Hepatitis C virus infection and co-infection with HIV among persons who inject drugs in 10 U.S. cities-National HIV Behavioral Surveillance, 2018.

BACKGROUND: Characterizing acute and chronic hepatitis C virus (HCV) infection and HIV/HCV co-infection among persons who inject drugs (PWID) can inform elimination efforts. METHODS: During 2018 National HIV Behavioral Surveillance in 10 U.S. metropolitan statistical areas (MSAs), PWID were recruited using respondent-driven sampling and offered a survey, HIV testing, and HCV antibody and RNA testing. We examined prevalence and associated characteristics of HCV infection and HIV/HCV co-infection. Associations were assessed using log-linked Poisson regression models with robust standard errors accounting for clustering by recruitment chain and adjusting for MSA and network size. RESULTS: Overall, 44.2% had current HCV infection (RNA detected), with 3.9% classified as acute infection (HCV antibody non-reactive/RNA detected) and 40.3% as chronic (HCV antibody reactive/RNA detected). Four percent had HIV/HCV co-infection. Current HCV infection was significantly higher among PWID who were male, White, injected >1 time/day, shared syringes in past year, and shared injection equipment in past year. PWID who were transgender, injecting >5 years, and most often injected speedball (heroin and cocaine together) or stimulants alone were more likely to have HIV/HCV co-infection. Among PWID who never previously had HCV infection, 9.9% had acute HCV infection. Among PWID who started injecting ≤5 years ago, 41.5% had already acquired HCV infection. CONCLUSIONS: Acute and chronic HCV infections were substantial among a sample of PWID in 10 U.S. MSAs. Accessibility to HCV RNA testing, promoting safer practices, and intervening early with harm reduction programs for recent injection initiates will be critical to disease elimination efforts for PWID.

HDV RNA assays: Performance characteristics, clinical utility, and challenges.

Coinfection with HBV and HDV results in hepatitis D, the most severe form of chronic viral hepatitis, frequently leading to liver decompensation and HCC. Pegylated interferon alpha, the only treatment option for chronic hepatitis D for many years, has limited efficacy. New treatments are in advanced clinical development, with one recent approval. Diagnosis and antiviral treatment response monitoring are based on detection and quantification of HDV RNA. However, the development of reliable HDV RNA assays is challenged by viral heterogeneity (at least 8 different genotypes and several subgenotypes), intrahost viral diversity, rapid viral evolution, and distinct secondary structure features of HDV RNA. Different RNA extraction methodologies, primer/probe design for nucleic acid tests, lack of automation, and overall dearth of standardization across testing laboratories contribute to substantial variability in performance characteristics of research-based and commercial HDV RNA assays. A World Health Organization (WHO) standard for HDV RNA, available for about 10 years, has been used by many laboratories to determine the limit of detection of their assays and facilitates comparisons of RNA levels across study centers. Here we review challenges for robust pan genotype HDV RNA quantification, discuss particular clinical needs and the importance of reliable HDV RNA quantification in the context of drug development and patient monitoring. We summarize distinct technical features and performance characteristics of available HDV RNA assays. Finally, we provide considerations for the use of HDV RNA assays in the context of drug development and patient monitoring.

Hepatitis B vaccine delivered by microneedle patch: Immunogenicity in mice and rhesus macaques.

Vaccination against hepatitis B using a dissolving microneedle patch (dMNP) could increase access to the birth dose by reducing expertise needed for vaccine administration, refrigerated storage, and safe disposal of biohazardous sharps waste. In this study, we developed a dMNP to administer hepatitis B surface antigen (HBsAg) adjuvant-free monovalent vaccine (AFV) at doses of 5 µg, 10 µg, and 20 µg, and compared its immunogenicity to vaccination with 10 µg of standard monovalent HBsAg delivered by intramuscular (IM) injection either in an AFV format or as aluminum-adjuvanted vaccine (AAV). Vaccination was performed on a three dose schedule of 0, 3, and 9 weeks in mice and 0, 4, and 24 weeks in rhesus macaques. Vaccination by dMNP induced protective levels of anti-HBs antibody responses (≥10 mIU/ml) in mice and rhesus macaques at all three HBsAg doses studied. HBsAg delivered by dMNP induced higher anti-HBsAg antibody (anti-HBs) responses than the 10 µg IM AFV, but lower responses than 10 µg IM AAV, in mice and rhesus macaques. HBsAg-specific CD4+ and CD8+ T cell responses were detected in all vaccine groups. Furthermore, we analyzed differential gene expression profiles related to each vaccine delivery group and found that tissue stress, T cell receptor signaling, and NFκB signaling pathways were activated in all groups. These results suggest that HBsAg delivered by dMNP, IM AFV, and IM AAV have similar signaling pathways to induce innate and adaptive immune responses. We further demonstrated that dMNP was stable at room temperature (20 °C-25 °C) for 6 months, maintaining 67 ± 6 % HBsAg potency. This study provides evidence that delivery of 10 µg (birth dose) AFV by dMNP induced protective levels of antibody responses in mice and rhesus macaques. The dMNPs developed in this study could be used to improve hepatitis B birth dose vaccination coverage levels in resource limited regions to achieve and maintain hepatitis B elimination.

