Presence of Merkel cell polyomavirus DNA and large-T antigen in keratinocyte carcinomas and its correlation with immunohistochemical markers p16, p53 and ki67.

BACKGROUND: Merkel cell polyomavirus (MCPyV), a human polyomavirus that is unequivocally linked to merkel cell carcinoma (MCC), has been found in association with keratinocytes carcinomas (KC), especially basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Nevertheless, there is scarce information about the possible involvement of MCPyV in the development of KC. OBJECTIVES: To assess the presence of MCPyV DNA and Large-T Antigen (LT-Ag) via Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC) in cases of KC, and to correlate its presence with immunohistochemical markers p16, p53, and ki67, tumor type and subtype, sun-exposed location, and epidemiological data. METHODS: The prevalence of MCPyV DNA, LT-Ag, and immunohistochemical markers p16, p53, and ki67 was assessed by PCR and Immunohistochemistry (IHC) in 127 cases of KC, these results were correlated with tumor type and subtype, sun-exposed location, and epidemiological data. RESULTS: The MCPyV DNA was detected in 42.57% (43 of 101) cases by PCR, the LT-Ag was detected in 16.4% (20 of 122) of cases, p16 in 81.5% (97 of 119), p53 in 66.4% (83 of 125), ki67 in 89% (73 of 82). No correlation between MCPyV LT-Ag and DNA confronted with tumor type, subtype, location site, and immunohistochemical markers was found. A single correlation between the MCPyV LT-Ag and cSCC tumors and peri-tumoral lymphocyte cells was noted. STUDY LIMITATIONS: Further steps need to be taken to better evaluate the MCPyV influence and its possible role in KC carcinogenesis, as the evaluation of the virus genome state, the gene sequence that encodes LT-Ag in the KC tumor cells, and in situ hybridization for viral DNA or RNA in these cells. CONCLUSIONS: Despite the frequent detection of MCPyV in KC, the data available so far does not support the hypothesis of a causal relationship between them.

Merkel cell polyomavirus and its etiological relationship with skin tumors.

Viruses have been frequently identified in several human neoplasms, but the etiological role of these viruses in some tumors is still a matter of controversy. Polyomaviruses stand out among the main viruses with oncogenic capacity, specifically the Merkel cell polyomavirus (MCPyV). Recent revisions in the taxonomy of polyomaviruses have divided the Polyomaviridae family into six genera, including 117 species, with a total of 14 currently known human-infecting species. Although the oncogenicity of polyomaviruses has been widely reported in the literature since 1950, the first description of a polyomavirus as an etiological agent of a neoplasm in humans was made only in 2008 with the description of MCPyV, present in approximately 80% of cases of Merkel cell carcinoma (MCC), with the integration of its genome to that of the tumor cells and tumor-specific mutations, and it is considered the etiological agent of this neoplasm since then. MCPyV has also been detected in keratinocyte carcinomas, such as basal cell carcinoma and squamous cell carcinoma of the skin in individuals with and without immunosuppression. Data on the occurrence of oncogenic viruses potentially involved in oncogenesis, which cause persistence and tissue injury, related to the Merkel cell polyomavirus are still scarce, and the hypothesis that the Merkel cell polyomavirus may play a relevant role in the genesis of other cutaneous carcinomas in addition to MCC remains debatable. Therefore, the present study proposes to explore the current knowledge about the presence of MCPyV in keratinocyte carcinomas.

