Detection of Pneumocystis carinii by DNA amplification in patients with connective tissue diseases: re-evaluation of clinical features of P. carinii pneumonia in rheumatic diseases
K. Saito,
S. Nakayamada,
K. Nakano,
M. Tokunaga,
S. Tsujimura,
K. Nakatsuka,
T. Adachi and
Y. Tanaka
First Department of Internal Medicine, University of Occupational and Environmental Health, School of Medicine, Fukuoka, Japan.
Correspondence to: Y. Tanaka, First Department of Internal Medicine, University of Occupational & Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishi, Kitakyushu 807-8555, Japan. E-mail: tanaka{at}med.uoeh-u.ac.jp
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Abstract
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Objectives. We evaluated the polymerase chain reaction (PCR) detection of Pneumocystis carinii DNA in induced sputum of patients with connective tissue diseases and assessed the clinical features of patients positive for P. carinii.
Methods. Sputum was induced by nebulization in 29 in-patients with various connective tissue diseases who presented with symptoms suggestive of P. carinii pneumonia (PCP), and was examined by PCR.
Results. Detection of P. carinii DNA by PCR was significantly more sensitive than cytology; 54.5% patients were positive by PCR and only 4.5% by cytology. The prevalence of PCP was higher than previously considered and was especially high in patients receiving > 30 mg/day prednisolone with or without other immunosuppressants. P. carinii-positive patients had significant lymphocytopenia and a low serum IgG level compared with P. carinii-negative patients. P. carinii disappeared within 710 days after therapy with trimethoprim/sulfamethoxazole.
Conclusion. We propose that the use of PCR for detection of P. carinii using induced sputum is a useful and non-invasive method that has high sensitivity and specificity for the early diagnosis of PCP.
KEY WORDS: Pneumocystis carinii pneumonia, Polymerase chain reaction, Rheumatic diseases, Steroids, Immunosuppressants, ß-D-glucan, Trimethoprim/sulfamethoxazole.
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Introduction
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Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in exclusively immunocompromised patients [1]. Host defence against P. carinii depends mainly on cellular immunity, and the depletion of CD4 T lymphocytes that is observed in patients with acquired immunodeficiency syndrome (AIDS) results in fatal pneumonia. More recently, an increasing number of reports have described the occurrence of PCP in patients with connective tissue diseases such as rheumatoid arthritis (RA), polymyositis (PM), systemic lupus erythematosus (SLE) and Wegener granulomatosis (WG) [2]. Several factors seem to be involved in the manifestation of PCP in patients with connective tissue diseases. These include impaired humoral and cellular immunity, which has been reported in autoimmune diseases, such as SLE, both by our laboratories and other investigators [35]. Furthermore, the use of immunosuppressive agents for the treatment of connective tissue diseases is closely related to the occurrence of PCP. At present, immunosuppressive agents may be used more commonly, in higher doses or in combinations, which may thus increase the susceptibility of patients to PCP.
P. carinii infection in immunocompromised hosts sometimes results in rapid and fatal pneumonia, and thus early and precise diagnosis is not easy. PCP patients usually present with non-productive cough and P. carinii is considered to adhere tightly to the alveolar wall with its pseudopodia. It is therefore difficult to obtain high-quality sputum for precise diagnosis. However, cytological examination of samples from the respiratory tract has been the mainstay of the laboratory diagnosis of PCP. P. carinii organisms have not yet been successfully cultured, and diagnostic identification of the organism depends on direct microscopy after Giemsa or silver staining of bronchoscope lavage material or induced sputum [6]. The development of assays that use monoclonal antibodies against P. carinii has improved specificity but the sensitivity remains uncertain [7]. The majority of studies in this area have been conducted in the context of HIV-induced immunosuppression, but the clinical presentation of PCP in patients with connective tissue diseases who are on immunosuppressants is different. In particular, disease is more rapid in onset and is often more severe, and there are generally far fewer organisms in induced sputum or bronchoalveolar lavage fluid [8]. Thus, there is a need for a diagnostic method that has high sensitivity and specificity and which is clinically applicable for the early diagnosis of PCP.
Recently, oligonucleotide primers and probes were used in a polymerase chain reaction (PCR) method designed to amplify P. carinii-specific DNA sequences from alveolar lavage samples and induced sputum. PCR is a highly sensitive and specific technique compared with conventional microscopic examination for the detection of P. carinii [9]. To our knowledge, however, there are no studies that have used PCR for the diagnosis of PCP in patients with connective tissue diseases, and to date the features of PCP have been evaluated mainly by conventional microscopic examination, which might result in the underevaluation of PCP or delay in its treatment.
