Absence of dihydropteroate synthase mutations in Pneumocystis jirovecii from Brazilian AIDS patients

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.     

Multilocus Genotyping of Pneumocystis jirovecii in Patients Developing Diverse Forms of Parasitism: Implication for a Wide Human Reservoir for the Fungus

ABSTRACT. Pneumocystis jirovecii ITS and DHPS genotypes were identified in 3 patient groups developing diverse forms of P. jirovecii infections: 13 patients with Pneumocystis pneumonia, 8 patients merely colonized by the fungus, and 19 immunocompetent infants with bronchiolitis developing mild P. jirovecii infection. Common P. jirovecii genotypes were found in the 3 patient groups, suggesting that common sources of P. jirovecii were involved in the fungus acquisition, and that transmission cycles of P. jirovecii infections in these patient groups are not independent. Parasitized patients, whatever the form of parasitism they present, may he part of a common reservoir for P. jirovecii.     

Use of restriction fragment length polymorphism to detect dihydropteroate synthase gene mutations in strains of Pneumocystis jirovecii isolated in Poland

Point mutation of the dihydropteroate synthase gene causes Pneumocystis jirovecii resistance to sulfa drugs. The RFLP method was used to search for DHPS polymorphism in P. jirovecii strains isolated in Poland. Positive results of DHPS gene fragment amplification using nested PCR were achieved in 25 out of the 30 examined Pneumocystis DNA samples. Two (8%) of the 25 isolates had point mutation at codon 55 (Thr55Ala). The mutation-positive strains were obtained from HIV positive (1/15) and HIV negative (1/10) patients. The observed DHPS gene polymorphism may point to the appearance of P. jirovecii strains resistant to sulfa drugs in Poland.     

Epidemiology and clinical relevance of Pneumocystis jirovecii Frenkel, 1976 dihydropteroate synthase gene mutations

A review was conducted to examine the published works that studied the prevalence of Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations in patients with P. jirovecii pneumonia (PcP), in develop and developing countries, and that focused the problem of the possible association of these mutations with exposure to sulpha or sulphone drugs and their influence in the PcP outcome. Studies conducted in United States of America presented higher P. jirovecii mutations rates, in comparison with European countries, and in developing countries, lower rates of DHPS mutations were reported, due to limited use of sulpha drugs. A significant association was reported between the use of sulpha or sulphone agents for PcP prophylaxis in HIV-infected patients and the presence of DHPS mutations. However these mutations were also detected in PcP patients who were not currently receiving sulpha or sulphone agents. The outcome and mortality of HIV-infected patients with PcP harbouring DHPS gene mutations were related primarily to the underlying severity of illness and the initial severity of PcP, more than to the presence of mutations.     

Transmission of Pneumocystis infection in humans

Is Pneumocystis pneumonia (PcP) a transmissible fungal disease? Does nosocomial PcP occur? Is there Pneumocystis transmission in the community? These questions, which could not be tackled before the 2000s, may at present be approached using either noninvasive detection methods or experimental transmission models. Represented by a unique entity (P.?carinii) for almost one century, the Pneumocystis genus was shown to contain several species, being P.?jirovecii the sole species identified in humans hitherto. Molecular methods combined with cross infection experiments revealed strong host specificity that precludes Pneumocystis inter-species transmission. In contrast, respiratory transmission between mammals of a same species is usually highly active, even between immunocompetent hosts. Other transmission ways could also exist. New data show that human being is the unique P.?jirovecii reservoir; it would constitute the sole infection source in both hospital and community.     

Comparative genomics suggests that the fungal pathogen pneumocystis is an obligate parasite scavenging amino acids from its host's lungs

Pneumocystis jirovecii is a fungus causing severe pneumonia in immuno-compromised patients. Progress in understanding its pathogenicity and epidemiology has been hampered by the lack of a long-term in vitro culture method. Obligate parasitism of this pathogen has been suggested on the basis of various features but remains controversial. We analysed the 7.0 Mb draft genome sequence of the closely related species Pneumocystis carinii infecting rats, which is a well established experimental model of the disease. We predicted 8'085 (redundant) peptides and 14.9% of them were mapped onto the KEGG biochemical pathways. The proteome of the closely related yeast Schizosaccharomyces pombe was used as a control for the annotation procedure (4'974 genes, 14.1% mapped). About two thirds of the mapped peptides of each organism (65.7% and 73.2%, respectively) corresponded to crucial enzymes for the basal metabolism and standard cellular processes. However, the proportion of P. carinii genes relative to those of S. pombe was significantly smaller for the "amino acid metabolism" category of pathways than for all other categories taken together (40 versus 114 against 278 versus 427, P<0.002). Importantly, we identified in P. carinii only 2 enzymes specifically dedicated to the synthesis of the 20 standard amino acids. By contrast all the 54 enzymes dedicated to this synthesis reported in the KEGG atlas for S. pombe were detected upon reannotation of S. pombe proteome (2 versus 54 against 278 versus 427, P<0.0001). This finding strongly suggests that species of the genus Pneumocystis are scavenging amino acids from their host's lung environment. Consequently, they would have no form able to live independently from another organism, and these parasites would be obligate in addition to being opportunistic. These findings have implications for the management of patients susceptible to P. jirovecii infection given that the only source of infection would be other humans.     

