Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.
Pneumocystis carinii pneumonia (PCP) is one of the most important causes of morbidity and mortality in patients with AIDS (13, 17, 19, 25). It is characterized clinically by dyspnea, tachypnea, and hypoxemia (5, 11, 12). The lung tissue is usually diffusely involved, with the alveoli becoming filled with a foamy, proteinaceous material (41). This foamy debris effec- tively diminishes the alveolar surface area available for gas exchange and therefore causes shunting of unoxygenized blood with a resultant disturbed ventilation-perfusion ratio and hy- poxemia (21). The alveolar debris contains cysts, trophozoites, some host-derived cells, and plasma proteins (44), providing the P. carinii organisms with the nutrients required for their life cycle and growth. Because it provides an intra-alveolar micromilieu conducive to continuing growth, the formation of such proteinaceous alveolar fluid is of high survival value for P. carinii. In patients with immunodeficiency, the interstitial in- flammatory response is relatively modest and consists mainly of the production of monocytes/macrophages and lymphocytes and of fibrosis (8, 44). In AIDS patients with opportunistic lung infections, elevated numbers of neutrophils in bronchoalveolar lavage fluid (BALF) are associated with a fatal outcome (1). The formation of the foamy alveolar exudate might involve local tissue destruction leading to the leaky alveoli typically seen in PCP (45). This might involve damage to the main
The frequency of Pneumocystis jiroveci (human-derived Pneumocystis) in immunocompetent infants develop- ing acute respiratory syndromes has recently been evaluated and has been shown to be close to 25%. Until now, there have been no data on the genomic characteristics of the fungus in these patients, while molecular typing of P. jiroveci organisms was mostly performed with samples from immunosuppressed patients with pneumo- cystosis (Pneumocystis carinii pneumonia [PCP]). The present report describes the genotypes of P. jiroveci organisms in 26 nonimmunosuppressed infants developing a mild Pneumocystis infection contemporaneously with an episode of bronchioloalveolitis. The typing was based on sequence analysis of internal transcribed spacers (ITSs) 1 and 2 of the rRNA operon, followed by the use of two typing scores. By use of the first score, 11 P. jiroveci ITS types were identified: 10 were previously reported in immunosuppressed patients with PCP, while 1 was newly described. By use of the second score, 13 types were identified, of which 2 were newly described. The most frequent type was identified as type B 1 a 3 (first score), which corresponds to type Eg
To evaluate the value of single and nested PCRs for diagnosis of Pneumocystis carinii pneumonia (PCP) in a variety of respiratorily distressed patient groups, 574 respiratory samples from 334 patients (89 human im- munodeficiency virus [HIV]-positive patients, 61 transplant recipients, 66 malignancy patients, 34 otherwise immunosuppressed patients, and 84 immunocompetent patients) were prospectively examined by microscopy and single and nested PCRs. The resulting data were correlated with clinical evidence of PCP. Microscopy and single PCR of bronchoalveolar lavage (BAL) specimens from HIV patients were 100% sensitive and specific in detecting PCP, whereas nested PCR, although being 100% sensitive, reached a specificity of only 97.5%. In the three non-HIV immunosuppressed patient groups, both single and nested PCR invariably produced lower pos- itive predictive values than microscopy. Among immunocompetent patients, the positive predictive values of both PCRs were 0%. Therefore, the diagnostic values of the PCR methods tested do not seem to offer any addi- tional advantage compared to that of conventional microscopy for these patient groups. However, nested PCR identified a significant percentage of clinically silent P. carinii colonizations in about 17 to 20% of immuno- competent and immunosuppressed non-HIV patients.
The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoal- veolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.
