Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes.

Human papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

Hepatitis B virus reactivation in HBsAg-negative patients is associated with emergence of viral strains with mutated HBsAg and reverse transcriptase.

BACKGROUND/AIMS: Virological factors associated with hepatitis B virus reactivation (HBV-R), following chemotherapy for cancer in hepatitis B surface antigen (HBsAg)-negative patients, are not well known. MATERIALS AND METHODS: HBV strains from 16 patients presenting HBV-R following chemotherapy were studied and compared to those obtained from 51 HBV chronically-infected patients. RESULTS: HBsAg variability was significantly increased within the major hydrophilic region, the a determinant and the C-terminal region. Amino acid substitutions were more frequently found in HBV-R patients as compared to controls at 17 and 11 positions within HBsAg and HBV-RT, respectively. This resulted in atypical serological testing in 56% of patients and detection of resistance mutation to nucleoside analogs in 12.5%. CONCLUSION: HBsAg and HBV-RT mutations are frequently encountered in patients with HBV-R, resulting in atypical serological testing and emergence of HBV strains resistant to nucleos(t)ides analogs.

Systematic serological testing for hepatitis E virus in kidney transplant recipients.

Hepatitis E virus (HEV) genotype 3 is endemic in Europe and hyperendemic in southern France. Recent reports of a high prevalence of HEV RNA in blood donations and in culinary specialties from this geographical area confirmed the endemicity of HEV and sources of viral transmission in this geographical area. HEV causes acute and chronic hepatitis in solid organ transplant recipients. Since March 2012, we have implemented systematic HEV serological testing in our cohort of kidney transplant recipients (KTRs) in Marseille in southeastern France. The aim of our study was to assess HEV exposure in this cohort between March 2012 and May 2014. During these 27 months, we found that 39% of the patients who underwent kidney transplantation had an anti-HEV IgG response using a sensitive microplate enzyme immunoassay. This seroprevalence was approximately 43% at both 1 and 8 years after, using the same assay. In addition, systematic HEV serological testing detected 6 cases of HEV infection among 578 KTRs (1%) during the 27 months of the study, with 5 at an acute stage and 1 at a chronic stage. In conclusion, continuous HEV monitoring in this population is useful for better understanding the epidemiology of HEV in France, because these patients are a well-monitored population. Moreover, HEV monitoring in KTRs is clinically relevant because HEV represents a clinical threat in these patients. Nevertheless, HEV serological testing may be more fruitful for identifying HEV infections when performed in cases of biological liver abnormalities than when performed systematically.

Hepatitis E in three immunocompromized children in southeastern France.

Hepatitis E virus (HEV) causes an emerging autochthonous disease in developed countries where links with a viral porcine reservoir have been evidenced. Moreover, chronic HEV infection and associated-cirrhosis have been described in severely immunocompromized patients. Nonetheless, only few studies have focused on pediatric HEV infections worldwide and only four autochthonous cases have been reported in children in developed countries. We describe here acute hepatitis E in three immunocompromized children. Case no. 1 was a 9-year-old liver transplant recipient girl in whom H1N1 2009 flu infection was diagnosed concurrently with hepatitis E. Case no. 2 was a 12-year-old boy presenting early medullar relapse of lymphoblastic leukemia of type B and in whom HEV RNA was detected over a 29-week period. Case no. 3 was a 9-year-old boy with a rare primary immunodeficiency due to XIAP deficiency. HEV infections were all autochthonously acquired and involved different viruses classified as subtype f, c, and e of genotype 3, which are those described in autochthonous cases in Europe. These three observations prompt to consider HEV as a causative agent of hepatitis in children in developed countries, and to perform particularly HEV testing in those severely immunocompromized who may develop chronic hepatitis E.

Evaluation of the clinical performance of the Beckman Coulter Access AbHBsII immunoassay for the detection of hepatitis B surface antibodies.

BACKGROUND: Hepatitis B virus (HBV) surface antibodies (anti-HBs) testing is useful in various clinical circumstances, including identification of HBV susceptible individuals in pre- and post-vaccination programs. OBJECTIVES: To assess the clinical performance of Beckman Coulter's anti-HBs chemiluminescence immunoassay (Access AbHBsII). STUDY DESIGN: Laboratory performances were evaluated on 1207 routine samples pre-screened with Abbott Axsym anti-HBs assay and divided into three different panels: vaccinated subjects (n=232), subjects with resolved HBV infection (n=150) and negative subjects for anti-HBs (n=825). Sera with discrepant results were resolved by an alternative method and further chart review. RESULTS: The overall concordance between Access and Axsym assays was 95.8%. The relative sensitivity, relative specificity, positive predictive value and negative predictive value were 97.8%, 98.1%, 96%, and 99%, respectively. Of the 51 discrepant results, eight were false negative by Access, fifteen were false positive by Access, including sera from seven pregnant women, two patients with acute leukemia, and four with inflammatory syndromes. CONCLUSIONS: The Beckman Coulter's Access AbHBsII assay displays satisfactory relative sensitivity and specificity performances. The assay has good precision and reliability and is technically simple and fast.