Dried blood spot is the feasible matrix for detection of some but not all hepatitis B virus markers of infection.

OBJECTIVE: Use of dried blood spots (DBS) for detection of hepatitis B virus (HBV) markers of infection has the potential to facilitate diagnosis of HBV infection especially in resource-limited countries. The aim of this study was to evaluate the feasibility of DBS for detection of various markers of HBV infections. RESULTS: Fifty-four DBS samples were engineered from well-characterized plasma samples. All DBS samples were tested for HBsAg, total anti-HBc and HBV DNA, 20 of 54 samples were also tested for HBeAg using commercially available assays. HBsAg was detected in 24 of 25 (96%), HBV DNA in 22 of 25 (88%), total anti-HBc in all 9 (100%), and HBeAg in all 7 (100%) DBS samples. The average difference in HBV DNA levels between DBS eluates and corresponding plasma samples was 2.7 log10 IU/mL. Fifteen DBS eluates positive for HBV DNA were sequenced and all of them belonged to HBV genotype A. Thirteen samples which were negative for all HBV markers showed HBeAg false positivity. Therefore, DBS is a reliable sample matrix for detection of HBsAg, total anti-HBc and HBV DNA, but not HBeAg. Further feasibility studies of DBS for diagnostic purposes and epidemiologic studies are warranted.

In vitro characterization of six hepatitis B virus genotypes from clinical isolates using transfecting linear HBV genomes.

Hepatitis B virus (HBV) infection is a global public health problem with about 257 million chronically infected people and over 887000 deaths annually. In this study, 32 whole HBV genomes of various genotypes were amplified from clinical isolates to create transfection clones. The clones were sequenced, and their biological properties characterized by transfecting linear HBV clones into HepG2 cells. We analysed the SPI and SPII promotor regions, X-gene, BCP/PC sequences, core, preS/S and HBV polymerase sequences. HBV clones analysed in this study revealed differential replication kinetics of viral nucleic acids and expression of proteins. Sequence analysis of HBV clones revealed mutations in preS1, preS2 and S genes; deletion and insertion and point mutations in BCP/PC region; including novel and previously reported mutations. Among the patient samples tested, HBV genotype B clones were more likely to have higher frequencies of mutations, while sub-genotype A1 and A2 clones tended to have fewer mutations. No polymerase drug resistant mutations were seen. HBeAg mutations were primarily in the BCP/PC region in genotype B, but core truncations were found in genotype E. S gene mutations affecting HBsAg expression and detection were seen in all genotypes except A2. Using an HBV clone with repetitive terminal sequences and a SapI restriction site allowed us to analyse HBV analyte production in cell culture and characterize the genetics of viral phenotypes using complete HBV genomes isolated from serum/plasma samples of infected patients.

Determination of potential biotin interference on accuracy of results of serologic assays for various viral hepatitis markers.

