Development of an enzyme-linked immunosorbent assay for diagnosis of human herpesvirus-7 infection

Abstract INTRODUCTION: Human herpesvirus (HHV)-7 establishes a latent infection during the lifetime of the host and can reactivate after the primary infection, leading to lytic replication in immunosuppressed patients. METHODS: This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) to identify HHV-7 serum antibodies and compare its performance with that of an indirect immunofluorescence assay (IFA). RESULTS: Serum samples (n=102) were tested by IgG-IFA and by ELISA. IFA and ELISA showed IgG-positive results in 77 and 73 samples, respectively. Qualitative concordance of 96% was demonstrated between the two techniques. CONCLUSIONS: ELISA may be useful to diagnose HHV-7 infection.

Influence of low-level laser irradiation on osteocalcin protein and gene expression in bone tissue1

Abstract Purpose: To evaluate osteocalcin gene and protein expression in vitro and in an in vivo model of ostectomy. Methods: Twenty Wistar rats were assigned into two groups A (n=10, laser) and B (n=10, control). Ostectomy was performed in the femur diaphysis; the twenty fragments removed, composed in vitro groups named as in vivo (A and B) and cultivated in CO2 atmosphere for thirteen days. Low-level laser irradiation was performed in groups A (in vivo and in vitro) by an GaAlAs device (λ=808 nm, dose of 2J/cm2, power of 200mW, power density of 0.2W/cm2, total energy of 1.25J, spot diameter of 0.02mm) for 5 seconds, at one point, daily. It was performed immunocytochemistry assays in vivo and in vitro groups. In vitro groups were also submitted to RNA extraction, cDNA synthesis and gene expression by quantitative PCR. Statistical analysis was realized with p<0.05. Results: Immunocytochemistry scores showed no significant differences between control and laser groups either in vivo and in vitro. Gene expression also showed no statistical differences. Conclusion: Low-level laser irradiation did not alter osteocalcin protein and gene expression in vivo and in vitro in the studied period but it may have been expressed them in an earlier period.

Comparison of five methods of extraction of Staphylococcus aureus DNA for molecular detection by PCR

Abstract INTRODUCTION Molecular techniques for the detection of pathogens have been shown to be effective diagnostic tools with high sensitivity and short turnaround times. METHODS This study compared five Staphylococcus aureus DNA extraction methods for detection by the polymerase chain reaction. RESULTS: The concentration and purity of the extracted DNA showed that the methods did not yield DNA of significant quality. However, most protocols yielded 100% positivity, even with low DNA concentrations. CONCLUSIONS: Although one protocol seemed more efficient than the others, PCR was sensitive enough to allow for detection of S. aureus with all the protocols.

Simultaneous monitoring of CMV and human herpesvirus 6 infections and diseases in liver transplant patients: one-year follow-up

OBJECTIVE: The aim of this study was to simultaneously monitoring cytomegalovirus and human herpesvirus 6 active infections using nested-polymerase chain reaction and, together with clinical findings, follow the clinical status of patients undergoing liver transplant. INTRODUCTION: The human β-herpesviruses, including cytomegalovirus and human herpesvirus 6, are ubiquitous among human populations. Active infections of human herpesvirus 6 and cytomegalovirus are common after liver transplantation, possibly induced and facilitated by allograft rejection and immunosuppressive therapy. Both viruses affect the success of the transplant procedure. METHODS: Thirty patients submitted to liver transplant at the Liver Transplant Unit, at the Gastro Center, State University of Campinas, SP, Brazil, were studied prospectively from six months to one year, nested-polymerase chain reaction for cytomegalovirus and human herpesvirus 6 DNA detections. Two or more consecutive positive nested-polymerase chain reaction were considered indicative of active infection. RESULTS: Active infection by cytomegalovirus was detected in 13/30 (43.3 percent) patients, median time to first cytomegalovirus detection was 29 days after transplantation (range: 0-99 days). Active infection by human herpesvirus 6 was detected in 12/30 (40 percent) patients, median time to first human herpesvirus 6 detection was 23.5 days after transplantation (range: 0-273 days). The time-related appearance of each virus was not statistically different (p = 0.49). Rejection of the transplanted liver was observed in 16.7 percent (5/30) of the patients. The present analysis showed that human herpesvirus 6 and/or cytomegalovirus active infections were frequent in liver transplant recipients at our center. CONCLUSIONS: Few patients remain free of betaherpesviruses after liver transplantation. Most patients presenting active infection with more than one virus were infected sequentially and not concurrently. Nested-polymerase chain reaction can be considered of limited value for clinically monitoring cytomegalovirus and human herpesvirus 6.

Detection of human herpesvirus-7 by qualitative nested-PCR: comparison between healthy individuals and liver transplant recipients / Detecção de herpesvirus humano-7 por nested-PCR qualitativo: comparação entre indivíduos sadios e receptores de transplante hepático

Diagnosis of human herpesvirus-7 active infection in transplant patients has proved difficult, because this virus is ubiquitous and can cause persistent infections in the host. The significance of viral DNA detected in leukocytes by PCR is unclear and cross-reaction in serological tests may occur. This study aimed to evaluate nested-PCR to detect human herpesvirus-7 active infection in liver transplant recipients compared to healthy individuals. human herpesvirus-7 nested-PCR was performed on leukocytes and sera of 53 healthy volunteers and sera of 29 liver transplant recipients. In healthy volunteers, human herpesvirus-7 was detected in 28.3 percent of leukocytes and 0 percent of serum. human herpesvirus-7 was detected in sera of 48.2 percent of the liver transplant recipients. Nested-PCR on DNA extracted from leukocytes detected latent infection and the study suggests that nested-PCR performed on serum could be useful to detect human herpesvirus-7 active infection in liver transplant recipients.

Diagnóstico da infecção ativa pelo herpesvirus humano-7 é difícil devido ao fato deste vírus ser ubíquo e poder causar infecção persistente no hospedeiro. O significado da detecção do DNA viral por reação em cadeia da polimerase não é claro e, reações cruzadas podem ocorrer em testes sorológicos. O objetivo deste estudo foi avaliar a nested-PCR para detectar infecção ativa pelo herpesvirus-7 em receptores hepáticos comparando com indivíduos sadios. Nested-PCR para herpesvirus-7 foi realizado em leucócitos e soro de 53 voluntários sadios e em soro de 29 receptores hepáticos. Nos voluntários sadios, herpesvirus-7 foi detectado em 28,3 por cento de leucócitos e 0 por cento de soro. herpesvirus-7 foi detectado em soro de 48,2 por cento de receptores hepáticos. Nested-PCR em DNA extraído de leucócitos detectou infecção latente e o estudo sugere que nested-PCR realizada em soro poderia ser útil para detectar infecção ativa por herpesvirus-7 em receptores de fígado.