Merkel cell polyomavirus and its etiological relationship with skin tumors

Abstract Viruses have been frequently identified in several human neoplasms, but the etiological role of these viruses in some tumors is still a matter of controversy. Polyomaviruses stand out among the main viruses with oncogenic capacity, specifically the Merkel cell polyomavirus (MCPyV). Recent revisions in the taxonomy of polyomaviruses have divided the Polyomaviridae family into six genera, including 117 species, with a total of 14 currently known human-infecting species. Although the oncogenicity of polyomaviruses has been widely reported in the literature since 1950, the first description of a polyomavirus as an etiological agent of a neoplasm in humans was made only in 2008 with the description of MCPyV, present in approximately 80% of cases of Merkel cell carcinoma (MCC), with the integration of its genome to that of the tumor cells and tumor-specific mutations, and it is considered the etiological agent of this neoplasm since then. MCPyV has also been detected in keratinocyte carcinomas, such as basal cell carcinoma and squamous cell carcinoma of the skin in individuals with and without immunosuppression. Data on the occurrence of oncogenic viruses potentially involved in oncogenesis, which cause persistence and tissue injury, related to the Merkel cell polyomavirus are still scarce, and the hypothesis that the Merkel cell polyomavirus may play a relevant role in the genesis of other cutaneous carcinomas in addition to MCC remains debatable. Therefore, the present study proposes to explore the current knowledge about the presence of MCPyV in keratinocyte carcinomas.

Human Bocavirus in Brazil: Molecular Epidemiology, Viral Load and Co-Infections.

Human bocavirus (HBoV) is an emerging virus and has been detected worldwide, especially in pediatric patients with respiratory and gastrointestinal infection. In this study, we describe HBoV prevalence, genotypes circulation and DNA shedding, in stool samples from children up to two years of age in Brazil. During 2016 and 2017, 886 acute gastroenteritis (AGE) stool samples from ten Brazilian states were analyzed by TaqMan®-based qPCR, to detect and quantify HBoV. Positive samples were genotyped by sequencing the VP1/2 overlap region, followed by phylogenetic analysis and co-infections were accessed by screening other gastroenteric viruses. HBoV was detected in 12.4% (n = 110) of samples, with viral load ranging from 1.6 × 102 to 1.2 × 109 genome copies per gram of stool. From these, co-infections were found in 79.1%, and a statistically lower HBoV viral load was found compared to viral loads of rotavirus, norovirus and adenovirus in double infected patients (p < 0.05). No significant differences were found between HBoV viral load in single or co-infections, age groups or genotypes. Phylogenetic analysis identified the circulation of HBoV-1 in 38%, HBoV-2 in 40% and HBoV-3 in 22%. Continuous HBoV monitoring is needed to clarify its role in diarrhea disease, especially in the absence of classic gastroenteric viruses.

Screening for BKV-DNAEMIA after renal transplantation in a resource limited setting.

Costs may hinder the implementation of BK polyomavirus (BKV)-DNAemia screening in resource-limited kidney transplant (KT) centers. We analyzed data from two studies to assess the performance and potential cost saving of a dual-step screening strategy based on the use of a preliminary qualitative semi-nested PCR (snPCR) assay followed by BKV-DNAemia quantification after KT. In the preliminary study, in which 130 samples from 33 KT recipients were screened for BKV-DNAemia, the estimated positive and negative predictive values of snPCR, as compared to quantitative PCR (qPCR), were 88% and 99%, respectively. In the second study, which included 84 KT recipients, BKV-DNAemia was detected by snPCR in 28/472 (5.9%) samples and confirmed by qPCR in 26 samples of 21 (25%) subjects. No graft loss occurred among KT recipients who developed BKV-DNAemia. Cost analyses suggested that this strategy might be a cost saving alternative for BKV-DNAemia screening for some resource-limited settings.

Impact assessment and investigation of factors associated with herpesviruses viremia in the first year of renal transplantation.