Biotin taken orally can interfere with some diagnostic immunoassays, including those for thyroid hormones, ferritin, and markers of infectious disease. Assays affected are ones that use streptavidin-biotin in their design. The goal of our study was to examine the effect of biotin concentrations of up to 1200 ng/mL on three serological assays performed on VITROS 3600 system, Immunoglobulin M (IgM) antibodies to hepatitis A virus (anti-HAV), total anti-HAV, and IgM antibodies to hepatitis B virus core antigen (anti-HBc), by spiking serum samples with variable amounts of biotin. No false-negative results were generated with either concentration of biotin for total anti-HAV (65/65). Likewise, biotin caused no false-positive IgM anti-HAV results (59/59) with either concentration of biotin; however, 6.7% false negativity was found for IgM anti-HAV when samples were spiked with 1200 ng/mL of biotin. Conversely, 100% false positivity (30/30) was produced by biotin interference in total anti-HAV negative specimens with both concentrations of biotin. False negativity rate was 87.5% in IgM anti-HBc positive samples when biotin levels were at 1200 ng/mL. These data show that individuals taking biotin-containing supplements may test false-positive in some serologic assays using streptavidin-biotin chemistries. Further studies are warranted to determine the extent of biotin interference resulting in false-positive and negative results and their impact, if any, on surveillance and diagnostic settings.

Integrated HIV surveillance finds recent adult hepatitis B virus (HBV) transmission and intermediate HBV prevalence among military in uncharacterized Caribbean country.

BACKGROUND: Guyana expanded its HIV response in 2005 but the epidemiology of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections has not been characterized. METHODS: The 2011 Seroprevalence and Behavioral Epidemiology Risk Survey for HIV and STIs collected biologic specimens with demographic and behavioral data from a representative sample of Guyana military personnel. Diagnostics included commercial serum: HIV antibody; total antibody to hepatitis B core (anti-HBc); IgM anti-HBc; hepatitis B surface antigen (HBsAg); anti-HBs; antibody to HCV with confirmatory testing; and HBV DNA sequencing with S gene fragment phylogenetic analysis. Chi-square, p-values and prevalence ratios determined statistical significance. RESULTS: Among 480 participants providing serologic specimens, 176 (36.7%) tested anti-HBc-positive. Overall, 19 (4.0%) participants tested HBsAg-positive; 17 (89.5%) of the HBsAg-positive participants also had detectable anti-HBc, including 1 (5.3%) IgM anti-HBc-positive male. Four (6.8%) females with available HBV testing were HBsAg-positive, all aged 23-29 years. Sixteen (16, 84.2%) HBsAg-positive participants had sufficient specimen for DNA testing. All 16 had detectable HBV DNA, 4 with viral load >2x104IU/ml. Sequencing found: 12 genotype (gt) A1 with 99.9% genetic identity between 1 IgM anti-HBc-positive and 1 anti-HBc-negative; 2 gtD1; and 2 with insufficient specimen. No statistically significant associations between risk factors and HBV infection were identified. CONCLUSIONS: Integrated HIV surveillance identified likely recent adult HBV transmission, current HBV infection among females of reproductive age, moderate HBV infection prevalence (all gtA1 and D1), no HCV infections and low HIV frequency among Guyana military personnel. Integrated HIV surveillance helped characterize HBV and HCV epidemiology, including probable recent transmission, prompting targeted responses to control ongoing HBV transmission and examination of hepatitis B vaccine policies.

Hepatitis B and C virus infections transmitted through organ transplantation investigated by CDC, United States, 2014-2017.

We evaluated clinical outcomes among organ recipients with donor-derived hepatitis B virus (HBV) or hepatitis C virus (HCV) infections investigated by CDC from 2014 to 2017 in the United States. We characterized new HBV infections in organ recipients if donors tested negative for total anti-HBc, HBsAg and HBV DNA, and new recipient HCV infections if donors tested negative for anti-HCV and HCV RNA. Donor risk behaviors were abstracted from next-of-kin interviews and medical records. During 2014-2017, seven new recipient HBV infections associated with seven donors were identified; six (86%) recipients survived. At last follow-up, all survivors had functioning grafts and five (83%) had started antiviral therapy. Twenty new recipient HCV infections associated with nine donors were identified; 19 (95%) recipients survived. At last follow-up, 18 (95%) survivors had functioning grafts and 14 (74%) had started antiviral treatment. Combining donor next-of kin interviews and medical records, 11/16 (69%) donors had evidence of injection drug use and all met Public Health Service increased risk donor (IRD) criteria. IRD designation led to early diagnosis of recipient infection, and prompt implementation of therapy, likely reducing the risk of graft failure, liver disease, and death.

Hepatitis B Virus Mutant Infections in Hemodialysis Patients: A Case Series.