In the present study, we used PCR to detect DNA of P. carinii in patients with connective tissue diseases suspected of having PCP based on the presence of progressive arterial hypoxaemia and interstitial pneumonitis on plain chest X-ray or computed tomography. Then we evaluated the clinical features of PCP in these patients. This is the first report that provides clinical assessment of patients with PCP complicating connective tissue diseases, and evaluates the risk factors for PCP, such as the daily dose of prednisolone, laboratory data, the clinical course and the prognosis after treatment based on DNA diagnosis using induced sputum.
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Patients and methods
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Clinical specimens
Sputum samples were obtained from hospitalized patients with connective tissue diseases who presented with a provisional diagnosis of PCP between September 1998 and February 2003 at the University Hospital of Occupational and Environmental Health. PCP was suspected in these patients because of the presence of all of the following parameters: (i) typical radiographic features of the lungs on plain chest X-ray or computed tomography (CT); and (ii) typical clinical signs and symptoms, such as non-productive cough, shortness of breath, and progressive hypoxia. In almost all patients, sputum was induced by nebulization with 10 ml of distilled water and then collected for examination. Then Grocott staining was performed as described in a previous paper [6]. Samples were obtained from 29 patients, comprising seven with SLE, five with PM/dermatomyositis (DM), nine with rheumatoid arthritis (RA), four with microscopic polyangiitis (MPA), three with mixed connective tissue diseases (MCTD) and one with Sjögren syndrome (SS). The samples were examined for P. carinii DNA by PCR. Each connective tissue disease was diagnosed based on previously proposed classification criteria for RA [10], SLE [11], PM/DM [12], SS [13], MCTD [14] and MPA [15]. Ten sputum samples from patients with idiopathic interstitial pneumonitis without connective tissue diseases were also examined. The study protocol was approved by the Human Ethics Review Committee of University of Occupational and Environmental Health, School of Medicine, and a signed consent form was obtained from each subject.
DNA amplification and detection of P. carinii
The sputum sample was diluted with distilled water (1:1), mixed with dithiothreitol to a final concentration of 5 mM and then vortexed vigorously. After 10 min incubation at 37°C, samples were centrifuged and pellets were washed three times with to phosphate-buffered saline (PBS). Then the pallets were resuspended in 1 ml of PBS, 50 µl of lysis buffer (10 mM TrisHCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.1 mg/ml of gelatin, 0.25% Tween-20, and 0.25% NP-40) and 5 µl of 20 mg/ml of proteinase K (Gibco BRL, MD, USA). The mixture was incubated for 6 h at 60°C and DNA was extracted with phenol/chloroform followed by ethanol precipitation. Finally DNA was dissolved in 20 µl of TE buffer, pH 8.0, and 5 µl of DNA was used as a template. Oligonucleotide primers (sense, 5'-AGTTACGGCCATACC TCAGA-3'; antisense, 5'-AAAGCTACAGCACGTCGTAT-3') were used at 100 pmol in 50 µl of amplification reaction mixture, with denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 90 s for 35 cycles using an iCycler Thermal cycler (Bio-Rad, Richmond, CA, USA) [16]. Negative controls with no added template and positive control DNA from a sample of definite PCP were included. The products (20 µl) were subjected to electrophoresis in 3% agarose gels and the presence of 124 base pair (bp) specific bands was confirmed.
Statistical analysis
Data are expressed as mean ± S.D. for the number of patients indicated. Differences between pairs of groups were examined for statistical significance with the MannWhitney U-test. The correlation coefficient was determined with Fisher's exact probability test. A P-value less than 0.05 denoted a statistically significant difference.
