J Infect Chemother 20 (2014) 678e681

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Original article

Serum (1 / 3) b-D-glucan assay for discrimination between Pneumocystis jirovecii pneumonia and colonization Sadatomo Tasaka a, *, Seiki Kobayashi b, Kazuma Yagi a, Takahiro Asami a, Ho Namkoong a, Wakako Yamasawa c, Makoto Ishii a, Naoki Hasegawa d, Tomoko Betsuyaku a a

Division of Pulmonary Medicine, Keio University School of Medicine, Tokyo, Japan Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo, Japan Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan d Center for Infection Control and Infectious Diseases, Keio University School of Medicine, Tokyo, Japan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 May 2014 Received in revised form 5 July 2014 Accepted 7 July 2014 Available online 24 July 2014

Polymerase chain reaction (PCR) technique is being increasingly used for the microbiological diagnosis of Pneumocystis pneumonia (PCP). As PCR is highly sensitive, it can be positive even in a patient with Pneumocystis colonization. In this study, we evaluated whether the b-D-glucan assay could be used to differentiate between PCP and Pneumocystis jirovecii colonization in immunocompromised patients with pulmonary infiltrates. We retrospectively evaluated data from 166 consecutive patients who underwent bronchoalveolar lavage for the diagnosis of PCP. Serum levels of b-D-glucan in the negative, colonization, probable PCP, and definite PCP groups were 20.2 ± 6.3, 48.8 ± 15.9, 89.9 ± 20.2, 224.9 ± 25.9 pg/mL, respectively. The b-D-glucan levels in the definite PCP group were significantly higher than those in the other 3 groups (p < 0.001). Serum b-D-glucan levels in patients with either definite or probable PCP (173.1 ± 18.8 pg/mL) were significantly greater than those in patients with colonization who had positive PCR results but improved without anti-PCP treatment (p < 0.002). The cut-off level for discrimination was estimated to be 33.5 pg/mL, with which the positive predictive value was 0.925. These results indicate that b-D-glucan is a useful marker to differentiate between PCP and Pneumocystis colonization. A positive b-D-glucan assay result might be a good indication to begin anti-PCP treatment. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Keywords: Pneumocystis pneumonia Colonization Polymerase chain reaction Bronchoalveolar lavage (1 / 3)-b-D-glucan

1. Introduction Pneumocystis pneumonia (PCP) remains one of the most frequent opportunistic infections in immunocompromised patients, including those infected with human immunodeficiency virus (HIV) [1]. Because Pneumocystis cannot readily be cultured in the laboratory, microscopic demonstration of the organism in respiratory specimens has been the gold standard for the diagnosis of PCP [1,2]. The sensitivity of microscopic visualization of Pneumocystis is inconsistent and depends on the skill and experience of the observer. In addition, nonHIV patients, who develop PCP, are characterized with a lower fungal burden than HIV-infected patients with PCP [3]. This lower fungal burden may result in false-negative results from microscopic

* Corresponding author. Division of Pulmonary Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: þ81 3 5363 3793; fax: þ81 3 3353 2502. E-mail address: [email protected] (S. Tasaka).

examination [4]. Because of these issues, the highly sensitive polymerase chain reaction (PCR) technique is being increasingly used for the microbiological diagnosis of PCP [5,6]. However, Pneumocystis colonization, defined as the detection of its DNA without signs or symptoms of pneumonia, has been reported to be highly prevalent even among the general population [7]. If a patient colonized with Pneumocystis jirovecii experiences pneumonia resulting from a different etiology, a positive Pneumocystis PCR result may mislead clinicians. In such cases, it is critical to distinguish Pneumocystis colonization from PCP, however, an algorithm for the discrimination between these two conditions remains to be established. (1 / 3)-b-D-glucan (b-D-glucan) is a major structural component of the cell wall of several fungi, including Pneumocystis [8]. Previous studies have shown that the serum b-D-glucan assay can be useful for the diagnosis of PCP [9,10]. However, there are few data available on the ability of this assay to detect Pneumocystis colonization or differentiate this colonization from PCP [11]. In this study, we evaluated whether b-D-glucan and other serum markers could be used to differentiate between PCP and P. jirovecii

http://dx.doi.org/10.1016/j.jiac.2014.07.001 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

S. Tasaka et al. / J Infect Chemother 20 (2014) 678e681

colonization in immunocompromised patients with pulmonary infiltrates. 2. Methods The ethical committee of Keio University School of Medicine approved the study protocol. 2.1. Patient selection We retrospectively evaluated data from 166 consecutive patients who underwent bronchoalveolar lavage (BAL) for the diagnosis of PCP at Keio University Hospital (Tokyo, Japan) during the period from April 2006 until November 2011. All of the patients had ground-glass opacities on chest computed tomography and clinical presentations such as fever, cough or dyspea. 2.2. Data collection We reviewed the medical records of all the patients evaluated for their clinical courses. In sera, five serum markers C-reactive protein (CRP), lactate dehydrogenase (LDH), KL-6, surfactant protein D (SP-D) and b-D-glucan were evaluated. The levels of b-Dglucan were measured with a kinetic turbidimetric assay using BetaeGlucan test WAKO™ (Wako Pure Chemical Industries, Tokyo, Japan).

