Introduction
Pneumocystis jiroveci pneumonia (PCP) is a
potentially life-threatening infection that occurs in
immunocompromised populations (1).
HIV-infected patients with a low CD4 count are at high risk of PCP.
Other individuals at substantial risk include hematopoietic stem
cell and solid organ transplant recipients, those with cancer and
those receiving corticosteroids, cytotoxic agents and other
immunosuppressive medications (2–5).
The detection of Pneumocystis jiroveci (P.
jiroveci) in individuals with no indication of PCP has been
defined as colonization. Although Pneumocystis colonization
was recently described using sensitive molecular techniques,
including polymerase chain reaction (PCR), the clinical
significance of Pneumocystis colonization is not yet fully
understood. It is unknown whether Pneumocystis colonization
leads to the development of PCP (6). In this study, we determined the
prevalence of Pneumocystis colonization in advanced lung
cancer patients in order to investigate the association between PCP
and Pneumocystis colonization.
Patients and methods
Patients
A total of 47 advanced lung cancer patients with no
indication of PCP from Kyoto University Hospital (Kyoto, Japan)
were evaluated between June 2009 and October 2009 to determine the
prevalence of Pneumocystis colonization. We also
retrospectively evaluated four advanced lung cancer patients with
PCP during the period between August 2002 and October 2009 at Kyoto
University Hospital. No patient was known to be HIV-positive.
Informed consent was obtained from all patients prior to the
study.
Samples
The investigation of patients with no indication of
PCP was performed using oral wash (OW) samples from the 47
patients. OW samples were obtained by rinsing and gargling oral
cavities with 20 ml of sterile saline for 30 sec. The investigation
of patients with PCP was performed using sections of
formalin-fixed, paraffin-embedded lung tissues obtained following a
diagnosis of lung cancer. Of the five patients, four had no
indication of PCP at the time of diagnosis. The sample from the one
patient who not analyzed further.
Sample analysis
Pneumocystis colonization was determined by a
PCR test for P. jiroveci. Formalin-fixed paraffin-embedded
tissue was cut into 5-μm sections and mounted onto
pre-treated glass slides. The slides were de-paraffinized and DNA
was extracted with phenol-chloroform and ethanol precipitation. The
PCR analysis for P. jiroveci was performed in 50 μl
of amplification reaction mixture with denaturation at 943°C for 90
sec, annealing at 503°C for 90 sec and extension at 723°C for 120
sec (40 cycles). The oligonucleotide primers used at 100 pmol were:
5′-GATGGCTGTTTCCAAGCCCA-3′ and 5′-GTGTAGGTTGCAAAGTACTC-3′. DNA
products of 376 bp were amplified from template DNAs. This analysis
was performed at SRL Inc. (Tachikawa, Tokyo, Japan). The details of
this method and its use in the detection of P. jiroveci were
described by Wakefield et al(7).
Results
Patients with no indication of PCP
The clinical characteristics of the 47 patients with
no indication of PCP are listed in Table I. Although corticosteroids or
cytotoxic agents were administered to certain patients, no P.
jiroveci was detected by PCR in the OW samples.
 | Table I.Clinical characteristics of the
patients without Pneumocystis jiroveci pneumonia. |
Table I.
Clinical characteristics of the
patients without Pneumocystis jiroveci pneumonia.
| Characteristics | No. of patients |
|---|
| Median age
(years) | 47 |
| Gender (%) | |
| Male | 36 (77) |
| Female | 11 (23) |
| Performance status
(%) | |
| 0 | 19 (40) |
| 1 | 15 (32) |
| 2 | 12 (26) |
| 3 | 1 (2) |
| Smoking status
(%) | |
| Never | 13 (28) |
| Former | 34 (72) |
| Comorbid lung
disease | |
| Chronic obstructive
pulmonary disease | 9 |
| Interstitial
pneumonia | 4 |
| Others | 6 (asthma 3,
radiation pneumonitis 1, bronchiectasis 1, silicosis 1) |
| Histology (%) | |
| Non-small cell lung
cancer | 30 (64) |
| Small cell lung
cancer | 17 (36) |
| Stage (%) | |
| IV | 27 (57) |
| IIIB | 18 (38) |
| rIV | 2 (5) |
| Treatment (%) | |
| None | 29 (62) |
| Cytotoxic
agents | 16 (34) |
| Others | 2 (4) (Gefitinib 1,
Stereotactic radiation 1) |
| Corticosteroid
(%) | |
| Oral | 4 (9) |
| Inhalation | 3 (6) |
| Peripheral blood
lymphocyte count (%) | |
|
<1500/μl | 24 (51) |
|
≥1500/μl | 23 (49) |
Patients with an indication of PCP
The clinical characteristics of the four patients
with PCP at the time of diagnosis of lung cancer are listed in
Table II. Neither corticosteroid
nor other immunosuppressive medication were administered to the
four patients upon diagnosis of lung cancer and no P.
