EXPERIMENTAL AND THERAPEUTIC MEDICINE 8: 1825-1830, 2014

Pneumocystis jirovecii dihydropteroate synthase gene mutations in a group of HIV‑negative immunocompromised patients with Pneumocystis pneumonia YINGJIAO LONG, CHENG ZHANG, LI SU and CHENGLI QUE Department of Pulmonary Medicine, First Hospital, Peking University, Beijing 100034, P.R. China Received March 5, 2014; Accepted September 4, 2014 DOI: 10.3892/etm.2014.2002 Abstract. The purpose of this study was to investigate dihydropteroate synthase (DHPS) mutations and their clinical context in non‑HIV‑infected patients with Pneumocystis pneumonia (PCP). DHPS genes in respiratory samples collected from HIV‑negative patients with PCP presented between January 2008 and April 2011 were amplified by polymerase chain reaction (PCR) and sequenced. Basic clinical data from the medical records of the patients were also reviewed. The most common point mutations, which result in Thr55Ala and Pro57Ser amino acid substitutions, were not detected in the Pneumocystis jirovecii sampled from the HIV‑negative patients. Two other point mutations, which result in nonsynonymous mutation, Asp90Asn and Glu98Lys, were identified in P. jirovecii from two patients. Among the patients, the levels of lactate dehydrogenase (LDH), C‑reactive protein (CRP) and plasma (1‑3) β‑D‑glucan were elevated in 75, 92.31 and 42.86% of patients, respectively. The percentage of circulating lymphocytes was significantly lower in non‑survivors than in survivors [4.2%, interquartile range (IQR) 2.4‑5.85 versus 10.1%, IQR 5.65‑23.4; P=0.019]. The neutrophil proportion in bronchoalveolar lavage fluid (BALF) was significantly higher in non‑survivors than in survivors (49.78±27.67 versus 21.33±15.03%; P=0.047). Thirteen patients had received adjunctive corticosteroids (1 mg/kg/day prednisone equivalent) and nine (69.23%) of them eventually experienced treatment failure. No common DHPS gene mutations of P. jirovecii were detected in the HIV‑negative PCP patients. However, other mutations did exist, the significance of which remains to be further identified. The elevation of neutrophil counts in BALF and reduction of the number of lymphocytes in peripheral blood may be associated with poor outcome. The efficacy

Correspondence to: Dr Chengli Que, Department of Pulmonary Medicine, First Hospital, Peking University, 8 Xishiku Street, Beijing 100034, P.R. China E‑mail: [email protected]

Key words: Pneumocystis jirovecii, drug resistance, dihydropteroate synthase gene, mutation

of adjunctive steroid therapy in HIV‑negative patients with P. jirovecii infection requires further investigation. Introduction Pneumocystis pneumonia (PCP), which is caused by Pneumocystis jirovecii (formerly called Pneumocystis carinii f. sp. hominis), is among the most prevalent opportunistic infections among immunocompromised patients, especially in patients with AIDS. In the past, PCP was generally indolent with latent onset; however, in HIV‑negative patients, it usually progresses rapidly to severe hypoxemia, or even respiratory failure (1) with high mortality. Currently, co‑trimoxazole, (a 1:5 mixture of trimethoprim and sulfamethoxazole; TMP‑SMZ) remains the first‑line and most effective regimen for anti‑Pneumocystis therapy and has been widely used for prophylaxis in patients at high risk for PCP since the 1990s. The widespread application of co‑trimoxazole has been implicated in the increases in sulfa drug‑resistant bacteria reported in HIV‑infected patients. It has also raised concerns about the possible selection of drug‑resistant Pneumocystis. In addition, a number of studies have confirmed that mutations in the Pneumocystis dihydropteroate synthase (DHPS) gene, which encodes the target enzyme that is combined with and inhibited by sulfamethoxazole, confer the sulfa drug resistance. The most frequently reported mutations are at positions corresponding to codons 55 and 57, which result in amino acid changes (2,3), that is, nonsynonymous mutation. Furthermore, there are also reports of mutations on codons 23 (2), 111 (2), 248 (2), 171 (4) and 60 (5). So far, genetic and epidemiological data concerning P. jirovecii infections in China are rather scarce, and mainly relate to isolates from HIV‑infected patients. Kazanjian et al (6) revealed a DHPS gene mutation rate of 7% (1/15) in AIDS patients with PCP in Beijing. However, Li et al  (7) did not find mutations in the DHPS gene in P. jirovecii in 10 HIV‑infected patients in Guangzhou, China. To the best of our knowledge, no data have been reported concerning Pneumocystis DHPS gene mutations in HIV‑negative patients in China. The objective of the present study was to investigate the presence of DHPS gene mutations in P. jirovecii from non‑HIV‑positive patients with PCP from a major general hospital in China. Clinical information, including certain laboratory parameters, treatment and outcome

