Pneumocystis Cysts Research Articles

Detection of Opportunistic Fungi from the Bronchoalveolar Lavage Specimens of Patients with Pulmonary Diseases.

Opportunistic fungi are a constantly evolving group of pathogens that become active when the immune system is compromised, begin to multiply, and soon overwhelm the weakened immune system. This study was performed to evaluate the number of opportunistic fungi in bronchoalveolar lavage (BAL) samples of patients with pulmonary diseases. After receiving patients' consent and demographic forms, a total of 120 BAL samples were taken by a pulmonary physician. The etiologic agents were identified by standard morphological and molecular methods. Yeast cells were counted on culture media, and direct smears were precisely examined for the presence of yeasts elements, Pneumocystis, and filamentous fungi. In this study, 29 (24.1%) patients showed positive direct smears for yeast elements in their BAL samples. The mean colony count of yeasts was 42,000 (CFU/mL) on culture media. Six (5%) species of filamentous fungi, including three (2.5%) isolates of Penicillium species (P. variabile, P. glabrum, and P. thomii), two (1.67%) Aspergillus species (A. flavus and A. fumigatus), 1 case (0.83%) Pseudallescheria boydii were detected. Seven cases (5.83%) of Pneumocystis cysts were observed in the direct smears stained with Giemsa. Identification of all fungi confirmed by molecular or sequencing methods. Due to the presence of a large number of fungi in the BAL samples and possible physical interference with the selected drugs for treatment, we draw the attention of pulmonologists to this important issue. Rapid diagnosis of fungal infections is essential to optimize treatments and outcomes.

Interstitial pneumonia in immunocompetent laboratory rats caused by natural infection with Pneumocystis carinii.

Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1,981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1,981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott’s methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.

Characterization of Pneumocystis murina Bgl2, an Endo-β-1,3-Glucanase and Glucanosyltransferase.

Glucan is the major cell wall component of Pneumocystis cysts. In the current study, we have characterized Pneumocystis Bgl2 (EC 3.2.1.58), an enzyme with glucanosyltransferase and β-1,3 endoglucanase activity in other fungi. Pneumocystis murina, Pneumocystis carinii, and Pneumocystis jirovecii bgl2 complementary DNA sequences encode proteins of 437, 447, and 408 amino acids, respectively. Recombinant P. murina Bgl2 expressed in COS-1 cells demonstrated β-glucanase activity, as shown by degradation of the cell wall of Pneumocystis cysts. It also cleaved reduced laminaripentaose and transferred oligosaccharides, resulting in polymers of 6 and 7 glucan residues, demonstrating glucanosyltransferase activity. Surprisingly, confocal immunofluorescence analysis of P. murina-infected mouse lung sections using an antibody against recombinant Bgl2 showed that the native protein is localized primarily to the trophic form of Pneumocystis in both untreated mice and mice treated with caspofungin, an antifungal drug that inhibits β-1,3-glucan synthase. Thus, like other fungi, Bgl2 of Pneumocystis has both endoglucanase and glucanosyltransferase activities. Given that it is expressed primarily in trophic forms, further studies are needed to better understand its role in the biology of Pneumocystis.

β-Glucans Are Masked but Contribute to Pulmonary Inflammation During Pneumocystis Pneumonia.

β-glucans, which can activate innate immune responses, are a major component in the cell wall of the cyst form of Pneumocystis In the current study, we examined whether β-1,3-glucans are masked by surface proteins in Pneumocystis and what role β-glucans play in Pneumocystis-associated inflammation. For 3 species, including Pneumocystis jirovecii, which causes Pneumocystis pneumonia in humans, Pneumocystis carinii, and Pneumocystis murina, β-1,3-glucans were masked in most organisms, as demonstrated by increased exposure following trypsin treatment. Using quantitative polymerase chain reaction and microarray techniques, we demonstrated in a mouse model of Pneumocystis pneumonia that treatment with caspofungin, an inhibitor of β-1,3-glucan synthesis, for 21 days decreased expression of a broad panel of inflammatory markers, including interferon γ, tumor necrosis factor α, interleukin 1β, interleukin 6, and multiple chemokines/chemokine ligands. Thus, β-glucans in Pneumocystis cysts are largely masked, which likely decreases innate immune activation; this mechanism presumably was developed for interactions with immunocompetent hosts, in whom organism loads are substantially lower. In immunosuppressed hosts with a high organism burden, organism death and release of glucans appears to be an important contributor to deleterious host inflammatory responses.