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2.6. Diagnosis of PCP The diagnosis of definite PCP was established by the microscopic identification of P. jirovecii in BAL fluid. A diagnosis of probable PCP was made when the patient did not have microscopically visible P. jirovecii but had a positive PCR results and a clinical presentation compatible with PCP, including complete resolution of pulmonary infiltrates after a full course of anti-PCP treatment [5]. The patients who had positive PCR results but improved without anti-PCP treatment were diagnosed as ‘pulmonary infiltration with P. jirovecii colonization’ (colonization). Those having neither observable P. jirovecii nor positive PCR results were classified as negative patients. 2.7. Statistical methods All data are expressed as the mean ± SE. The data were analyzed by one-way analysis of variance with Scheffe's post hoc test using SPSS Windows 15.0 statistical analysis software (SPSS, Inc., Chicago, IL). In patients with PCP (either definite or prpbable), serum b-Dglucan levels were compared between HIV and non-HIV subjects using unpaired t-test. To evaluate the sensitivity and specificity of b-D-glucan assay, a receiver operating characteristic (ROC) curve was constructed. Statistical significance was defined as p < 0.05. 3. Results 3.1. Patient characteristics

2.3. BAL procedure Informed consent to conduct BAL was obtained from either the patient or surrogate. In most cases, BAL was targeted toward affected lung segments as noted on chest CT and performed with 50 mL of 0.9% saline solution per lavage. Usually, three lavages were performed, and the lavage fluid was immediately placed on ice. 2.4. BAL fluid processing for analysis The BAL fluid was pooled, filtered through sterile gauze to remove mucous strands, and centrifuged at 200  g for 5 min at 4  C. The cell pellets were used for the differential counts on Wright-Giemsa-stained preparations. For the detection of P. jirovecii, a 10 mL aliquot of BAL fluid was centrifuged at 1875  g for 10 min, and a smear was microscopically examined for the presence of P. jirovecii with Grocott-Gomori methenamine stain (GMS) and Calcofluor white stain (Fungifluor; Polysciences, Inc., Warington, PA), following the manufacturer's recommendations. 2.5. Detection of Pneumocystis DNA Template DNAs were extracted from BAL fluid samples by means of proteinase K digestion and phenol/chloroform extraction and subsequently subjected to PCR as templates. The PCR analysis for Pneumocystis DNA was performed in 50 ml of amplification reaction mixtures, with denaturation at 94  C for 90 s, annealing at 50  C for 90 s, and extension at 72  C for 2 min (40 cycles). The following oligonucleotide primers were used at 100 pmol: 50 -GAT GGC TGT TTC CAA GCC CA-30 and 50 -GTG TAC GTT GCA AAG TAC TC-30 . DNA products with lengths of 376 bp were amplified from template DNAs. This analysis was done at SRL Inc., (Tachikawa, Japan). The details of this method were described by Wakefield and colleagues [12].

During the study period, 64 patients, of whom 16 (25.0%) were HIV-positive, were diagnosed with definite PCP. Another 38 patients had probable PCP, of whom 6 (15.8%) were HIV-positive. Twenty-four patients were colonized with P. jirovecii; none of the colonized patients were HIV-positive. Forty patients were negative for P. jirovecii, of which 1 (2.5%) was HIV-positive. The patient characteristics and laboratory data are summarized in Table 1. The patients with definite PCP were significantly younger than those of other 3 groups (p < 0.001). As an underlying disease, 22 of the patients with definite and probable PCP had HIV infection, whereas none of those with Pneumocystis colonization did. A chisquare test showed heterogeneity of the underlying diseases across the groups (p < 0.02).

Table 1 Patient's characteristics and laboratory data.

