1 Equipe de Mycologie, UMR 956 INRA-AFSSA-ENVA-UPVM Biologie Moléculaire et Immunologie Parasitaires et Fongiques, Ecole Nationale Vétérinaire d'Alfort, 7 Avenue du Général de Gaulle, 94704 Maisons-Alfort, France
2 Centre de Primatologie, ULP Strasbourg, Fort Foch, Niederhausbergen, France
3 Laboratoire de Parasitologie, Université Pierre et Marie Curie, Paris VI, France
4 Laboratoire d'Ecologie des Sols Tropicaux, UMR 137 BioSol, IRD/Paris VI, 32 avenue Henri Varagnat, 93143 Bondy Cedex, France
5 EA3609-Parasitologie-Mycologie, Faculté de Médecine et CHRU de Lille and IFR-17-Ecologie du Parasitisme, Institut Pasteur de Lille, France
Correspondence
Jacques Guillot
jguillot{at}vet-alfort.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In humans, reports of pneumocystosis outbreaks among immunosuppressed patients in hospitals suggest that the main source of Pneumocystis is patients with a Pneumocystis infection (Helweg-Larsen et al., 1998; Hennequin et al., 1995
; Hocker et al., 2005
; Rabodonirina et al., 2004
; Singer et al., 1975
). The presence of Pneumocystis DNA has been reported in air samples collected from hospital rooms of patients with pneumocystosis (Bartlett et al., 1994
, 1997
). Detection of Pneumocystis DNA in the air is also consistent with the hypothesis that transmission occurs from person to person. Recent studies have shown that Pneumocystis DNA can also be detected in immunocompetent humans after close contact with patients with pneumocystosis (Miller et al., 2001
; Vargas et al., 2000
). In these cases, Pneumocystis DNA detection was transient and might have been the result of continuous inhalation of Pneumocystis cells, indicating upper respiratory surface contamination rather than an active infectious process. The actual role of transiently parasitized immunocompetent hosts as a source of infective elements has been demonstrated in the mouse model (Dumoulin et al., 2000
) and warrants further investigations in humans. Recent studies on mice suggested that transmission from healthy host to healthy host, as an asymptomatic or minimally symptomatic infection, could be a way to maintain Pneumocystis organisms in the environment (Chabé et al., 2004
; Gigliotti et al., 2003
).
The interest of non-human primate models relies on the phylogenetic closeness between monkeys and humans and therefore between Pneumocystis in monkeys and Pneumocystis in humans (Demanche et al., 2001; Hugot et al., 2003
). In the present study, we describe for the first time the circulation of Pneumocystis organisms within a social organization of crab-eating macaques (Macaca fascicularis) living in a natural setting in France. These conditions simulate, with good fidelity, real conditions in wildlife, while reducing uncontrolled biotic and abiotic parameters. The aims of the study were to identify potential sources of infection, to assess Pneumocystis circulation within the colony and to test the hypothesis of airborne transmission. Deep nasal swab and blood samples were collected monthly from each animal. Environmental air samples were also examined every month. Since known Pneumocystis species have been analysed at the mitochondrial large subunit (mtLSU) rRNA gene (Demanche et al., 2001
; Wakefield, 1998
), which is considered as a sensitive and robust target for Pneumocystis PCR detection (Tsolaki et al., 1999
; Wakefield et al., 1990
), this locus was used for detecting Pneumocystis organisms in both macaque respiratory specimens and environmental air samples.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Samples for analysis.
Deep nasal swabs and blood samples were collected from each animal under general anaesthesia induced by the administration of 10 mg ketamine (Imalgene) kg1 by an intramuscular route. A moist sterile cotton-swab was introduced deep into each nasal cavity, left there for 5 s, rotated and withdrawn (Vargas et al., 2000). A blood sample (35 ml) was collected in a serum separator tube by puncturing a femoral vein. After centrifugation, serum samples were stored at 20 °C.
Air samples from the environment of the colony were obtained by using the CAP (Capteur Atmosphérique de Poussières) device (Arelco) as previously described by Guillot et al. (1999b). This device sampled airborne particles with a flow rate of 10 litres min1. Particles were impacted on the surface of a rotative cup. Every month, air sampling was performed for 1 week in the centre of the park where the macaques lived. Deep nasal swab and blood samples were collected from the animals the last day of the air-sampling week.
