Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sing, A. Articles by Heesemann, J. Search for Related Content PubMed PubMed Citation Articles by Sing, A. Articles by Heesemann, J.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 1999, p. 3409-3410, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Pneumocystis carinii Carriage in Immunocompetent Patients with Primary Pulmonary Disorders as Detected by Single or Nested PCR

Andreas Sing,^* Andreas Roggenkamp, Ingo B. Autenrieth, and Jürgen Heesemann

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Munich, Germany

Received 14 April 1999/Returned for modification 15 May 1999/Accepted 2 July 1999

	ABSTRACT

Top Abstract Text References

Ninety-five bronchoalveolar lavage specimens from 63 immunocompetent adult patients with primary pulmonary disease were analyzed for Pneumocystis carinii colonization by primary and nested PCR. Twelve of 63 patients (19%) were PCR positive. None of them developed P. carinii pneumonia. These results suggest that P. carinii carriage may exist in immunocompetent patients with underlying pulmonary disease.

	TEXT

Top Abstract Text References

Pneumocystis carinii is an opportunistic pathogen causing serious and even life-threatening pneumonia (P. carinii pneumonia [PCP]) in immunosuppressed patients. PCP is speculated to result either from a de novo infection or from reactivation of a latent childhood infection. Seroconversion usually happens during early childhood, leading to high rates of seroprevalence (9). Autopsy studies using microscopy or immunofluorescence, however, revealed no evidence or only a very low rate of prevalence of P. carinii (less than 1%) in adults without predisposing diseases (6, 7, 10). More-sensitive methods like PCR may be able to detect even low numbers of P. carinii organisms in clinically silent, but colonized, persons. It is conceivable that lung tissue already damaged by different conditions such as bacterial pneumonia, fibrosis, and chronic obstructive pulmonary disease is at increased risk for P. carinii colonization. In an autopsy study using PCR, however, no P. carinii DNA could be isolated (8).

To evaluate the prevalence of P. carinii colonization in immunocompetent patients with primary pulmonary diseases, 95 bronchoalveolar lavage (BAL) specimens of 63 immunocompetent patients (21 females, 42 males; median age, 53 years [Table 1]) suffering from primary acute (37 patients) or chronic (26 patients) lung disease were examined microscopically and by primary and nested PCRs. Inclusion criteria were the absence of underlying malignant disease (except bronchial carcinoma), systemic disease, and human immunodeficiency virus (HIV) infection; no use of immunosuppressive or cytotoxic medication; and normal immunological function. None of the patients had received antipneumocystic chemoprophylaxis.

View this table: [in this window] [in a new window]   TABLE 1.   Characteristics of 63 immunocompetent patients with primary pulmonary disease

Clinical specimens were centrifuged at 4,000 rpm for 10 min in an Omnifuge (2.0 RS; Heraeus, Munich, Germany). Portions of the pellets were smeared on slides and Giemsa and Grocott stained for microscopy. The other parts of the pellets were stored at 20°C until PCR analysis. Following proteinase K digestion, DNA was extracted with a Qiagen (Hilden, Germany) tissue kit. A two-step protocol with the external primers pAZ 102E and pAZ 102H (11) and the nested primers pLE1 and pLE2 (12) was applied as described elsewhere (12). Products of both primary and nested PCRs were investigated by agarose gel electrophoresis, stained with ethidium bromide, and analyzed under UV light. Precautions against contamination included the use of aerosol barrier pipette tips and the performance of the steps of the procedure (master mix preparation, DNA extraction, PCR, and specimen detection) in separate rooms. Several positive (from BAL specimens of PCP patients) and negative (autoclaved water and the PCR mixture minus the DNA template) controls were tested simultaneously. All experiments were performed at least twice. Nested-PCR-positive samples were identified as P. carinii specific following DNA sequencing of the amplicons and sequence comparison with the BLAST program (1). To further assess the specificities of the PCR primers, the nested PCR was performed on human and Candida albicans DNAs, yielding negative results.