Cesarean sections. Three gravid and two nongravid female Long Evans rats (controls) were chosen for cesarean section analysis to explore the route of transplacental transmission of Pneumocystis in rats. At 10 days to 4 weeks of gestation, oral swabs were collected from each gravid dam. Subsequently, each dam was sacrificed by intraperitoneal injection of approximately 500 mg of chloral hydrate (Major Pharmaceuticals, Livonia, Mich.) and immediately placed under a laminar flow hood (The Germfree Laboratories, Inc.) which had previ- ously been wiped down with DNA AWAY. All instruments used for dissection were treated with a solution of DNA AWAY after standard cleaning regimens and were autoclaved in separate pouches. Prior to dissection, each rat was sprayed with 70% alcohol followed by a spray of DNA AWAY solution. One set of instruments (forceps and scissors) was used to open the animal’s abdomen. A separate set was used for organ removal. The intact uterus was removed from the abdominal cavity and placed in a large, sterile plastic petri plate. The lungs, liver, and spleen were removed from each dam and placed in individual sterile tubes. For the gravid females, each pup was removed from the uterus, the placenta (including the deciduae) was removed from each pup, and then each pup, each placenta, and the uterus were placed in individual sterile tubes. Three sets of internal negative controls were performed by treating three sterile tubes like all other samples, minus tissue, controlling for reagent contamination. All samples, including the three internal negative controls, were digested in 5 to 10 ml of proteinase K solution (0.5 mg of proteinase K/ml of DNA extraction buffer; Roche, Indianapolis, Ind.) overnight at 55°C. DNA was extracted from 250 l of each sample by a phenol-chloroform extraction method (10) and was stored in TE at ⫺20°C until analyzed by PCR.
Pneumocystis pneumonia is caused in humans by the recently renamed Pneumocystis jiroveci (Frenkel 1999), previously known as Pneumocystis carinii and now thought to be related to fungi on the basis of DNA analy- sis, despite morphological similarities to protozoa . The organism shows significant genetic divergence and is host specific with no cross-species infectivity. There has been considerable debate about the nature of the relation- ship between humans and P. jiroveci. At one time it was hypothesised that infection occurred through reactivation of colonisation acquired in childhood, as specific anti- body was found in seven out of eight normal adults . This suggestion has been refuted by the absence of detect- able P. jiroveci in bronchoalveolar lavage specimens from healthy volunteers, despite amplification by the polymer- ase chain reaction . In addition, genotypic analysis of P. jiroveci from infected adults identified strains found in patients' place of residence, rather than their place of birth . Investigation of apparent clusters has shown different genetic strains affecting most cases, indicating that trans- mission from affected cases to susceptible persons does not account for the majority of infections [8,9], despite the recognised transmission of Pneumocystis DNA from affected patients to their immunocompetent contact health care workers to produce colonisation . As P. jiroveci has been isolated in samples of air  and pond water , it is likely that the environment represents the main source of infection for most patients. Nevertheless, isolation of known cases of PCP is advisable.
Characterization of P. carinii myo-inositol transport and comparisons to mammalian and fungal transport systems. PcITR1, as well as other ITRs analyzed in this work, appears to belong to the major facilitator superfamily (MFS) of membrane proteins that are expressed ubiquitously across all phyla and are involved in the transport of various small-molecule compounds (32). The superfamily comprises at least 17 distinct families that generally show specificity to a single class of compounds, in- cluding simple sugars (hexoses, pentoses, and disaccharides), inositols, amino acids, nucleosides, various organic and inor- ganic cations and anions, and even some drugs. The MFS fold consists of two 6-transmembrane helix bundles that are con- nected by intracellular helices or extended loops. These two 6-helix bundles can adopt a clamshell-like outward opening with a hydrophilic cavity, where the amino acids lining this cavity determine the transporter’s specificity for a certain class of substrates (19, 33-35). Once a substrate is docked into the cavity, the MFS transporter changes the conformation of the 6-helix bundles to open the cavity inward and simultaneously close the outward cavity through a so-called “rocker switch” mechanism (also known as the alternating access mode of op- eration), which prevents a continuous permeability across the membrane (34, 35). In this work, we characterized the kinetics, substrate specificity, and possible inhibition of PcITR1 to help define the function of this essential system.