The increased risk for opportunistic infections after a renal transplant requires monitoring of viral infections to avoid future complications. Our goal was to investigate the impact and factors associated with Epstein-Barr virus (EBV), human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) viremia in renal transplant recipients. Whole blood samples were collected monthly from 82 patients during the first semester and then quarterly up to 1 year after transplantation. EBV, HCMV, and HHV-6 were detected and quantified by TaqMan real-time polymerase chain reaction. The results showed that EBV and HCMV viremia were detected in 32 patients (39% each), while HHV-6 viremia in only 3 patients (3.7%). EBV was significantly associated with age (P = .050), thymoglobuline induction (P = .019), mTOR inhibitor-based therapy (P = .003), and female gender (P = .044). HCMV was significantly associated with basiliximab induction (P = .015), mycophenolate mofetil (MMF)-based therapy (P = .003) and allograft acute rejection (P = .033). Moreover, HCMV-disease was correlated with MMF-based therapy (P = .021) and female gender (P = .003). In conclusion, EBV and HCMV viremia were associated with different immunosuppressive induction and maintenance strategies. Additionally, higher HCMV viremia (> 10 4 copies/mL) was related to acute allograft rejection.

Investigation of three oncogenic epitheliotropic viruses shows human papillomavirus in association with non-melanoma skin cancer.

Non-melanoma skin cancers (NMSC) share similar risk factors with other virus-related cancers, despite the lack of proved causal association between viral infection and NMSC development. We investigated the presence of Merkel cell polyomavirus (MCPyV), Epstein-Barr virus (EBV), and human papillomavirus (HPV) DNA in 83 NMSC fresh-frozen and 16 non-cancerous skin biopsies and evaluated viral infection according to demographical data, histopathological diagnosis, and ultraviolet exposure. Our results showed that 75% of NMSC biopsies were positive for at least one out of three viruses, whereas only 38% of non-cancerous skin biopsies were positive (p = 0.02). Notably, HPV detection was frequent in NMSC (43%) and nearly absent (one sample, 6.7%) in non-cancerous biopsies (p = 0.007). MCPyV was associated with sites of higher exposure to ultraviolet radiation (p = 0.010), while EBV was associated with a compromised immune system (p = 0.032). Our study showed that HPV was strongly associated with NMSC while EBV and MCPyV with other risk factors. Though further studies are required to elucidate the role of viral infection in NMSC development and management, this study supports the possible role of oncogenic viruses in skin cancers, especially HPV.

A globally applicable PCR-based detection and discrimination of BK and JC polyomaviruses.

BKV and JCV belong to the Polyomaviridae family and are opportunistic agents associated with complications in immunocompromised individuals. Although a single screening assay for both viruses would be convenient, the diversity of BKV and JCV serotypes and genotypes is a methodological challenge. In this paper, we developed a PCR method able to detect and segregate BKV and JCV, despite these genetic discrepancies. A duplex semi-nested PCR (duplex snPCR) was designed to target a conserved region (639nt-1516nt) within the VP2 gene. In the first PCR, a primer set common to all BKV and JCV serotypes/ genotypes was used, followed by a semi-nested PCR with internal primers for BKV and JCV segregation. The limit of detection of the duplex snPCR was as low as 10 copies of BKV or JCV plasmids/µL. Specific products were observed when JCV and BKV plasmids were mixed in the same reaction. In field sample testing, the duplex snPCR detected and distinguished both viruses in different biological samples. Results were confirmed by Sanger's sequencing. The geographical complexity of BKV and JCV serotypes and genotypes imposes limits to a simple and universal method that could detect each virus. However, we describe here a sensitive and reliable PCR technique for BKV and JCV diagnosis that overcomes these limitations and could be universally applied.

BK polyomavirus nephropathy in two kidney transplant patients with distinct diagnostic strategies for BK virus and similar clinical outcomes: two case reports.