Pneumocystis encodes a functional endo-β-1,3-glucanase that is expressed exclusively in cysts.

β-1,3-glucan is a major cell wall component of Pneumocystis cysts. We have characterized endo-β-1,3-glucanase (Eng) from 3 species of Pneumocystis. The gene eng is a single-copy gene that encodes a protein containing 786 amino acids in P. carinii and P. murina, and 788 amino acids in P. jirovecii, including a signal peptide for the former 2 but not the latter. Recombinant Eng expressed in Escherichia coli was able to solubilize the major surface glycoprotein of Pneumocystis, thus potentially facilitating switching of the expressed major surface glycoprotein (Msg) variant. Confocal immunofluorescence analysis of P. murina-infected mouse lung sections localized Eng exclusively to the cyst form of Pneumocystis. No Eng was detected after mice were treated with caspofungin, a β-1,3-glucan synthase inhibitor that is known to reduce the number of cysts. Thus, Eng is a cyst-specific protein that may play a role in Msg variant expression in Pneumocystis.

Diagnosis of the primary infection by pneumocystis in autopsy specimens from two infants using lung impression smears (touch preps)

The primary infection by Pneumocystis of normal, healthy infants is asymptomatic and goes undiagnosed. Microscopy diagnosis of Pneumocystis was sought in lung impression smears (LIS) from two ~3-month-old infants dying unexpectedly in the community. Pneumocystis nuclei and cysts were identified using Hema-Gurr with subsequent Gomori–Grocott staining in the same spot documenting that these stains may be complementary. LIS provide for an observer–dependent, inexpensive, and ready-available method for detection of Pneumocystis in infant lungs.

Histopathology of Pneumocystis carinii pneumonia in immunocompetent laboratory rats.

The occurrence of idiopathic pulmonary lesions in laboratory rats, characterized by lymphohistiocytic interstitial pneumonia with dense perivascular lymphoid cuffs, has been reported over the past decade. Although the term rat respiratory virus (RRV) was adopted to confer a putative viral etiology to the idiopathic pulmonary lesions, the etiology of this disease remains to be elucidated. Recently, inflammatory lesions have been observed in the lungs of immunocompetent laboratory rats similar to those previously described. Based on the latest evidence indicating that Pneumocystis carinii (P. carinii), and not putative RRV, causes infectious interstitial pneumonia in laboratory rats, the present study investigated whether the pulmonary lesions observed were caused by P. carinii infection. Male Sprague-Dawley rats, free of known pathogens, were introduced into a rat colony positive for RRV-type lesions. Routine histopathological examinations were performed on the rat lung tissues following exposure. The presence of Pneumocystis organisms was confirmed using Grocott’s methenamine silver (GMS) staining. At week 3 following introduction, a few small lymphoid aggregates were located adjacent to the edematous vascular sheath. By week 5, foci of dense perivascular lymphoid cuffing were observed. Multifocal lymphohistiocytic interstitial pneumonia and prominent lymphoid perivascular cuffs were observed between week 7 and 10. GMS staining confirmed the presence of Pneumocystis cysts. Thus, the results of the present study demonstrated that P. carinii caused lymphohistiocytic interstitial pneumonia in a group of laboratory rats. The observations strongly support the conclusion that P. carinii infection in immunocompetent laboratory rats causes the lung lesions that were previously attributed to RRV.