DNA extraction from nasal swab and air samples.
Just after sampling, from December 2000 to October 2001, deep nasal swabs were placed in a sterile tube containing 500 µl extraction buffer (10 mM Tris, 0·5 % SDS, 25 mM EDTA, 0·1 M NaCl). For air samples, the rotative cup from CAP apparatus was washed with 600 µl of the same extraction buffer. DNA was prepared by proteinase K digestion (Boehringer Mannheim) at a final concentration of 0·28 mg ml1, followed by phenol/chloroform extraction with a final precipitation in ethanol. From November 2001 to November 2002, deep nasal swab DNA extraction was performed by using a Qiagen kit (DNeasy tissue kit).
Primers and PCR amplification.
The presence of Pneumocystis DNA in swabs was assessed by nested-PCR at the mtLSU rRNA gene. We used the primer sets pAZ102-H/pAZ102-E (5'-GATGGCTGTTTCCAAGCCCA-3'/5'-GTGTACGTTGCAAAGTACTC-3') and pAZ102-X/R1/pAZ102-Y/R1 (5'-GGGAATTCGTGAAATACAAATCGGACTAGG-3'/5'-GGGAATTCTCACTTAATATTAATTGGGGAGC-3') (Wakefield, 1998). The thermocycling conditions for the first round of PCR were as follows: each cycle consisted of denaturation for 30 s at 94 °C, annealing for 1 min at 50 °C and extension for 2 min at 72 °C for 30 cycles. The second round of PCR was performed with 5 % (v/v) of the first round mix. The thermocycling conditions for the second PCR round were as follows: each cycle consisted of denaturation for 30 s at 94 °C, annealing for 1 min at 55 °C and extension for 2 min at 72 °C for 30 cycles. For both PCR reactions, the initial denaturation was performed at 94 °C for 10 min and the final extension step at 72 °C for 20 min.
Negative controls were included in each experiment, in both DNA extraction and PCR amplification, to monitor for possible contamination. One negative control was tested per five experimental tubes. A laminar flow was used and PCR products were not manipulated in the 2 days preceding PCR reactions.
We selected some amplification products corresponding to long-carriage periods (for animals F14 and F17) or to periods where amplifications were positive for both mothers (F5 and F7) and their respective babies (F16, F19 and F18). These amplification products were purified in a 2 % agarose gel (Tris borate EDTA buffer) and extracted with PCR purification kit (Qiagen). Amplification products were directly sequenced from both ends using sets of internal primers on an automated DNA sequencer (GenomeExpress). The mtLSU sequences were aligned with already known Pneumocystis sequences using the computer program CLUSTAL X (version 1.63b) (Thompson et al., 1997).
When direct sequencing failed, positive amplification products were cloned with pGEM-T vector system II kit (Promega). Four separate colonies were selected from the transformant plates and examined for each positive sample. DNA extraction was performed by Wizard Plus minipreps DNA purification system kit (Promega). We used the primer set Sp6/T7 vector specific for the fragment size, and we used the primer set pAZ102-X/R1/pAZ102-Y/R1 (5'-GGGAATTCGTGAAATACAAATCGGACTAGG-3'/5'-GGGAATTCTCACTTAATATTAATTGGGGAGC-3') (Wakefield, 1996) for the fragment specificity. Thermocycling conditions were as follows: each cycle consisted of denaturation for 30 s at 94 °C, annealing for 1 min at 50 °C and extension for 1 min at 72 °C for 30 cycles. Amplification products were sequenced from both ends on an automated DNA sequencer (Qiagen).
Serum anti-Pneumocystis antibody detection.