BACKGROUND: BK polyomavirus-associated nephropathy is an important cause of post-transplantation renal failure. We present two cases of BK polyomavirus-associated nephropathy who were submitted to contrasting strategies of clinical follow-up to BK polyomavirus reactivation, but progressed to a similar final outcome. CASE PRESENTATION: Case 1 is a 37-year-old white man whose graft had never presented a good glomerular filtration rate function, with episodes of tacrolimus nephrotoxicity, and no urinary monitoring for BK polyomavirus; stage B BK polyomavirus-associated nephropathy was diagnosed by biopsy at 14 months post-transplant. Despite clinical treatment (dosage decrease and immunosuppressive drug change), he progressed to stage C BK polyomavirus-associated nephropathy and loss of graft function 30 months post-transplant. Case 2 is a 49-year-old mulatto man in his second renal transplantation who was submitted to cytological urinary monitoring for BK polyomavirus; he presented early, persistent, and massive urinary decoy cell shedding and concomitant tacrolimus nephrotoxicity. Even with decreasing immunosuppression, he developed BK polyomavirus-associated nephropathy 1-year post-transplant. Loss of graft function occurred 15 months post-transplant. CONCLUSIONS: Cytological urinary monitoring was an efficient strategy for monitoring BK virus reactivation. Decoy cell shedding may be related to BK polyomavirus-associated nephropathy when extensive and persistent. The presence of associated tacrolimus nephrotoxicity may be a confounding factor for the clinical diagnosis of BK polyomavirus-associated nephropathy.

Interaction Between Human Polyomavirus BK and Hypoxia Inducible Factor-1 alpha.

BK polyomavirus (BKV) has a worldwide seroprevalence of approximately 90%. After primary infection, BKV establishes a life-long latency within the urogenital tract. The severe immunological impairment occurring in renal transplant recipients leads to BKV reactivation, which may result in polyomavirus associated nephropathy (PVAN). While the transplanted kidney is transiently unperfused, Hypoxia Inducible Factors (HIFs) mediate the cellular response to hypoxia. The α-subunit of HIF isoform 1 (HIF-1α) may interact with several viruses, but until now, there has been no information regarding the interaction between BKV and HIF-1α. The aim of this study is to investigate the possible interaction between HIF-1α and BKV and its potential effect on the pathogenesis of PVAN. Screening of 17 kidney tissue samples revealed that HIF-1α expression was 13.6-fold higher in PVAN tissues compared to control tissues. A luminometric assay in co-transfected African green monkey kidney cells (VERO) demonstrated BKV promoter activation ranging from two to sixfold (P < 0.05) when HIF-1α was over-expressed. A Chromatin ImmunoPrecipitation (ChIP) assay showed structural binding between the BKV promoter and HIF-1α. The amount of BKV DNA increased by threefold in VERO infected cells that were exposed to simulated hypoxia, compared to the cells not subjected to hypoxia. Both ex vivo and in vitro interactions between HIF-1α and BKV were observed, suggesting that HIF-1α, stabilized during transplantation, may be able to bind the BKV promoter and enhance BKV replication. Thus, hypoxia should be considered a risk factor for the development of PVAN in kidney transplant recipients.

Adenovirus species C detection in children under four years of age with acute bronchiolitis or recurrent wheezing.

BACKGROUND: Lower respiratory tract viral infection is an important cause of morbidity and mortality in children worldwide. Among viral etiological agents the human Adenovirus (AdV) has been associated to mild or severe respiratory tract infection. OBJECTIVE: To detect the presence of human Adenovirus (AdV) in children with acute bronchiolitis or recurrent wheezing, describing their clinical features and determining Adenovirus species and AdV association to Respiratory Syncytial Virus (RSV), Human Metapneumovirus (MPV) and Parainfluenza virus (PIV). STUDY DESIGN: A total of 155 children bellow 48 months of age with acute bronchiolitis or recurrent wheezing were investigated for the presence of AdV, RSV, MPV and PIV in nasopharyngeal aspirate, by real-time PCR method. RESULTS: AdV, predominantly of species C, has been detected as the unique pathogen (AdVi) or in association to other pathogens (AdVa.), in 39/155 samples. Crackles were more frequent in children with AdV. RSVi was detected predominantly in children with acute bronchiolitis while AdVi and AdVa were detected more frequently in patients with recurrent wheezing. CONCLUSION: A small outbreak of AdV species C was observed in 2012 and 2013. AdV was detected more frequently in children with recurrent wheezing while RSVi was more frequent in infants with acute bronchiolitis.