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LONG et al: DHPS GENE MUTATIONS IN NON-HIV IMMUNOCOMPROMISED PATIENTS WITH PCP IN CHINA

were also reviewed, to determine the effect of mutation of the Pneumocystis DHPS gene on clinical outcome in HIV‑negative patients. Methods Patients. In this retrospective study, 22 non‑HIV‑positive patients with PCP, which were confirmed by Gomori methenamine silver (GMS) staining of respiratory samples, were included. The patients attended Peking University First Hospital, a 1,368‑bed teaching hospital in Beijing, China between January 2008 and April 2011. The present study was approved by the Institutional Review Board of Peking University and was performed in accordance with the recommendations of the Helsinki Declaration of 1975. Written informed consent was obtained from all patients. Materials. A total of 24 respiratory samples, comprising 22 bronchoalveolar lavage fluid (BALF) and two sputum samples, were obtained from the 22 HIV‑negative patients with confirmed PCP. The patients' medical records were also reviewed and the outcome was followed up. Sample processing and DNA extraction. All the samples were dissolved with dithiothreitol (DTT) first, and then filtered with a nylon mesh and centrifugation. Part of each sample was stained and demonstrated to be GMS positive, and the remainder was stored at -20˚C. DNA extraction was performed using E.Z.N.A. Blood DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA). Polymerase chain reaction (PCR). PCR was performed to analyze the DHPS gene of Pneumocystis, using the previously reported primers DHPS‑3 and DHPS‑4 (8). The reaction mixture contained 2.5 µl DNA template, 12.5 µl Taq PCR Master Mix (Qiagen, Valencia, CA, USA), 1.5 µl (10 µM) forward primer, 1.5 µl (10 µM) reverse primer and sterile water, making a total volume of 25 µl. PCR was used to amplify the samples, yielding a 370‑bp fragment. A hot‑start step at 94˚C for 5 min was followed by the following for 45 cycles: DNA denaturation at 94˚C for 30 sec, annealing at 61˚C for 30 sec and extension at 72˚C for 30 sec. This was followed by a final extension step at 72˚C for 5 min. The amplification products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide, and the bands were visualized with ultraviolet light. To prevent contamination, all PCR procedures were performed with a negative control of sterile water. Sequencing. The PCR products were sent to a genomic company (SinoGenoMax Co., Ltd, Beijing, China) and sequenced using an automated DNA sequencer (ABI 3730xl; Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast. cgi) and Clustal W (http://www.ebi.ac.uk/Tools/msa/) software. Statistical analysis. Clinical information, including laboratory results, therapy and outcome of patients were collected from medical records. SPSS software, version 17.0 (SPSS,

Table I. Patient demographics at diagnosis of Pneumocystis pneumonia in 18 patients. Characteristic Number Age, years (range)

51 (23-77)

Male gender (%)

12 (67)

Underlying disease (%) Inflammatory disease Pemphigus erythematosus ANCA‑associated systemic vasculitis Connective tissue disease Chronic glomerulonephritis Malignancy Solid tumor Hematologic malignancy Organ transplantation Bone marrow Kidney Other

1 (6) 3 (17) 3 (17) 1 (6) 3 (17) 2 (11) 1 (6) 3 (17) 2 (11)

Some patients had two or more underlying conditions. ANCA, ant‑neutrophil cytoplasmic antibody.

Inc., Chicago, IL, USA) was used to analyze data, and the Student's t‑test was used to assess significant differences in continuous data with Gaussian distribution, while the Mann‑Whitney U-test was used for non‑normal distribution. Proportions between groups were compared using the χ2 test. A P‑value of 240 IU/l (LLN) (%) β-D-glucan, pg/ml (%) β-D-glucan >50 pg/ml 10 pg/ml< β-D-glucan 50 pg/ml (%) β-D-glucan >10 pg/ml (%) PaO2/FiO2 Lymphocytes in BALF, % Neutrophils in BALF, %

52.89±10.89 49.22±5.18 0.479 5 (55.6) 7 (77.8) 0.331 91.24±5.18 75.44±17.18 0.003a 4.2 (2.4-5.85) 10.1 (5.65-23.4) 0.019a 416±84.29 435±355.81 0.908 93 (54-209) 97 (39-165.80) 0.703 69.1 (13.5-140) 55.15 (26.1-89.55) 0.454 4/6 (66.6) 2/8 (25.0) 0.170 5/6 (83.3) 5/8 (62.5) 0.580 164.79±78.44 245.73±114.32 0.106 18.44±18.60 31.00±11.80 0.107 49.78±27.67 21.33±15.03 0.047a

Multivariate analysis cannot be achieved due to small sample size. BALF, bronchoalveolar lavage fluid; PaO2, partial pressure of oxygen in arterial blood; FiO2, fraction of inspired oxygen. a

Table IV. Pneumocystis pneumonia treatment and outcome. Treatment TMP-SMZ TMP-SMZ + caspofungin Primaquine + clindamycin Adjunctive corticosteroids

Total

Non-survivors

Survivors

16 8 8 6 3 2 13 9

Mortality (%)

8 50 2 75 1 66.67 4 69.23

TPM, trimethoprim; SMZ, sulfamethoxazole.

prophylaxis and the occurrence of mutations in the P. jirovecii gene coding for DHPS, especially at codons 55 and 57, either as single mutations or combined (15,16). One study reported that DHPS mutations were significantly more common in patients who had previous exposure to sulfa drugs (18/29; 62%) than in those who had no exposure (13/123, 10.5%; P

Pneumocystis jirovecii dihydropteroate synthase gene mutations in a group of HIV-negative immunocompromised patients with Pneumocystis pneumonia.

The purpose of this study was to investigate dihydropteroate synthase (DHPS) mutations and their clinical context in non-HIV-infected patients with Pn...
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