Age (year) Male/female Underlying disease HIV infection Hematological malignancy Collagen vascular disease Solid tumor Organ transplantation Others Serum marker CRP (mg/dL) LDH (IU/L) KL-6 (U/mL) SP-D (ng/mL) b-D-glucan (pg/mL) PaO2/FIO2 (mmHg)

Definite PCP (n ¼ 64)

Probable PCP Colonization Negative (n ¼ 38) (n ¼ 24) (n ¼ 40)

47 ± 2* 37/27

59 ± 3 22/16

66 ± 3 11/13

60 ± 3 20/20

16 15

6 6

0 7

1 8

15

14

6

12

9 6 3

4 1 7

4 1 6

9 1 9

6.87 ± 0.74 436 ± 24 1.216 ± 203 291 ± 117 224.9 ± 25.9* 270 ± 19

6.73 ± 0.91 352 ± 28 1037 ± 161 268 ± 71 89.9 ± 20.2 308 ± 18

5.14 ± 0.89 397 ± 38 791 ± 180 212 ± 36 48.8 ± 15.9 269 ± 21

5.63 ± 0.83 339 ± 24 588 ± 66 181 ± 49 20.2 ± 6.3 318 ± 18

Mean ± SE. *p < 0.001 vs other 3 groups.

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S. Tasaka et al. / J Infect Chemother 20 (2014) 678e681

The final diagnosis of the colonized patients included druginduced pneumonitis (n ¼ 8), alveolar hemorrhage (n ¼ 5), cytomegalovirus pneumonia (n ¼ 3), and lung involvement of the underlying disease (n ¼ 2). In 6 patients with P. jirovecii colonization, the final diagnosis remained undetermined. 3.2. Levels of serum markers and oxygenation Serum levels of CRP, LDH, KL-6, SP-D and b-D-glucan were summarized in Table 1. The levels of CRP, LDH, KL-6 and SP-D were elevated above the normal range but did not differ between the patient groups. Serum levels of b-D-glucan in the negative, colonization, probable PCP, and definite PCP groups were 20.2 ± 6.3 pg/mL, 48.8 ± 15.9 pg/mL, 89.9 ± 20.2 pg/mL, 224.9 ± 25.9 pg/mL, respectively (Fig. 1). The b-D-glucan levels in the definite PCP group were significantly higher than those in the other 3 groups (p < 0.001). Although the b-D-glucan levels were not different between the probable PCP and colonization groups, the average b-Dglucan level in the 102 patients with either definite or probable PCP was 173.1 ± 18.8 pg/mL, which was significantly greater than that in the colonization group (p < 0.002). In HIV patients with PCP (either definite or probable), the average b-D-glucan level was 240.3 ± 43.1 pg/mL, which was greater than in non-HIV patients with PCP (152.8 ± 20.4 pg/mL). There was a significant difference in the b-D-glucan levels between HIV and non-HIV patients with PCP (p < 0.05). In non-HIV patients, serum b-D-glucan levels were 22.5 ± 9.4 pg/ mL, 48.8 ± 15.9 pg/mL, 94.5 ± 23.7 pg/mL, 233.7 ± 30.2 pg/mL in the negative, colonization, probable PCP, and definite PCP groups, respectively. When only non-HIV patients were subjected, the b-Dglucan levels in the definite PCP group were still significantly higher than those in the other 3 groups (p < 0.001). Oxygenation index that was defined as PaO2/FIO2 ratio did not differ between the patient groups. 3.3. Discrimination of Pneumocystis pneumonia and colonization by serum b-D-glucan An ROC curve of the serum b-D-glucan assay was constructed for discrimination between PCP, either definite or probable, and P. jirovecii colonization (Fig. 2). The area under the curve value was

Fig. 1. Serum levels of b-D-glucan. The b-D-glucan levels in the negative, colonization, probable PCP, and definite PCP groups were 20.2 ± 6.3 pg/mL, 48.8 ± 15.9 pg/mL, 89.9 ± 20.2 pg/mL, 224.9 ± 25.9 pg/mL, respectively. The b-D-glucan levels in the definite PCP group were significantly higher than those in the other 3 groups (p < 0.001).

Fig. 2. A receiver operating characteristic curve for serum b-D-glucan assay for discrimination between PCP and Pneumocystis colonization. The areas under the curve value is 0.870.

