Le Infezioni in Medicina, n. 1, 57-61, 2014 Casi clinici Usefulness of (1→3)-b-D glucan in early diagnosing Case reports Pneumocystis jirovecii pneumonia: a case report Utilità del (1→3)-b-D glucano nella diagnosi precoce di polmonite da Pneumocystis jirovecii: un caso clinico Mustafa Altay Atalay1, Ayse Nedret Koc1, Leyla Gul Kaynar2, Melek Inci3, Fatma Filiz Kasap Tekinsen1, Bulent Eser2 1Department of Clinical Microbiology, Faculty of Medicine, Erciyes University, Kayseri, Turkey; 2Department of Hematology and Oncology, Faculty of Medicine, Erciyes University, Kayseri, Turkey: 3Department of Clinical Microbiology, Faculty of Medicine, MustafaKemal University, Hatay, Turkey n INTRODUCTION measure serum BDG and galactomannan levels in immunocompromised patients and start pre- neumocystis jirovecii pneumoniae (PJP) is emptive therapy based on the results. We re- a well-known opportunistic infection af- port an HIV-seronegative patient with acute P fecting immunocompromised hosts, es- lymphoblastic leukaemia (ALL) who devel- pecially patients infected with HIV. However, oped PJP, correlated with increasing serum with the rising number of patients receiving im- BDG levels. munosuppressive therapy, PJP is being increas- ingly recognized in immunosuppressed hosts who are not infected with HIV [1]. Invasive di- n CASE REPORT agnostic techniques such as BAL or trans- bronchial lung biopsy and specific staining of A 23-year-old male was admitted to the haema- cyst and/or trophozoites are the gold standard tological ward of the Erciyes University Hospi- for PJP diagnosis [2]. tal in September 2011 with fever and neutrope- BDG, one of the major components of the cyst nia and diagnosed with acute lymphoblastic wall of P. jirovecii, can be a serological marker leukaemia (ALL). The patient was treated with for the diagnosis and monitoring of PJP; some cycles of four Hyper-CVAD/ MTX-Ara-C (cy- reports have also supported this evidence [2-4]. clophosphamide, vincristine, doxorubicin, and (1→3) b-D-glucan (BDG) composes a portion of dexamethasone/methotrexate-cytarabine) the cell wall of most fungi, including Candida, chemotherapy and achieved complete remis- Aspergillus, and other yeasts and moulds such sion. Two years after the diagnosis, he com- as Trichosporon, Fusarium, Penicillium, plained of progressive dyspnea for three Cephalosporium, and Pneumocystis jiroveci [5]. months and fever with chills for two days. The serum level of BDG may be useful as a di- Cough and sputum were observed. Physical ex- agnostic and therapeutic indicator [6]. For the amination revealed a temperature of 38.6 ºC, early detection of fungal infection, we routinely respiratory rate of 25 breaths·min-1 and diffuse rales predominantly in the middle and lower chest. On laboratory examination, there was a Corresponding author severe neutropenia (white blood cell count Mustafa Altay Atalay [WBC] 1.07/mm3 and absolute neutrophil E-mail: [email protected] count 430/mm3). Initial chest film showed bilat- 57 2014 were sterile; smears for acid-fast bacilli and tests for viral antibodies were both negative. Serology for HIV was negative. A bron- choscopy with bronchoalveolar lavage was per- formed, but biopsy could not be carried out be- cause of decreasing oxygen saturation. My- cobacterial and mycologic cultures of BAL were negative. Giemsa and immunofluorescence staining of the BAL detected the existence of P. jiroveci in the lungs. The patient was therefore diagnosed with PJP. For the detection of P. jirovecii, a 10-mL BAL fluid was centrifuged at 1,875 g for 10 min., and a smear was microscopically examined for the presence of P. jirovecii with Giemsa stain and immunofluorescence stain (Cellabs Diagnostics Figure 1 - Giemsa staining (x 1000) of P. jirovecii cyst Pty. Ltd. Sydney, Australia) following the rec- containing up to 8 intracystic bodies. ommendations of the manufacturer. Seven days after admission, in Giemsa staining we saw P. jirovecii cysts containing up to eight in- tracystic bodies (Figure 1) and in immunofluo- rescence staining P. jirovecii exhibited bright, apple-green fluorescence of the round, thick- walled cysts and pleomorphic trophozoites in contrast to the reddish-brown colour of coun- terstained material (Figure 2). Laboratory values showed rising LDH with a maximum of 621 IU/L (normal 100-245 IU/L). One day and five days after admission, the serum level of BDG was >500 pg/mL (normal range <60 pg/mL) and galactomannan assay in serum was negative. After detection of P. jirovecii, TMP-SMX was added intravenously. The high serum level of BDG was considerably reduced by treatment Figure 2 - Immunofluorescence staining (x 400) of P. with TMP-SMX. The fever subsided the follow- jirovecii, apple-green fluorescence of the round, thick-walled