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Journal of Clinical Microbiology, March 2000, p. 1307-1308, Vol. 38, No. 3
0095-1137/00/$04.00+0
LETTERS TO THE EDITOR
Very Low Frequence of Pneumocystis carinii DNA
Detection by PCR in Specimens from Patients with Lung Damage
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LETTER |
Data reported by Dr. Sing et al. (3) about
Pneumocystis carinii carriage in immunocompetent adults with
primary pulmonary disorders differ partially from our results. We have
analyzed 81 bronchoalveolar lavage fluid samples (BALs) from 78 patients (37 male and 41 female; mean age, 57.8 years; range, 35 to 84 years) who underwent bronchoscopy from November 1997 to June 1998. Forty-four patients had experienced a worsening of their chronic obstructive lung disease (COLD), 15 patients had suspected pulmonary carcinoma (definitive diagnosis of lung neoplasm in 10 cases), and 18 patients had a respiratory syndrome with abnormal chest X ray
(bacterial pneumonia in 15 cases and mycobacterial infection in 3 cases). All samples were directly treated for microbiological and
cytological analysis; we tested for P. carinii by indirect immunofluorescence (IF) assay and by PCR. Twenty-microliter samples of
BAL were digested with proteinase K and then amplified; a nested PCR
was performed using mitochondrial LSU region primers (5). All experiments were repeated at least three times, and multiple negative controls were included in each amplification run.
All specimens were negative for P. carinii by IF assay and
upon the first amplification; only two samples (2.5%) were positive for P. carinii by nested PCR. The definitive diagnoses for
the two patients who supplied these samples were, respectively, COLD and microcitoma. The patients were not receiving corticoid therapy or
anti-P. carinii prophylaxis or therapy, and they did not
develop P. carinii pneumonia (PCP) within an 18-month
surveillance period.
None of the specimens in our study were positive for P. carinii after the first amplification. In our experience PCP is
defined by an acute respiratory syndrome that is confirmed by positive morphological staining or a positive result upon the first cycle of
amplification, and under any of these conditions anti-P.
carinii treatment is necessary for the recovery of the patients;
moreover, previous data (2) have demonstrated that
positivity upon the first amplification is consistent with the
diagnosis of PCP.
Only 2.5% of our samples were P. carinii positive by nested
PCR compared to the 19% described by Dr. Sing. We hypothesize that
some technical differences between our methods exist. We did not
perform DNA extraction, but samples were digested with proteinase K
without any loss in sensitivity, as previously reported (4).
Our nested PCR was performed with different internal primers, but both
primer pairs belong to the mtLSU-rRNA region, and it seems unlikely
that there would be a different sensitivity. Moreover, our nested PCR
showed a sensitivity of 100% and a specificity of 62% in the
diagnosis of PCP from BALs of human immunodeficiency virus
(HIV)-positive patients; in addition, acute PCP is rarely defined
solely on the basis of positive nested PCR results (E. Visconti et al.,
Conf. Rec. 12th World AIDS Conf., abstr. 22184, p. 299, 1998).
In conclusion, we question whether subclinical colonization by P. carinii occurs in HIV-positive and elderly patients. It may occur
in a small number of immunocompetent adults with lung damage due to
COLD or lung carcinoma; further studies need to define the role of
P. carinii carriage in the diffusion of P. carinii and the possibility, as previously described
(1), that a positive nested PCR could precede the debut of
acute PCP even though none of our patients developed PCP within 18 months of follow-up.
| |
REFERENCES |
| 1.
|
Elvin, K.,
M. Olsson,
C. Lidman, and A. Bjorkman.
1996.
Detection of asymptomatic Pneumocystis carinii infection by polymerase chain reaction: predictive for subsequent pneumonia.
AIDS
10:1296-1297[Medline].
|
| 2.
|
Peters, S. E.,
A. E. Wakefield,
S. Banerji, and J. M. Hopkin.
1992.
Quantification of the detection of Pneumocystis carinii by DNA amplification.
Mol. Cell. Probes
6:115-117[CrossRef][Medline].
|
| 3.
|
Sing, A.,
A. Roggenkamp,
I. B. Autenrieth, and J. Heesemann.
1999.
Pneumocystis carinii carriage in immunocompetent patients with primary pulmonary disorders as detected by single or nested PCR.
J. Clin. Microbiol.
37:3409-3410[Abstract/Free Full Text].
|
| 4.
|
Tamburrini, E.,
P. Mencarini,
A. De Luca,
G. Maiuro,
G. Ventura,
A. Antinori,
A. Ammassari,
E. Visconti,
L. Ortona,
A. Siracusano,
E. Ortona, and G. Vicari.
1993.
Diagnosis of Pneumocystis carinii pneumonia: specificity and sensitivity of polymerase chain reaction in comparison with immunofluorescence in bronchoalveolar lavage specimens.