Anti-Pneumocystis IgG titre was assessed on serum samples by indirect immuno-fluorescence assay (IFA). Antigen was a suspension of Pronase-treated rabbit-derived Pneumocystis organisms that was prepared as follows: infected rabbit lungs were cut into small pieces in Hanks' solution without Ca2+/Mg2+ and homogenized either with a magnetic stirrer (4 °C, 90 min) or squeezed through a stainless steel mesh. The homogenate was poured through gauze and centrifuged (2900 g, 10 min, 4 °C). The pellet was resuspended in a 0·08 % Pronase solution in Hanks' medium with Ca2+/Mg2+ and homogenized for 3 h at 37 °C under magnetic stirring. After incubation, the suspension was centrifuged and Pneumocystis organisms were washed three times with Hanks' medium without Ca2+/Mg2+. The pellet was resuspended in a known volume of Ca2+/Mg2+-free Hanks' medium in order to obtain a suspension of 106 Pneumocystis cysts ml1. Quantification of Pneumocystis cysts was performed on toluidine blue O (TBO) stained dry smears. Then, 10 µl of the suspension was spotted into each well of multiwell immuno-fluorescence slides (10 wells 5 mm black; lames epoxy noir, Polylabo). Slides were dried, fixed in cold acetone, wrapped individually in aluminium foil and stored at 20 °C until use (Soulez et al., 1989).
Macaque serum samples were diluted (1/200, 1/400 and 1/800) in PBS pH 7·2 (bioMerieux) and dropped on the antigen-fixed immuno-fluorescence slides. After incubation for 30 min at 37 °C, the reaction was revealed by using fluorescent-conjugated anti-monkey immunoglobulin antibody IgG() (Kirkegaard and Perry Laboratories). Titres of 1/200 or higher were considered positive (Soulez et al., 1989
).
Statistical analyses.
Carriage of Pneumocystis and antibodies were analysed separately at each month. The detection of antibodies was treated by presence (titre >1/200)absence (titre 1/200). In all cases, hypotheses were tested by using
2 tests on 2x2 table of contingency (Everitt, 1977
; Sokal & Rohlf, 1995
). Observed probability distributions were compared with the excepted distributions corresponding to the case where the stated null hypotheses would have been true. The first error type rate was fixed at 0·05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
For each animal, the apparent duration of Pneumocystis DNA carriage varied from less than 1 month (one positive amplification) to 6 months (positive amplifications on seven consecutive sampling dates for F14 and F17). For the longest carriage periods, the same sequence type (accession no. AY265385) (for F14 from November 2001 to April 2002) or closely related sequence types (for F17 from January to June 2002) were detected. Considering only consecutive amplifications, Pneumocystis DNA carriage duration averaged 2 months. All the monkeys were positive at least once during the study. No difference was observed according to the sex of the macaques (30·5 and 34·3 % positive samples from males and females, respectively). During the first year, Pneumocystis DNA was detected in 30 % of the samples from unweaned macaques, in 30·9 % of the samples from subadult macaques and 50·5 % of the samples from adults. The statistical analysis performed on adults, subadults and young showed that prevalence of Pneumocystis DNA detection tends to increase in association with the age of the animals. During the second year of the study eight births occurred, and the trend observed for the first year of the survey was found to be inverted: the proportion of animals with detectable Pneumocystis DNA was significantly higher in unweaned macaques (48·3 %) than in subadults (28·9 %) and adults (18·9 %). However, the proportion of unweaned macaques within the whole population was not correlated to the probability to carry fungal DNA within adults and subadults individuals. When Pneumocystis DNA was amplified from an unweaned macaque, parasite DNA was not systematically amplified from the corresponding mother, and when a positive amplification occurred from a mother, Pneumocystis DNA was not systematically detected in her baby. When a mother and her baby were shown to harbour Pneumocystis DNA, the Pneumocystis sequence type was not the same. In January 2002, a mtLSU sequence from group 2 was detected in the mother F5 (B966), whereas a mtLSU sequence from group 1 was observed in her baby F16 (Mf101). The same observation occurred in October 2002, in mother F7 (C971) (mtLSU sequence from group 1) and in her unweaned macaque F18 (Mf202) (mtLSU sequence from group 2) (Tables 1 and 2). The two babies, F14 (Mf001) and F17 (Mf102), of mother F4 (D823), harboured closely related Pneumocystis sequence types (from group 2) during the study, except in January 2002 when F17 was co-infected with Pneumocystis isolates corresponding to two distinct mtLSU sequence types from groups 1 and 2 (Table 2
).
Pneumocystis DNA detection in air samples
During the study, a total number of 11 air samples were collected. Using nested-PCR with primers pAZ102-H/pAZ102-E (first round) and pAZ102-X/R1/pAZ102-Y/R1 (second round), Pneumocystis DNA was not detected in these environmental samples.
