cercaries but, in the four strains of snails, the level of the issued cercaries is quite the same. 3. many planorbid snails in the groups studied during more than 2 months, showed significant periodic variations in the emission of cercaries, We thought that those variations might be caused by the alternate maturations of sporocysts born from the same miracidium or from different miracidia. 4. Infestation by S. mansoni had a strong effect on fecondity of the snails but the laid eggs had a normal development.