0.870. Based on the ROC curve, the discriminatory cut-off level of bwas estimated to be 33.5 pg/mL. With this cut-off point, the sensitivity and specificity values of b-D-glucan were 80.3% and 83.3%, respectively. The positive predictive value (PPV) was 0.925, and the negative predictive value (NPV) was 0.625. D-glucan

4. Discussion In the present study, we evaluated whether the serum b-Dglucan assay is useful to differentiate between PCP and P. jirovecii colonization. Although the b-D-glucan level did not differ significantly between the probable PCP and the colonization groups, the level was significantly higher in patients with either definite or probable PCP than in those with colonization. With a cut-off level of 33.5 pg/mL, the PPV was as high as 0.925. These results indicate that a positive b-D-glucan assay result might be a good indication to begin anti-PCP treatment. Because of invasiveness of BAL and imperfect specificity of PCR, there was a need to investigate the utility of serological diagnosis of PCP [9]. b-D-glucan is a major component of the yeast cell wall, and measurements of serum b-D-glucan levels have been used for the diagnosis of invasive deep mycosis such as candidiasis and aspergillosis [8,13]. Although the assay is not specific for Pneumocystis, the measurement of serum b-D-glucan level has been used as an adjunctive tool for the diagnosis of PCP [9e11,14]. We have previously reported that serum b-D-glucan is a reliable marker for the diagnosis of PCP [9]. Because a meta-analysis revealed high sensitivity of the b-D-glucan assay for PCP, it could be useful at least for screening for the disease, especially for patients with severe respiratory failure [10]. In a previous investigation, we compared serum levels of b-D-glucan between patients with PCP diagnosed microscopically and those without PCP [9]. The results indicated that a cut-off value of 31.1 pg/mL had a sensitivity of 92% and a specificity of 86% for detecting PCP [9]. In another study including 111 patients with AIDS, Watanabe and coworkers evaluated the diagnostic value of the assay and described the use of a cut-off value of 23.2 pg/mL, which is a lower cut-off than the value we had reported [14]. The difference in the cut-off values might be due to differences in the proportions of HIV-positive patients included in the 2 studies. It has been reported that PCP patients without HIV infection have a lower burden of Pneumocystis than those with AIDS. This can lead to difficulty in detecting the organisms by

S. Tasaka et al. / J Infect Chemother 20 (2014) 678e681

microscopic observation in non-HIV patients [3]. In our previous study, only 3 patients had HIV infection among 222 patients who were microscopically negative. This might have led to some PCP patients having false-negative results, as determined by microscopy, and in turn, this could have led to the determination of a higher cut-off value, one that is closer to the cut-off level between PCP and Pneumocystis colonization determined in the present study [9]. In this study, 25% of patients with definite PCP had HIV infection, whereas the rate was only 2.5% in the negative group. A metaanalysis by Lu and colleagues showed that PCR had a sensitivity of 99% and a specificity of 91% for diagnosing PCP in an AIDS population [6]. They also showed that PCR had a sensitivity of 99% and a specificity of 92% for diagnosing PCP in a non-AIDS population, indicating that the PCR assay is a powerful tool for the diagnosis of PCP, especially in those without HIV infection [6]. Because of its high sensitivity, PCR is now widely used in clinical practice, although it lacks the necessary specificity to distinguish PCP from colonization [15,16]. Because the b-D-glucan assay might complement the use of PCR in the diagnosis of PCP, we consider that the cut-off level of 33.5 pg/mL for discrimination between PCP and Pneumocystis colonization could have clinical significance. In HIV patients with PCP, the b-D-glucan levels were significantly higher than in non-HIV patients with PCP, which is compatible with the observation by Nakamura and colleagues [17]. This result might be associated with the fact that the number of P. jirovecii in the lungs of HIV-related PCP patients is significantly increased as compared with that in non-HIV PCP patients [3]. In a previous report, Matsumura and colleagues evaluated whether b-D-glucan assays could differentiate PCP from P. jirovecii colonization [11]. They revealed that the sensitivity and specificity for discriminating probable PCP from colonization were 76.2% and 73.3%, respectively, at a cut-off value of 6.0 pg/mL. In comparison with their values, our results showed higher sensitivity and specificity with higher cut-off level of b-D-glucan. These differences might be owing to the higher percentage of PCP patients in our study subjects. In the study by Matsumura and colleagues, 53 of 147 study subjects (36%) had PCP [11], while 61% of our patients had PCP. In the present study, all of the study subjects underwent BAL and sensitive Calcofluor white stain was used to detect P. jirovecii, which might result in the higher percentage of PCP diagnoses. Recently, Damiani and coworkers reported that combination of quantitative PCR and serum b-D-glucan assay could efficiently differentiate PCP and colonization [18]. Although quantitative PCR assays have been reported to be more promising for the diagnosis of PCP than conventional PCR [15], the DNA sequences targeted and the cut-off values used in quantitative PCR assays remain to be standardized. Because both conventional PCR and b-D-glucan assays are standardized and commercially available in many countries, the results of the present study might be applicable to the clinical practice. In conclusion, we compared serum b-D-glucan levels in patients with PCP with those in patients with Pneumocystis colonization and

