Lilia Ganova-Raeva

Assuring the quality of next-generation sequencing in clinical laboratory practice

Amy S Gargis, Centers for Disease Control and Prevention Lisa Kalman, Centers for Disease Control and Prevention Meredith W Berry, SeqWright Inc David P Bick, Medical College of Wisconsin David P Dimmock, Medical College of Wisconsin Tina Hambuch, Illumina Clinical Services Fei Lu, SeqWright Inc Elaine Lyon, University of Utah Karl V Voelkerding, University of Utah Barbara Zehnbauer, Emory University

Good laboratory practice for clinical next-generation sequencing informatics pipelines

Amy S Gargis, Centers for Disease Control & Prevention Lisa Kalman, Centers for Disease Control & Prevention David P Bick, Medical College of Wisconsin Cristina da Silva, Emory University David P Dimmock, Medical College of Wisconsin Birgit H Funke, Partners Healthcare Personalized Medicine Sivakumar Gowrisankar, Partners Healthcare Personalized Medicine Madhuri Hegde, Emory University Shashikant Kulkarni, Washington University Christopher E Mason, Cornell University

Epidemic History and Evolutionary Dynamics of Hepatitis B Virus Infection in Two Remote Communities in Rural Nigeria

Background In Nigeria, hepatitis B virus (HBV) infection has reached hyperendemic levels and its nature and origin have been described as a puzzle. In this study, we investigated the molecular epidemiology and epidemic history of HBV infection in two semi-isolated rural communities in North/Central Nigeria. It was expected that only a few, if any, HBV strains could have been introduced and effectively transmitted among these residents, reflecting limited contacts of these communities with the general population in the country. Methods and Findings Despite remoteness and isolation, ∼11% of the entire population in these communities was HBV-DNA seropositive. Analyses of the S-gene sequences obtained from 55 HBV-seropositive individuals showed the circulation of 37 distinct HBV variants. These HBV isolates belong predominantly to genotype E (HBV/E) (n = 53, 96.4%), with only 2 classified as sub-genotype A3 (HBV/A3). Phylogenetic analysis showed extensive intermixing between HBV/E variants identified in these communities and different countries in Africa. Quasispecies analysis of 22 HBV/E strains using end-point limiting-dilution real-time PCR, sequencing and median joining networks showed extensive intra-host heterogeneity and inter-host variant sharing. To investigate events that resulted in such remarkable HBV/E diversity, HBV full-size genome sequences were obtained from 47 HBV/E infected persons and P gene was subjected to Bayesian coalescent analysis. The time to the most recent common ancestor (tMRCA) for these HBV/E variants was estimated to be year 1952 (95% highest posterior density (95% HPD): 1927–1970). Using additional HBV/E sequences from other African countries, the tMRCA was estimated to be year 1948 (95% HPD: 1924–1966), indicating that HBV/E in these remote communities has a similar time of origin with multiple HBV/E variants broadly circulating in West/Central Africa. Phylogenetic analysis and statistical neutrality tests suggested rapid HBV/E population expansion. Additionally, skyline plot analysis showed an increase in the size of the HBV/E-infected population over the last ∼30–40 years. Conclusions Our data suggest a massive introduction and relatively recent HBV/E expansion in the human population in Africa. Collectively, these data show a significant shift in the HBV/E epidemic dynamics in Africa over the last century.

End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: Performance under optimal and suboptimal conditions

An approach for determination of hepatitis C virus (HCV) quasispecies by end-point limiting-dilution real-time PCR (EPLD-PCR) is described. It involves isolation of individual coexisting sequence variants of the hypervariable region 1 (HVR1) of the HCV genome from serum specimens using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak study provided insights into the epidemiological relationships between incident and chronic cases. When applied to samples from a longitudinal study of infected patients, HVR1 sequences from each sampling time-point were observed to group as distinct phylogenetic clusters. Melting peak analysis conducted on EPLD-PCR products generated from these patients could be used for evaluation of HVR1 sequence heterogeneity without recourse to clonal sequencing. Further, to better understand the mechanism of single-molecule PCR, experiments were conducted under optimal and suboptimal annealing temperatures. Under all temperature conditions tested, HVR1 variants from the major phylogenetic clusters of quasispecies could be amplified, revealing that successful HVR1 quasispecies analysis is not contingent to dilution of starting cDNA preparations to a single-molecule state. It was found that EPLD-PCR conducted at suboptimal annealing temperatures generated distributions of unique-sequence variants slightly different from the distribution obtained by PCR conducted at the optimal temperature. Hence, EPLD-PCR conditions can be manipulated to access different subpopulations of HCV HVR1 quasispecies, thus, improving the range of the quasispecies detection. Although EPLD-PCR conducted at different conditions detect slightly different quasispecies populations, as was shown in this study, the resulted samples of quasispecies are completely suitable for molecular epidemiological investigation in different clinical and epidemiological settings.

Laboratory-based Surveillance for Hepatitis E Virus Infection, United States, 2005–2012

Clinicians should consider this virus in the differential diagnosis of hepatitis, regardless of patient travel history.

Convergence and coevolution of Hepatitis B virus drug resistance

Treatment with lamivudine of patients infected with hepatitis B virus (HBV) results in a high rate of drug resistance, which is primarily associated with the rtM204I/V substitution in the HBV reverse transcriptase domain. Here we show that the rtM204I/V substitution, although essential, is insufficient for establishing resistance against lamivudine. The analysis of 639 HBV whole-genome sequences obtained from 11 patients shows that rtM204I/V is independently acquired by more than one intra-host HBV variant, indicating the convergent nature of lamivudine resistance. The differential capacity of HBV variants to develop drug resistance suggests that fitness effects of drug-resistance mutations depend on the genetic structure of the HBV genome. An analysis of Bayesian networks that connect rtM204I/V to many sites of HBV proteins confirms that lamivudine resistance is a complex trait encoded by the entire HBV genome rather than by a single mutation. These findings have implications for public health and offer a more general framework for understanding drug resistance.