Serum anti-Pneumocystis antibody assessment
A total number of 468 blood samples were collected. Anti-Pneumocystis antibody was detected in 238 samples (50·8 %). Titres remained quite stable throughout the study (Table 1). Seven monkeys were found seropositive (M1, F1, F2, F9, F10, F11 and F16). Two of them showed a high and constant rate of anti-Pneumocystis antibody (1/800) (F2 and F10). The remaining animals either were seronegative or had low levels of anti-Pneumocystis antibody (1/200) from time-to-time. Anti-Pneumocystis antibody was detected in monkeys younger than 1 year (F17, F16, M4 and M5). Seroconversion occurred in 1-year-old macaques F14 and M3 and in the 14-month-old macaque F15 (Table 1
).
Relationships between the presence of antibodies and the likelihood to carry Pneumocystis DNA
Throughout the study, anti-Pneumocystis IgG titres were less variable than results of Pneumocystis DNA amplification. There was no statistical difference in PCR amplification results between samples from seropositive and seronegative animals (Fig. 1). Similarly, no statistical difference was observed when the analysis was made independently on the following three categories: seronegative macaques (n=8), animals in which a seroconversion was detected during the study (n=13), and seropositive animals from the beginning to the end of the study (n=7). For instance, Pneumocystis DNA was detected in 10 nasal swab samples from the adult female F3 and F5, and in nine samples from adult females F8 (Table 1
), although these animals remained seronegative. Conversely, Pneumocystis DNA was detected in only three nasal samples from the seropositive female F10.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We first demonstrated a relatively high prevalence of Pneumocystis DNA in the upper respiratory tract of healthy macaques (33·6 % of PCR-positive nasal swabs). However, marked variations were observed in the number of animals with detectable Pneumocystis DNA during the experiment. Also, each month, a positive amplification occurred systematically from nasal samples from at least one animal in the colony. The mean duration for Pneumocystis DNA carriage in PCR-positive monkeys was relatively short (2 months). By using immunohistochemical methods to detect the presence of Pneumocystis in fixed lung tissues, Vogel et al. (1993) reported that latent Pneumocystis infection was uncommon in rhesus macaque. To account for the apparent discrepancy between our results and those from Vogel et al. (1993)
, it may be hypothesized that PCR-positive results occurred in transient carriers, which were not colonized by Pneumocystis organisms. Essentially, it was not clearly established if the finding of Pneumocystis DNA in nasal samples provided information about the actual presence of cysts or trophic forms in lung alveoli or whether these parasite stages were viable or infective if present. Recent experiments, however, showed that healthy BALB/c mice transiently colonized with Pneumocystis parasites were able to transmit them by an airborne route either to severe combined immunodeficiency (SCID) (Dumoulin et al., 2000
) or to BALB/c mice, which showed seroconversion (Chabé et al., 2004
; Gigliotti et al., 2003
). In addition, parasites were detected histologically in the lungs of healthy BALB/c mice that contracted their infection from SCID mice with pneumocystosis (Chabé et al., 2004
). What is more, in these experiments, healthy mice infected by the aerial way by co-housing with transiently infected healthy mice were able to transmit the infection to SCID mice (Chabé et al., 2004
; Gigliotti et al., 2003
).
In the present work, the detection of anti-Pneumocystis antibody may help the interpretation of PCR results. Throughout the study, two groups of macaques could be distinguished according to serological results. A first group included animals (n=7), which either were seronegative or showed fluctuating but low anti-Pneumocystis antibody titres, suggesting transient colonization or intermittent contact with an infective source. Monkeys of the second group (n=8) showed a constant level of anti-Pneumocystis antibody, which might indicate a durable colonization. However, the prevalence of Pneumocystis DNA detection was not higher in seropositive animals than in seronegative ones. This apparent divergence between PCR and serological results may indicate that high levels of anti-Pneumocystis antibody do not necessarily reveal colonization. An alternative explanation would be that the deep nasal sampling method used in this work was not invasive enough for the detection of Pneumocystis carriage.