Outbreak-Causing Fungi: Pneumocystis jirovecii

Mycopathologia - Pneumocystis jirovecii pneumonia (PCP) is an important cause of morbidity in immunocompromised patients, with a higher mortality in non-HIV than in HIV patients. P. jirovecii is...     

Isolation of the Pneumocystis carinii dihydrofolate synthase gene and functional complementation in Saccharomyces cerevisiae

The Pneumocystis carinii gene encoding the enzyme dihydrofolate synthase (DHFS), which is involved in the essential biosynthesis of folates, was isolated from clones of the Pneumocystis genome project, and sequenced. The deduced P. carinii DHFS protein shares 38% and 35% identity with DHFS of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. P. carinii DHFS expressed from a plasmid functionally complemented a S. cerevisiae mutant with no DHFS. Comparison of available DHFSs with highly similar folylpolyglutamate synthases allowed the identification of potential signatures responsible for the specificities of these two classes of enzymes. The results open the way to experimentally analyse the structure and function of P. carinii mono-functional enzyme DHFS, to investigate a possible role of DHFS in the resistance to antifolates of P. jirovecii, the species infecting specifically humans, and to develop a new class of antifolates.     

Pneumocystis jirovecii colonization in chronic pulmonary disease

Pneumocystis jirovecii causes pneumonia in immunosuppressed individuals. However, it has been reported the detection of low levels of Pneumocystis DNA in patients without signs and symptoms of pneumonia, which likely represents colonization. Several studies performed in animals models and in humans have demonstrated that Pneumocystis induces a local and a systemic response in the host. Since P jirovecii colonization has been found in patients with chronic pulmonary diseases it has been suggested that P jirovecii may play a role in the physiopathology and progression of those diseases. In this report we revise P. jirovecii colonization in different chronic pulmonary diseases such us, chronic obstructive pulmonary disease, interstitial lung diseases, cystic fibrosis and lung cancer.     

Molecular diagnosis of Pneumocystis pneumonia

The detection of Pneumocystis DNA in clinical specimens by using PCR assays is leading to important advances in Pneumocystis pneumonia (PcP) clinical diagnosis, therapy and epidemiology. Highly sensitive and specific PCR tools improved the clinical diagnosis of PcP allowing an accurate, early diagnosis of Pneumocystis infection, which should lead to a decreased duration from onset of symptoms to treatment, a period with recognized impact on prognosis. This aspect has marked importance in HIV-negative immunocompromised patients, who develop often PcP with lower parasite rates than AIDS patients. The specific amplification of selected polymorphous sequences of Pneumocystis jirovecii genome, especially of internal transcribed spacer regions of the nuclear rRNA operon, has led to the identification of specific parasite genotypes which might be associated with PcP severity. Moreover, multi-locus genotyping revealed to be a useful tool to explore person-to-person transmission. Furthermore, PCR was recently used for detecting P. jirovecii dihydropteroate synthase gene mutations, which are apparently associated with sulfa drug resistance. PCR assays detected Pneumocystis-DNA in bronchoalveolar lavage fluid or biopsy specimens, but also in oropharyngeal washings obtained by rinsing of the mouth. This non-invasive procedure may reach 90%-sensitivity and has been used for monitoring the response to treatment in AIDS patients and for typing Pneumocystis isolates.     

艾滋病合并肺孢子菌肺炎1例

本文报道1例通过肺组织活检明确诊断的艾滋病合并肺孢子菌肺炎(Pneumocystis carinii pneumonia,PCP)病例,结合文献复习,分析艾滋病合并PCP的病理学特点及临床诊治措施。本例患者经实验室检查确诊为艾滋病,通过气管镜肺活检取得肺组织标本,组织病理学诊断为PCP,给予复方磺胺甲唑治疗后病情好转。PCP多见于艾滋病等免疫缺陷患者,临床上表现为间质性肺炎,提高对该病的认识并尽早进行病原学检测是确诊的关键。尽早使用复方磺胺甲唑等有效药物是改善预后的主要措施。     

Dynamics of Pneumocystis carinii-specific immune complexes in AIDS patients with P. carinii pneumonia

Pneumocystis carinii-specific immune complexes were detected by immunoblot and enzyme-linked immunosorbent assay (ELISA) in 53% of sera from Acquired Immunodeficiency Syndrome (AIDS) patients with P. carinii pneumonia (PCP). Resolution of glycoprotein antigenemia (50-55 kd = dominant species) appears to correlate with successful PCP drug therapy and recovery. An epitope map has been constructed from immunoblots of P. carinii hydrolysates and from human and murine serum containing P. carinii antigens.     