Epidemic history of hepatitis C virus infection in two remote communities in Nigeria, West Africa.

We investigated the molecular epidemiology and population dynamics of HCV infection among indigenes of two semi-isolated communities in North-Central Nigeria. Despite remoteness and isolation, ~15% of the population had serological or molecular markers of hepatitis C virus (HCV) infection. Phylogenetic analysis of the NS5b sequences obtained from 60 HCV-infected residents showed that HCV variants belonged to genotype 1 (n=51; 85%) and genotype 2 (n=9; 15%). All sequences were unique and intermixed in the phylogenetic tree with HCV sequences from people infected from other West African countries. The high-throughput 454 pyrosequencing of the HCV hypervariable region 1 and an empirical threshold error correction algorithm were used to evaluate intra-host heterogeneity of HCV strains of genotype 1 (n=43) and genotype 2 (n=6) from residents of the communities. Analysis revealed a rare detectable intermixing of HCV intra-host variants among residents. Identification of genetically close HCV variants among all known groups of relatives suggests a common intra-familial HCV transmission in the communities. Applying Bayesian coalescent analysis to the NS5b sequences, the most recent common ancestors for genotype 1 and 2 variants were estimated to have existed 675 and 286 years ago, respectively. Bayesian skyline plots suggest that HCV lineages of both genotypes identified in the Nigerian communities experienced epidemic growth for 200-300 years until the mid-20th century. The data suggest a massive introduction of numerous HCV variants to the communities during the 20th century in the background of a dynamic evolutionary history of the hepatitis C epidemic in Nigeria over the past three centuries.

Accurate Genetic Detection of Hepatitis C Virus Transmissions in Outbreak Settings

Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections are associated with unsafe injection practices, drug diversion, and other exposures to blood and are difficult to detect and investigate. Here, we developed and validated a simple approach for molecular detection of HCV transmissions in outbreak settings. We obtained sequences from the HCV hypervariable region 1 (HVR1), using end-point limiting-dilution (EPLD) technique, from 127 cases involved in 32 epidemiologically defined HCV outbreaks and 193 individuals with unrelated HCV strains. We compared several types of genetic distances and calculated a threshold, using minimal Hamming distances, that identifies transmission clusters in all tested outbreaks with 100% accuracy. The approach was also validated on sequences obtained using next-generation sequencing from HCV strains recovered from 239 individuals, and findings showed the same accuracy as that for EPLD. On average, the nucleotide diversity of the intrahost population was 6.2 times greater in the source case than in any incident case, allowing the correct detection of transmission direction in 8 outbreaks for which source cases were known. A simple and accurate distance-based approach developed here for detecting HCV transmissions streamlines molecular investigation of outbreaks, thus improving the public health capacity for rapid and effective control of hepatitis C.

Hepatitis B outbreak associated with a hematology-oncology office practice in New Jersey, 2009

BACKGROUND Transmission of bloodborne pathogens due to breaches in infection control is becoming increasingly recognized as greater emphasis is placed on reducing health care-associated infections. Two women, aged 60 and 77 years, were diagnosed with acute hepatitis B virus (HBV) infection; both received chemotherapy at the same physicians office. Due to suspicion of health care-associated HBV transmission, a multidisciplinary team initiated an investigation of the hematology-oncology office practice. METHODS We performed an onsite inspection and environmental assessment, staff interviews, records review, and observation of staff practices. Patients who visited the office practice between January 1, 2006 and March 3, 2009 were advised to seek testing for bloodborne pathogens. Patients and medical providers were interviewed. Specimens from HBV-infected patients were sent to the Centers for Disease Control and Prevention for HBV DNA testing and phylogenic analysis. RESULTS Multiple breaches in infection control were identified, including deficient policies and procedures, improper hand hygiene, medication preparation in a blood processing area, common-use saline bags, and reuse of single-dose vials. The office practice was closed, and the physicians license was suspended. Out of 2,700 patients notified, test results were available for 1,394 (51.6%). Twenty-nine outbreak-associated HBV cases were identified. Specimens from 11 case-patients demonstrated 99.9%-100% nucleotide identity on phylogenetic analysis. CONCLUSION Systematic breaches in infection control led to ongoing transmission of HBV infection among patients undergoing invasive procedures at the office practice. This investigation underscores the need for improved regulatory oversight of outpatient health care settings, improved infection control and injection safety education for health care providers, and the development of mechanisms for ongoing communication and cooperation among public health agencies.

Hepatitis B Virus and Hepatitis C Virus Infections in United States-Bound Refugees from Asia and Africa

The aim of this study was to determine the prevalence of active hepatitis B and C virus infections among refugees from various countries in Africa and Asia. Pre-admission serum samples collected during 2002-2007 from refugees originating from Bhutan (N = 755), Myanmar (N = 1076), Iraq (N = 1137), Laos (N = 593), Thailand (N = 622), and Somalia (N = 707) were tested for hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA. The HBV DNA (genotypes A, B, C, and G) was detected in 12.1% of samples negative for anti-HBs. Highest HBV prevalence was found among Hmong; lowest among Bhutanese. The HCV RNA (genotypes 1a, 1b, 1c, 3b, 6n, and 6m) was detected in 1.3% of the samples. Highest HCV prevalence was found among Hmong from Thailand; lowest among Iraqis. Screening specific refugee groups at high risk for viral hepatitis infections will identify infected individuals who could benefit from referral to care and treatment and prevent further transmissions.