With the BAL specimens of 3 of the 63 patients, both primary and nested PCR yielded a positive result, while with the specimens of 9 patients, only the more sensitive nested PCR could detect P. carinii-specific DNA. In none of the 12 PCR-positive specimens could P. carinii organisms be visualized microscopically. The characteristics of the 12 PCR-positive patients are summarized in Table 2. None of them presented with PCP according to criteria of the Centers for Disease Control and Prevention (4) or received antipneumocystic chemoprophylaxis and/or therapy. They either improved with adequate treatment aimed at the underlying pulmonary disease or were not curable. In the latter cases, pulmonary material obtained intra vitam or at autopsy did not justify assignment of the cause of death to PCP. Improved patients did not develop PCP within the follow-up period indicated in Table 2. Of the 12 patients with PCR-positive BAL specimens, 6 suffered from chronic pulmonary disease, with preexisting long-term lung tissue damage, and 6 were afflicted with more acute respiratory diseases of both infectious and noninfectious natures.

View this table: [in this window] [in a new window]   TABLE 2.   Characteristics of 12 immunocompetent patients with primary pulmonary disease and P. carinii colonization as detected by single and nested PCRs

The percentage of PCR-positive patients among the 63 immunocompetent patients without clinical PCP, 19%, is relatively high. In a study of 50 patients with chronic bronchial disease, 10% were found to be P. carinii positive by sputum sampling (3). However, it was not stated whether P. carinii contributed to or even was responsible for pulmonary disease in these patients. Moreover, P. carinii carriage in these patients was defined by the simultaneous determination of positivity by four techniques less sensitive than PCR with sputum samples, which usually yield fewer positive results than more invasively obtained material. These results might suggest at least some pathological role for P. carinii in these patients. Furthermore, three of the five P. carinii patients had received inhalative corticoid therapy, which may have led to some degree of local immunosuppression among elderly patients. Therefore, a higher rate of prevalence might have been found in this study by a more sensitive PCR on material from deeper airways.

An even lower rate of prevalence of P. carinii carriage was found by Armbruster et al. (2). In 5 of 77 (6.5%) HIV-negative immunocompetent patients with acute respiratory illness P. carinii colonization was detected by immunofluorescence and PCR of microscopically negative BAL specimens. However, underlying pulmonary disease in four of these five patients was a rather short-term condition. In contrast, the P. carinii DNA-positive patients described here were more equally distributed among those with acute and those with chronic underlying pulmonary diseases, possibly explaining the higher level of prevalence of P. carinii carriage in our study population.

A level of prevalence of P. carinii colonization similar to that found in our study was found among immunocompetent HIV-negative children with chronic respiratory disorders (5). Seven of 28 (25%) patients had PCR-positive nasopharyngeal aspirates. In at least five of them, P. carinii may have contributed to pulmonary disease since antipneumocystic therapy led to clinical and microbiological improvement. Those authors concluded that the pathologic changes in chronic lung diseases might permit colonization of P. carinii and that the pathogenic role of P. carinii in exacerbations of chronic lung diseases needs further clarification.

In contrast, our study suggests a less severe pathogenic effect of P. carinii in immunocompetent patients with underlying pulmonary diseases, as the 12 P. carinii-colonized patients either recovered without antipneumocystic therapy or died from non-PCP-related diseases. None of the seven P. carinii-colonized patients not lost to follow-up developed PCP in a mean period of 5 months.

In conclusion, we found a relatively high percentage of P. carinii DNA-positive BAL specimens among immunocompetent patients with primary respiratory diseases, suggesting that lung tissue damage may favor P. carinii colonization. The pathogenic impact of colonization on immunocompetent patients, however, may not be significant as long as the immunostatus of the patient has not deteriorated dramatically. In this scenario, reactivation might occur. In addition, it can be speculated that patients with underlying pulmonary disease might serve as carrier reservoirs for P. carinii.

	ACKNOWLEDGMENTS

We thank Karin Tybus and Friederike Pfaff for expert technical assistance.

	FOOTNOTES

^* Corresponding author. Mailing address: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Pettenkoferstraße 9a, 80336 Munich, Germany. Phone: 49-89-5160-5293. Fax: 49-89-5160-5223. E-mail: sing{at}m3401.mpk.med.uni-muenchen.de.

