Carinii Organisms Research Articles

Binding of Pneumocystis carinii to the lung epithelial cell receptor HSPA5 (GRP78).

The importance of lung macrophages in Pneumocystis-host interaction is well known, but little is known about the initial binding/colonization of the airway epithelium. Our prior studies have documented cell-signalling events that occur following binding of the organisms to lung epithelial cells; however, the receptors that mediate Pneumocystis attachment to lung surfaces have not yet been fully defined. Using affinity chromatography, we identified heat shock protein 5 (HSPA5), also known as GRP78, as a potential host receptor that may have relevance in Pneumocystis lung colonization. Pneumocystis carinii (Pc) organisms not only bound HSPA5 on a rat lung epithelial cell line, but also on primary rat airway epithelial cells (AECs). Furthermore, Pc bound CHO1 cells overexpressing HSPA5 more than the CHO1 parent line alone, supporting a role for Pc-HSPA5 protein interaction in mediating organism attachment. These results provide new insights into the interactions of Pneumocystis with host lung epithelium.

Uptake and degradation of Pneumocystis carinii by macrophages in vitro.

Pneumocystis carinii organisms, isolated from lungs of rats with glucocorticoid-induced pneumocystosis, were ingested and degraded in vitro by alveolar and peritoneal macrophages obtained from healthy rats and mice. Uptake of the organisms was inhibited at 4 degrees C and by treatment of macrophages with iodoacetate. Neither uptake nor degradation was inhibited by pretreatment of macrophages with hydrocortisone sodium phosphate in vitro. Although peritoneal macrophages of any age in vitro were capable of ingesting Pneumocystis, alveolar macrophages that had been cultivated in vitro for less than 2 days were incapable of phagocytosis of the organism, a functional defect that corrected itself spontaneously during cultivation in vitro.

No occurrence ofPneumocystis jiroveci (carinii)pneumonia in 120 adults undergoing myeloablative unrelated cord blood transplantation

The incidence of pneumonia caused by Pneumocystis carinii (PCP) (organism now renamed Pneumocystis jiroveci) during the early period after cord blood transplantation (CBT) was studied in 120 adults. Initially 89 patients (74%) received oral administration of 2 single-strength trimethoprim-sulfamethoxazole (TMP-SMZ) tablets twice daily from day -21. In 45 of 89 patients (51%), TMP-SMZ administration for a scheduled duration was completed. In the remaining 44 patients (49%), however, TMP-SMZ administration was discontinued prior to day -3 because of toxicity. Among these patients, 42 subsequently received aerosolized pentamidine (AP) on a median of day -13 (range, -20 to -6). Thirty-one patients (26%) received AP without TMP-SMZ administration on a median of day -14 (range, -21 to -9). None of the 120 patients were diagnosed with PCP within 100 days or 2 years after CBT; however, one patient who received AP before CBT but no prophylaxis after CBT developed cerebral toxoplasmosis on day +91. Pre-transplant prophylaxis against PCP did not significantly affect transplantation-related mortality or disease-free survival at 2 years after CBT. The results suggest that PCP during the early period after CBT can be effectively prevented by any pre-transplant prophylactic method.

Role of Naso-Gastric Aspirate in HIV-Positive Children Presenting with Respiratory Symptoms

