Search

Several studies from developed countries have documented the association between trimethoprim-sulfamethoxazole prophylaxis failure and mutations in the Pneumocystis jirovecii gene coding for dihydropteroate synthase (DHPS). DNA was extracted from Giemsa-stained smears of 70 patients with P. jirovecii pneumonia seen in Porto Alegre, Brazil, from 1997 to 2004. Successful PCR amplification of the DHPS locus was obtained in 57 of 70 cases (81.4%), including five cases (8.7%) that had used sulfa prophylaxis. No DHPS gene mutations were seen. These results suggest that DHPS mutations are currently as rare in Brazil as in other developing countries.

Pneumocystis, an AIDS-associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several delta(7) 24-alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24-alkylsterols are synthesized by the action of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C-24 position of the sterol side chain and the cells accumulate C28 24-alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C28 and C29 24-alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C-5 of the sterol nucleus and C-22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C30 lanosterol differs from that of homologous enzymes in any other organisms studied. C31 24-Methylenelanosterol and C32 pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human-derived Pneumocystis jiroveci populations.

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.

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.

In recent years the clinical face of the Acquired Immune Deficiency Syndrome has changed significantly as a consequence of use of prophylaxis against Pneumocystis carinii pneumonia and combination antiretroviral therapy. In this context several opportunistic pathogens have emerged as causes of clinically important disease. Many of these infective agents have previously been defined by specific geographical locations. Their clinical presentation frequently mimics other (non) opportunistic infections with which they may co-exist. The diagnosis is frequently delayed as the diagnostic possibility may not be in the clinician's differential diagnosis. Invasive procedures are frequently required in order to secure a diagnosis. Despite treatment, prognosis is often poor. Clinicians should be aware of these opportunistic pathogens in order that a timely diagnosis may be made and appropriate therapy given.

Most species of Penicillium are considered relatively benign with respect to causing human disease. However, one species, P. marneffei, has emerged as a significant pathogen particularly among individuals who live in Southeast Asia and are concurrently infected with the human immunodeficiency virus. While environmental and epidemiological studies have yet to resolve the reason for the heightened virulence of P. marneffi, one characteristic does distinguish this fungus from other Penicillium species. Whereas the latter grow as monomorphic moulds bearing typical asexual propagules (conidia), P. marneffei is thermally dimorphic. At room temperature, P. marneffei exhibits the morphology characteristic of the genus. In contrast to other Penicillia, though, P. marneffei grows as a yeast-like entity (arthroconidium) when found in diseased tissue or cultivated at 37 degrees C. Studies in our laboratory have focused on the differential gene expression between the mould and arthroconidial phases. Many of the genes whose expression differs during mould-to-arthrocondium transition are related to energy metabolism. A better understanding of gene expression during morphogenesis in P. marneffei may help detect unique target sites or cellular processes that can be exploited in the development of antifungal agents or immunomodulation therapies.

To understand the way of reinfection of Pneumocystis carinii we have analyzed the genetic variation at the internal transcribed spacer (ITS) in DNA samples from bronchoalveolar lavage fluid of Italian HIV patients who had multiple episodes of P.carinii pneumonia. The presence of the same and/or a new type in both episodes suggest the possible occurrence of both reactivation of a previously acquired infection and reinfection from an exogenous source. Furthermore the occurrence of two different types in the same episode indicate that a mixed infection is common.

Oropharyngeal washings (Ophs) from 27 HIV infected patients (18 with P. carinii pneumonia, PCP, and 9 without PCP) were examined for P. carinii using morphological staining and DNA amplification with PCR-SHELA and nested PCR methods. The comparison of these techniques shows that 1. the amplification of P. carinii DNA is more sensitive than (and as specific as) morphological staining; 2. PCR-SHELA is less sensitive than (and as specific as) nested PCR.

P.carinii molecular epidemiology appears a new interesting investigational field to understand distribution and incidence of isolates from different geographical locations. Recently a typing system, the Type Specific Oligoblotting (TSO) based on 6 different sequences of the Internal Transcribed Spacers (ITSs) of P.carinii rRNA has been developed [1]. By using P.carinii ITSs nested PCR followed by TSO hybridization we have typed 55 lung derived specimens collected in Italy, The Netherlands and sub-Saharian Africa from pts with microscopically detected P.carinii pneumonia.

Pneumocystis carinii pneumonia (PCP) is usually diagnosed by examination of BAL, a sample often unpleasant to be collected from immunocompromised host affected by acute respiratory disease. We studied by the Internal Transcribed Spacers (ITSs) nested PCR the presence of P.carinii DNA in serum and Peripheral Blood Mononuclear Cells (PBMC) during acute episodes of PCP to test blood as a possible noninvasive diagnostic tool.

The DNA sequences of a portion of the 5-enolpyruvyl shikimate phosphate synthase domain of the arom gene, encoding the pentafunctional AROM protein, were determined from isolates of Pneumocystis carinii from five mammalian host species (rat, human, ferret, rabbit and mouse). High levels of genetic divergence were found among P. carinii derived from different host species, 7-22% at the DNA sequence level, and 7-26% at the derived amino acid sequence level. Two separate and distinct sequences were isolated from infected ferret lungs. Low levels of divergence were seen in human-derived organisms.