Detection of Cytomegalovirus in Urine Specimen of Cholestatic Infants by Polymerase Chain Reaction

ABSTRACT: Cholestatic infants are associated with congenital abnormalities or viral infections, such as cytomegalovirus (CMV) infection. CMV can be detected by polymerase chain reaction (PCR) in body fluids, including urine which can be obtained easily and is non-invasive. The objective was to detect CMV in urine specimens of cholestasis infants and to analyze its correlation with serological status. This was a descriptive observational study with the cross-sectional approach, used urine from 39 cholestatic infants who meet the inclusion and exclusion criteria and have been approved by Ethics Committee. The nested-PCR was performed from extracted urine and unextracted direct urine. Serological data of immunoglobulin (Ig) M and IgG data were collected. Data were analyzed by Chi-square. Detection of CMV from extracted urine by PCR showed positive in 87.2% patients and from unextracted urine was positive in 48.7% patients. Serological status showed that IgM was positive in 41.0% patients and IgG was positive in 89.7% patients. The acute infection (IgM+ IgG+) was found in 41.0% patients, past infection (IgM-IgG+) was 48.7% patients, and not infected (IgM-IgG-) was in 10.3% patients. The acute infection (IgM+ IgG+), past infection (IgM-IgG+) and not infected (IgM-IgG-) was found in 41.0%, 48.7%, and 10.3% patients, respectively. The correlation between PCR CMV from extracted urine with serological CMV was moderate, while the unextracted urine was low. It indicates that to detect the infection of CMV, PCR technique is more accurate than serological testing, and the extracted urine is more appropriate specimen as PCR template than direct urine

Hepatitis B Virus X Gene Mutation With Predominance A1762T+G1764A Double Mutation on Chronic Liver Disease Patients in Surabaya, Indonesia

Background: HBV infection is a major problem worldwide, especially in developing countries including Indonesia. Mutations in the HBV gene X are commonly found in patients with CLD, especially in cirrhosis and hepatocellular carcinoma. Mutations in the X gene can cause loss of stability, increased transactivation function, and decreased anti-apoptotic ability of HBx protein. Aim: The aim of this study was to detect HBV X gene mutations in CLD patients in Surabaya. Methods: This was a cross sectional research taking samples at Dr. Soetomo General Hospital, Surabaya, Indonesia. This study used nested PCR by targeting HBV X gene. Samples showing positive HBV DNA PCR results were followed by sequencing and X gene mutation analysis by comparing sequencing results with reference strains. Results: In this study, 30 samples of CLD patients with positive HBsAg in Dr. Soetomo Surabaya were obtained. From the results of the multiple alignments, 12/30 samples (40%) had mutations on HBV X region which overlapped with Core Promoter region. There were 3 types of substitution mutations on HBV X gene (C1632T, T1753A/C/G, A1762T, and G1764A) with the dominant mutation types were A1762T and G1764A mutations, in which both mutations were found together as double mutation. Conclusion: X gene mutations were found in 40% CLD patients in Surabaya with the dominant mutation was in the form of double mutation A1762T and G1764A in 30% CLD patients in this study. The mutation was found mostly in advance stage of CLD

Intact interferon signaling in peripheral blood leukocytes of high-grade osteosarcoma patients

High-grade osteosarcoma has a poor prognosis with an overall survival rate of about 60 percent. The recently closed European and American Osteosarcoma Study Group (EURAMOS)-1 trial investigates the efficacy of adjuvant chemotherapy with or without interferon-α. It is however unknown whether the interferon-signaling pathways in immune cells of osteosarcoma patients are functional. We studied the molecular and functional effects of interferon treatment on peripheral blood lymphocytes and monocytes of osteosarcoma patients, both in vivo and ex vivo. In contrast to other tumor types, in osteosarcoma, interferon signaling as determined by the phosphorylation of signal transducer and activator of transcription (STAT)1 at residue 701 was intact in immune cell subsets of 33 osteosarcoma patients as compared to 19 healthy controls. Also, cytolytic activity of interferon-α stimulated natural killer cells against allogeneic (n = 7 patients) and autologous target cells (n = 3 patients) was not impaired. Longitudinal monitoring of three osteosarcoma patients on interferon-α monotherapy revealed a relative increase in the CD16-positive subpopulation of monocytes during treatment. Since interferon signaling is intact in immune cells of osteosarcoma patients, there is a potential for indirect immunological effects of interferon-α treatment in osteosarcoma

Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell–mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell–mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell–mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient–derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell–activating agents in patients with high-grade osteosarcoma

Presentation of Human Cytomegalovirus (HCMV) in Liver Tissues of Cholestatic Infants with Extrahepatic and Non-Extrahepatic Biliary Atresia.

ABSTRACT: Introduction: Human cytomegalovirus (HCMV) is associated with cholestasis in infants. Diagnosis of HCMV infection is most often based on serological anti-HCMV. Identification of HCMV in liver tissue has been rarely reported. The aims of this study were to determine the presentation of HCMV in liver tissues and to analyze its association with serological anti-HCMV of cholestatic infants with extrahepatic and non-extrahepatic biliary atresia. Methods: This observational study was performed during December 2017- December 2018 with ethics from our institutions. The parents or guardians of subjects signed the informed consent. Anti-HCMV serological data were collected from patient medical records. Histopathological diagnosis and polymerase chain reaction (PCR) for HCMV were performed from liver biopsy tissues. The data were analyzed by Chi-square. Results: There were 47 cholestatic infants, 38.3% EBA and 61.7% non-EBA. Anti-HCMV IgM was positive in 38.3% patients and IgG was positive in 91.5% patients. Acute infection or recent infection were 38.3%, past or not acute infection were 53.1%, and uninfected or early infection were 8.5% patients. The presentation of HCMV in liver tissues was 68.1% patients, consisting of 11/18 EBA and 21/29 non-EBA and negative in 31.9% patients, consisting of 7/18 EBA and 8/29 non-EBA. There was no association between serological anti-HCMV and PCR HCMV with histopathological features. Conclusion: It suggests that PCR can be used as a routine tool to detect the presentation of HCMV DNA in liver tissue. Type of cholestasis in infants, both EBA and non-EBA, cannot be determined based on the serological and PCR examination, but based on histopathological features

Presentation of Human Cytomegalovirus (HCMV) in Liver Tissues of Cholestatic Infants with Extrahepatic and Non-Extrahepatic Biliary Atresia.

