Pneumocystis pneumonia (PCP) is a well-known opportunistic infection that affects patients infected with human immunodeficiency virus (HIV) [1]. PCP, the incidence of which continues to increase, also occurs in immunocompromised patients without HIV, such as those with hematological malignancies or solid tumors; those who have undergone organ transplantation; and those receiving immunosuppressants, including corticosteroids [2-4]. Pneumocystis jirovecii may be present in the respiratory tracts of patients without clinical pneumonia. P. jirovecii polymerase chain reaction (PCR) positivity could reflect PCR contamination, colonization, or transient carriage of a very small number of P. jirovecii organisms, which has been defined as Pneumocystis colonization [5].

In immunocompromised patients without HIV, PCP progresses rapidly and causes severe respiratory failure. However, it is often difficult to diagnose. Standard microscopic examinations of sputum or bronchial specimens for P. jirovecii organisms have low sensitivity due to the low bacterial burden, and although lung biopsy can be confirmative, it is quite invasive [6,7]. Moreover, the rate of colonization is high in these patients. A recent study documented Pneumocystis colonization in induced sputum from 16% of patients with various autoimmune diseases [8]. The molecular detection tool P. jirovecii PCR provides a higher degree of sensitivity than do standard methods and can be useful for the clinical diagnosis of PCP [9]. However, its use is limited by P. jirovecii colonization, resulting in lower specificity. The clinical value of P. jirovecii PCR in diagnosing PCP in patients with systemic diseases has not been defined clearly.

The aim of this study was to evaluate the prevalence of P. jirovecii PCR positivity in respiratory samples from immunocompromised patients without HIV who underwent bronchoscopy due to suspected respiratory infection. Furthermore, we attempted to determine the risk of PCP development in these patients by comparing the treatment group (patients diagnosed with PCP) with the non-treatment group (those with suspected colonization).

We collected and analyzed the results of P. jirovecii PCR performed on bronchial washing fluid or bronchoalveolar lavage fluid (BALF) from adult immunocompromised patients without HIV who were suspected of having respiratory infection at a 1,200-bed tertiary university teaching hospital between May 2009 and September 2012. Medical records of the patients with positive P. jirovecii PCR results were reviewed retrospectively. The study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, which permitted evaluation and publishing of information from the patients’ records. The requirement for informed consent was waived because of the retrospective nature of the study.

Definite PCP is diagnosed in the presence of a compatible clinical presentation of PCP and a positive immunofluorescent test in BALF [1,10]. In this study, we defined the presence of PCP as P. jirovecii PCR positivity and treatment for PCP based on respiratory symptoms and the detection of new pulmonary infiltrates on chest radiography or computed tomography. Patients with PCP were treated with trimethoprim-sulfamethoxazole (TMP-SMX) for 21 days; treatment with other anti-infective agents was withheld, except for patients with coinfections. Co-infected patients were additionally treated with appropriate empirical antibiotics, voriconazole, amphotericin, or ganciclovir, according to the coinfection diagnosed. Adjuvant corticosteroid therapy was administered to patients with partial pressure of arterial oxygen (PaO₂) values < 60 mmHg, as in the recommended regimen [11]. Patients without PCP (non-PCP group) were defined as P. jirovecii PCR-positive patients who did not require treatment for PCP, suggesting colonization or subclinical infection, rather than PCP.

The baseline clinical data included demographic characteristics, presence of underlying disease, previous use of medications (steroids and other immunosuppressants), and chemotherapy in the previous month; bronchoscopy results, including those from bronchoalveolar lavage, transbronchial lung biopsy (TBLB), and evaluation with special stains (e.g., methenamine silver or Giemsa); laboratory findings, such as complete blood count and blood chemistry; and symptoms, pneumonia severity index (PSI), and radiological findings at the time of P. jirovecii PCR testing.

We investigated the clinical outcomes of the two groups regarding the decision to treat (or not to treat) PCP and in-hospital mortality.

We defined improvement as clinical recovery and discharge within 1 month after treatment. Causes of death were divided into four categories: acute respiratory distress syndrome (ARDS), sepsis, multiorgan failure, and cardiac arrest. We used the Berlin definition to diagnose ARDS [12]. Sepsis was defined as the presence (probable or documented) of infection together with systemic manifestations of infection [13]. Organ dysfunction was defined according to Marshall et al. [14]. Cardiac arrest was defined as the cessation of cardiac mechanical activity with loss of effective circulation, as determined by the absence of a palpable central pulse [15].