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The possible transmission of Pneumocystis carinii f. sp. hominis from patients with P. carinii pneumonia to asymptomatic health care workers (HCW), with or without occupational exposure to human immunodeficiency virus (HIV)-infected patients with P. carinii pneumonia, was examined. HCW in a specialist inpatient HIV-AIDS facility and a control group in the general medical-respiratory service in the same hospital provided induced sputum and/or nasal rinse samples, which were analyzed for the presence of P. carinii f. sp. hominis DNA by using DNA amplification (at the gene encoding the mitochondrial large subunit rRNA [mt LSU rRNA]). P. carinii f. sp. hominis DNA was detected in some HCW samples; those with the closest occupational contact were more likely to have detectable P. carinii DNA. P. carinii DNA was detected in one HCW who carried out bronchoscopy over a 2-year period. P. carinii-positive samples were genotyped by using DNA sequence varia- tions at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, along with bronchoalveolar lavage samples from patients with P. carinii pneumonia hospitalized at the same time. Genotyping identified 31 different P. carinii f. sp. hominis ITS genotypes, 26 of which were found in the patient samples. Five of the eight ITS genotypes detected in HCW samples were not observed in the patient samples. The results suggested that HCW in close occupational contact with patients who had P. carinii pneumonia may have become colonized with P. carinii. Carriage was asymptomatic and did not result in the development of clinical disease.
universally derive from patients in well-‐resourced settings without immunocompromise. 92,93 Given differences in demographics, comorbidity profile, and disease aetiology, extrapolation beyond these cohorts should be cautioned. Relevant data from sub-‐Saharan Africa are limited. In a small cohort of 88 patients from Nigeria, 30-‐day mortality rose with CURB65 and at a threshold score of 3 or more, reported sensitivity and specificity were 80% and 97%, respectively. 94 In a well-‐characterised cohort of 280 HIV-‐infected patients with radiographic CAP from South Africa, Albrich et al. found CURB65 was not a useful discriminator of in-‐hospital mortality for either all CAP patients or those with confirmed pneumococcal pneumonia. 22 Similarly, CRB65 (the abbreviated version of CURB65 that
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PJP remains a still major health problem in both HIV and other immuno-compromised individuals. Improper treatment for PJP infection may lead to mortality. HIV cases with HAART may reduce the risk of PJP. Mortality is associated with PJP in both HIV and non-HIV patients. The rate of infection as well as mortality rates was increasing year by year. Major problem in AIDS patients with Pneumocystis infection leads to increasing death rate due to failure of drug therapy. Finally, further advance research is required for strategies development as well as to stop further increase of resistant strains. New diagnostic methods and usage of non-invasive respiratory specimens such as oral washes for diagnosis of PJP should be expanded. PCR is the most sensitive method compared to other conventional and serological methods. Using PCR and Sequence analysis can be helpful for estimation of prevalence of disease as well as epidemiological purposes. Following evidence based guidelines will be helpful for giving proper treatment and can reduce the improper usage of drugs and decrease drug resistance. Conducting programmes to public, regarding disease will be helpful for patient management particularly in HIV people suffering with opportunistic infections. There is an urgent need to develop newer drugs and vaccines to eradicate the PJP disease burden.
Although PCR detection of Pneumocystis carinii DNA has been described, little is known about the sensitivity or specificity of the assay in routine laboratory practice. We had the unique opportunity to use a bronchoal- veolar lavage (BAL) specimen bank with samples for which the direct examination results for P. carinii were known. DNA purified from 129 selected specimens was amplified by using the primers described previously (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moton, and J. M. Hopkin, Mol. Biochem. Parasitol. 43:69–76, 1990). Of the 129 specimens, 37 were positive for P. carinii by direct examination. All 37 specimens were positive for P. carinii by PCR, yielding a 100% sensitivity and 100% negative predictive value for the assay. An additional 23 specimens were repeatedly positive for P. carinii by PCR but were not positive by direct examination. Review of the patient charts for these specimens with discordant results demonstrated that five of the patients were actually positive for P. carinii, as determined by either biopsy or examination of repeat or prior BAL specimens. A response to empiric therapy for P. carinii pneumonia was seen in an additional two patients. Of the remaining specimens, 8 produced no significant isolates other than P. carinii, while 12 contained culture-confirmed significant respiratory pathogens in addition to P. carinii (two fungal, nine bacterial, and one viral pathogen). Cytomegalovirus, which was of unknown significance, was isolated from 16 additional specimens. Overall, the specificity of the PCR assay was 79.3% compared to the results of direct examination. We hypothesized that the apparently poor specificity of the PCR assay was due to the increased sensitivity of the assay compared to that of direct examination. The sensitivity of the PCR assay was therefore assessed with BAL specimens containing P. carinii cysts. Serial dilutions of this prepara- tion were evaluated by direct examination and PCR. PCR was found to be 100-fold more sensitive than direct examination, which detected one to two cysts per amplification. No false-positive results were detected in controls containing no DNA or by using target DNA from various fungal, viral, or bacterial respiratory pathogens. We conclude that PCR detection of P. carinii in BAL specimens is very sensitive and should be considered for patients whose specimens do not yield a diagnosis. The increased sensitivity of the PCR assay may help to identify those patients with low-titer infections who might benefit from directed antibiotic therapy for P. carinii and would otherwise be missed by direct examination alone.
