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The occurrence of polyomaviruses WUPyV and KIPyV among patients with severe respiratory infections

In 2007, the new polyomaviruses WUPyV and KIPyV were identified in patients with acute respiratory infections. The aim of this study was to investigate these viruses in hospitalized patients with severe acute respiratory infection (SARI). A retrospective study was conducted with 251 patients, from April 2009 to November 2010, using nasopharyngeal aspirates, naso- and oropharyngeal swab samples from hospitalized patients (children < 12 years and adults) who had SARI within 7 days of the onset of symptoms, including fever (> 38.8 °C), dyspnea, and cough. Clinical and epidemiological information was obtained through standardized questionnaire. Enrolled patients were initially suspected to have influenza A(H1N1)pdm09 infections. WUPyV and KIPyV were detected by real-time PCR. Samples were also tested for influenza A and B viruses, human respiratory syncytial virus, rhinovirus, metapneumovirus, coronavirus, adenovirus, and parainfluenza viruses. WUPyV and KIPyV were detected in 6.77% (4.78% and 1.99%, respectively) of hospitalized patients with SARI. All samples from children showed coinfections (rhinovirus was the most commonly detected). Six adults had polyomavirus infection and four (1.6%) had monoinfection. Of them, 3 reported comorbidities including immunosuppression and 1 patient had worse outcome, requiring ICU admission. These preliminary data may suggest a possible role of polyomaviruses in SARI among immunocompromised adult patients.

Coinfection with other pathogens has been reported in 74% of patients with KIPyV, 68 to 79% of patients with WUPyV, and 10% of coinfection with KIPyV and WUPyV in the absence of other respiratory viruses [21]. In Brazil, KIPyV and WUPyV were found in the saliva of immunocompromised and immunocompetent patients [22,23]. In this study, we investigated the PyV KI and WU in the occurence of severe respiratory diseases in hospitalized patients with acute respiratory infection (SARI).

Materials and methods
A retrospective study was conducted from April 2009 to November 2010 (19 months) using samples from hospitalized patients who had presented with SARI within 7 days of the onset of symptoms, including fever (> 38.8°C), dyspnea, and cough. Children (< 12 years) and adults were included. The clinical and epidemiological information was obtained through a standardized questionnaire. Informed consent was previously obtained from all enrolled patients initially suspected to have influenza A(H1N1)pdm09 infections. This study was approved by the Ethics Committee of Sao Paulo Federal University (CEP 0090/2016). Nasopharyngeal aspirates were obtained from children under 2 years old. For the other patients, naso-and oropharyngeal swabs were collected, according to the Brazilian Ministry of Health Protocol for management of influenza A (H1N1) 2009 pandemics, and stored in a freezer at − 80°C [24]. Total nucleic acids were extracted using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer instructions. The specimens were then tested for the presence of respiratory viruses according to PCR protocols available at our laboratory for influenza A (FLU A) and B (FLU B) viruses [25,26], human respiratory syncytial virus (HRSV) [27], human rhinovirus (HRV) [28], human metapneumovirus (hMPV) [29], human coronavirus (HCoV) [30], human adenovirus (HAdV) [31,32], and parainfluenza virus (PIV 1,2,3,4) [33,34].
For the WUPyV and KIPyV specific detection, qualitative real-time PCR was performed with the primers PyVF (5'-TTGGATGAAAATGGCATTGG-3′) and PyVR (5'-TAACCCTTCTTTGTCTAAARTGTAGCC-3′), that amplify a fragment of the gene encoding the capsid protein VP1 only common to KIPyV and WUPyV, and two specific probes, WUPyVp (5'-FAM-CATAACTTGTGCTGACCTTT TGGGAGTTAAC-BHQ1-3′) and KIPyVp (3'-VIC-ACATTACTTGTGCAGATATGCTTGGAACAGC-BHQ1-3′), both within the VP1 gene, to differentiate the WUPyV and KIPyV viruses [35]. The concentration of primers and probes was 800 nM and 200 nM, respectively, in 25 μL of total reaction volume, with the use of AgPath-ID™ One-Step RT-PCR reagents (Applied Biosystems, Austin, USA) and addition of 5 μL of extracted nucleic acid. In parallel reactions, primers and specific probe were used to amplify a fragment of the gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous control and to assess the quality of the samples as described by Bergallo et al. [16] Thermocycling conditions were performed in an Applied Biosystems 7500 Real-Time PCR System and involved 10 min at 95°C followed by 45 cycles of 10 s at 95°C and 1 min at 55°C (data collection). For both reactions, the cycle threshold value (Ct) of 39 was adopted as a cut-off. Chi-square analysis and Fisher's exact test were used to analyze the occurrence of the investigated PyV. Student's t test for independent samples and analysis of variance (ANOVA) were used to compare the mean ages. All statistical analyses were performed using OpenEpi version 2.3.1 (http://www.openepi.com), and a p value < 0.05 was considered statistically significant.  Table 1 compares the viral detection patterns within the 251 patients included in the study. Among the patients with viral infection, 18.5% (29/157) had a coinfection with 2 different viruses and 6.4% (10/157) with 3. Of the population with double viral coinfections, 44.8% (13/29) were also infected with WUPyVor KIPyV and 4 out of the 10 patients with triple coinfections were also positive for WUPyV or KIPyV. However, coinfection with WUPyV or KIPyV and other viruses was not correlated with severity of symptoms.