ABSTRACT: Introduction: Human cytomegalovirus (HCMV) is associated with cholestasis in infants. Diagnosis of HCMV infection is most often based on serological anti-HCMV. Identification of HCMV in liver tissue has been rarely reported. The aims of this study were to determine the presentation of HCMV in liver tissues and to analyze its association with serological anti-HCMV of cholestatic infants with extrahepatic and non-extrahepatic biliary atresia. Methods: This observational study was performed during December 2017- December 2018 with ethics from our institutions. The parents or guardians of subjects signed the informed consent. Anti-HCMV serological data were collected from patient medical records. Histopathological diagnosis and polymerase chain reaction (PCR) for HCMV were performed from liver biopsy tissues. The data were analyzed by Chi-square. Results: There were 47 cholestatic infants, 38.3% EBA and 61.7% non-EBA. Anti-HCMV IgM was positive in 38.3% patients and IgG was positive in 91.5% patients. Acute infection or recent infection were 38.3%, past or not acute infection were 53.1%, and uninfected or early infection were 8.5% patients. The presentation of HCMV in liver tissues was 68.1% patients, consisting of 11/18 EBA and 21/29 non-EBA and negative in 31.9% patients, consisting of 7/18 EBA and 8/29 non-EBA. There was no association between serological anti-HCMV and PCR HCMV with histopathological features. Conclusion: It suggests that PCR can be used as a routine tool to detect the presentation of HCMV DNA in liver tissue. Type of cholestasis in infants, both EBA and non-EBA, cannot be determined based on the serological and PCR examination, but based on histopathological features

Presentation of Human Cytomegalovirus (HCMV) in Liver Tissues of Cholestatic Infants with Extrahepatic and Non-Extrahepatic Biliary Atresia

ABSTRACT: Introduction: Human cytomegalovirus (HCMV) is associated with cholestasis in infants. Diagnosis of HCMV infection is most often based on serological anti-HCMV. Identification of HCMV in liver tissue has been rarely reported. The aims of this study were to determine the presentation of HCMV in liver tissues and to analyze its association with serological anti-HCMV of cholestatic infants with extrahepatic and non-extrahepatic biliary atresia. Methods: This observational study was performed during December 2017- December 2018 with ethics from our institutions. The parents or guardians of subjects signed the informed consent. Anti-HCMV serological data were collected from patient medical records. Histopathological diagnosis and polymerase chain reaction (PCR) for HCMV were performed from liver biopsy tissues. The data were analyzed by Chi-square. Results: There were 47 cholestatic infants, 38.3% EBA and 61.7% non-EBA. Anti-HCMV IgM was positive in 38.3% patients and IgG was positive in 91.5% patients. Acute infection or recent infection were 38.3%, past or not acute infection were 53.1%, and uninfected or early infection were 8.5% patients. The presentation of HCMV in liver tissues was 68.1% patients, consisting of 11/18 EBA and 21/29 non-EBA and negative in 31.9% patients, consisting of 7/18 EBA and 8/29 non-EBA. There was no association between serological anti-HCMV and PCR HCMV with histopathological features. Conclusion: It suggests that PCR can be used as a routine tool to detect the presentation of HCMV DNA in liver tissue. Type of cholestasis in infants, both EBA and non-EBA, cannot be determined based on the serological and PCR examination, but based on histopathological features

Detection of Cytomegalovirus in Urine Specimen of Cholestatic Infants by Polymerase Chain Reaction

ABSTRACT: Cholestatic infants are associated with congenital abnormalities or viral infections, such as cytomegalovirus (CMV) infection. CMV can be detected by polymerase chain reaction (PCR) in body fluids, including urine which can be obtained easily and is non-invasive. The objective was to detect CMV in urine specimens of cholestasis infants and to analyze its correlation with serological status. This was a descriptive observational study with the cross-sectional approach, used urine from 39 cholestatic infants who meet the inclusion and exclusion criteria and have been approved by Ethics Committee. The nested-PCR was performed from extracted urine and unextracted direct urine. Serological data of immunoglobulin (Ig) M and IgG data were collected. Data were analyzed by Chi-square. Detection of CMV from extracted urine by PCR showed positive in 87.2% patients and from unextracted urine was positive in 48.7% patients. Serological status showed that IgM was positive in 41.0% patients and IgG was positive in 89.7% patients. The acute infection (IgM+ IgG+) was found in 41.0% patients, past infection (IgM-IgG+) was 48.7% patients, and not infected (IgM-IgG-) was in 10.3% patients. The acute infection (IgM+ IgG+), past infection (IgM-IgG+) and not infected (IgM-IgG-) was found in 41.0%, 48.7%, and 10.3% patients, respectively. The correlation between PCR CMV from extracted urine with serological CMV was moderate, while the unextracted urine was low. It indicates that to detect the infection of CMV, PCR technique is more accurate than serological testing, and the extracted urine is more appropriate specimen as PCR template than direct urine