Human immunodeficiency virus (HIV) infection predisposes to various opportunistic infections like tuberculosis, Pneumocystis carinii (P. carinii) pneumonia, toxoplasmosis and fungal infections. Naso-gastric aspirate (NGA) is utilized to demonstrate acid-fast-bacilli (AFB); similarly P. carinii was isolated with the help of naso-gastric aspirate in HIV positive children. Prospective study. Tertiary care center. To study the role of naso-gastric aspirate in HIV positive patients presenting with respiratory symptoms. HIV positive patients less than 5 years age (n = 35) presenting with respiratory symptoms of cough, breathlessness, feeding difficulties and/or tachypnoea were studied. Gastric aspirate was obtained on three consecutive mornings after overnight fasting. 1 volume of dithiotretiol (mucolytic agent) was added to 2 volumes of gastric aspirate. The mixture was incubated for 15 min followed by centrifugation at 1500 r.p.m. for 5 min; the supernatant was decanted. The sediment was re-suspended in phosphate buffer saline with pH of 6.8 and re-centrifuged at 1500 r.p.m. for 5 min. The final sediment was again suspended in phosphate buffer saline followed by addition of 6 drops of 30% bovine serum albumin. Smears were prepared from the sample followed by Zeihl-Neelsen stain for AFB; toluidine blue, Giemsa stain and Gomori's methenamine stain for identification of trophozoite and cyst forms of P. carinii. P. carinii organism was identified in 17 patients (48.57%) with 11 patients having only P. carinii while 6 patients having isolated both P. carinii and AFB. 1 patient had isolated only AFB. The total number of AFB isolated was 7 patients (20.0%). Pulmonary Koch was identified in 17 patients; however AFB was identified in 7 cases (a yield of 41.18%). No complications as a result of the procedure were identified in any of the patients. Naso-gastric aspirate is a simple, convenient, relatively non-invasive and economical technique for identification of AFB and/or P. carinii organisms in HIV-positive patients presenting with respiratory symptoms.

Inhibition of surfactant activity byPneumocystis cariniiorganisms and components in vitro

This study examines the direct inhibitory effects of Pneumocystis carinii (Pc) organisms and chemical components on the surface activity and composition of whole calf lung surfactant (WLS) and calf lung surfactant extract (CLSE) in vitro. Incubation of WLS suspensions with intact Pc organisms (10(7) per milligram of surfactant phospholipid) did not significantly alter total phospholipid levels or surfactant protein A content. Incubation with intact Pc organisms also did not impair dynamic surface tension lowering in suspensions of WLS or centrifuged large surfactant aggregates on a bubble surfactometer (37 degrees C, 20 cycles/min, 0.5 and 2.5 mg phospholipid/ml). However, exposure of WLS or CLSE to disrupted (sonicated) Pc organisms led to severe detriments in activity, with minimum surface tensions of 17-19 mN/m vs. <1 mN/m for surfactants alone. Extracted hydrophobic chemical components from Pc (98.8% lipids, 0.1 mM) reduced the surface activity of WLS and CLSE similarly to sonicated Pc organisms, whereas extracted hydrophilic chemical components from Pc (primarily proteins) had only minor effects on surface tension lowering. These results indicate that in addition to surfactant dysfunction induced by inflammatory lung injury and edema-derived inhibitors in Pc pneumonia, disrupted Pc organisms in the alveolar lumen also have the potential to directly inhibit endogenous and exogenous lung surfactants in affected patients.

Noninvasive method for monitoring Pneumocystis carinii pneumonia.

The progression of Pneumocystis carinii pneumonia was temporally monitored and quantified by real-time polymerase chain reaction of P. carinii–specific DNA in oral swabs and lung homogenates from infected rats. DNA levels correlated with the number of P. carinii organisms in the rats’ lungs, as enumerated by microscopic methods. This report is the first of a noninvasive, antemortem method that can be used to monitor infection in a host over time.