	REFERENCES

Top Abstract Text References

1.	Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402[Abstract/Free Full Text].
2.	Armbruster, C., A. Hassl, and S. Kriwanek. 1997. Pneumocystis carinii colonization in the absence of immunosuppression. Scand. J. Infect. Dis. 29:591-593[Medline].
3.	Calderon, E. J., C. Regordan, F. J. Medrano, M. Ollero, and J. M. Varela. 1996. Pneumocystis carinii infection in patients with chronic bronchial disease. Lancet 347:977[Medline].
4.	Centers for Disease Control and Prevention. 1992. Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Morbid. Mortal. Weekly Rep. 41(RR-17):1-19.
5.	Contini, C., M. P. Villa, R. Romani, R. Merolla, S. Delia, and R. Ronchetti. 1998. Detection of Pneumocystis carinii among children with chronic respiratory disorders in the absence of HIV infection and immunodeficiency. J. Med. Microbiol. 47:329-333[Abstract/Free Full Text].
6.	Esterly, J. A. 1968. Pneumocystis carinii in lungs of adults at autopsy. Am. Rev. Respir. Dis. 97:935-937[Medline].
7.	Millard, P. R., and A. R. Heryet. 1988. Observations favouring Pneumocystis carinii pneumonia as a primary infection: a monoclonal antibody study on paraffin sections. J. Pathol. 154:365-370[Medline].
8.	Peters, S. E., A. E. Wakefield, K. Sinclair, P. R. Millard, and J. M. Hopkin. 1992. A search for Pneumocystis carinii in post-mortem lungs by DNA amplification. J. Pathol. 166:195-198[Medline].
9.	Pifer, L. L., W. T. Hughes, S. Stagno, and D. Woods. 1978. Pneumocystis carinii infection: evidence for high prevalence in normal and immunosuppressed children. Pediatrics 61:35-41[Abstract/Free Full Text].
10.	Vogel, C. L., M. H. Cohen, R. D. Powell, Jr., and V. T. DeVita. 1968. Pneumocystis carinii pneumonia. Ann. Intern. Med. 68:97-108.
11.	Wakefield, A. E., F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin. 1990. Detection of Pneumocystis carinii with DNA amplification. Lancet 336:451-453[Medline].
12.	Weig, M., H. Klinker, M. Wilhelm, K. Lemmer, and U. Gross. 1996. Correlation of Pneumocystis carinii PCR with clinical diagnosis in immunocompromised patients. Lancet 347:1266[Medline].

