Stuart C. Ray

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Protection against persistence of hepatitis C

BACKGROUND Neither previous hepatitis C virus (HCV) infection nor vaccination with HCV-derived antigens protects against reinfection. However, HCV infection and vaccination in chimpanzees has been shown to reduce the magnitude and duration of viraemia with re-challenge. We aimed to establish whether similar immunity could be achieved in man. METHODS From a study of injecting drug users, we identified 164 people who had no evidence of previous HCV infection and 98 individuals who had been previously, but were not currently, infected with HCV. We compared the incidence and persistence of HCV viraemia in these two groups over four consecutive 6-month periods. FINDINGS Of participants without previous infection, the incidence of HCV infection was 21% (35/164). By contrast, people previously infected were half as likely to develop new viraemia (12% [12/98]), even after accounting for risk behaviour (hazard ratio, 0.45; 95% CI 0.23-0.88). Furthermore, in HIV-1-negative people, those previously infected were 12 times less likely than people infected for the first time to develop persistent infection (odds ratio 0.05, 95% CI 0.01-0.30), and median peak HCV RNA concentration was two logs lower. HCV persisted in six of six HIV-1-positive people, even in one man who had previously cleared HCV infection when he was HIV-1 negative. INTERPRETATION There is an alarming frequency of HCV infection and persistence among injecting drug users. Our data suggest that immunity against viral persistence can be acquired, and that vaccines should be tested to reduce the burden of HCV-related liver disease.

Residual Human Immunodeficiency Virus Type 1 Viremia in Some Patients on Antiretroviral Therapy Is Dominated by a Small Number of Invariant Clones Rarely Found in Circulating CD4+ T Cells

ABSTRACT Antiretroviral therapy can reduce human immunodeficiency virus type 1 (HIV-1) viremia to below the detection limit of ultrasensitive clinical assays (50 copies of HIV-1 RNA/ml). However, latent HIV-1 persists in resting CD4+ T cells, and low residual levels of free virus are found in the plasma. Limited characterization of this residual viremia has been done because of the low number of virions per sample. Using intensive sampling, we analyzed residual viremia and compared these viruses to latent proviruses in resting CD4+ T cells in peripheral blood. For each patient, we found some viruses in the plasma that were identical to viruses in resting CD4+ T cells by pol gene sequencing. However, in a majority of patients, the most common viruses in the plasma were rarely found in resting CD4+ T cells even when the resting cell compartment was analyzed with assays that detect replication-competent viruses. Despite the large diversity of pol sequences in resting CD4+ T cells, the residual viremia was dominated by a homogeneous population of viruses with identical pol sequences. In the most extensively studied case, a predominant plasma sequence was also found in analysis of the env gene, and linkage by long-distance reverse transcriptase PCR established that these predominant plasma sequences represented a single predominant plasma virus clone. The predominant plasma clones were released for months to years without evident sequence change. Thus, in some patients on antiretroviral therapy, the major mechanism for residual viremia involves prolonged production of a small number of viral clones without evident evolution, possibly by cells other than circulating CD4+ T cells.

Spontaneous Control of Primary Hepatitis C Virus Infection and Immunity against Persistent Reinfection

BACKGROUND & AIMS We followed patients with ongoing hepatitis C virus (HCV) exposure following control of an initial HCV infection to determine whether primary control conferred protection against future persistent infections. METHODS Twenty-two active injection drug users (IDU) who had cleared a primary hepatitis C viremia for at least 60 days were monitored monthly. Reinfection was defined as the detection of a new HCV infection. Protection was assessed based on the magnitude and duration of viremia following reinfection and generation of T-cell and neutralizing antibody (nAb) responses. RESULTS Reinfection occurred in 11 IDU (50%) who previously spontaneously controlled primary HCV infection. Although viral clearance occurs in approximately 25% of patients with primary infections, spontaneous viral clearance was observed in 83% of reinfected patients. The duration and maximum level of viremia during subsequent episodes of reinfection were significantly decreased compared with those of the primary infection in the same subjects. In contrast to chronic infection, reinfection was associated with a significant increase in the breadth of T-cell responses. During acute infection, nAbs against heterologous viral pseudoparticles were detected in 60% of reinfected subjects; cross-reactive nAbs are rarely detected in patients who progress to chronic infection. CONCLUSIONS HCV reinfection is associated with a reduction in the magnitude and duration of viremia (compared with the initial infection), broadened cellular immune responses, and generation of cross-reactive humoral responses. These findings are consistent with development of adaptive immunity that is not sterilizing but protects against chronic disease.