RATIONALE & OBJECTIVE: Hepatitis B virus (HBV) transmission in hemodialysis units has become a rare event since implementation of hemodialysis-specific infection control guidelines: performing hemodialysis for hepatitis B surface antigen (HBsAg)-positive patients in an HBV isolation room, vaccinating HBV-susceptible (HBV surface antibody and HBsAg negative) patients, and monthly HBsAg testing in HBV-susceptible patients. Mutations in HBsAg can result in false-negative HBsAg results, leading to failure to identify HBsAg seroconversion from negative to positive. We describe 4 unique cases of HBsAg seroconversion caused by mutant HBV infection or reactivation in hemodialysis patients. STUDY DESIGN: Following identification of a possible HBsAg seroconversion and mutant HBV infection, public health investigations were launched to conduct further HBV testing of case patients and potentially exposed patients. A case patient was defined as a hemodialysis patient with suspected mutant HBV infection because of false-negative HBsAg testing results. Confirmed case patients had HBV DNA sequences demonstrating S-gene mutations. SETTING & PARTICIPANTS: Case patients and patients potentially exposed to the case patient in the respective hemodialysis units in multiple US states. RESULTS: 4 cases of mutant HBV infection in hemodialysis patients were identified; 3 cases were confirmed using molecular sequencing. Failure of some HBsAg testing platforms to detect HBV mutations led to delays in applying HBV isolation procedures. Testing of potentially exposed patients did not identify secondary transmissions. LIMITATIONS: Lack of access to information on past HBsAg testing platforms and results led to challenges in ascertaining when HBsAg seroconversion occurred and identifying and testing all potentially exposed patients. CONCLUSIONS: Mutant HBV infections should be suspected in patients who test HBsAg negative and concurrently test positive for HBV DNA at high levels. Dialysis providers should consider using HBsAg assays that can also detect mutant HBV strains for routine HBV testing.

Evaluation of performance characteristics of hepatitis B e antigen serologic assays.

BACKGROUND: Hepatitis B e antigen (HBeAg) is considered an indicator of high hepatitis B virus (HBV) replication. Performance characteristics of commercially available HBeAg assays have not been determined, thus it is unknown whether lack of HBeAg detection is because of test sensitivity or HBV basal core promoter and precore mutations. OBJECTIVES: We studied the correlation between HBeAg reactivity with HBV DNA levels in three commercially available HBeAg assays using 335 HBsAg and HBV DNA positive serum/plasma samples. STUDY DESIGN: Diagnostic sensitivity was determined by serial dilutions of a WHO HBeAg standard. The limit of HBeAg detection estimated through regression was 1 IU/mL (Centaur), 97 IU/mL (DiaSorin) and 129 IU/mL (Vitros). Of these 335 samples, enough sample volume remained in 253 samples for head-to-head comparison of the assays. RESULTS: 81 (32%), 41 (16%) and 36 (14%) of the samples were HBeAg positive by the Centaur, DiaSorin and Vitros assays, respectively. Compared to the FDA-approved Centaur assay the specificity of the other two assays was 98%, while sensitivity was 47% for the DiaSorin assay and 41% for the Vitros assay. Significant association was found between HBeAg positive samples and HBV DNA levels >20,000 IU/mL; 31% of HBeAg negative samples (Centaur) had HBV DNA levels >20,000 IU/mL, 26% of HBeAg positive samples had HBV DNA levels <20,000 IU/mL and 5 HBeAg positive samples had HBV DNA levels <2000 IU/mL. CONCLUSION: Discordance was seen between these HBeAg assays, indicating reliance on HBeAg alone as a marker of high HBV replication can be misleading. Detection and quantification of HBV DNA remains the accurate and reliable marker of HBV replication.

Hepatitis C virus transmission in a skilled nursing facility, North Dakota, 2013.

BACKGROUND: From March-May 2013, 3 cases of acute hepatitis C virus (HCV) infection were diagnosed among elderly patients residing at the same skilled nursing facility (facility A) and who received health care at hospital X during their likely exposure period. METHODS: We performed HCV testing of at-risk populations; quasispecies analysis was performed to determine relatedness of HCV in persons with current infection. Infection control practice assessments were conducted at facility A and hospital X. Persons residing in facility A on September 9, 2013, were enrolled in a case-control study to identify risk factors for HCV infection. RESULTS: Forty-five outbreak-associated infections were identified. Thirty cases and 62 controls were enrolled in the case-control study. Only podiatry (odds ratio, 11.6; 95% confidence interval, 2.4-57.2) and international normalized ratio monitoring by phlebotomy (odds ratio, 6.7; 95% confidence interval, 1.7-26.6) at facility A were significantly associated with case status. Infection control lapses during podiatry and point-of-care testing procedures at facility A were identified. CONCLUSIONS: HCV transmission was confirmed among residents of facility A. The exact mode of transmission was not able to be identified, but infection control lapses were likely responsible. This outbreak highlights the importance of prompt reporting and investigation of incident HCV infection and the need for adherence to basic infection control procedures by health care personnel.