Complementation and characterization of the Pneumocystis carinii MAPK, PCM

Mitogen-activated protein kinase (MAPK) pathways transfer environmental signals into intracellular events such as proliferation and differentiation. Fungi utilize a specific pheromone-induced MAPK pathway to regulate conjugation, formation of an ascus, and entry into meiosis. We have previously identified a MAPK, PCM, from the fungal opportunist Pneumocystis, responsible for causing severe pneumonia in patients with AIDS. In order to gain insight into the function of PCM, we expressed it in Saccharomyces cerevisiae deficient in pheromone signaling and tested activation and inhibition of this MAPK pathway. PCM restored pheromone signaling in S. cerevisiae fus3Δ kss1Δ mutants with α-factor pheromone (six-fold increase) and was not activated by osmotic stress. Signaling through this pathway decreased 2.5-fold with 10 μM U0126, and was unaffected with SB203580. We evaluated the conditions for native PCM kinase activity isolated from Pneumocystis carinii organisms and found that 0.1 mM MgCl 2, pH 6.5, temperature 30–35°C, and 10 μM ATP were optimal. The activity of PCM is significantly elevated in P. carinii trophic forms compared to cysts, implicating a role for PCM in the life cycle transition of P. carinii from trophic forms to cysts.

T cell costimulatory molecule function determines susceptibility to infection with Pneumocystis carinii in mice.

Loss of T cell number and function during HIV infection or secondary to pharmacologic immunosuppression renders individuals susceptible to opportunistic infections, including Pneumocystis carinii pneumonia. Because costimulatory receptors are critical for optimal T cell function, we hypothesized that these proteins would regulate susceptibility to opportunistic infections. We found that despite normal T cell numbers, mice deficient in the costimulatory molecules CD2 and CD28 spontaneously developed P. carinii pneumonia. In experiments using intratracheal injection of P. carinii organisms to induce infection, the loss of CD28 alone was sufficient to render mice susceptible to acute infection; however, the organism was eventually cleared. Examination of inflammatory responses to P. carinii revealed that mice deficient in both CD2 and CD28 accumulated CD8(+) T cells in their lungs in response to infection and demonstrated markedly reduced specific Ab titers. Analysis of cytokine profiles suggested that regulation of IL-10 and IL-15 may be important elements of the response to this pathogen. Thus, costimulatory molecule function is critical in determining the initial susceptibility to infection with P. carinii. Analysis of immunologic responses in these mice may provide important insights into the defects that render individuals susceptible to opportunistic infection, and provide opportunities for novel immunologically based therapies.

Decrease in alveolar macrophage number during Pneumocystis carinii infection.

The alveolar macrophage is an important effector cell in the host response to Pneumocystis. The alveolar macrophage rapidly phagocytoses the organism under normal conditions, but is defective in animals infected with Pneumocystis carinii. Rats with Pneumocystis pneumonia (PcP) phagocytose 90% fewer radiolabeled P. carinii organisms and latex beads in vitro [4] and produce less nitric oxide [2]. As with humans [5], the number of alveolar macrophages decreases during Pneumocystis infection in hosts with defective immune systems, whereas normal animals respond to the organism by increasing their alveolar macrophage numbers [3]. Numbers of alveolar macrophages can decrease for several reasons, including increased cell death, increased cell migration out of the lung, decreased monocyte recruitment, or decreased differentiation of monocytes to macrophages. This study investigated the nature of the decrease in alveolar macrophage number in rats during PcP in the rat.

Environmental stress and encystment of Pneumocystis carinii.

