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Andrew H. Limper, MD, FCCP
Author and Funding Information

From the Division of Pulmonary and Critical Care Medicine, Mayo Clinic College of Medicine.

Correspondence to: Andrew H. Limper, MD, FCCP, Division of Pulmonary and Critical Care Medicine, Mayo Clinic, 8-24 Stabile Bldg, 200 First St, Rochester, MN 55905; e-mail: limper.andrew@mayo.edu


Financial/nonfinancial disclosures: The author has reported to CHEST the following conflicts: Dr Limper is coinventor of the pjcdc2 diagnostic PCR assay discussed in this report and holds a patent on this method.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2014;145(3):664. doi:10.1378/chest.13-2747
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To the Editor:

I thank Dr Farkas and colleagues for their interest in our study on the potential risks of developing Pneumocystis pneumonia (PcP) in patients with compromised immune systems receiving rituximab.1 The letter raises the important question about whether the three patients reported in the case series might actually represent colonization rather than PcP. This question arose because the cases were detected by polymerase chain reaction (PCR) assay. We believe that these cases represent true PcP rather than colonization; thus, further clarification is needed.

Many PCR assays reported in the literature amplify mitochondrial DNA sequences.2 Hence, they amplify multicopy Pneumocystis target genes, significantly heightening detection sensitivity. In addition, these assays often rely on nested PCR approaches where the initial amplification products are subsequently reamplified, yielding even greater sensitivity.3 Indeed, such approaches may be overly sensitive for many routine clinical applications. Accordingly, previously reported PCR assay approaches are known to detect Pneumocystis colonization in addition to invasive infections.4 However, the Pneumocystis PCR assay used in our clinical microbiology laboratory has unique features that circumvent many of these issues.5 To address the issues of colonization, we use a diagnostic Pneumocystis PCR assay that uses a single-copy target gene from Pneumocystis jirovecii, namely pjcdc2.5 We also perform quantitative amplification with real-time PCR and do not reamplify the reaction products with nested PCR.5 With this PCR assay, we have observed an increase (approximately 7%) in total diagnostic sensitivity over smeared stains alone. In addition, in now > 200 consecutive cases without clinical evidence of infection, we have not detected a PCR signal. Hence, the assay used in this study was specifically designed to detect PcP rather than colonization.5 On this basis, our laboratory now routinely uses this PCR assay rather than microscopic examination for the diagnosis of Pneumocystis infection. Moreover, it must be noted that the patients in the current report had active lung infiltration as well as signs and symptoms of infection. In each patient, there was no evidence of any other significant alternative organism, despite routine comprehensive culture, antigen detection, and molecular diagnostic assays performed to rigorously detect typical and atypical bacteria, viruses, and other fungi. Thus, in all three patients, the clinical evidence pointed to active PcP rather than to colonization.

References

Martin-Garrido I, Carmona EM, Specks U, Limper AH. Pneumocystispneumonia in patients treated with rituximab. Chest. 2013;144(1):258-265. [CrossRef] [PubMed]
 
Ribes JA, Limper AH, Espy MJ, Smith TF. PCR detection ofPneumocystis cariniiin bronchoalveolar lavage specimens: analysis of sensitivity and specificity. J Clin Microbiol. 1997;35(4):830-835. [PubMed]
 
Krajicek BJ, Thomas CF Jr, Limper AH. Pneumocystispneumonia: current concepts in pathogenesis, diagnosis, and treatment. Clin Chest Med. 2009;30(2):265-278. [CrossRef] [PubMed]
 
Morris A, Sciurba FC, Lebedeva IP, et al. Association of chronic obstructive pulmonary disease severity andPneumocystiscolonization. Am J Respir Crit Care Med. 2004;170(4):408-413. [CrossRef] [PubMed]
 
Wilson JW, Limper AH, Grys TE, Karre T, Wengenack NL, Binnicker MJ. Pneumocystis jiroveciitesting by real-time polymerase chain reaction and direct examination among immunocompetent and immunosuppressed patient groups and correlation to disease specificity. Diagn Microbiol Infect Dis. 2011;69(2):145-152. [CrossRef] [PubMed]
 

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References

Martin-Garrido I, Carmona EM, Specks U, Limper AH. Pneumocystispneumonia in patients treated with rituximab. Chest. 2013;144(1):258-265. [CrossRef] [PubMed]
 
Ribes JA, Limper AH, Espy MJ, Smith TF. PCR detection ofPneumocystis cariniiin bronchoalveolar lavage specimens: analysis of sensitivity and specificity. J Clin Microbiol. 1997;35(4):830-835. [PubMed]
 
Krajicek BJ, Thomas CF Jr, Limper AH. Pneumocystispneumonia: current concepts in pathogenesis, diagnosis, and treatment. Clin Chest Med. 2009;30(2):265-278. [CrossRef] [PubMed]
 
Morris A, Sciurba FC, Lebedeva IP, et al. Association of chronic obstructive pulmonary disease severity andPneumocystiscolonization. Am J Respir Crit Care Med. 2004;170(4):408-413. [CrossRef] [PubMed]
 
Wilson JW, Limper AH, Grys TE, Karre T, Wengenack NL, Binnicker MJ. Pneumocystis jiroveciitesting by real-time polymerase chain reaction and direct examination among immunocompetent and immunosuppressed patient groups and correlation to disease specificity. Diagn Microbiol Infect Dis. 2011;69(2):145-152. [CrossRef] [PubMed]
 
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