In human communities, infants could constitute a major infectious reservoir for Pneumocystis organisms. Pneumocystis DNA has been frequently detected in nasopharyngeal aspirates from immunocompetent infants (Nevez et al., 2001; Vargas et al., 2001
), suggesting that colonization may occur at higher rates in healthy children than in healthy adults. Similar observations were made in different animal species. In wild rabbits, positive amplification systematically occurred with samples collected from 1-month-old or younger animals (Guillot et al., 1999a
). A large retrospective study concerning Pneumocystis infection in pigs indicated that animals from herds where adult and young pigs shared the same air space were more heavily infected than those from herds in which adults and weaners were reared separately (Kondo et al., 2000
). In the present study, the proportion of animals with detectable Pneumocystis DNA was significantly higher in young macaques but only during the second year when eight births occurred. The detection of antibodies to Pneumocystis in the serum of the unweaned macaque suggested that seroconversion took place very early in life. However, when positive amplification occurred from unweaned macaque samples, Pneumocystis DNA was not systematically detected in the mother samples. When mothers and their unweaned monkeys were PCR-positive their respective strains did not correspond to the same sequence type, excluding a possible transmission from the young macaques to their mothers and conversely.
Many observations suggested that infection from undefined environmental sources of Pneumocystis organisms was possible (Hughes, 1982). In humans, the risk of pneumocystosis has been linked to the degree of soil exposure (Navin et al., 2000
). Wakefield (1996)
was able to detect DNA from rat- and human-derived Pneumocystis in air samples from rural locations in the UK. This result suggested that Pneumocystis organisms might be current components of the air spora. In the present study, we found a constant circulation of Pneumocystis organisms within the members of the colony but Pneumocystis DNA could not be detected in air. Reasons for this could be that the time sampling was probably too short (168 h vs 240 h in the study from Wakefield, 1996
) or that the device for air sampling was not used at convenient places.
In conclusion, we used an original model for the study of Pneumocystis transmission in a group of non-immunocompromised animals. Throughout the study, the presence of Pneumocystis DNA was frequently detected from nasal swab samples. Anti-Pneumocystis antibodies were also detected from sera but serological titres could not be clearly correlated with the detection of Pneumocystis DNA in the upper respiratory tract. These results indicated a constant and intensive circulation of Pneumocystis organisms within the community. However, both occurrence of Pneumocystis carriage and colonization level varied significantly throughout the study in the members of this colony of apparently immunocompetent monkeys. DNA sequencing demonstrated that closely related animals (unweaned monkeys and their mothers) frequently harboured different Pneumocystis strains. This result does not contradict the hypothesis that young animals may represent a source of infection in a community of immunocompetent individuals. However, other infection sources could operate during the follow-up of the colony. Further studies including quantification of Pneumocystis DNA, and a more discriminative genotyping of isolates from monkeys should clarify this complex picture and help to elucidate both routes of transmission and carriage of Pneumocystis in non-human primates.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartlett, M. S., Lu, J. J., Lee, C. H., Durant, P. J., Queener, S. F. & Smith, J. W. (1996). Types of Pneumocystis carinii detected in air samples. J Eukaryot Microbiol 43, 44S.[Medline]
Ceré, N. & Polack, B. (1999). Animal pneumocystosis: a model for man. Vet Res 30, 126.[Medline]
Chabé, M., Dei-Cas, E., Creusy, C., Fleurisse, L., Respaldiza, N., Camus, D. & Durand-Joly, I. (2004). Immunocompetent hosts as a reservoir of Pneumocystis organisms: histological and RT-PCR data demonstrate active replication. Eur J Clin Microbiol Infect Dis 23, 8997.[CrossRef][Medline]
Cushion, M. (1998). Chapter 34: Pneumocystis carinii. In Topley and Wilson's Microbiology and Microbial Infections, vol. 4 Mycology, 9th edn, pp. 645683. Edited by L. Ajello & R. J. Hay. London: Arnold.
Cushion, M. T. (2004). Pneumocystis: unraveling the cloak of obscurity. Trends Microbiol 12, 243249.[CrossRef][Medline]
Dei-Cas, E., Mazars, E. E., Aliouat, E. M., Nevez, G., Cailliez, J. C. & Camus, D. (1998). The host-specificity of Pneumocystis carinii. J Mycol Méd 8, 16.
Demanche, C. Berthelemy M., Petit, T., Polack, B., Dei-Cas, E., Wakefield, A. E., Dei-Cas, E. & Guillot, J. (2001). Phylogeny of Pneumocystis carinii from 18 primate species confirms host specificity and suggests coevolution. J Clin Microbiol 39, 21262133.