The structural elements and molecular events associated with encystment of Pneumocystis carinii are poorly understood. The cyst form of P. carinii is present in the lungs of infected hosts in low numbers as compared to the trophozoite; as few as 1/100 of the total organism number in severely infected animals are cysts [3]. It is not known if the cyst form is infectious or the degree to which it participates in eliciting immune responses from the host. Studies using drugs, that specifically prevent formation or destabilization of the cyst wall result in lower numbers of both trophozoites and cysts, suggesting that the cyst is an integral life cycle stage of P. carinii [1,4]. The environmental or organism signals that trigger encystment of P. carinii are unknown. Cysts appear late in the infection [3], suggesting that damage done to the host pulmonary system may create an inhospitable environment or reduce nutritional resources for the trophozoite, thereby triggering formation of the more environmental resistant and quiescent form, the cyst. This study sought to determine if a specific source of stress in the trophozoites’ environment is associated with encystment. MATERIALS AND METHODS Female Sprague Dawley rats (colony 202) were obtained from Harlan (Indianapolis, Ind.) and housed in an AAALAC-approved facility. Rats were immunosuppressed with dexamethasone (Dex) (0.36 mg/liter) in their drinking water for seven days. Then P. carinii organisms were introduced into rat lungs by transtracheal inoculation as previously described [2]. Broncheoalveolar lavage (BAL) fluid collection was performed as described previously [3]. Briefly, the trachea was exposed by blunt dissection. The thoracic cavity was opened by incisions through the ribcage and removing the ribcage to allow full expansion of the lungs during lavage. A 14-gauge Angiocath (Becton Dickinson, Infusion Therapy Systems, Sandy, UT) with a 10-mL syringe was inserted into the trachea, and the lungs were lavaged with aliquots of sterile, pyrogen-free saline solution at 378C, pH 7.3. The saline was recovered from the lung by withdrawing the plunger of the syringe in a deliberate manner. In some cases, only 5 mL of lavage fluid was instilled and recovered. Some lavage samples for pH determination were obtained from animals that were incubated at 48C for 24 hr after cardiac exsanguination. In these cases, lavage was performed as described above, with special attention to slow instillation and removal of lavage fluid to account for the increased friability of the tissue. Severity of P. carinii infection was determined by scoring numbers of organisms on histochemically stained impression smears of lung tissue using the semi-logarithmic scale of Barlett et al. [2]. The smears were stained with Wright’s-Giemsa stain for both trophozoite and cysts and with Grocott methenamine-silver nitrate stain for cyst forms [2]. For culture experiments, human embryonic lung (HEL 299) cells of fibroblast morphology (CCL-137; ATCC, Manassas, VA) were grown as monolayers on 24-well culture dishes using minimum essential medium (MEM) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 10% fetal calf serum (FCS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Prior to inoculation of monolayers with P. carinii, the pH of the MEM was adjusted using 1 N HCl to 7.4, 7.3, 7.2, 7.1, 7.0, or 6.9. P. carinii samples were diluted to ten trophozoites per 10003 field to seed wells in the different pH media. Every other day, four individual wells were mixed using a Pasteur pipet, and 10-lL aliquots were

Sequence diversity of transcripts from Pneumocystis carinii gene families MSR and PRT1.

