Metagenomic Next-Generation Sequencing vs. Traditional Microbiological Tests for Diagnosing Varicella-Zoster Virus Central Nervous System Infection.

BACKGROUND: Unbiased metagenomic next-generation sequencing (mNGS) detects pathogens in a target-independent manner. It is not well-understood whether mNGS has comparable sensitivity to target-dependent nucleic acid test for pathogen identification. METHODS: This study included 31 patients with chickenpox and neurological symptoms for screening of possible varicella-zoster virus (VZV) central nervous system (CNS) infection. Microbiological diagnosing of VZV cerebrospinal fluid (CSF) infection was performed on stored CSF samples using mNGS, quantitative and qualitative VZV-specific PCR assays, and VZV IgM antibodies test. RESULTS: The median age was 30.0 [interquartile range (IQR), 24.3-33.3] years. 51.6% of the patients were men. About 80.6% of the patients had normal CSF white blood cell counts (≤ 5 × 106/L). VZV IgM antibodies presented in 16.1% of the CSF samples, and nucleic acids were detectable in 16.1 and 9.7% using two different VZV-specific real-time PCR protocols. Intriguingly, maximal identification of VZV elements was achieved by CSF mNGS (p = 0.001 and p = 007; compared with qualitative PCR and VZV IgM antibody test, respectively), with sequence reads of VZV being reported in 51.6% (16/31) of the CSF samples. All VZV PCR positive samples were positive when analyzed by mNGS. Of note, human betaherpesvirus 6A with clinical significance was unexpectedly detected in one CSF sample. CONCLUSIONS: Our study suggests that CSF mNGS may have higher sensitivity for VZV detection than CSF VZV PCR and antibody tests, and has the advantage of identifying unexpected pathogens.

Occult hepatitis B virus infection among people with a family history of chronic hepatitis B virus infection.

UNLABELLED: The prevalence of occult hepatitis B virus infection (OBI) among people with a family history of chronic hepatitis B virus (HBV) infection is unclear. Serum samples were collected from 747 hepatitis B surface antigen (HBsAg)-negative people with a family history of HBV infection and 579 HBsAg-negative volunteer blood donors. The presence of HBV DNA was evaluated using nested PCR with primers specific for the X, S, and C regions of HBV. The Pre-S1/Pre-S2/ S region PCR products for the OBI group and their family members with chronic HBV infection (control group) were sequenced and compared. The prevalence of OBI was 8.0% (60/747) among HBsAg-negative people with a family history of chronic HBV infection, compared to 2.6% (15/579) among the blood donors (P < 0.05). The prevalence of HBV genotype B infection was lower in the OBI group than in the control group (P = 0.031). The substitution rates in the major hydrophilic region and the "a" determinant seemed to be higher in the OBI group (0.893 vs. 0.507; 1.042 vs. 0.403, respectively), and stop codon mutations more frequent in the OBI sequences (OBI: 2/26, 7.7% vs. CONTROL: 0/31, 0%). However, none of these differences was statistically significant (P = 0.237, 0.199, 0.201, respectively). In summary, the prevalence of OBI among HBsAg-negative people with a family history of chronic HBV infection was significantly higher than that in Chinese blood donors. However, S region mutations and the escape mechanism are not likely to be the major causes of increased prevalence of OBI.

Models for predicting hepatitis B e antigen seroconversion in response to interferon-α in chronic hepatitis B patients.

AIM: To develop models to predict hepatitis B e antigen (HBeAg) seroconversion in response to interferon (IFN)-α treatment in chronic hepatitis B patients. METHODS: We enrolled 147 treatment-naïve HBeAg-positive chronic hepatitis B patients in China and analyzed variables after initiating IFN-α1b treatment. Patients were tested for serum alanine aminotransferase (ALT), hepatitis B virus-DNA, hepatitis B surface antigen (HBsAg), antibody to hepatitis B surface antigen, HBeAg, antibody to hepatitis B e antigen (anti-HBe), and antibody to hepatitis B core antigen (anti-HBc) at baseline and 12 wk, 24 wk, and 52 wk after initiating treatment. We performed univariate analysis to identify response predictors among the variables. Multivariate models to predict treatment response were constructed at baseline, 12 wk, and 24 wk. RESULTS: At baseline, the 3 factors correlating most with HBeAg seroconversion were serum ALT level > 4 × the upper limit of normal (ULN), HBeAg ≤ 500 S/CO, and anti-HBc > 11.4 S/CO. At 12 wk, the 3 factors most associated with HBeAg seroconversion were HBeAg level ≤ 250 S/CO, decline in HBeAg > 1 log10 S/CO, and anti-HBc > 11.8 S/CO. At 24 wk, the 3 factors most associated with HBeAg seroconversion were HBeAg level ≤ 5 S/CO, anti-HBc > 11.4 S/CO, and decline in HBeAg > 2 log10 S/CO. Each variable was assigned a score of 1, a score of 0 was given if patients did not have any of the 3 variables. The 3 factors most strongly correlating with HBeAg seroconversion at each time point were used to build models to predict the outcome after IFN-α treatment. When the score was 3, the response rates at the 3 time points were 57.7%, 83.3%, and 84.0%, respectively. When the score was 0, the response rates were 2.9%, 0.0%, and 2.1%, respectively. CONCLUSION: Models with good negative and positive predictive values were developed to calculate the probability of response to IFN-α therapy.

In vitro and in vivo replication of a chemically synthesized consensus genome of hepatitis B virus genotype B.

Hepatitis B virus (HBV) genotypes vary in their geographical distribution and virological features. Previous investigations have shown that HBV genotype B is a predominant HBV genotype in China. Studies on HBV concerning different isolates frequently meet the question about the HBV reference strain and its representativeness. Although HBV consensus sequences can be generated easily by sequence alignment, they may not exist in nature or could not usually be isolated from patient samples. Thus, the construction of a consensus HBV genome has been proposed. In this study, an HBV genotype B consensus sequence was established by comparing 42 full-length HBV genotype B sequences and the genome was generated by chemical synthesis. This consensus genome was fully replication competent by in vitro transfection into hepatoma cells. The plasmid pHBV1.3B carrying a 1.3× full-length HBV consensus genome was hydrodynamically injected into Balb/c mice. HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc detection indicated expression and replication of this HBV genome in mice, similar to other HBV isolates. This approach represents a strategy to design and create consensus HBV genomes for future studies.