High hepatitis C virus (HCV) prevalence among men who have sex with men (MSM) in Vietnam and associated risk factors: 2010 Vietnam Integrated Behavioural and Biologic Cross-Sectional Survey.

BACKGROUND: Hepatitis C virus (HCV) is an increasing health issue among key populations such as men who have sex with men (MSM). We sought to assess the burden of and risk factors for HCV among MSM in Vietnam. METHODS: We analysed behavioural and demographic data and stored specimens from MSM surveyed in four provinces through Vietnam's 2009-2010 Integrated Biologic and Behavioural Survey, which used probability-based, respondent-driven sampling. Commercial hepatitis B surface antigen (HBsAg) and HCV/antibody (HCV Ag/Ab) testing were performed on archived sera with follow-up PCR for HCV RNA and genotype determination. RESULTS: Among the 1588 MSM surveyed, the median (range) frequency, by province, of HCV Ag/Ab detection was 28.4% (13.7%-38.8%); 84.5% (83.1%-100%) among HIV-infected and 21.9% (8.9%-28.2%) among HIV-uninfected. HCV prevalence was higher in northern Hanoi and Hai Phong provinces than in southern Ho Chi Minh City and Chan Tho provinces. Among a convenience sample of 67 HCV Ag/Ab+ MSM, 67.2% were HCV RNA+; of 41 genotyped, 73.2% were genotype 1. HBsAg prevalence varied from 8.5% to 27.4%. In the multivariable logistic regression analysis, being HIV-infected (adjusted OR (aOR) 19.0; 7.0-51.9), ever having used injected drugs (aOR 4.4; 1.6-12.4) and age >25 years were significant risk factors for testing HCV Ag/Ab+. CONCLUSIONS: HCV infection in Vietnam appears to be high among MSM, particularly among HIV-infected MSM, with a north-south gradient. Given overlapping risk behaviours and associations between HCV and HIV, integrating HIV and HCV programme services to prevent both HIV and HCV transmission among MSM is indicated.

Notes from the Field: Health Care-Associated Hepatitis A Outbreak - Texas, 2015.

On August 27-28, 2015, the Texas Department of State Health Services received calls from Fort Bend County and Harris County health departments requesting postexposure prophylaxis (PEP) recommendations for contacts of two nurses (patients A and B) with confirmed hepatitis A virus (HAV) infection. Both nurses had symptom onset during August 15-19 and worked for the same pediatric home health care agency in another jurisdiction. Because of the proximity of the onset dates, a common source exposure was suspected. The state and local health departments began an investigation to identify potentially exposed patients, their families, and other agency personnel; offer PEP; and identify the source of exposure.

Notes from the Field: Hepatitis C Outbreak in a Dialysis Clinic--Tennessee, 2014.

Outbreaks of hepatitis C virus (HCV) infections can occur among hemodialysis patients when recommended infection control practices are not followed (1). On January 30, 2014, a dialysis clinic in Tennessee identified acute HCV in a patient (patient A) during routine screening and reported it to the Tennessee Department of Health. Patient A had enrolled in the dialysis clinic in March 2010 and had annually tested negative for HCV (including a last HCV test on December 19, 2012), until testing positive for HCV antibodies (anti-HCV) on December 18, 2013 (confirmed by a positive HCV nucleic acid amplification test). Patient A reported no behavioral risk factors, but did have multiple health care exposures.

Performance of ARCHITECT HCV core antigen test with specimens from US plasma donors and injecting drug users.