BK polyomavirus in kidney transplant recipients: screening, monitoring and clinical management.

BK polyomavirus (BKPyV) is a causal agent of nephropathy, ureteral stenosis and hemorrhagic cystitis in kidney transplant recipients, and is considered an important emerging disease in transplantation. Regular screening for BKPyV reactivation mainly during the first 2 years posttransplant, with subsequent pre-emptive reduction of immunosuppression is considered the best option to avoid disease progression, since successful clearance or reduction of viremia is achieved in the vast majority of patients within 6 months. The use of drugs with antiviral properties for patients with persistent viremia has been attempted despite unclear benefits. Clinical manifestations of BKPyV nephropathy, current strategies for diagnosis and monitoring of BKPyV infection, management of immunosuppressive regimen after detection of BKPyV reactivation and the use of antiviral drugs are discussed in this review.

BK polyomavirus in Kidney transplant recipients: screening, monitoring and clinical management / BK poliomavírus em receptores do transplante renal: rastreamento, monitoramento viral e manuseio clínico

BK polyomavirus (BKPyV) is a causal agent of nephropathy, ureteral stenosis and hemorrhagic cystitis in kidney transplant recipients, and is considered an important emerging disease in transplantation. Regular screening for BKPyV reactivation mainly during the first 2 years posttransplant, with subsequent pre-emptive reduction of immunosuppression is considered the best option to avoid disease progression, since successful clearance or reduction of viremia is achieved in the vast majority of patients within 6 months. The use of drugs with antiviral properties for patients with persistent viremia has been attempted despite unclear benefits. Clinical manifestations of BKPyV nephropathy, current strategies for diagnosis and monitoring of BKPyV infection, management of immunosuppressive regimen after detection of BKPyV reactivation and the use of antiviral drugs are discussed in this review.

BK Poliomavírus (BKPyV) é um agente causal de nefropatia, estenose ureteral e cistite hemorrágica em receptores de transplante renal, sendo considerado uma importante doença emergente na transplantação. Rastreamento regular para reativação do BKPyV, principalmente nos dois primeiros anos pós-transplante, com subsequente redução preemptiva da imunossupressão é considerada a melhor conduta para evitar a progressão da doença, já que a eliminação ou redução da viremia é alcançada na grande maioria dos pacientes dentro de 6 meses. O uso de drogas com propriedades antivirais para os pacientes com viremia persistente tem sido tentado, embora sem benefícios claros. As manifestações clínicas da nefropatia por BKPyV, as estratégias para o diagnóstico e monitoramento da infecção por BKPyV, o manejo do regime de imunossupressão após a detecção da reativação do BKPyV e o uso de drogas antivirais são discutidas nesta revisão.

Detection of Merkel cell polyomavirus in oral samples of renal transplant recipients without Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine cancer, with approximately 80% of cases associated with Merkel cell polyomavirus (MCPyV). The lack of information concerning its occurrence in non-MCC immunosuppressed populations led to the investigation of MCPyV DNA in saliva and oral biopsies from 60 kidney allograft recipients and 75 non-transplanted individuals (control group). In contrast to herpesviruses, which was also investigated (CMV, HHV-6A, and B, HHV-7) MCPyV was detected predominantly in patients with oral lesions (gingivitis and/or periodontitis) of both transplanted and non-transplanted groups (P=0.016) and in the saliva of the transplanted group (P=0.009). MCPyV co-detection with CMV (P=0.048), and HHV-6 (P=0.020) in the saliva of transplanted patients requires further investigation on a possible role of co-infection.

A qualitative seminested PCR assay as an alternative to urine cytology for BK polyomavirus screening after renal transplantation.