jiroveci was detected by PCR in the lung tissues of the
patients. At the diagnosis of PCP, corticosteroids were
administered to all four patients. Of the four patients, three
received cytotoxic agents following a diagnosis of lung cancer with
PCP.
| Table II.Clinical characteristics of the
patients with Pneumocystis jiroveci pneumonia upon the
diagnosis of lung cancer. |
Table II.
Clinical characteristics of the
patients with Pneumocystis jiroveci pneumonia upon the
diagnosis of lung cancer.
| Case 1 | Case 2 | Case 3 | Case 4 |
|---|
| Age (years) | 78 | 47 | 75 | 66 |
| Gender | Male | Male | Male | Male |
| Performance
status | 1 | 0 | 0 | 0 |
| Smoking status | Former | Former | Former | Former |
| Comorbid lung
disease | COPD | None | COPD | COPD, IP |
| Histology | Sq | Ad | Ad | Sq |
| Stage | IIIB | IV | IV | IIIA |
|
Immunosuppression | No | No | No | No |
| Corticosteroid | No | No | No | No |
| Lymphocyte
(cells/μl) | 1800 | 1600 | 2600 | 3900 |
Discussion
In this study, no P. jiroveci was detected by
PCR in OW samples of the 47 patients with no indication of PCP. In
addition, no P. jiroveci was detected by PCR in the lung
tissues of the four patients with PCP. These results indicate that
PCP was not associated with Pneumocystis colonization.
The most significant risk factors for PCP in
patients without HIV infection are corticosteroid use and defects
in cell-mediated immunity (5,8–10).
Cancer is also one of the significant risk factors for PCP and
cancer patients often receive cytotoxic agents or corticosteroids
(11). In a retrospective study of
80 PCP cases in 79 cancer patients, PCP occurred more often in
patients with hematological malignancies (66%), the majority of
whom had either leukemia or lymphoma. In addition, 25 patients had
solid tumors and 23 patients had undergone hematopoietic stem cell
transplantation (11). There were
no data with regard to what type of solid cancer tumors were
complicated with PCP in this study. In this retrospective study, 5
of 505 patients (1.0%) with advanced lung cancer had complications
with PCP. Therefore, PCP is considered to be a rare complication in
patients with advanced lung cancer.
The primary mode of transmission of P.
jiroveci is uncertain. Primary exposure to P. jiroveci
is common in young children, as is demonstrated by the increase in
anti-Pneumocystis antibody titers during the first few years
of life (7,12). These primary infections were
presumed to be asymptomatic and the organism was believed to remain
in a latent state unless the patient became immunosuppressed.
However, this theory of latency has come under scrutiny.
Experiments in rats or mice with severe combined immunodeficiency
and macaques infected with simian immunodeficiency virus all show
that these hosts are capable of clearing the organism (13–15).
Pneumocystis colonization is different from this latent
infection in that P. jiroveci is detected directly. The
prevalence of Pneumocystis colonization among healthy adults
without immunosuppressive conditions or lung disease in previous
studies has ranged from 0 to 20% (6). Certain risk factors, including low CD4
cell count, corticosteroid, smoking and COPD appear to affect the
risk of Pneumocystis colonization (6). Of the 47 patients with advanced lung
cancer included in our study, no Pneumocystis colonization
was detected although there were some patients with these risk
factors. Maskell et al detected six Pneumocystis
colonizations in bronchoalveolar lavage from 35 patients with lung
cancer (16). The differences in
these studies may be explained by varying geographic exposure in
the populations, differences in experimental techniques or
differing subject characteristics.
Certain diseases, including SIDS and COPD, are
associated with a high prevalence of Pneumocystis
colonization, but the association between Pneumocystis
colonization and development of these diseases is unclear (6). In our retrospective study, no P.
jiroveci was detected by PCR in the past lung tissues of the
four advanced lung cancer patients with PCP, which indicated that
PCP was not associated with Pneumocystis colonization.
However, this study is limited as it is a retrospective study. To
fully assess the association, a prospective study is needed.
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