P. carinii has three gene families, MSG, PRT1, and MSR, the members of which are interspersed at the ends of chromosomes. Only one of these families, MSG, has been characterized with respect to transcription. It appears that only one MSG gene is transcribed per organism at any given time because MSG transcripts come from a unique locus called the UCS. This locus encodes the Upstream Conserved Sequence (UCS) present at the beginning of messenger RNA molecules that encode different MSG proteins [4–6]. The UCS in messenger RNA molecules encodes the signal peptide motif needed to direct nascent MSG proteins into the endoplasmic reticulum, the first step in their journey to the cell surface. MSG on the surface lacks the UCS-encoded peptide, suggesting that it is removed by proteolytic cleavage. A possible site for such processing is marked by a conserved lysine-arginine motif, which would presumably be a target for socalled dibasic processing proteases (DPP), which are also known as subtilisin-like proteases. Interestingly, the PRT1 family encodes DPPs. The number of PRT1 genes has not been determined, but most if not all of the members of this gene family are linked to MSG genes, and 80% of characterized telomeric gene clusters contain at least one PRT1 gene. These clusters average 1.5 PRT1 genes each. There are approximately 34 telomeres in the P. carinii genome. Therefore, there would appear to be between 34 and 51 PRT1 genes. Based on antibody studies, at least one PRT1 protein appears to be located on the surface of the microbe [2]. P. carinii telomeric gene clusters also contain members of a third gene family, MSR, the members of which encode peptides that are related to MSG. MSR and MSG genes are distinguished by dependence, or lack thereof, on the UCS locus for expression [3]. MSG genes that are not linked to the UCS locus appear to be silent, but MSR genes have not been found at the UCS, and MSR messenger RNA molecules lack the UCS. Instead of the UCS, MSR messenger RNA molecules begin with sequences that map to MSR genes. These regions of MSR messenger RNA molecules encode a motif that would be expected to direct MSR proteins into the secretory pathway. Therefore, it seems probable that MSR proteins either are secreted or are on the surface of P. carinii. There are probably between 34 and 51 copies of the MSR gene family. Previous studies of RNA made from P. carinii organisms from the lungs of immunosuppressed rats have established that transcripts from multiple MSG, PRT1, and MSR genes are often present in a single animal. While these observations show that multiple genes can be expressed in a population of P. carinii, they do not show that multiple genes are expressed in an individual organism. In the case of MSG, the involvement of the UCS provided a molecular tag that could be used to discern transcriptional regulation even though all of the organism populations analyzed were expressing more than one MSG. In these studies, populations of P. carinii were found to differ with respect to the MSG genes that were present at the UCS locus. Comparisons of UCS-linked genes and MSG messenger RNA molecules in different populations indicated that genes at the UCS are represented by mRNA, while genes not at the UCS are not. This correlation can be explained with a simple model whereby linkage to the UCS locus is necessary and sufficient for MSG gene transcription. Recently, we discovered that the number of MSG genes at the UCS locus could be drastically reduced by restricting the number of organisms that contribute to the original infection [1]. We found that immunosuppression of a P. carinii-colonized rat that has been kept in isolation since weaning and remains in isolation during immunosuppression produced P. carinii populations expressing one MSG in at least 90% of organisms. The discovery of these populations provided an opportunity to detect co-regulation, if it occurs. In the present study, we used a P. carinii population dominated by organisms with one UCS-MSG sequence to address two questions. (1) Do transcripts encoding MSG have the same sequence as the UCS locus? (2) Is transcription of the other two gene families, MSR and PRT, coregulated with MSG?

Evidence for the requirement of T cell costimulation in the pathogenesis of natural Pneumocystis carinii pulmonary infection

Pneumocystis carinii pneumonia (PCP) is a frequent and serious opportunistic infection in immunocompromized patients. Although the pathogenesis of PCP-mediated lung injury is poorly understood, a central involvement of host inflammatory responses has been implicated. We have found that while the loss of specific T cell costimulatory signals increases susceptibility to the spontaneous pneumocystis infection, PCP-induced pulmonary injury (and subsequent morbidity and mortality) involves other intact costimulatory pathways. Mice that are genetically deficient for the costimulatory receptor CD154 (CD154 knockout (ko) mice) spontaneously developed PCP, consistent with the increased susceptibility of X-linked hyper IgM syndrome patients (caused by CD154 gene mutations) to P. carinii infection. In these mice PCP was manifested by progressive weight loss, dyspnea and death. In contrast, CD154 ko mice also genetically lacking ICAM1 (CD154 ko×ICAM1 ko) or CD28 (CD154 ko×CD28 ko) costimulatory receptors had later onset of weight loss and significantly prolonged survival. Although onset of infection and age-matched P. carinii organism burden were equivalent, the CD154 single knockout mice had evidence of greater pulmonary inflammation vs. the double ko's. These findings suggest that costimulation-dependent T cell-mediated inflammation plays an important role in both susceptibility to and pathogenesis of PCP, and may identify potential molecular targets for novel immunomodulatory treatment approaches.