Demanche, C., Wanert, F., Herrenshmidt, N., Moussu, C., Durand-Joly, I., Dei-Cas, E., Chermette, R. & Guillot, J. (2003). Influence of climatic factors on Pneumocystis carriage within a socially organized group of immunocompetent macaques (Macaca fascicularis). J Eukaryot Microbiol 50, S611S613.[CrossRef]
Dumoulin, A., Mazars, E., Seguy, N., Gargallo-Viola, D., Vargas, S., Cailliez, J. C., Aliouat, E. M., Wakefield, A. E. & Dei-Cas, E. (2000). Transmission of Pneumocystis carinii disease from immunocompetent contacts of infected hosts to susceptible hosts. Eur J Clin Microbiol Infect Dis 19, 671678.[CrossRef][Medline]
Everitt, B. S. (1977). The Analysis of Contingency Tables. New York: Chapman & Hall.
Gigliotti, F., Harmsen, A. G. & Wright, T. W. (2003). Characterization of transmission of Pneumocystis carinii f. sp. muris through immunocompetent BALB/c mice. Infect Immun 71, 38523856.
Guillot, J., Chevalier, V., Queney, G., Berthelemy, M., Polack, B., Lacube, P., Roux, P. & Chermette, R. (1999a). Acquisition and biodiversity of Pneumocystis carinii in a colony of wild rabbits (Oryctolagus cuniculus). J Eukaryot Microbiol 46, 100S101S.
Guillot, J., Berthelemy, M., Polack, B., Lainé, V., Lacube, P., Chermette, R. & Roux, P. (1999b). Impaction versus filtration for the detection of Pneumocystis carinii DNA in air. J Eukaryot Microbiol 46, 94S.[Medline]
Guillot, J., Demanche, C., Hugot, J. P., Berthelemy, M., Wakefield, A. E., Dei-Cas, E. & Chermette, R. (2001). Parallel phylogenies of Pneumocystis species and their mammalian hosts. J Eukaryot Microbiol (Suppl), 113S115S.
Guillot, J., Demanche, C., Norris, K., Wildschutte, H., Wanert, F., Berthelemy, M., Tataine, S. E., Dei-Cas, E. & Chermette, R. (2004). Phylogenetic relationships among Pneumocystis from Asian macaques inferred from mitochondrial rRNA sequences. Mol Phylogenet Evol 31, 988996.[CrossRef][Medline]
Helweg-Larsen, J., Tsolaki, A. G., Miller, R. F., Lundgren, B. & Wakefield, A. E. (1998). Clusters of Pneumocystis carinii pneumonia: analysis of person-to-person transmission by genotyping. Q J Med 91, 813820.
Hendley, J. O. & Weller, T. H. (1971). Activation and transmission in rats of infection with Pneumocystis. Proc Soc Exp Biol Med 137, 14011404.
Hennequin, C., Page, B., Roux, P., Legendre, C. & Kreis, H. (1995). Outbreak of Pneumocystis carinii pneumonia in a renal transplant unit. Eur J Clin Microbiol Infect Dis 14, 122126.[Medline]
Hocker, B., Wendt, C., Nahimana, A., Tonshoff, B. & Hauser, P. M. (2005). Molecular evidence of Pneumocystis transmission in pediatric transplant unit. Emerg Infect Dis 11, 330332.[Medline]
Hughes, W. T. (1982). Natural mode of acquisition for de novo infection with Pneumocystis carinii. J Infect Dis 145, 842848.[Medline]
Hugot, J. P., Demanche, C., Barriel, V., Dei-Cas, E. & Guillot, J. (2003). Phylogenetic systematics and evolution of primate-derived Pneumocystis based on mitochondrial or nuclear DNA sequences comparison. Syst Biol 52, 735744.[CrossRef][Medline]
Kondo, H., Hikita, M., Ito, M. & Kadota, K. (2000). Immunohistochemical study of Pneumocystis carinii infection in pigs: evaluation of Pneumocystis pneumonia and a retrospective investigation. Vet Rec 147, 544549.
Miller, R. F., Ambrose, H. E. & Wakefield, A. E. (2001). Pneumocystis carinii f. sp. hominis DNA in immunocompetent health care workers in contact with patients with P. carinii pneumonia. J Clin Microbiol 39, 38773882.