Results
The proportion of children with WUPyV or KIPyV was 7.3% (11/150), and all samples had multiple detections. The mean age of the children coinfected with polyomaviruses was 1.6 years (median 1 year; range 0-4 years); of these 7 had WUPyV infection and 4 had KIPyV infection. The children coinfected with respiratory viruses other than PyV were younger than those coinfected with PyV, with rates among patients up to 6 months of age being 1/11 (9.1%) and 12/24 (50.0%), respectively (p < 0.027).
Two adults (aged 33 and 91 years) showed coinfections with WUPyV or KIPyV, respectively. Monoinfections were detected in 4 adults, and 3 of them had WUPyV infection (median age 30 years, range 21-51 years). These 4 patients were admitted with fever, cough, and dyspnea, and the symptoms prevailed for up to 4 days. Radiological chest examination, bacterial hemoculture and systemic antibiotic prophylaxis were conducted according to hospital proceedings for clinical bacterial pneumonia investigation and current guidelines. Three patients reported comorbidities and presented with different states of immunosuppression. The length of the period of hospitalization and ICU admission are shown in Table 2. The patient with systemic lupus erythematosus (SLE) was admitted to the ICU for mechanical ventilation due to nosocomial pneumonia with bacterial coinfection. The 28-year-old patient without comorbidities was hospitalized for 24 h and was discharged upon resolution of the dyspnea.

Discussion
The role of the PyV in the pathogenesis of respiratory infections is not well understood. Some studies have demonstrated the presence of both PyV WU and KI in the asymptomatic population [36,37]. However, the rate of polyomavirus infection in adult patients hospitalized with SARI has not been established.
The debate about the role of these viruses in respiratory infections is ongoing because of the high rates of coinfection with other respiratory viruses (approximately 80%) [9,11,12,38,39]. Recently, high viral loads of KIPyV were observed in children with severe or very severe pneumonia in the absence of other viral or bacterial respiratory infection, suggesting a potential pathogenic role for KIPyV in these patients [12].
A coinfection rate of 76.5% was observed in the present study, mainly among children, with the most common types being WUPyV/HRV in 43% of cases, KIPyV/HRV in 60% and KIPyV/AdV in 67%, which is in accordance with the results by Allander et al. [1] and Gaynor et al. [2] Nonetheless, the frequent coinfections with WUPyV and KIPyV with other respiratory viruses and variations in clinical presentation preclude any conclusion about the association between infection and disease in children. Of interest is the finding of lower rates among very young children that may posit the later acquisition of polyomavirus infection by older children as suggest by serological and molecular studies [19,[40][41][42].
The finding of monoinfection by polyomaviruses in 4 adult patients hospitalized with SARI may suggest a potential pathogenic role. Moreover, 3 out of these 4 patients were immunocompromised, and one patient with SLE had a severe outcome and required ICU admission and mechanical ventilation. We performed a retrospective study. Therefore, it was not   possible to obtain samples from the lower respiratory tract from the immunocompromised patients for immunohistochemistry or in situ hybridization. Further studies are needed in order to understand the possible role of viral latency and reactivation of these DNA viruses in adult patients, especially among those who are immunocompromised. In this regard, prospective longitudinal studies with large sample sizes, including immunocompetent, immunocompromised, symptomatic, and asymptomatic patients could better answer some of these questions.