cysts and pleomorphic trophozoites. ing day and the clinical condition of the patient increasingly improved. He was discharged from the hospital and clinical follow up was eral infiltrates without evidence of pleural effu- suggested. sion. Bone marrow aspirate showed myeloid suppression and no significant increase in blast cells. n DISCUSSION He was treated initially with supplemental oxy- gen and intravenous clarithromycin plus ce- We reported herein a case of PJP occurring two fepime for seven days, but clarithromycin was years after a patient was diagnosed as having changed to levofloxacin because of allergic re- ALL, in which the serum level of BDG was use- action. However, his condition deteriorated ful as an early diagnostic and therapeutic indi- and fever persisted. cator. The patient received treatment with granulo- Invasive fungal diseases, as opportunistic infec- cyte colony stimulating factor (GCSF) until his tions, are common among haematological ma- neutrophil count recovered. Intravenous dex- lignancy and AIDS patients and account for a amethasone with aminophylline was adminis- growing number of nosocomial infections, par- tered intermittently to relieve his dyspnea and ticularly among organ transplant recipients and wheezing. Sputum, blood, and urine cultures other patients receiving immunosuppressive 58 2014 treatments [7]. PJP in humans is caused by P. patient, one day after admission, both serum jirovecii, which has recently been reclassified as levels of LDH and BDG were significantly high- a fungus because its cell wall composition and er. As P. jirovecii is more closely related to fun- nucleotide sequences are more similar to those gi than to protozoa, the measurement of plasma of fungi [8]. PJP may occur in a variety of BDG, which is a cell wall constituent of fungi, haematological diseases, including non- may be useful for both the diagnosis and the Hodgkin’s lymphoma, chronic lymphocytic therapeutic monitoring of PCP [14]. The BDG leukaemia, acute leukaemia, chronic myeloid assay we used (Fungitell; Associates of Cape leukaemia, multiple myeloma, myelodysplastic Cod) is made from Limulus polyphemus found syndrome, Hodgkin’s disease and thrombocy- on the Atlantic coast of North America between topenia [9]. Florida and the Gulf of Maine. Our patient was diagnosed as having ALL ap- Certain fungal species produce very low levels proximately two years before and was admit- of BDG and are not detected by the Fungitell as- ted to hospital with progressive dyspnea and say. These include the genus Cryptococcus as fever with chills. well as Zygomycetes such as Absidia, Mucor and Though fever, dry cough and progressive dys- Rhizopus [15]. Persat et al. reported that the pnea with pulmonary infiltrates of an alveolar BDG test would be the first valuable non-inva- pattern are typical of PJP, these manifestations sive serodiagnostic tool for P. jiroveci pneumo- are non-specific [10]. nia and would be particularly useful for pa- PJP may be difficult to diagnose owing to non- tients for whom bronchoscopy was sometimes specific symptoms and signs, concurrent use of problematic [3]. prophylactic drugs, and simultaneous infection Compared with infection caused by other with various organisms. Since Pneumocystis pathogens, PJP can be significantly overlooked, cannot be cultured, the diagnosis of PJP re- especially in the presence of neutropenia. Our quires microscopic examination in order to case developed PJP in the setting of febrile neu- identify P. jirovecii from a clinically relevant tropenia. source such as specimens of induced sputum, The patient received broad-spectrum antibi- BAL fluid or lung tissue [11]. BAL has tradi- otics as the initial empirical treatment. tionally been the definitive clinical sample. In conclusion, PJP should be considered in the However, the procedure to obtain the specimen early phase of febrile neutropenia in patients is invasive while the induced sputum sample is with haematological malignancies. We experi- not sensitive compared to BAL and lung tissue enced a case of PJP that was diagnosed by for detection of P. jiroveci [12]. Giemsa and immunofluorescence stains in We detected the existence of P. jiroveci in the BAL; also, the patient’s serum level of BDG was lung by Giemsa and immunofluorescence stain- useful as diagnostic and therapeutic indicator. ing of the BAL. With Giemsa stain P jirovecii We suggest that measuring BDG could be con- cysts are variably stained and intracystic bodies sidered as a primary modality for early diagno- are present in different numbers within the sis of PJP, especially for patients in whom bron- cysts. Mature cysts have eight intracystic bod- choscopy is problematic because of respiratory ies. Some cysts appear empty. Serum testing