OBJECTIVE: Polyomavirus-associated nephropathy (PVAN) is a major cause of graft dysfunction after kidney transplantation. Therefore, routine screening for BK polyomavirus (BKV) infection with urine cytology or quantitative PCR-based assays has been recommended. Although less expensive than quantitative tests, qualitative PCR assays are not recommended for screening based on the assumption that their diagnostic accuracy is inferior to urine cytology. However, studies comparing the performance of both methods are scarce. METHODS: We compared the accuracy between a qualitative seminested PCR (snPCR) assay and urine cytology for the screening of BKV viruria in 104 renal transplant recipients. RESULTS: The snPCR assay was more sensitive than cytology (100 and 61%, respectively), yielding better negative predictive value (100 vs. 90%). In 7 (39%) of the 18 PVAN cases, BKV infection was detected exclusively by snPCR. Although the specificity of snPCR (63%) was lower than cytology (74%), their positive predictive values were similar (36 vs. 33%, respectively). In ROC curve analysis, the accuracy of snPCR was significantly higher (p = 0.03). CONCLUSION: This qualitative snPCR assay was more accurate than urine cytology for the detection of BKV viruria in renal transplant patients.

Acyclovir susceptibility of herpes simplex virus isolates from transplanted and nontransplanted patients in Rio de Janeiro, Brazil.

Herpes simplex virus (HSV) is prevalent worldwide and known as a notorious opportunistic pathogen ofimmunocompromised patients. During the course of HSV treatment, acyclovir (ACV)-resistant HSV strains may emerge, causing many clinical complications. Because few studies in this area showing the presence and/or frequency ofACV-resistant HSV are available, the authors evaluated the sensitivity of HSV isolated from 3 hematopoietic stem cell transplant recipients and 24 normal patients observed in a University Hospital in Rio de Janeiro, Brazil. Twenty-seven HSV isolated in VERO cells and typed by the direct immunofluorescence assay were tested for their susceptibility to various concentrations ofACV by the dye-uptake method. The susceptibility of the HSV strains was expressed as ED50 (the concentration of drug reducing viral cytophatic effect by 50%). The sensitivity to ACV by the dye-uptake assay revealed that 25 samples (92.6%) were sensitive to ACV concentrations < 1.5 microg/mL. One sample (3.7%) showed intermediate susceptibility (1.56 microg/mL) and one other sample (3.7%) showed resistance to ACV concentrations above the cut-off (2 microg/mL). The presence of resistance in immunocompetent patients could be the result of the frequent use of ACV for the treatment of recurrence. The routine use of HSV susceptibility testing is fundamental in the clinical suspicion of resistance not only for the knowledge of the incidence of HSV resistance in Brazil, but also to understand the mechanism of HSV resistance.

Molecular characterization of BK polyomavirus subtypes in renal transplant recipients in Brazil.

BK polyomavirus (BKV) is highly prevalent in the world population. Different reports indicate that BKV subtypes and subgroups present an uneven geographical distribution which might be correlated with human migration. However, there is a lack of data on the BKV subtype distribution in the South American population. The occurrence of BKV subtypes and subgroups detected in 51 kidney transplant recipients in Rio de Janeiro, Brazil is described. According to genetic studies, the population in this region descends mainly from European or African immigrants, with a relatively low genetic background from the Amerindians. By sequencing the VP1 region of BKV, subgroups Ib1 and Ia of subtype I were found in 34 (67%) and 15 (29%), respectively, of samples, while subtype II was present in 2 (4%) of the samples. Subtypes III and IV were not detected. Phylogenetic analysis indicated similarities between Brazilian BKV subgroup Ia and East African lineages; and subgroup Ib-1 with Asian and North American lineages, while subtype II samples were similar to sequences from Japan and the UK. This is the first report that describes distribution of BKV subtypes in South America. The high prevalence of BKV subgroup Ia probably reflects the high proportion of African descendants in this population. On the other hand, the predominance of subgroup Ib-1 and the absence of Ib-2 in an area with a high proportion of European ancestry was unexpected. Further studies in South American populations are needed to provide a better understanding of the epidemiology of BKV in this region.