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proposed a cut-off level of 33.5 pg/mL to differentiate between PCP and colonization. Serum b-D-glucan is a useful marker for discriminating between PCP and Pneumocystis colonization. Conflict of interest None. References [1] Thomas Jr CF, Limper AH. Pneumocystis pneumonia. N Engl J Med 2004;350: 2487e98. [2] Catherinot E, Lanternier F, Bougnoux ME, Lecuit M, Couderc LJ, Lortholary O. Pneumocystis jirovecii pneumonia. Infect Dis Clin North Am 2010;24:107e38. [3] Limper AH, Offord KP, Smith TF, Martin 2nd WJ. Pneumocystis carinii pneumonia. Differences in lung parasite number and inflammation in patients with and without AIDS. Am Rev Respir Dis 1989;140:1204e9. [4] Reid AB, Chen SC, Worth LJ. Pneumocystis jirovecii pneumonia in non-HIVinfected patients: new risks and diagnostic tools. Curr Opin Infect Dis 2011;24:534e44. [5] Azoulay E, Bergeron A, Chevret S, Bele N, Schlemmer B, Menotti J. Polymerase chain reaction for diagnosing pneumocystis pneumonia in non-HIV immunocompromised patients with pulmonary infiltrates. Chest 2009;135:655e61. [6] Lu Y, Ling G, Qiang C, Ming Q, Wu C, Wang K, et al. PCR diagnosis of pneumocystis pneumonia: a bivariate meta-analysis. J Clin Microbiol 2011;49: 4361e3. [7] Ponce CA, Gallo M, Bustamante R, Vargas SL. Pneumocystis colonization is highly prevalent in the autopsied lungs of the general population. Clin Infect Dis 2010;50:347e53. [8] Obayashi T, Yoshida M, Mori T, Goto H, Yasuoka A, Iwasaki H, et al. Plasma (1– >3)-beta-D-glucan measurement in diagnosis of invasive deep mycosis and fungal febrile episodes. Lancet 1995;345:17e20. [9] Tasaka S, Hasegawa N, Kobayashi S, Yamada W, Nishimura T, Takeuchi T, et al. Serum indicators for the diagnosis of pneumocystis pneumonia. Chest 2007;131:1173e80. [10] Onishi A, Sugiyama D, Kogata Y, Saegusa J, Sugimoto T, Kawano S, et al. Diagnostic accuracy of serum 1,3-b-D-glucan for Pneumocystis jiroveci pneumonia, invasive candidiasis, and invasive aspergillosis: systematic review and meta-analysis. J Clin Microbiol 2012;50:7e15. [11] Matsumura Y, Ito Y, Iinuma Y, Yasuma K, Yamamoto M, Matsushima A, et al. Quantitative real-time PCR and the (1,3)-b-D-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization. Clin Microbiol Infect 2012;18:591e7. [12] Wakefield AE, Pixley FJ, Banerji S, Sinclair K, Miller RF, Moxon ER, et al. Detection of Pneumocystis carinii with DNA amplification. Lancet 1990;336: 451e3. [13] Matsumoto Y, Matsuda S, Tegoshi T. Yeast glucan in the cyst wall of Pneumocystis carinii. J Protozool 1989;36:21Se2S. [14] Watanabe T, Yasuoka A, Tanuma J, Yazaki H, Honda H, Tsukada K, et al. Serum (1–>3) b-D-glucan as a noninvasive adjunct marker for the diagnosis of pneumocystis pneumonia in patients with AIDS. Clin Infect Dis 2009;49: 1128e31. [15] Huggett JF, Taylor MS, Kocjan G, Evans HE, Morris-Jones S, Gant V, et al. Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients. Thorax 2008;63:154e9. [16] Maskell NA, Waine DJ, Lindley A, Pepperell JC, Wakefield AE, Miller RF, et al. Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study. Thorax 2003;58:594e7. [17] Nakamura H, Tateyama M, Tasato D, Haranaga S, Yara S, Higa F, et al. Clinical utility of serum b-D-glucan and KL-6 levels in Pneumocystis jirovecii pneumonia. Intern Med 2009;48:195e202. [18] Damiani C, Le Gal S, Da Costa C, Virmaux M, Nevez G, Totet A. Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1->3)-bD-glucan for differential diagnosis of Pneumocystis pneumonia and Pneumocystis colonization. J Clin Microbiol 2013;51:3380e8.