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Results
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PCR analysis of P. carinii in connective tissue diseases
PCR was performed for detection of P. carinii in induced sputum samples from 29 patients with connective tissue diseases. Table 1 summarizes the clinical characteristics of patients with suspected diagnosis of PCP, including diagnosis, recent involvement within the preceding 4 weeks and current treatment. Almost all patients with connective tissue diseases, except those with RA, were being treated with more than 25 mg/day of prednisolone with or without other immunosuppressants. All patients presented with suspicious clinical signs of PCP, such as non-productive cough, shortness of breath and progressive hypoxia, as well as ground glass appearance on plain chest X-ray or CT. They had no previous manifestation of PCP and no prophylactic treatment for PCP. DNA was extracted from the sputum obtained after nebulization with distilled water, and PCR using specific oligonucleotides for P. carinii was carried out. The results showed a 124 bp single band in 3% agarose gel containing ethidium bromide (Fig. 1). Based on the results of PCR, 19 out of 29 patients (65.5%) were confirmed to have PCP. Among the connective tissue diseases, five out of seven patients with SLE, five of five with PM/DM, two of three with MCTD, four of nine with RA, two of four with MPA, and one of one with SS were PCR-positive for P. carinii. However, 10 sputum samples were also examined from patients with idiopathic interstitial pneumonitis and healthy controls, all of whom were negative for P. carinii.
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TABLE 1. Clinical characteristics of patients with suspected diagnosis of P. carinii infection and results of PCR detection
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FIG. 1. PCR detection of P. carinii in induced sputum from patients with connective tissue diseases. DNA was extracted from the sputum as described in Patients and methods. PCR products (20 µl) were subjected to electrophoresis in 3% agarose gels and the presence of 124 bp specific bands was confirmed. Results of a representative examination are shown. N, negative controls with no added template; P, positive control DNA from a definitive case of PCP; M, molecular size marker; ¢X 174 HaeIII digestion.
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Comparison of PCR with Grocott staining detection for P. carinii using the same specimens
Both PCR and conventional microscopic examination by Grocott staining were simultaneously performed on 22 samples. As shown in Table 2, positive results were noted in 54.5% of the samples by PCR and in only 4.5% by cytology. Eleven samples negative by cytology were positive by PCR. In contrast, none of the samples that were positive by cytology were PCR-negative. Detection of P. carinii DNA by PCR was significantly more sensitive than cytology.
Clinical background of PCR-positive patients with P. carinii
Because PCP is one of the most well-known opportunistic infections, we evaluated the relation between the occurrence of PCP and immunosuppressive agents used by our patients. As shown in Figure 2, the oral dose of steroids, converted into prednisolone, correlated closely with PCP. The mean dose of prednisolone was significantly higher in patients with PCP (48.7 mg/day) than in PCP-negative patients (15.6 mg/day). The frequency of PCP was markedly higher in patients who were taking > 30 mg prednisolone daily (16 out of 19; 84.2%). Furthermore, 15 out of 20 patients (excluding those with RA) were also receiving oral or intravenous (i.v.) cyclophosphamide (CY), azathioprine (AZA) or cyclosporin A (CSA) because of disease severity. Even in patients on < 10 mg prednisolone, combination therapy with other immunosuppressants seemed to increase the risk of PCP. In contrast, all patients who were taking prednisolone at < 50 mg/day alone without other immunosuppressants were free of P. carinii infection.

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FIG. 2. Daily dose of prednisolone in patients positive or negative for P. carinii as determined by PCR. Open circles, daily dose of prednisolone when the patients were suspected having P. carinii infection and were treated with steroids alone; closed circles, intravenous pulse therapy; open squares, oral cyclophosphamide; closed squares, intravenous cyclophosphamide (IV-CY); open triangles, cyclosporin A (CSA); closed triangles, methotrexate (MTX); open diamonds, azathioprine (AZA). Horizontal bars represent mean values. Data for different groups were compared using the MannWhitney U-test.
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Figure 3A shows the relation between white blood cell (WBC) count and PCP. The WBC count was significantly lower in patients positive for P. carinii (< 10 000/mm3) than in patients free of PCP. In fact, none of the patients infected with P. carinii had a leucocyte count above 15 000 mm3. Furthermore, PCP was associated with severe lymphocytopenia (280/mm3) and low serum IgG (843 mg/dl) compared with PCP-negative patients (lymphocyte count 711/mm3, IgG 1189 mg/dl) (Fig. 3B and C). These results suggest that a lymphocyte count <400/mm3 and serum IgG <1000 mg/dl appeared to increase the risk of PCP. The proportions of P. carinii-positive patients with less than the above threshold values for lymphocytes and IgG were 89% (16/18) and 82% (14/17) respectively.

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FIG. 3. Numbers of (A) WBC and (B) lymphocytes and (C) serum IgG levels of individual patients positive and negative for P. carinii as determined by PCR. Horizontal bars represent mean values. Data for different groups were compared using the MannWhitney U-test.