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

Pneumocystis causes a type of pneumonia in immunodeficient mammals, such as AIDS patients. Mammals cannot alkylate the C-24 position of the sterol side chain, nor can they desaturate C-22. Thus, the reactions leading to these sterol modifications are particularly attractive targets for the development of drugs against fungal and protozoan pathogens that make them. In the present study, the definitive structures of 43 sterol molecular species in rat-derived Pneumocystis carinii were elucidated by nuclear magnetic resonance spectroscopy. Ergosterol, Δ5,7 sterols, trienes, and tetraenes were not among them. Most (32 of the 43) were 24-alkylsterols, products of S-adenosyl-l-methionine:C-24 sterol methyl transferase (SAM:SMT) enzyme activity. Their abundance is consistent with the suggestion that SAM:SMT is highly active in this organism and that the enzyme is an excellent anti-Pneumocystis drug target. In contrast, the comprehensive analysis strongly suggest that P. carinii does not form Δ22 sterols, thus C-22 desaturation does not appear to be a drug target in this pathogen.The lanosterol derivatives, 24-methylenelanost-8-en-3β-ol and (Z)-24-ethylidenelanost-8-en-3β-ol (pneumocysterol), previously identified in human-derived Pneumocystis jiroveci, were also detected among the sterols of the rat-derived P. carinii organisms.

Pneumocystis carinii

Pneumocystis carinii is an atypical fungus that causes pneumonia in immunocompromised individuals. P. carinii comprises a heterogeneous group of organisms that have been isolated from a wide range of mammalian host species. P. carinii infection is host species specific, the P. carinii organisms that infect humans have only been found in humans. This review discusses the application of molecular techniques to the study of the biology and epidemiology of P. carinii infection. It addresses the use of DNA amplification for the detection and diagnosis of P. carinii pneumonia. Studies investigating the reservoir of infectious P. carinii organisms, the routes of transmission of the infection, and the emergence of drug resistant strains of P. carinii are also discussed.

Passive intranasal monoclonal antibody prophylaxis against murine Pneumocystis carinii pneumonia.

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.

Parallel phylogenies of Pneumocystis species and their mammalian hosts.

The single name Pneumocystis carinii consists of an heterogeneous group of specific fungal organisms that colonize a very wide range of mammalian hosts. In the present study, mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences of P. carinii organisms from 24 different mammalian species were compared. The mammals were included in six major groups: Primates (12 species), Rodents (5 species), Carnivores (3 species), Bats (1 species), Lagomorphs (1 species), Marsupials (1 species) and Ungulates (1 species). Direct sequencing of PCR products demonstrated that specific mtSSU and mtLSU rRNA Pneumocystis sequence could be attributed to each mammalian species. No animal harbored P. carinii f. sp. hominis. Comparison of combined mtLSU and mtSSU aligned sequences confirmed cospeciation of P. carinii and corresponding mammalian hosts. P. carinii organisms isolated from mammals of the same zoological group systematically clustered together. Within each cluster, the genetic divergence between P. carinii organisms varied in terms of the phylogenetic divergence existing among the corresponding host species. However, the relative position of P. carinii groups (rodent, camivore or primate-derived P. carinii) could not be clearly determined. Further resolution will require the integration of additional sequence data.

Pneumocystis carinii pneumonia alters expression and distribution of lung collectins SP-A and SP-D