---

Journal of Clinical Microbiology, October 1999, p. 3409-3410, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Azoulay, E., Bergeron, A., Chevret, S., Bele, N., Schlemmer, B., Menotti, J. (2009). Polymerase Chain Reaction for Diagnosing Pneumocystis Pneumonia in Non-HIV Immunocompromised Patients With Pulmonary Infiltrates. Chest 135: 655-661 [Abstract] [Full Text]  
• Schmoldt, S., Schuhegger, R., Wendler, T., Huber, I., Sollner, H., Hogardt, M., Arbogast, H., Heesemann, J., Bader, L., Sing, A. (2008). Molecular Evidence of Nosocomial Pneumocystis jirovecii Transmission among 16 Patients after Kidney Transplantation. J. Clin. Microbiol. 46: 966-971 [Abstract] [Full Text]  
• Tasaka, S., Hasegawa, N., Kobayashi, S., Yamada, W., Nishimura, T., Takeuchi, T., Ishizaka, A. (2007). Serum Indicators for the Diagnosis of Pneumocystis Pneumonia. Chest 131: 1173-1180 [Abstract] [Full Text]  
• Huang, L., Morris, A., Limper, A. H., Beck, J. M., on behalf of the ATS Pneumocystis Workshop Partici, (2006). An Official ATS Workshop Summary: Recent Advances and Future Directions in Pneumocystis Pneumonia (PCP). Proc Am Thorac Soc 3: 655-664 [Abstract] [Full Text]  
• Linssen, C. F. M., Jacobs, J. A., Beckers, P., Templeton, K. E., Bakkers, J., Kuijper, E. J., Melchers, W. J. G., Drent, M., Vink, C. (2006). Inter-laboratory comparison of three different real-time PCR assays for the detection of Pneumocystis jiroveci in bronchoalveolar lavage fluid samples.. J Med Microbiol 55: 1229-1235 [Abstract] [Full Text]  
• Miller, R F, Lindley, A R, Copas, A, Ambrose, H E, Davies, R J O, Wakefield, A E (2005). Genotypic variation in Pneumocystis jirovecii isolates in Britain. Thorax 60: 679-682 [Abstract] [Full Text]  
• Miller, R, Huang, L (2004). Pneumocystis jirovecii infection. Thorax 59: 731-733 [Full Text]  
• Morris, A., Sciurba, F. C., Lebedeva, I. P., Githaiga, A., Elliott, W. M., Hogg, J. C., Huang, L., Norris, K. A. (2004). Association of Chronic Obstructive Pulmonary Disease Severity and Pneumocystis Colonization. Am. J. Respir. Crit. Care Med. 170: 408-413 [Abstract] [Full Text]  
• Flori, P., Bellete, B., Durand, F., Raberin, H., Cazorla, C., Hafid, J., Lucht, F., Sung, R. T. M. (2004). Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens. J Med Microbiol 53: 603-607 [Abstract] [Full Text]  
• Totet, A., Duwat, H., Magois, E., Jounieaux, V., Roux, P., Raccurt, C., Nevez, G. (2004). Similar genotypes of Pneumocystis jirovecii in different forms of Pneumocystis infection. Microbiology 150: 1173-1178 [Abstract] [Full Text]  
• Maskell, N A, Waine, D J, Lindley, A, Pepperell, J C T, Wakefield, A E, Miller, R F, Davies, R J O (2003). Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study. Thorax 58: 594-597 [Abstract] [Full Text]  
• Totet, A., Pautard, J.-C., Raccurt, C., Roux, P., Nevez, G. (2003). Genotypes at the Internal Transcribed Spacers of the Nuclear rRNA Operon of Pneumocystis jiroveci in Nonimmunosuppressed Infants without Severe Pneumonia. J. Clin. Microbiol. 41: 1173-1180 [Abstract] [Full Text]  
• Turner, D., Schwarz, Y., Yust, I. (2003). Induced sputum for diagnosing Pneumocystis carinii pneumonia in HIV patients: new data, new issues. Eur Respir J 21: 204-208 [Abstract] [Full Text]  
• Nevez, G., Totet, A., Jounieaux, V., Schmit, J.-L., Dei-Cas, E., Raccurt, C. (2003). Pneumocystis jiroveci Internal Transcribed Spacer Types in Patients Colonized by the Fungus and in Patients with Pneumocystosis from the Same French Geographic Region. J. Clin. Microbiol. 41: 181-186 [Abstract] [Full Text]  
• Larsen, H. H., Masur, H., Kovacs, J. A., Gill, V. J., Silcott, V. A., Kogulan, P., Maenza, J., Smith, M., Lucey, D. R., Fischer, S. H. (2002). Development and Evaluation of a Quantitative, Touch-Down, Real-Time PCR Assay for Diagnosing Pneumocystis carinii Pneumonia. J. Clin. Microbiol. 40: 490-494 [Abstract] [Full Text]  
• 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: 3877-3882 [Abstract] [Full Text]  
• Sing, A., Geiger, A. M., Hogardt, M., Heesemann, J. (2001). Pneumocystis carinii Carriage among Cystic Fibrosis Patients, as Detected by Nested PCR. J. Clin. Microbiol. 39: 2717-2718 [Abstract] [Full Text]  
• Stringer, J. R., Keely, S. P. (2001). Genetics of Surface Antigen Expression in Pneumocystis carinii. Infect. Immun. 69: 627-639 [Abstract] [Full Text]  
• Palmer, R. J., Settnes, O. P., Lodal, J., Wakefield, A. E. (2000). Population Structure of Rat-Derived Pneumocystis carinii in Danish Wild Rats. Appl. Environ. Microbiol. 66: 4954-4961 [Abstract] [Full Text]  
• Visconti, E., Marinaci, S., Zolfo, M., Mencarini, P., Tamburrini, E., Pagliari, G., Ortona, E., Siracusano;, A., Sing, A. (2000). Very Low Frequence of Pneumocystis carinii DNA Detection by PCR in Specimens from Patients with Lung Damage. J. Clin. Microbiol. 38: 1307-1308 [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sing, A. Articles by Heesemann, J. Search for Related Content PubMed PubMed Citation Articles by Sing, A. Articles by Heesemann, J.