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Measurement of plasma ß-D-glucan has been proposed as a useful non-invasive diagnostic test for P. carinii infection [17]. The mean serum level of ß-D-glucan was significantly higher in patients with PCP compared with non-PCP patients (Fig. 4A).

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FIG. 4. (A) Serum ß-D-glucan, (B) LDH and (C) KL-6 levels of individual patients positive and negative for P. carinii as determined by PCR. Horizontal bars represent mean values.
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Lactate dehydrogenase (LDH) and KL-6 have been considered as markers of pulmonary interstitial damage [1824]. KL-6 is a mucinous glycoprotein expressed on type II pneumocytes, and is considered to be a clinical marker for interstitial pneumonia (IP) in various collagen diseases, including RA, SS and PM/DM [19, 20, 22, 23]. Furthermore, the serum KL-6 level increases with deterioration of IP, while successful treatment of IP results in significant decreases in the level [23]. Serum levels of KL-6 and LDH have been reported to be useful markers of PCP but not bacterial infection, and were considered to be associated with the clinical severity of PCP [1824]. Accordingly, we determined the levels of LDH and KL-6 in patients with PCP (Fig. 4B and C). Although the mean serum concentrations of these factors were elevated in PCP patients compared with the PCP-negative group, the difference between the two groups was not statistically significant.
Clinical course of patients with PCP
As shown in Fig. 5, specimens could be obtained from 11 patients over the periods indicated after positive results for PCP, allowing us to examine the time course of detectability of P. carinii DNA in sputum samples. P. carinii DNA diminished within 710 days after commencement of trimethoprim/sulfamethoxazole (TMP/SMX) treatment in almost all PCP patients. This finding might have excluded false-positive results in the present study. Once the DNA had been reduced by TMP/SMX treatment for 14 days, recurrence was not observed in these patients. Among the 19 patients diagnosed with PCP by PCR detection, four died within 30 days after commencement of TMP/SMX treatment. One patient with DM died of fatal arrhythmia and one patient with RA died of pneumonia caused by Pseudomonas and Aspergillus infection. However, two patients with SLE died of bacterial sepsis; an indirect effect of PCP could not be excluded as we could not confirm the total disappearance of P. carinii DNA. The prolonged presence of P. carinii DNA in the respiratory tract seems to reflect a serious opportunistic state.

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FIG. 5. Effects of treatment with TMP/SMX on PCR for P. carinii. In 11 patients, repeated specimens were obtained over the indicated periods after the first positive PCR for P. carinii. Results of PCR for P. carinii are indicated as positive (+) or negative (-). Shaded bars denote periods of treatment with TMP/SMX. #Patient numbers were identical to those in Table 1; day of death.
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Discussion
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To determine the aetiology of progressive hypoxia and interstitial pneumonia is a very important issue in the treatment of patients with connective tissue diseases. In the present study, we used non-invasive sampling (induced sputum) and successfully detected P. carinii at a high rate in patients with connective tissue diseases using PCR. The PCR method used in the present study allowed early diagnosis; the results were available within 1224 h. In comparison, conventional microscopic detection using Grocott staining, which was performed simultaneously with PCR, was associated with poor detection of PCP compared with PCR. These results indicate that negative results obtained by microscopic examination of sputum without bronchoalveolar lavage do not always exclude the presence of P. carinii. Although the risk of spread of pathological microorganisms is taken into account when collecting bronchoalveolar lavage material, it seems essential to use bronchoalveolar lavage for the accurate microscopic detection of P. carinii. Induced sputum is easily obtained and often saves seriously unwell patients from the risk of bronchoscopy and bronchoalveolar lavage. Our results also showed that none of the patients who were considered negative for PCP by PCR showed deterioration of their clinical condition without TMP/SMX treatment, suggesting the lack of false-negative cases in the present study.
In general, overdiagnosis has to be excluded in the case of PCR diagnosis because of the extensive amplification, especially if P. carinii is present as part of the normal flora in healthy people. In children older than 2 yr, antibodies to P. carinii were found in >85% of the specimens, suggesting that P. carinii probably spread by airborne infection [25]. Although other studies suggested possible colonization of P. carinii in alveolar tissue of healthy individuals [1], P. carinii could be detected with 100% specificity by PCR [26, 27]. Our study also showed no positive cases out of 10 individuals among healthy controls and those with irrelevant diseases, such as idiopathic interstitial pneumonia. Therefore, the oligonucleotides and the number of cycles of PCR in this study did not detect a small population of P. carinii, even if present, in normal lungs. Furthermore, when patients with PCR-diagnosed PCP were followed by frequent examination, clinical improvement in response to treatment with TMP/SMX was associated with eradication of P. carinii and negative PCR, indicating no false-positive results in the present study. Oz and Hughes [28] demonstrated that all patients with PCP and none without PCP had P. carinii DNA in samples from 31 patients with pneumonitis and AIDS or cancer, supporting our present results.