Surfactant proteins SP-A and SP-D, members of the collectin family, have been shown to play a significant role in lung host defense. Both proteins selectively bind Pneumocystis carinii (PC) organisms and modulate the interaction of this pathogen with alveolar macrophages. We hypothesized that the expression and distribution of lung collectins SP-A and SP-D is altered by PC lung infection. PC organisms (2 × 105) were inoculated intratracheally into C.B–17 scid/scid mice that do not require steroids for immunosuppression. Four weeks after inoculation, bronchoalveolar lavage (BAL) fluid was fractionated into three fractions—cell pellet, large aggregate (LA), and small aggregate (SA) surfactant—and each fraction was analyzed for the expression of surfactant components. In uninfected mice, the majority of SP-A (62% ± 10%) was found in association with lipids in the LA fraction, while 55% ± 14% of SP-D was distributed in the SA fraction. In contrast, both hydrophobic proteins SP-B and SP-C were associated exclusively with LA. PC infection resulted in major changes in the expression of all surfactant components. Total protein content of LA was unchanged by PC infection (115% ± 18% of control), whereas SA protein content markedly increased (240% ± 18% of control level, P < .001). In contrast, the phospholipid content of LA was significantly decreased (53% ± 5% of control level, P < .001), whereas the SA phospholipid content of infected mice was increased (172% ± 16% of control level, P < .001). By Western blotting, PC pneumonia (PCP) induced a 3-fold increase in the total alveolar SP-D protein that was reflected mainly in increases in SA SP-D (454% ± 135% of control, P < .05). The total alveolar SP-A protein content was also increased in PCP because of a large increase in SP-A in SA (720% ± 115% of control, P < .05); SP-A levels in LA were unchanged. The increases in lung collectin expression were selective, because PCP resulted in the down-regulation of both SP-B and SP-C in LA (5% ± 2% and 13% ± 2% of control, respectively, P < .001). We conclude that PCP induces marked elevations in alveolar collectin levels because of increased expression and accumulation of SP-A and SP-D protein in SA surfactant. (J Lab Clin Med 2001;137:429-39)

This month in J Lab Clin Med: Issue highlights for June 2001

This month in J Lab Clin Med: Issue highlights for June 2001

Phylogeny of Pneumocystis carinii from 18 primate species confirms host specificity and suggests coevolution.

Primates are regularly infected by fungal organisms identified as Pneumocystis carinii. They constitute a valuable population for the confirmation of P. carinii host specificity. In this study, the presence of P. carinii was assessed by direct examination and nested PCR at mitochondrial large subunit (mtLSU) rRNA and dihydropteroate synthetase (DHPS) genes in 98 lung tissue samples from captive or wild nonhuman primates. Fifty-nine air samples corresponding to the environment of different primate species in zoological parks were also examined. Cystic forms of P. carinii were detected in smears from 7 lung tissue samples corresponding to 5 New World primate species. Amplifications at the mtLSU rRNA gene were positive for 29 lung tissue samples representing 18 different primate species or subspecies and 2 air samples corresponding to the environment of two simian colonies. Amplifications at the DHPS gene were positive for 8 lung tissue samples representing 6 different primate species. Direct sequencing of nested PCR products demonstrated that a specific mtLSU rRNA and DHPS sequence could be attributed to each primate species or subspecies. No nonhuman primate harbored the human type of P. carinii (P. carinii f. sp. hominis). Genetic divergence in primate-derived P. carinii organisms varied in terms of the phylogenetic divergence existing among the corresponding host species, suggesting coevolution.

Pulmonary colonisation with Pneumocystis carinii in an immunosuppressed HIV-negative patient: detection and typing of the fungus by PCR.

Mostly Pneumocystis carinii isolates from patients with acute pneumocystosis (PCP) have been typed until now. This report describes the typing of P. carinii organisms obtained from an HIV-negative patient without PCP. The patient underwent a broncho-alveolar lavage (BAL) to investigate an abnormal chest X-ray. He was diagnosed with sarcoidosis. However, a low level of P. carinii organisms undetectable by microscopy was detected in BAL fluid by two subsequent nested PCR assays: one assay amplifying a portion of the mitochondrial large subunit RNA gene and a second one amplifying the internal transcribed spacers (ITS) 1 and ITS 2 of the nuclear rRNA operon. This low level of the fungus did not reflect acute PCP. Indeed, the clinical outcome was improvement despite the absence of specific treatment. The patient was considered to be only colonised by the fungus. Analysis of sequences of ITS PCR products led to identification of genotype Gg. This information constitutes the first data concerning P. carinii ITS genotype from a patient without acute PCP and HIV. This type has been described previously in AIDS patients diagnosed with PCP. These results show that PCR and ITS genotyping could represent efficient tools for the further investigation of the role played by HIV-negative patients with pulmonary colonisation in the human reservoir of P. carinii.