Pneumocystis jirovecii is an ascomycetous fungus that causes opportunistic infections, and its life cycle remains unknown because it cannot be consistently cultured [17, 18]. Furthermore, the epidemiology of human PCP is un- clear yet [17, 19]. Due to little knowledge about Pneumo- cystis jirovecii, it is necessary to investigate whether the long term effect of PCP on allografts is due to the infec- tion alone or with chronic inflammatory process and other unknown mechanisms. Although PCP is relatively com- mon after kidney transplantation, its clinical implications, especially in relation to graft outcomes, have not been fully evaluated. The results of the present study demon- strated that male gender and CMV positivity were risk
Trimethoprim-sulfamethoxazole (TMP-SMX) is the drug of choice for pneumocystis. TMP-SMX is made up of two antimicrobial agents that act synergistically against a wide variety of bacteria. Although trimethoprim has been com- bined with other sulfonamides, TMP-SMX is by far the most widely used. TMP and SMX are individually weak bacteri- cidal agents, but when given together, they become highly bactericidal against many bacteria. Maximum inhibition of most susceptible bacteria occurs when the TMP-SMX con- centration ratio is 1:20. The drug combination is prepared in a higher fixed ratio of 1:5; however, peak serum concentrations of 1:20 are still achieved by both oral and intravenous therapy because of the wider volume of distribution of TMP.
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Specific antimicrobial prophylaxis, by itself or in conjunction with antiretroviral therapy, can reduce the substantial morbidity and mortality caused by opportunistic infections in patients with HIV infection. We are in a fortunate period in which the effects of opportunistic infections can be dramatically decreased for patients with access to comprehensive care in which durable immune reconstitution is induced by highly active antiretroviral therapy. Understanding and applying measures to prevent opportunistic infections has had, and will continue to have, a critical role in the treatment of patients with HIV infection. Several HIV-related infections (including tuberculosis, bacterial pneumonia, malaria, septicaemia and PCP) can be prevented using drugs. This is known as drug prophylaxis 18 .
Pneumocystis jirovecii is an important opportunistic pathogen in immunocompromised patients. Molecular typing is employed to study this pathogen, as no culture system exists. No Australian P. jirovecii strains have been previously studied. Direct sequencing, targeting the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, the mitochondrial large-subunit rRNA (mt LSU rRNA), and the dihydropteroate synthase (DHPS) gene, was performed on 68 Australian samples, collected between 2001 and 2007. Seven novel Australian ITS haplotypes (a composite of the ITS1 and ITS2 regions) were identified (SYD1m, SYD1g, Isyd2, Esyd3, Osyd4, Ag, and Hc). A dendrogram of published ITS haplotypes revealed that of the seven novel haplotypes, three (SYD1m, SYD1g, and Osyd4) are closely related to the haplotype Eg. Applying statistical parsimony, an Australian haplotype network was constructed which identified Eg as the ancestral haplotype, with two unresolved loops encountered. This suggests that the ITS lacks the resolution required for evolu- tionary analysis. Only two mt LSU rRNA genotypes were detected, with genotype 1 predominating. Mutant DHPS genotypes were present in 13% (8/60) of the samples. The novel haplotype Isyd2 was associated with less severe disease than the other Australian haplotypes. In contrast, patients with mutant DHPS genotypes were more likely to have severe disease, require invasive ventilation, and have a poor outcome than patients with wild-type DHPS genotypes. In conclusion, genetic clinical correlates continue to be found for Pneumocystis pneumonia; however, they remain controversial and warrant further study.