Low incidence of Pneumocystis jirovecii pneumonia in an unprophylaxed liver transplant cohort

Liver transplant recipients are managed with a range of immunosuppressive regimens that place them at heightened risk of life-threatening opportunistic infections such as Pneumocystis jirovecii pneumonia (PJP). No routine PJP prophylaxis is used at out institution. We reviewed the incidence of PJP in this cohort of unprophylaxed liver transplant recipients. We examined all liver transplants performed between January 2000 and January 2012 in Ireland's National Liver Transplant Centre, St. Vincent's University Hospital, Dublin. Cases were identified through a computerized database and through the histopathology and microbiology registration system. The diagnosis of PJP was confirmed by identification of Pneumocystis cysts in bronchoalveolar lavage (BAL) fluid or on autopsy specimens using Grocott-Gomori methenamine-silver nitrate or modified Wright-Giemsa staining methods. During the study period, 687 liver transplants were performed. We found 7 cases of PJP with an incidence rate of 0.84 per 1000 person transplant years. Five cases occurred within 12months of transplant with 2 cases occurring at 56 and 60months, respectively. Two cases were diagnosed at postmortem; 1 previously had negative cytology from BAL, while the other could not be bronchoscoped because of rapid deterioration in the clinical condition. Three of the 5 treated patients died. The incidence of PJP in this cohort was very low, but the case fatality rate was high. Two cases occurred well after the usual recommended period of prophylaxis. In institutions with a very low risk of infection, targeted rather than universal prophylaxis may be reasonable.

Evidence for DNA Synthesis in Pneumocystis carinii Trophozoites Treated with the β‐1,3‐Glucan Synthesis Inhibitor Pneumocandin L‐693,989

Pneumocandins inhibit beta-1,3-glucan synthesis preventing the development of Pneumocystis cysts that are absent from the lungs of treated rats. To determine whether treated trophozoites are capable of DNA replication, cytochemical analyses were performed on 4',6-diamidino-2-phenylindole (DAPI)- and DB181-stained Pneumocystis carinii isolated from pneumocandin L-693-989-treated rats. Fluorescence intensities of trophozoite nuclei from drug-treated rats were greater than those of untreated controls, suggesting that DNA replication was not inhibited but that cytokinesis and perhaps karyokinesis were blocked.

Molecules involved in the NFκB signaling pathway are down regulated in neonatal alveolar macrophages resulting in reduced response to Pneumocystis infection (37.7)

Abstract Neonates are unable to clear Pneumocystis (PC) infection with the same efficiency as adults. The alveolar macrophage (AM) phenotype of neonates during infection varies from that of adults, leading to a delayed inflammatory response and a slower clearance of infection. Upon infection with PC, neonatal AMs appear to maintain an alternatively activated phenotype, where as adults tend to adopt a classical AM phenotype. Previous data from our lab has shown that an elevated expression of TGFβ in neonatal lungs suppresses the ability of AMs to respond to PC. Use of a conditional smad4 KO model in which TGFβ signaling is defective in macrophages allowed us to show that blocking the TGFβ signaling pathway improves clearance of PC in neonatal mice. To further examine the response of neonatal AMs, in vitro immunoflourescence assays were performed to examine NFκB translocation to the nucleus. We found that, while neonatal AMs translocate NFκB in response to zymosan, they remain unresponsive to PC cysts. This data suggests that the unresponsiveness of neonatal AMs to PC infection may be due in part to reduced response of macrophages to PC cysts, in addition to the immunosuppressed lung environment. We hypothesize that the down regulation of molecules involved with NFκB translocation to the nucleus results in the reduced response of AMs to PC.