Dynamics of Pneumocystis carinii-Specific Immune Complexes in AIDS Patients with P. carinii Pneumonia

Pneumocystis carinii-specitic immune complexes were detected by immunoblot and enzyme-linked immunosorbent assay (ELISA) in 53% of sera from Acquired Immunodeficiency Syndrome (AIDS) patients with P. carinii pneumonia (PCP). Resolution of glycoprotein antigenemia (50–55 kd = dominant species) appears to correlate with successful PCP drug therapy and recovery. An epitope map has been constructed from im-munoblots of P. carinii hydrolysates and from human and murine scrum containing P. carinii antigens.     

Population structure of rat-derived Pneumocystis carinii in Danish wild rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia

Pneumocystis jirovecii is a common cause of life-threatening pneumonia among immunocompromised patients. Using 400 fresh bronchoalveolar lavage samples, we compared prospectively routine direct immunofluorescence assay (DFA) and a real-time PCR assay, performed on a LightCycler system, for the detection of P. jirovecii. Among the 66 PCR positive samples, 31 were positive by DFA. No patient was found as having the pattern "PCR--ve/DFA+ve". The semi-quantification of the P. jirovecii DNA was represented by the cycle threshold (Ct). Using DFA as the gold standard, the sensitivity of the PCR was 100% for Ct>/=28 and the specificity was 100% for Ct<22. Between these two points, the results could be discrepant. The patients of the "22/=28" group, and presented less frequently with HIV-infection and elevated lactate dehydrogenase (LDH) assay than in the "Ct<22" group. A negative PCR allowed us to exclude the P. jirovecii pneumonia. The real-time PCR assay seems to be an accurate diagnosis method and could replace the DFA. The semi-quantitative results should be helpful to distinguish colonized, subclinically infected and P. jirovecii pneumonia patients.     

Molecular basis for atovaquone resistance in Pneumocystis jirovecii modeled in the cytochrome bc(1) complex of Saccharomyces cerevisiae

Atovaquone is a substituted hydroxynaphthoquinone that is widely used to prevent and clear Plasmodium falciparum malaria and Pneumocystis jirovecii pneumonia. Atovaquone inhibits respiration in target organisms by specifically binding to the ubiquinol oxidation site at center P of the cytochrome bc(1) complex. The failure of atovaquone treatment and mortality of patients with malaria and P. jirovecii pneumonia has been linked to the appearance of mutations in the cytochrome b gene. To better understand the molecular basis of atovaquone resistance, we have introduced seven of the mutations from atovaquone-resistant P. jirovecii into the cytochrome b gene of Saccharomyces cerevisiae and thus obtained cytochrome bc(1) complexes resistant to inhibition by atovaquone. In these enzymes, the IC(50) for atovaquone increases from 25 nm for the enzyme from wild-type yeast to >500 nm for some of the mutated enzymes. Modeling of the changes in cytochrome b structure and atovaquone binding with the mutated bc(1) complexes provides the first quantitative explanation for the molecular basis of atovaquone resistance.     

Phylogenetic systematics and evolution of primate-derived Pneumocystis based on mitochondrial or nuclear DNA sequence comparison

Previous studies have demonstrated that the agent of Pneumocystis pneumonia (PcP), Pneumocystis carinii, is actually a complex of eukaryotic organisms, and cophylogeny could explain the distribution of the hosts and parasites. In the present work, we tested the hypothesis of cophylogeny between the primate-derived Pneumocystis group and their hosts. Specific strains isolated from 20 primate species, including humans, were used to produce a phylogeny of the parasites. Aligned sequences corresponding to DNA sequences of three genes (DHPS, mtSSU-rRNA, and mtLSU-rRNA) were separately analyzed and then combined in a single data set. The resulting parasite phylogeny was compared with different controversial phylogenies for the hosts. This comparison demonstrated that, depending upon which topology is accepted for the hosts, at least 61% and perhaps 77% of the homologous nodes of the respective cladograms of the hosts and parasites may be interpreted as resulting from codivergence events. This finding and the high specificity of these parasites suggests that cophylogeny may be considered the dominant pattern of evolution for Pneumocystis organisms, representing a new example of parallel evolution between primates and their specific parasites. Because the phylogeny of Pneumocystis followed very closely the differentiation of their hosts at the species level, the study of the parasites could provide valuable information on the phylogeny of their hosts. We used this information to explore controversial hypotheses of the phylogeny of the Platyrrhini by comparison with the phylogeny of their specific Pneumocystis parasites. If these organisms were closely associated as lung parasites with primates through the ages, the hypothesis of the Pneumocystis spp. being new pathogenic agents could be refuted. However, these organisms are opportunistic symbionts, becoming pathogenic whenever the immunological defences of their hosts decline. This study also provides support for the hypothesis that the different Pneumocystis species are genetically independent organisms, helping to clarify their taxonomic status.