Use of oral fluid samples for the investigation of outbreaks of human parvovirus B19 infection.

The use of oral fluid (OF) samples for serological diagnosis of parvovirus B19 infection during outbreaks of erythema infectiosum had already been demonstrated, but the feasibility of using OF for the characterization of B19 genotypes circulating during outbreaks has not been described. The aim of this study was to assess the use of "in-house" PCR-based assays as a powerful tool for a rapid diagnosis and molecular characterization of B19 strains in OF samples during outbreaks. Paired serum and OF samples collected from anti-B19 IgM-positive patients, during two outbreaks of ertythema infectiosum (1999-2000 and 2004-2005), were tested by conventional (cPCR) and quantitative PCR (qPCR). qPCR was more sensitive than cPCR for detecting B19-DNA in both OF and serum. Overall, OF presented lower viral load (9.97 × 106 UI/mL) than serum (2.42 × 1010 UI/mL) and this difference was statistically significant. All OF samples obtained from patients in the age group < 14 years presented low viral load (< 104 IU/mL). No correlation was found between viral load and the number of days of onset of rash. Sequence analysis from PCR positive OF samples confirmed the circulation of subgenotype 1a (G1a) during these outbreaks. Our findings indicate that PCR-based assays may fail to detect B19-DNA in approximately 50% of OF compared to serum samples. Nevertheless, our study has shown for the first time that the genome sequence of the amplicon from non-invasive clinical sample is useful for molecular genotyping and may be a tool to clarify the genetic diversity of B19 strains circulating in distinct outbreaks.

Evaluation of Molecular Test for the Discrimination of "Naked" DNA from Infectious Parvovirus B19 Particles in Serum and Bone Marrow Samples.

Low levels of parvovirus B19 (B19V) DNA can be detected in the circulation and in different tissue of immunocompetent individuals for months or years, which has been linked to inflammatory diseases such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply that infectious virions are present. This study aimed to evaluate the method based on the Benzonase® treatment for differentiation between the infectious virions from "naked" DNA in serum and bone marrow (BM) samples to be useful for the B19V routine diagnosis. In addition, we estimated the period of viremia and DNAemia in the sera and bone marrow of nonhuman primates experimentally infected with B19V. Serum samples from ten patients and from four cynomolgus monkeys experimentally infected with B19V followed up for 60 days were used. Most of the human serum samples became negative after pretreatment; however, only decreased viral DNA loads were observed in four patients, indicating that these samples still contained the infectious virus. Reduced B19V DNA levels were observed in animals since 7th dpi. At approximately 45th dpi, B19V DNA levels were below 105 IU/mL after Benzonase® pretreatment, which was not a consequence of active B19V replication. The test based on Benzonase® pretreatment enabled the discrimination of "naked DNA" from B19V DNA encapsidated in virions. Therefore, this test can be used to clarify the role of B19V as an etiological agent associated with atypical clinical manifestations.

Beyond arboviruses: A multicenter study to evaluate differential diagnosis of rash diseases and acute febrile illness cases in Rio de Janeiro, Brazil.

INTRODUCTION: A wide variety of viruses can cause rash diseases (RDs) or acute febrile illness (AFIs) in children, adolescents and adults; however, approximately 19% of RD cases and 40% of AFI cases remain without a defined etiology. Parvovirus B19 (B19V) and herpesvirus infection can also cause RD and/or AFI, and in some risk groups, these infections can become persistent (or latent) and may require hospital treatment. Since these infections do not have mandatory reporting, they can be hidden by other diseases, such as those caused by arboviruses (e.g., dengue virus). In this context, the aim of this study was to pursue the differential laboratory diagnoses of B19V and herpesvirus infections in patients with RD and AFI, without a defined etiology, seen in hospitals and/or reference centers for infectious diseases in Rio de Janeiro. METHODS: A total of 114 participants were enrolled in the study, including 54 children and 60 adults. B19V infection was assessed by real-time PCR (qPCR) and ELISA (anti-B19V IgM and IgG). EBV was assessed through qPCR, and betaherpesviruses (HCMV, HHV-6 and HHV-7) were assessed through multiplex qPCR. Sociodemographic and clinical data were obtained from the medical record data of these participants. RESULTS: The median age of children with RD was 2 years (interquartile range (IQR): 5), and 55.6% were male. Among adults with AFI, the median age was 38 years (IQR: 21), and 56.7% were female. Regarding RD patients, viral prevalence (and load) were 5.5%(104IU/mL), 3.4%(104IU/mL), 5.5%(104IU/mL) and 11.1%(105IU/mL) for B19V, EBV, HCMV and HHV-6 infection, respectively, and in AFI patients they were 6.6%(105IU/mL), 1.6%(103IU/mL), 3.3%(104IU/mL) for B19V, HCMV and HHV-6, respectively. HHV-7 was not detected in RD or AFI patients. CONCLUSION: These results suggest the importance of including B19V and herpesviruses in the differential laboratory diagnoses for patients with RD and AFI, not only for epidemiological purposes but also for the proper management of the patient.