Characterization of a Novel ADAM Protease Expressed byPneumocystis carinii

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Recent evidence has suggested that unidentified proteases are involved in Pneumocystis life cycle regulation. Proteolytically active ADAM (named for "a disintegrin and metalloprotease") family molecules have been identified in some fungal organisms, such as Aspergillus fumigatus and Schizosaccharomyces pombe, and some have been shown to participate in life cycle regulation. Accordingly, we sought to characterize ADAM-like molecules in the fungal opportunistic pathogen, Pneumocystis carinii (PcADAM). After an in silico search of the P. carinii genomic sequencing project identified a 329-bp partial sequence with homology to known ADAM proteins, the full-length PcADAM sequence was obtained by PCR extension cloning, yielding a final coding sequence of 1,650 bp. Sequence analysis detected the presence of a typical ADAM catalytic active site (HEXXHXXGXXHD). Expression of PcADAM over the Pneumocystis life cycle was analyzed by Northern blot. Southern and contour-clamped homogenous electronic field blot analysis demonstrated its presence in the P. carinii genome. Expression of PcADAM was observed to be increased in Pneumocystis cysts compared to trophic forms. The full-length gene was subsequently cloned and heterologously expressed in Saccharomyces cerevisiae. Purified PcADAMp protein was proteolytically active in casein zymography, requiring divalent zinc. Furthermore, native PcADAMp extracted directly from freshly isolated Pneumocystis organisms also exhibited protease activity. This is the first report of protease activity attributable to a specific, characterized protein in the clinically important opportunistic fungal pathogen Pneumocystis.

Acute fibrinous and organizing pneumonia as a rare presentation of abacavir hypersensitivity reaction

Abacavir, an HIV nucleoside analogue reverse transcriptase inhibitor, can cause hypersensitivity reactions in 3–5% of patients started on the drug, and 90% of cases occur within the first 6 weeks after initiation (median time 11 days). The most common symptoms of abacavir hypersensitivity are fever, rash, and gastrointestinal symptoms. Respiratory symptoms such as dyspnea, cough, and pharyngitis are prominent in 30% of cases and mimic pneumonia [1]. Characteristically, symptoms appear suddenly and worsen over just a few days when the use of abacavir is continued. Very rarely hypotension, renal insufficiency, and bronchoconstriction have resulted in death, especially after the rechallenge of the drug. If the use of abacavir is discontinued in time to prevent the development of hypotension, the hypersensitivity reaction resolves completely in a few days [2]. Herring and Krieger [3] recently reported a patient with fulminant pneumonitis leading to acute respiratory syndrome (ARDS) after 11 days of therapy with abacavir. ARDS resolved within 3 days after the discontinuation of abacavir. The lung tissue was not available in that report. A 52-year-old woman infected with HIV (CD4 cell count 231 cells/μl) presented to our institution with a worsening of dyspnea and productive cough for a day. She had been diagnosed 4 years earlier, and antiretroviral therapy (ART) had been started 2 weeks before she presented. Her ART regimen consisted of abacavir, lamivudine, and ritonavir boosted with atazanavir. Examination revealed bilateral expiratory wheezes. Partial pressure of oxygen in the blood was 49 mmHg and the alveolar–artery oxygen gradient was elevated at 63. Chest radiograph showed diffuse reticular interstitial infiltrates. As Pneumocystis jiroveci pneumonia and atypical pneumonia were considered most likely, trimethoprim/sulfamethoxazole, azithromycin, and corticosteroids were started. Dyspnea and hypoxia and bilateral infiltrates worsened progressively, in spite of treatment. On day 4, ART was discontinued as immune reconstitution inflammatory syndrome was considered to be the cause of the worsening. On day 5, she was intubated and bronchoscopy was performed. Cytology of bronchoalveolar lavage did not reveal Pneumocystis cysts. Over the course of the next few days, there was a remarkable improvement in the condition of the patient, with dramatic resolution of the diffuse bilateral infiltrates (Fig. 1a–c). Thoracoscopic lung biopsy performed on day 10 revealed patchy areas of organizing pneumonia with fibrin balls within alveolar spaces, which was compatible with acute fibrinous and organizing pneumonia (AFOP; Fig. 1d, e).Fig. 1: Chest radiographs. (a) On admission; (b) day 5: intubation; (c) day 10: lung biopsy of left lower lobe; (d) organizing pneumonia; (e) fibrin ball.AFOP was first described by Beasley et al. [4] in 2002, as a new pattern of lung injury, showing prominent intra-alveolar fibrin deposition (fibrin balls) and organizing pneumonia. The absence of hyaline membrane, eosinophils, and granulomatous inflammation are important features that differentiate it from diffuse alveolar damage, hypersensitivity pneumonitis, eosinophilic pneumonia, and cryptogenic organizing pneumonia, which was previously known as bronchiolitis obliterans organizing pneumonia. AFOP appears to have two distinct patterns of disease progression and outcome. Fulminant AFOP rapidly progresses to death. In contrast, subacute AFOP resembles cryptogenic organizing pneumonia (bronchiolitis obliterans organizing pneumonia) and recovers without mechanical ventilation. Beasley et al. [4] noted several conditions associated with AFOP, including collagen-vascular disease, zoological work, coal mining, construction work, hairspray exposure, high-grade lymphoma, altered immune status, the use of amiodarone, and infections caused by Haemophilus influenzae and Acinetobacter baumannii. After the original report by Beasley et al. [4], reports of AFOP in patients with severe acute respiratory syndrome (SARS), juvenile dermatomyositis, and acute lymphoblastic leukemia have been described [5–7]. Our patient represents the first case of AFOP occurring as a hypersensitivity reaction to abacavir. From our observation, abacavir can cause acute lung injury clinically presenting as ARDS, presumed to be a hypersensitivity reaction and pathologically showing AFOP. The early recognition and discontinuation of the drug is the only established treatment. None of the patients with fulminant AFOP recovered in the previous studies, but AFOP secondary to abacavir hypersensitivity was reversible in our patient. This highlights the characteristic of abacavir hypersensitivity: fulminant exacerbation with continuation of the drug and rapid recovery after discontinuation of the drug. If respiratory symptoms worsen progressively within a few weeks after the initiation of ART, the differential diagnosis includes new infection, immune reconstitution inflammatory syndrome, and drug-induced hypersensitivity. The only way to differentiate this is by lung biopsy, as in our case. Conflicts of interest: None.