Morris, A., Lundgren, J. D., Masur, H., Walzer, P. D., Hanson, D. L., Frederick, T., Huang, L., Beard, C. B. & Kaplan, J. E. (2004). Current epidemiology of Pneumocystis pneumonia. Emerg Infect Dis 10, 17131720.[Medline]
Navin, T. R., Rimland, D., Lennox, J. L., Jernigan, J., Cetron, M., Hightower, A., Roberts, J. M. & Kaplan, J. E. (2000). Risk factors for community-acquired pneumonia among persons infected with human immunodeficiency virus. J Infect Dis 181, 158164.[CrossRef][Medline]
Nevez, G., Totet, A., Pautard, J. C. & Raccurt, C. (2001). Pneumocystis carinii detection using nested-PCR in nasopharyngeal aspirates of immunocompetent infants with bronchiolitis. J Eukaryot Microbiol (Suppl), 122S123S.
Olsson, M., Sukura, A., Lindberg, L. A. & Linder, E. (1996). Detection of Pneumocystis carinii DNA by filtration of air. Scand J Infect Dis 28, 279282.[Medline]
Rabodonirina, M., Vanhems, P., Couray-Targe, S. & 10 other authors (2004). Molecular evidence of interhuman transmission of Pneumocystis pneumonia among renal transplant recipients hospitalized with HIV-infected patients. Emerg Infect Dis 10, 17661773.[Medline]
Singer, C., Armstrong, D., Rosen, P. P. & Schottenfeld, D. (1975). Pneumocystis carinii pneumonia: a cluster of eleven cases. Ann Intern Med 82, 772777.[Medline]
Sokal, R. R. & Rohlf, F. J. (1995). Biometry: the Principles and Practice of Statistics in Biological Research. New York: Freeman & Company.
Soulez, B., Dei-Cas, E., Charet, P., Mougeot, G., Caillaux, M. & Camus, D. (1989). The young rabbit: a non-immunosuppressed model for Pneumocystis carinii pneumonia. J Infect Dis 160, 355356.[Medline]
Soulez, B., Palluault, F., Cesbron, J. Y., Dei-Cas, E., Capron, A. & Camus, D. (1991). Introduction of Pneumocystis carinii in a colony of SCID mice. J Protozool 38, S123S125.
Thompson, J. D., Gibson, T. S., Plewniak, F., Jeanmougin, F. & Higgings, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 48764882.
Tsolaki, A. G., Miller, R. F. & Wakefield, A. E. (1999). Oropharyngeal samples for genotyping and monitoring response to treatement in AIDS patients with Pneumocystis carinii pneumonia. J Med Microbiol 48, 897905.[Abstract]
Vargas, S. L., Ponce, C. A., Gigliotti, F., Ulloa, A. V., Prieto, S., Muñoz, M. P. & Hughes, W. T. (2000). Transmission of Pneumocystis carinii DNA from a patient with P. carinii pneumonia to immunocompetent contact health care workers. J Clin Microbiol 38, 15361538.
Vargas, S. L., Hughes, W. T., Santolaya, M. E., Ulloa, A. V., Ponce, C. A., Cabrera, C. E., Cumsille, F. & Gigliotti, F. (2001). Search for primary infection by Pneumocystis carinii in a cohort of normal, healthy infants. Clin Infect Dis 32, 855861.[CrossRef][Medline]
Vogel, P., Miller, C. J., Lowenstine, L. L. & Lackner, A. A. (1993). Evidence of horizontal transmission of Pneumocystis carinii pneumonia in simian immunodeficiency virus-infected rhesus macaques. J Infect Dis 168, 836843.[Medline]
Wakefield, A. E. (1996). DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora. J Clin Microbiol 34, 17541759.[Abstract]
Wakefield, A. E. (1998). Genetic heterogeneity in Pneumocystis carinii: an introduction. FEMS Immunol Med Microbiol 22, 513.[CrossRef][Medline]
Wakefield, A. E., Pixley, F. J., Banerji, S., Sinclair, K., Miller, R. F., Moxon, E. R. & Hopkin, J. M. (1990). Detection of Pneumocystis carinii with DNA amplification. Lancet 336, 451453.[CrossRef][Medline]
Received 22 March 2005;
revised 6 June 2005;
accepted 13 June 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |