Pneumocystis jirovecii Colonization and Its Association with Pulmonary Diseases Based on a Multicenter Study by an Improved Loop-Mediated Isothermal Amplification Assay CURRENT

Abstract Background Pneumocystis jirovecii an fungus and the The aim of present study is to develop an improved loop-mediated isothermal amplification (LAMP) assay for detection of Pneumocystis jirovecii (P. jirovecii) DNA and use it to examine the prevalence and association of P. jirovecii colonization among non-HIV patients with various pulmonary diseases. Methods We modified the previously reported LAMP assay for P. jirovecii by adding real-time detection. This method was used to detect P. jirovecii colonization in respiratory samples collected from 403 non-HIV patients with various respiratory diseases enrolled from 5 hospitals in China. We determined the prevalence of P. jirovecii colonization in 7 types of pulmonary diseases and assessed the association of P. jirovecii colonization with clinical characteristics of these diseases. Results The new LAMP assay showed no cross-reactivity with other common respiratory microbes and was 1,000 times more sensitive than that of conventional PCR. Using the new LAMP assay, we detected P. jirovecii colonization in 281 (69.7%) of the 403 patients enrolled. P. jirovecii colonization was more common in interstitial lung diseases than in chronic obstructive pulmonary disease (COPD) (84.6% vs 64.5%, P < 0.05). Patients with acute exacerbation of COPD had a higher prevalence of P. jirovecii colonization compared to patients with stabilized COPD (67.4% vs 43.3%, P < 0.05). P. jirovecii colonization was associated with decreased pulmonary function, increased levels of 1,3-β-D-glucan and C-reactive protein, and decreased levels of CD4+ T-cell counts (P < 0.05 for each). Approximately 70% of P. jirovecii colonized patients had confections with other fungi or bacteria. Conclusions We developed an improved LAMP assay for detecting P. jirovecii. Our multi-center study of 403 patients supports that P. jirovecii colonization is a risk factor for the development of pulmonary diseases and highlights the need to further study the pathogenesis and transmission of P. jirovecii colonization in pulmonary diseases.


Introduction
Pneumocystis jirovecii (P. jirovecii)is a unicellular parasitic fungus and an opportunistic pathogen almost exclusively infecting the lungs [ 1,2]. P. jirovecii infection may lead to fatal P. jirovecii pneumonia (PJP) in immunocompromised patients, particularly in those infected with human immunodeficiency virus (HIV). While PJP incidence in HIV-infected patients has been dramatically decreased due to the widespread combination antiretroviral therapy, there are growing reports of PJP cases among HIVnegative individuals [3][4][5][6][7]. In addition, asymptomatic P. jirovecii colonization is being increasingly identified in immunocompetent individuals [ 2,8]. In recent years, the role of P. jirovecii colonization in respiratory diseases has become a popular focus, especially in those with chronic obstructive pulmonary disease (COPD) and sudden infant death syndrome [ 5,[8][9][10][11]. These asymptomatic PJP individuals carrying P. jirovecii may act as important reservoirs of nosocomial infections. Therefore, it is necessary to clarify the role of P. jirovecii colonization in the pathogenesis of respiratory diseases.
Various molecular methods for P. jirovecii detection have been described previously, including conventional polymerase chain reaction (PCR) and real-time quantitative PCR [12][13][14][15]. Loop-mediated isothermal amplification (LAMP), originally developed by Notomi et al [ 16], is a novel, highly sensitive and specific method to diagnose infectious diseases. There has been one report on the application of LAMP to the detection of P. jirovecii in patients with chronic pulmonary diseases [ 5]. However, this method suffers several drawbacks, including subjective end-point visual detection of the LAMP amplification products, and potential inhibition of the LAMP amplification due to the presence of SYBR Green I [ 17]. The aims of this study were to develop an improved real-time fluorescence LAMP assay for P. jirovecii and then use it to examine the prevalence and association of P. jirovecii colonization among non-HIV patients with various pulmonary diseases.  [ 14,18]. Besides considering the predisposing factors for the disease, the respiratory diseases were diagnosed not only based on the clinical manifestations of respiratory tract such as the symptoms including cough, fever, expectoration, wheezing, chest pain, chest tightness, hemoptysis, and dyspnea, but also chest imaging test, bronchoscopic examination, and laboratory test, primarily including COPD (including the stable stage of COPD and acute exacerbations of (AECOPD), acute exacerbations of chronic bronchitis (AECB), interstitial lung diseases (ILDs), bronchiectasis, bronchial asthma, invasive pulmonary aspergillosis (IPA), and type I respiratory failure. a), regarding the To collect sputum samples, patients gargled with saline solution approximately three times to prevent oral microbial contamination before coughing up their morning sputa from the deep respiratory tract.

Materials And Methods
Sputa were collected in sterile containers and sent to clinical laboratories for bacterial and/or fungal culture and identification respectively using the VITEC-2 system (ATB system, BioMérieux, Marcyl'Étoile, France) and the Sensititre™, Aris 2X, system (Thermo Fisher Scientific, USA). The remaining of sputum samples were used to exact DNA and make slide smear to identify P. jirovecii microscopically following GMS and Giemsa staining.
Bronchoscopy examination was performed for 79 patients. BALF was collected using strict aseptic technique and filtered through a 2-layer nylon gauze to remove mucus. The filtered BALF was divided for DNA extraction (described below) and slide smear staining by GMS and Giemsa staining.
DNA extraction BALF samples were centrifuged at 1,500 rpm for 15 minutes; cell pellets were collected. The sputa were pretreated with 4% NaOH (w/v %) and then centrifuged at 12,000 rpm for 10 minutes. Cell pellets were collected. All cell pellets were washed three times with PBS and then subjected to DNA extraction following the traditional protocol involving proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. The DNA extracts were quantitated using the NanoDrop UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) and stored at -80°C before use.
Real-time fluorescence LAMP assay

Statistical analysis
All data were analyzed using the statistical analysis software SPSS (version 20.0, IBM Corporation).
Counting variables are represented as frequencies or percentages. Continuous variables are represented as the means ± SD. Data related to clinical findings, imaging examination; serologic parameters and other indicators were compared using the 2 test. Fisher's exact testing was used for the clinical data analysis when the number of samples was less than 40 or the expected value was less than 1. P values less than 0.05 were considered statistically significant.

Results
Development of an improved real-time fluorescence LAMP assay for P. jirovecii detection We developed a novel real-time fluorescence LAMP assay of P. jirovecii. This assay showed no crossreactivity with 10 other common respiratory pathogenic microorganisms tested, suggesting its high specificity (Fig 1). The sensitivity of this assay was compared with that of conventional PCR using serial dilutions of recombinant plasmid DNA containing P. jirovecii 18S rRNA gene. As shown in Fig 2, the lowest detection threshold was 1×10 copies μl -1 for the LAMP assay, which was 1,000 times more sensitive than that of conventional PCR (1×10 4 copies μl -1 ).
We also compared the sensitivity of the new LAMP assay with conventional PCR using clinical specimens of patients with various respiratory diseases. The positive rate of P. jirovecii was 69.7 % (281/403) by the LAMP assay and 40.5 % (163/403) by conventional PCR. In only one case positive by both assays, cyst forms ofP. jirovecii were detected by GMS and Giemsa staining. There was a good coincidence of the P. jirovecii detection rate between the LAMP and conventional PCR. The positive specimens detected by conventional PCR were also all positive in LAMP and vice versa. All these data indicate that our modified real-time fluorescence LAMP method is reliable for the detection of P.
jirovecii. Association of P. jirovecii colonization with clinical characteristics of patients with various pulmonary diseases.

Prevalence of P. jirovecii colonization in sputa and BALF from patients with various respiratory diseases
As illustrated in Table 5, no significant differences in PCT, LDH and ESR measurements were observed between patients with P. jirovecii colonization and patients without (P > 0.05). However, patients with P. jirovecii colonization showed increased levels of BDG and CRP, decreased levels of pulmonary function and CD4+ T-cell counts compared to patients without P. jirovecii colonization (P < 0.05). In addition, compared to patients without P. jirovecii colonization, patients with P. jirovecii colonization had more abnormal findings by thoracic HRCT imaging, including reticulation opacities, cystic lesions, septal thickening, nodules, irregular linear opacities and consolidation (P < 0.05).  9,29]. In the present multicenter study of 403 patients, we found that the prevalence of P.

Discussion
jirovecii colonization is high and associated with some clinical and laboratory characteristics of patients with respiratory diseases, supporting the involvement of P. jirovecii colonization in respiratory diseases.
To better assess the prevalence of P. jirovecii colonization, we developed an improved real-time fluorescence LAMP assay for the detection of P. jirovecii, which showed high sensitivity and specificity.
To avoid the inhibitory effect of SYBR Green I on LAMP amplification [ 17,30] , we chose to use SYTO-9 fluorescent dye (Thermo Fisher Scientific), which can be added into the reaction mixture without detectable inhibitory effect. This advantage allowed us to add real-time detection capacity to the LAMP assay, which showed higher amplification efficiency and sensitivity compared to the previously described LAMP assay with end-point detection [ 5]. (Fig 3).
The overall frequency of colonization by P. jirovecii observed in this study (69.7%) appears to be higher than that reported in previous studies (16%-63.3%) [ 5,31]. And the prevalence of P. jirovecii colonization in BALF of patients in this study (75.9%) is higher than that previously reported (21/30 or 70%) [ 29 ]. The reasons for this difference are unclear but may be related to different detection methods or different patient populations tested. Another possibility is the high-dose, long-term steroid treatment in our patient population. P. jirovecii cysts were detected by GMS and Giemsa in BALF from only one of the four IPA patients. This patient had received prolonged steroid treatment (~ 4 months) due to multiple recurrent infections with other fungi and bacteria. Although the steroid treatment is helpful to some respiratory diseases, especially COPD, IPA and bronchial asthma, high-dose steroids for a longer time lead to compromised lung function and immunity in patients, which might promote the colonization of P. jirovecii or the occurrence of PJP. The high prevalence of P. jirovecii colonization we observed is consistent with elevated serum beta-glucan levels in patients with P. jirovecii colonization compared to patients without P. jirovecii colonization ( Patients with chronic pulmonary diseases suffer frequently from various comorbidities diseases (Table   3), which may also contribute to the severity of diseases. In this study, we found a high rate of underlying autoimmune diseases in patients with P. jirovecii colonization (  (Table 4). Further studies are needed of the differential contributions of these pathogens to the respiratory diseases.
Our study did have certain limitations. First, there were certain heterogeneities in the sample types (sputa and BALF samples) and underlying clinical conditions, which may cause biases in data interpretation. Second, we did not subtract coinfection with other fungi or bacteria when assessing the association of P. jirovecii colonization with clinical characteristics due to the small sample size after subtraction. Third, sputum specimens are not the best specimens to examine the prevalence of coinfection with other fungi or bacteria in the lungs due to unavoidable contamination with normal microbial flora in the oral cavity and nasopharynx. We used a large number of sputum specimens in this study since the limited availability of BALF samples.
In summary, we have developed an improved LAMP assay for detecting P. jirovecii. Our multi-center study of 403 patients using this assay has provided additional new evidence for the involvement of P.
jirovecii colonization in the development of pulmonary diseases, and highlights the need to further study the pathogenesis and transmission of P. jirovecii colonization in pulmonary diseases.

Acknowledgement
This study was supported by Natural Science Foundation of China (Project-81370189). We would like to thank for the assistance from many healthcare workers from the following units including the    Tables.doc