With regard to the background of the patients, our results showed that the use of immunosuppressive agents was the most important factor associated with the development of PCP. Our study demonstrated that treatment with prednisolone at a dose > 30 mg/day was a risk factor. The frequency of PCP was markedly higher in patients who were taking a daily dose of prednisolone of >30 mg (16 out of 19; 84.2%). In addition, the risk was higher in patients receiving combination therapy comprising steroid and other immunosuppressants, such as cyclophosphamide, CSA and methotrexate (MTX), even when the dose of prednisolone was low. In contrast, prednisolone at low doses (< 10 mg) without any immunosuppressive agents was not associated with the development of PCP. Godeau et al. [29] reported that most patients with PCP were treated with corticosteroids at a mean prednisone equivalent dose of 1.2 mg/kg/day at the time of diagnosis of PCP by bronchoscopy in 24/34 (71%) of cases treated with immunosuppressants. In the present study, we found that patients were being treated with about 50 mg/day of prednisolone and 74% (14/19) of patients with PCP were treated with immunosuppressive agents. However, these results were obtained by PCR detection using induced sputum and not bronchoalveolar lavage fluid, and resembled closely the profile of patients with PCP. It should be noted that one patient with RA who had been treated with methotrexate was diagnosed as having PCP. Although complications of immunosuppression are thought to be rare with the use of low-dose pulse MTX for RA, there is increasing evidence of PCP in RA patients treated with MTX and other immunosuppressants because of the impairment of cellular immunity, including T-cell function, by MTX [3033]. Such cases suggest that MTX should be used cautiously in patients with rheumatic disease that is being concomitantly treated with other medications. In this regard, a well known adverse effect of methotrexate is interstitial pneumonia [34, 35]. This makes the differential diagnosis between interstitial pneumonia caused by MTX and P. carinii infection a very important issue, in addition to the treatment of RA. PCR detection of P. carinii appears to be a useful method for the differentiation of PCP from pneumonitis caused by MTX.
An increase in serum LDH activity often supports the provisional diagnosis of PCP. Serum LDH activity is increased in > 90% of AIDS-related cases of PCP [18]. Previous studies have also shown a positive correlation between high serum LDH and KL-6 levels and the degree of radiographic abnormalities in PCP [23]. However, our results showed that LDH and KL-6 are not good markers of PCP in patients with connective tissue disease. These markers were high in patients with connective tissue diseases with or without PCP. On the other hand, ß-D-glucan constitutes part of the cell wall of most fungi and is detected in the serum of patients with organic mycosis. The serum level of ß-D-glucan correlates with the severity of disease and is used as a serological marker of such infections [36]. A recent genetic analysis suggested that P. carinii is a fungus [37], and ß-D-glucan has been detected in the cell wall of this organism [38]. ß-D-glucan is detected in the serum of patients with PCP and it is a clinically useful serological marker for monitoring the disease during treatment [39]. In our study, serum ß-D-glucan levels closely correlated with the occurrence of PCP and we consider it to be a useful marker of PCP and useful for distinguishing PCP from IP in patients with connective tissue diseases.
In conclusion, we emphasize the usefulness of PCR-based detection of P. carinii using induced sputum because of its high detectability and specificity. This technique could improve the diagnosis of PCP from respiratory material obtained non-invasively from patients with connective tissue diseases, and could thus lead to early administration of appropriate treatment and improvement of the prognosis. Recently, anti-TNF-
monoclonal antibody, soluble TNF receptor and anti-IL-6 receptor antibody, which have strong immunosuppressive effects, were used for refractory rheumatic diseases. The use of these biological agents might result in an increased incidence of opportunistic infections, including PCP. It is very important to consider the management of these infections simultaneously with the development of such an epoch-making therapy. To establish easy and early diagnostic assays for these infections is the responsibility of physicians.
The authors have declared no conflict of interest.
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Submitted 29 May 2003;
Accepted 13 October 2003