In the current study, we divided the P. jirovecii PCR-positive population into two groups—patients with clinical diagnoses of PCP who received TMP-SMX treatment, and those diagnosed simply with colonization—and compared the two groups to evaluate the risk factors for PCP. The results showed that PCP was more common in P. jirovecii PCR-positive patients aged < 60 years; those with hematological malignancies and solid tumors; those who were solid organ transplant recipients; those with low albumin levels; and those with symptoms of dyspnea. Moreover, colonization was detected in a considerable percentage of P. jirovecii PCR-positive immunocompromised patients, indicating that they did not require treatment.

The diagnosis of PCP in clinical practice is challenging. First, the diagnostic yields of direct staining and lung biopsy are low, ranging from 38% to 53% [6,7]; values are lower for immunocompromised patients without HIV than for patients with HIV. Second, some methods, such as TBLB, are invasive and often inaccessible in a clinical setting. Lastly, the symptoms and radiological findings at the time of PCP development are frequently nonspecific and ambiguous. Recently, the P. jirovecii PCR method has helped to improve the diagnostic sensitivity of PCP diagnosis from BALF and induced sputum. However, the differentiation of infection and colonization in the PCR-based diagnosis of PCP can be difficult, particularly in patients without HIV [18]. The extent of colonization has been debated, with different rates of P. jirovecii carriage reported. Nevez et al. [19] reported that 20% of 169 HIV-negative patients with moderate or severe immunosuppression showed P. jirovecii positivity according to nested PCR; none of these patients developed PCP. In the current study, 37% of patients with hematological malignancies, 37% of those with solid tumors, 21% of solid organ transplant recipients, 56% of patients with autoimmune diseases, and 81% of patients with other diseases had P. jirovecii colonization. Most (83.5%, n = 66) patients in the non-PCP group, who were P. jirovecii PCR-positive according to analysis of bronchoscopic specimens, recovered without treatment of PCP, indicating that they were colonized or had subclinical infection. This finding suggests that a fair number of patients with P. jirovecii PCR positivity do not need to be treated, despite their low immune status. On the other hand, P. jirovecii colonization has several clinical implications. It may increase the risk of acute PCP in susceptible hosts, or result in the transmission of P. jirovecii to others [16,20]. Nevertheless, the duration of colonization before the development of PCP and the efficacy of treating colonization to prevent disease are unknown [21].

In our study, we attempted to determine the predictive factors of PCP among P. jirovecii-positive patients by comparing the PCP and non-PCP groups. The PCP group contained significantly larger percentages of patients with hematological malignancies, those with solid tumors, and solid organ transplant recipients among various underlying immunocompromising diseases. The higher incidence of PCP among these patients might be due primarily to a higher net state of immunosuppression caused by the underlying disease and the intensity of chemotherapy or immunosuppressant use in these patients [22].

Intriguingly, the current study showed that patients in the PCP group were significantly younger than those in the non-PCP group. Previous reports showed an association between age > 60 years and pulmonary Pneumocystis colonization, especially in patients with rheumatoid arthritis [23,24]. Advanced age itself may compromise the immune system, leads to increased Pneumocystis colonization. This hypothesis should be tested in future studies.

Dyspnea symptoms were significantly more common in the PCP group than in the non-PCP group. However, interestingly, PaO₂ did not differ between groups. P. jirovecii PCR-positive patients with subjective symptoms of dyspnea despite no evidence of hypoxia should be evaluated carefully for evidence of PCP. Significant hypoalbuminemia, suggesting compromised nutritional status, was noted in the PCP group compared with the non-PCP group. Hypoalbuminemia has been reported as a poor prognostic factor for PCP [25].

Regarding mortality, whereas 34 patients (38%) in the PCP group died despite treatment, 66 patients (84%) in the non-PCP group improved without treatment of PCP. In the clinical PCP group, the main causes of death were ARDS, multiorgan failure, sepsis, and cardiac arrest. Some patients may have died due to conditions other than PCP, whereas others may have died due to refractoriness to PCP therapy. In contrast, in the non-PCP group, most patients survived with adequate treatment other than PCP therapy. This finding suggests a considerable incidence of colonization in patients with P. jirovecii PCR positivity, despite their significant immunodeficient status.

The limitations of this study were its retrospective nature and the use of a clinical diagnosis as the gold standard for PCP, as opposed to the performance of lung biopsy in all patients. However, lung biopsy is quite invasive and often extremely risky in these patients. In addition, some studies have failed to show benefits in terms of PCP diagnosis [26].

Another limitation was that we used conventional PCR methods instead of quantitative real-time PCR assays, which have been reported to be more reliable for the diagnosis of PCP [27,28]. Quantitative real-time PCR assays, along with the use of serum markers such as ß-D glucan, enable differentiation between PCP and Pneumocystis colonization [27-30]. However, these assays are costly and their cut-off values are not standardized; therefore, their application in clinical practice remains limited.