J. Med. Microbiol.
38:449-453[Abstract/Free Full Text].
|
| 5.
|
Wakefield, A. E.,
F. J. Pixley,
S. Banerji,
K. Sinclair,
R. F. Miller,
E. R. Moxon, and J. M. Hopkin.
1990.
Amplification of mitochondrial ribosomal RNA sequences for Pneumocystis carinii DNA of rat and human origin.
Mol. Biochem. Parasitol.
43:69-76[CrossRef][Medline].
|
| | | | |
Elena Visconti
Salvatore Marinaci
Maria Zolfo
Paola Mencarini
Enrica Tamburrini
Clinic of Infectious Diseases
Università Cattolica del S. Cuore
Rome, Italy
|
| | | | |
Gabriella Pagliari
Clinic of Pulmonary Diseases
Università Cattolica del S. Cuore
Rome, Italy
|
| | | | |
Elena Ortona
Alessandra Siracusano
Laboratory of Immunology
Istituto Superiore di Sanità
Rome, Italy
|
| |
AUTHOR'S REPLY |
I read with great interest the letter by Dr. Visconti et al. on our
study of P. carinii carriage in immunocompetent patients with primary pulmonary disorders as detected by single or nested PCR
(3). We agree with the authors that PCP is defined as an acute respiratory syndrome that is confirmed by positive morphological staining of P. carinii organisms on respiratory samples.
However, the inclusion of a positive first cycle of amplification
(single PCR) in their PCP definition has to be restricted, in our
opinion, to defined patient groups, since in our experience of a
yet-unpublished study with 334 patients of different immunostatus
specificity rates of single PCR on BALs vary between HIV positive and
immunosuppressed HIV negative (e.g., transplant patients or patients
suffering from malignancies) patient groups. According to these
results, the proposed inclusion of a positive first cycle of
amplification (single PCR) in the definition of PCP is justified only
for HIV-positive patients. This conclusion may also be drawn from the
study by Weig et al. who found nested PCR useful only in HIV-positive
patients, i.e., not in otherwise-immunocompromised HIV-negative
patients (4). Therefore, we feel comfortable recommending
anti-P. carinii treatment only when conventional staining is
positive; however, we report a positive PCR result to the clinician
immediately and discuss the laboratory finding and the management of
the patient. We agree with the authors that acute PCP is rarely defined
solely on the basis of positive nested PCR results.
Dr. Visconti and colleagues report a very low frequency of P. carinii DNA in the BALs of 78 patients, as detected by a nested PCR using mtLSU-rRNA region primers after proteinase K digestion without a DNA extraction step. The technical differences might contribute to their differing results. However, we did not test for the
influence of a DNA extraction step on the sensitivity of the nested PCR
we performed (4). In our opinion, the sensitivity of the
different primers used can only be compared by performing a study with
both PCR methods on the same samples.
The nested PCR we used showed a sensitivity of 100% and a specificity
of 97.9% on BAL samples of HIV-positive PCP patients. Weig et al.
(4) found a 45.4% rate of positivity by nested PCR for BALs
from HIV-negative immunosuppressed patients without clinical proof of
PCP, suggesting a high sensitivity of their nested PCR. As discussed in
our paper (3), several other studies on different
HIV-negative immunocompetent patient groups with underlying pulmonary
diseases found P. carinii carriage rates of 6.5%
(1) to 25% (2) as shown by PCR.
Additionally, differences in the incidence of PCR positive samples
might be explained by different patient collectives or geographical factors.
We agree with Dr. Visconti and colleagues that further studies are
needed to define the role of P. carinii carriage in the diffusion of P. carinii.
| |
REFERENCES |
| 1.
|
Armbruster, C.,
A. Hassl, and S. Kriwanek.
1997.
Pneumocystis carinii colonization in the absence of immunosuppression.
Scand. J. Infect. Dis.
29:591-593[Medline].
|
| 2.
|
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].
|
| 3.
|
Sing, A.,
A. Roggenkamp,
I. B. Autenrieth, and J. Heesemann.
1999.
Pneumocystis carinii carriage in immunocompetent patients with primary pulmonary disorders as detected by single or nested PCR.
J. Clin. Microbiol.
37:3409-3410.
|
| 4.
|
Weig, M.,
H. Klinker,
B. H. Bogner,
A. Meier, and U. Gross.
1997.
Usefulness of PCR for diagnosis of Pneumocystis carinii pneumonia in different patient groups.
J. Clin. Microbiol.
35:1445-1449[Abstract].
|
| | | | |
Andreas Sing
Max von Pettenkofer-Institut für Hygiene und Medizinische
Mikrobiologie
Ludwig-Maximilians-Universität
80336 Munich, Germany
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Journal of Clinical Microbiology, March 2000, p. 1307-1308, Vol. 38, No. 3
0095-1137/00/$04.00+0
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