The changing spectrum of pulmonary disease in patients with HIV infection on antiretroviral therapy

The pulmonary manifestations of HIV disease in 2005 can be divided into two sets of presentations. For patients who do not have access to care, and who are not taking antiretroviral or chemoprophylactic drugs, opportunistic infections and neoplasms continue to occur. Pulmonary disease caused by Streptococcus pneumoniae, Pneumocystis carinii (now known as Pneumocystis jiroveci pneumonia; PCP), Mycobacterium tuberculosis, lymphoma, and Kaposi’s sarcoma present much as they did in the 1980s. Management has improved in terms of new diagnostic, therapeutic, and preventive strategies, as reviewed in guidelines issued jointly by the National Institutes of Health, Centers for Disease Control and Prevention, and Infectious Disease Society of America (available online at http://www.aidsinfo.nih.gov).

Cell wall antigens of Pneumocystis carinii trophozoites and cysts: purification and carbohydrate analysis of these glycoproteins.

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose, and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanvalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host.

Differences exist in the immunoblotting profiles of cyst and trophozoite antigens of Pneumocystis carinii.

The antigenic profiles of Pneumocystis carinii trophozoites and cysts were compared by immunoblotting with hyperimmune rat sera against cyst and trophozoite antigens. Strong bands corresponding to proteins of 50-60 kDa and 104 kDa were demonstrated in cyst and trophozoite antigens by all antisera. Additional prominent proteins of 81 and 63 kDa and less prominent proteins of 88, 73, 69 and 37 kDa were found only in trophozoite antigen. The latter proteins were recognised by anti-trophozoite and anti-cyst antisera but the 81- and 63-kDa proteins were associated specifically with trophozoites. With cyst-rich antigen, antibodies to the 50-60-kDa protein were detected in only two of 14 sera from P. carinii pneumonia (PCP)-positive rats. With trophozoite-rich antigen, 11 of 24 rats with PCP and one of 18 PCP-negative animals had antibodies to both the 50-60 kDa and 104-kDa antigens. Antibodies to the 81- or 63-kDa antigens were demonstrated in 15 of 24 PCP-positive animals and none of the PCP-negative animals. The use of trophozoites rather than cysts increased the sensitivity of immunoblotting. As trophozoites predominate in PCP, antibody to trophozoite-specific antigens rather than common cyst and trophozoite antigens is likely to be a more useful marker of current infection.

