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.

[Cardiac arrhythmia observed in 400 workers without obvious heart disease by Holter monitoring]. / Troubles du rythme cardiaque observés chez 400 salariés sans cardiopathie évidente par l'enregistrement Holter.

To obtain information on the cardiac rhythm characteristics of subjects without heart disease during their daily work, we examined the continuous 24-hour electrocardiographic recordings of 400 workers from 4 factories in Eastern France presenting with criteria of cardiac normality. Sex, age and socio-professional category were taken into account. Supraventricular extrasystoles were extremely common, being observed in 52 p. 100 of men and 39 p. 100 of women. In men, the frequency of these extrasystoles increased with age (p less than 0.001), and their number was less than 20 per 24 hours in 90 p. 100 of the cases. Ventricular extrasystoles were detected in 40 p. 100 of men and 32 p. 100 of women. They too were age-related, though not significantly. Their number was less than 10 per 24 hours in 68 p. 100 of the cases and 10 to 50 per 24 hours in 28 p. 100. These ventricular extrasystoles usually were monomorphous, regularly coupled (92 p. 100) and isolated. However, 2 attacks of tachycardia were discovered. Episodes of bradyarrhythmia (RR superior or equal to 1500 ms) were present in 25 p. 100 of men and 19 p. 100 of women. They occurred more frequently before the age of 35 than later (p less than 0.001) and the recordings confirmed that they were predominantly nocturnal. Recordings without "disorders of rhythm" were relatively rare (20 p. 100 of men, 28.5 p. 100 of women). Finally, there was no clear-cut correlation between the prevalence or characteristics of these various "rhythmic abnormalities" and the type of professional activity.

[Continuous electrocardiographic registration in occupational medicine]. / Enregistrement électrocardiographique continu en médecine du travail.

Continuous 24 hour electrocardiography (Holter method) was carried out during work time in 64 workers. They were divided into two groups: the first group comprised 34 subjects with either organic heart disease (coronary artery disease, valvular heart disease, operated coarctation, hypertrophic cardiomyopathy) or a documented arrhythmia without proven underlying cardiac disease; the second group comprised 30 subjects without known cardiac disease but complaining of symptoms suspected to be of cardiac origin or with isolated electrocardiographic abnormalities. At the end of the study we concluded that Holter monitoring is possible in subjects performing physical occupations even in difficult conditions. The trends of heart rate, especially mean heart rate calculated over 10 minute periods, confirmed the relationship between heart rate and the intensity of the physical activity. Atrial extrasystoles and episodes of supraventricular tachycardia were as common in the first as in the second group (20% and 18% respectively). This did not apply to ventricular extrasystoles: they were observed in both groups but were significantly more common in the first group (55% compared to 33%); ventricular extrasystoles, usually of a single configuration and isolated, were more common in the first group, especially amongst the coronary patients. Sinus node dysfunction was only observed in the second group (12.5%), in young subjects, and this occurred with only one exception at night. These findings support previous reports in the literature. However, the interpretation of these results is difficult because of the absence of well established normal values.