BACKGROUND: Hepatitis C virus (HCV) core antigen is a serological marker of current HCV infection. OBJECTIVES: The aim of this study was mainly to evaluate the performance characteristics of the ARCHITECT HCV core antigen assay with specimens from US plasma donors and injecting drug users. STUDY DESIGN: A total of 551 serum and plasma samples with known anti-HCV and HCV RNA status were tested for HCV core antigen using the Abbott ARCHITECT HCV core antigen test. RESULTS: HCV core antigen was detectable in 100% of US plasma donor samples collected during the pre-seroconversion phase of infection (anti-HCV negative/HCV RNA positive). Overall sensitivity of the HCV core antigen assay was 88.9-94.3% in samples collected after seroconversion. The correlation between HCV core antigen and HCV RNA titers was 0.959. CONCLUSIONS: HCV core antigen testing may be reliably used to identify current HCV infection.

Disparate detection outcomes for anti-HCV IgG and HCV RNA in dried blood spots.

Dried blood spots (DBS) expedite the collection, storage and shipping of blood samples, thereby facilitating large-scale serologic studies. We evaluated the sensitivity of anti-HCV IgG testing and HCV-RNA quantitation using freshly prepared and stored DBS derived from HCV-infected patients. Protocols for elution were optimized using DBS prepared from plasma of 52 HCV-infected persons and 51 uninfected persons (control DBS), then applied to DBS from 33 chronic hepatitis C patients that had been stored at -20°C for 5 years (stored DBS). Control and stored DBS, and their corresponding plasma, were processed for anti-HCV IgG testing using the VITROS chemiluminescence assay (CIA) and the HCV 3.0 enzyme immunoassay (EIA) (Ortho-Clinical Diagnostics), and for HCV RNA quantitation by quantitative (q) RT-PCR. HCV genotyping was conducted by nucleotide sequencing. The sensitivity of CIA and EIA in control DBS was 92% and 90%, respectively, compared to 100% and 97%, respectively, in stored DBS. The sensitivity of HCV RNA detection was 88% in control DBS, compared to 36% in stored DBS. Specificity was 100% for all the assays in both control and stored DBS. Genotypes 1, 2 and 3 were detected in 16 (62%), 6 (23.1%), and 4 (15.3%) samples, respectively. Sequences generated from DBS and their corresponding plasma samples were identical. Whereas the sensitivity of anti-HCV IgG detection in stored DBS was equivalent to that in recently prepared DBS, the sensitivity of HCV RNA detection was markedly lower in stored DBS compared to recently prepared DBS. Stored DBS may be reliably used for anti-HCV detection but for HCV-RNA-based testing freshly prepared DBS is preferable to stored DBS.

Rapid and sensitive approach to simultaneous detection of genomes of hepatitis A, B, C, D and E viruses.

BACKGROUND: Five viruses have been etiologically associated with viral hepatitis. Nucleic acid testing (NAT) remains the gold standard for diagnosis of viremic stages of infection. NAT methodologies have been developed for all hepatitis viruses; however, a NAT-based assay that can simultaneously detect all five viruses is not available. OBJECTIVES: We designed TaqMan card-based assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. STUDY DESIGN: The performances of individual assays were evaluated on TaqMan Array Cards (TAC) for detecting five viral genomes simultaneously. Sensitivity and specificity were determined by testing 329 NAT-tested clinical specimens. RESULTS: All NAT-positive samples for HCV (n = 32), HDV (n = 28) and HEV (n = 14) were also found positive in TAC (sensitivity, 100%). Forty-three of 46 HAV-NAT positive samples were also positive in TAC (sensitivity, 94%), while 36 of 39 HBV-NAT positive samples were positive (sensitivity, 92%). No false-positives were detected for HBV (n = 32), HCV (n = 36), HDV (n = 30), and HEV (n = 31) NAT-negative samples (specificity 100%), while 38 of 41 HAV-NAT negative samples were negative by TAC (specificity 93%). CONCLUSIONS: TAC assay was concordant with corresponding individual NATs for hepatitis A-E viral genomes and can be used for their detection simultaneously. The TAC assay has potential for use in hepatitis surveillance, for screening of donor specimens and in outbreak situations. Wider availability of TAC-ready assays may allow for customized assays, for improving acute jaundice surveillance and for other purposes for which there is need to identify multiple pathogens rapidly.

Characterization of the bacterial communities of life stages of free living lone star ticks (Amblyomma americanum).

The lone star tick (Amblyomma americanum) is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females) from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5-3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii) and an obligate Coxiella symbiont, together accounting for 6.7-100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001), but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002) and structure (p = 0.002) of their bacterial communities. Adults differed only in their community structure (p = 0.002) with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of the bacterial community change during the tick life cycle and that some sex-specific attributes may be detectable in nymphs.