Decreased Yield of Pneumocystis carinii from Cortisonized Rats

Thirty-five male Sprague-Dawley rats were divided into 3 groups, including 1 control and 2 experimental groups, in order to compare the efficacy of using cortisone acetate alone or in addition to intranasal inoculation of Pneumocystis carinii organisms for the purpose of inducing acute P. carinii pneumonia. The presence of P. carinii was monitored in nasal secretions on a weekly basis and in lungs at autopsy. Titers of IgG antibody were also monitored by enzyme-linked immunosorbent assay. No rat receiving cortisone acetate injections alone and only 2 of the rats receiving both cortisone and intranasal inoculation of P. carinii organisms showed Pneumocystis organisms in the lungs. However, Pneumocystis cysts did appear in the nasal secretions of 3 of the 5 control rats, all 8 rats receiving cortisone acetate injections only, and 12 of 18 rats receiving both cortisone acetate injections and an intranasal inoculum. IgG titers of both cortisonized groups remained less than 1:4 throughout the course of the experiment. The titer of the control group increased from negative to 13 (geometric mean).

Prognostic factors and life expectancy of patients with acquired immunodeficiency syndrome and Pneumocystis carinii pneumonia.

To assess determinants of prognosis for 43 patients with the acquired immunodeficiency syndrome (AIDS) and Pneumocystis carinii pneumonia, objective clinical and histopathologic characteristics were analyzed for acute and long-term prognostic significance. Severe abnormalities on initial chest radiographs and alveolar-arterial oxygen differences (AaPO2) greater than 30 mm Hg were associated with higher mortality during the period of treatment for the acute episode (p less than 0.05). Decreased long-term survival after the diagnosis of Pneumocystis pneumonia correlated with the severity of interstitial edema (a component of diffuse alveolar damage) on initial transbronchial biopsy and elevation of AaPO2 at the time of diagnosis (Cox proportional hazards analysis, p less than 0.05). The persistence of Pneumocystis cysts after 3 wk of therapy was associated with significantly decreased long-term survival (p less than 0.05) when follow-up biopsy was performed in 27 of the patients. Patients with a diagnosis of Pneumocystis pneumonia before July 1985 had more advanced disease at the time of diagnosis and a worse prognosis than did those in whom the diagnosis was made after July 1985 (p less than 0.05). This study demonstrates that important prognostic information can be derived from information obtained at initial presentation and follow-up bronchoscopic evaluation in patients with AIDS and Pneumocystis carinii pneumonia, and suggests that early detection and initiation of therapy may improve chances for survival.

Pneumocystis carinii pneumonia. An approach to rapid laboratory diagnosis.

Laboratory procedures used to establish the diagnosis of Pneumocystis carinii pneumonia were evaluated using an experimental murine model. Touch preparations and suspension smears were prepared from lung tissue know to contain Pneumocystis cysts. These preparations were stained by a variety of methods known to demonstrate either cyst forms or sporozoites and trophozoites. Suspension smears proved to be superior to touch preparations in terms of cyst content and homogeneity of staining. Also, methods that stain cyst forms were superior to those that stain sporozoites and trophozoites for location and identification of organisms. The authors believe that suspension smears prepared from lung tissue and stained with toluidine blue O should be examined initially as a rapid screening method for Pneumocystis cysts. When the results of this initial screen are negative or inconclusive, additional suspension smears stainded by the modified Gomori methenamine silver nitrate method should be examined, pending availability of histologic sections.

A preliminary report of an indirect fluorescent antibody test for detecting antibodies to cysts of Pneumocystis carinii in human sera.

An indirect immunofluorescence screening test for detecting antibodies to Pneumocystis carinii in human serum is described. The antigen used was prepared from isolated pneumocystis cysts separated from human pulmonary tissue. Of 89 sera from patients with confirmed or provisionally diagnosed pneumocystis pneumonia in the United States, 46% were positive. Of 85 sera from healthy contacts in the home or hospital, 14% were positive serologically. None of 50 sera from healthy young Americans was reactive.