Quantitative real-time PCR for differential diagnostics of parvovirus B19 infection in acute liver failure patients.

BACKGROUND: Human Parvovirus B19 (B19V) is a common pathogen worldwide. After primary infection, B19V-DNA may permanently persist in non-erythroid tissues, including the liver of patients with acute liver failure (ALF). OBJECTIVE: To validate a real-time PCR (qPCR) for the quantification of B19V-DNA, in order to establish a differential diagnosis for B19V infection in ALF patients. METHODS: The qPCR techniques were based on Sybr Green® and TaqMan® methodologies. To evaluate the quality parameters of both methods, samples from patients with or without B19V infection were tested. The diagnostic utility of qPCR in the detection B19V-DNA in patients with ALF was evaluated by testing archived serum and hepatic tissue explants from 10 patients. RESULTS: The Sybr Green® methodology showed 97% efficiency, the limits of detection and quantification were 62.6 and 53,200 copies/mL, respectively. The TaqMan® methodology showed 95% efficiency, the limits of detection and quantification were 4.48 and 310 copies/mL, respectively. A false positive result was found only with the Sybr Green® methodology. Among ALF patients without defined etiology, three (30%) were positive for B19V DNA in serum and liver. CONCLUSION: The qPCR methods validated here were effective in clarifying uncommon cases of B19V-related ALF and are fit for differential diagnosis of ALF causes.

Molecular diversity of human parvovirus B19 during two outbreaks of erythema infectiosum in Brazil.

This study was conducted to provide information on the genetic diversity of human parvovirus B19 (B19V) circulating in the municipality of Niterói, Rio de Janeiro, Southeast Brazil during 1996-2006, a period with two distinct outbreaks of B19V infection: 1999-2000 and 2004-2005. A total of 27 sera from patients with erythema infectiosum and five sera from HIV-infected patients that tested positive for B19V DNA during the study period were analyzed. To genotype B19V strains, a semi-nested PCR for partial amplification of the capsid gene was performed and sequence analysis revealed that 31 sequences belonged to subgenotype 1a (G1a) of the main genotype 1 and one sequence was characterized as subgenotype 3b (G3b). The phylogenetic tree supported the division of the G1a into two well-defined clades with 1.3% of divergence. The low diversity of the G1a strains may be explained by the fact that all patients had acute B19V infection and 30/32 sera were collected during two distinct outbreaks. The G3b strain was from an HIV-infected patient who seroconverted to anti-B19 IgG antibodies in September/2005. This is the first report of G3b in the state of Rio de Janeiro.

Canine parvovirus infection in puppies with gastroenteritis in Niterói, Rio de Janeiro, Brazil from 1995 to 1997

Fecal samples from puppies with gastroenteritis less than 7 months old were examined for canine parvovirus infection (CPV-2) by hemagglutination (HA) and subsequent hemagglutination-inhibition (HI) tests. Forty of the 79 samples collected from April 1995 to June 1997 were found to be positive. About 70 percent of these samples were from 2 to 4 months old puppies, age in which they are at increased risk of developing CPV-2 infection, despite of vaccination. No seasonal distribution of canine parvovirus cases was found and it was supported by the results of a retrospective study realized at PolVet-UFF, which showed that gastroenteritis cases occurred throughout the year, for a six-year period (1991-97) in Niterói, Rio de Janeiro