Human Immunodeficiency Virus-Related Microbial Translocation and Progression of Hepatitis C

BACKGROUND & AIMS Human immunodeficiency virus (HIV)-1 infection has been associated with enhanced microbial translocation, and microbial translocation is a mechanism through which alcohol and some enteric conditions cause liver disease. We hypothesized that HIV promotes liver disease by enhancing microbial translocation. METHODS We studied human cohorts in which hepatitis C virus (HCV) and HIV outcomes were carefully characterized. RESULTS HIV-related CD4(+) lymphocyte depletion was strongly associated with microbial translocation as indicated by elevated levels of circulating lipopolysaccharide (LPS), LPS-binding protein, soluble CD14, and fucose-binding lectin (AAL) reactive to immunoglobulin G specific for the alpha-galactose epitope and suppressed levels of endotoxin core antibodies (EndoCAb IgM) in HIV-infected subjects compared with the same persons before they had HIV infection and compared with HIV-uninfected subjects. The same measures of microbial translocation were strongly associated with HCV-related liver disease progression (cirrhosis), eg, LPS, odds ratio, 19.0 (P = .002); AAL, odds ratio, 27.8 (P < .0001); in addition, levels of LPS were elevated prior to recognition of cirrhosis. CONCLUSIONS Microbial translocation may be a fundamental mechanism through which HIV accelerates progression of chronic liver disease.

Acute hepatitis C

Symptomatic acute hepatitis C occurs in only about 15% of patients who are infected with hepatitis C virus (HCV). Acute hepatitis C is most often diagnosed in the setting of post-exposure surveillance, or seroconversion in high-risk individuals (eg, health-care professionals or injecting drug users) previously known to be seronegative. Although transmission via transfusion and injecting drug use has declined in developed countries, unsafe blood products and medical practices continue to increase transmission of HCV in many developing countries. Clinically, acute hepatitis C can increase concentrations of alanine aminotransferase to ten times the upper limit of normal but almost never causes fulminant hepatic failure. Diagnosis of HCV infection in the acute phase is difficult since production of antibodies against HCV can be delayed by up to 12 weeks, and about a third of infected individuals might not have detectable antibody at the onset of symptoms. Therefore, testing for HCV RNA by PCR is the only reliable test for the diagnosis of acute infection. Symptomatic patients with jaundice have a higher likelihood of spontaneous viral clearance than do asymptomatic patients, and thus should be monitored for at least 12 weeks before initiating antiviral therapy. By contrast, asymptomatic patients have a much lower chance of spontaneous clearance, and might benefit from early antiviral therapy. Antiviral therapy for 12 weeks is generally effective in treating patients who are HCV RNA negative after 4 weeks of treatment; lengthier courses could be needed for those who relapse or fail to show early virological clearance.

Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C

We comprehensively studied the cellular immune response during acute human hepatitis C virus (HCV) infection by monthly prospective sampling of persons at high risk of infection. In 19 of 23 subjects, interferon‐gamma–secreting T cells specific for 1 or more peptides spanning the entire HCV polyprotein were detected 1 to 3 months after infection. The median time to development of interferon gamma responses to HCV peptides was 33 days (range, 29‐50 days), and these responses peaked between 180 and 360 days. Nineteen subjects had sufficient follow‐up to determine outcome, with 15 (79%) developing persistent viremia and 4 (21%) clearing viremia spontaneously. Of those with progression to chronic infection and detectable T cell responses, all lost recognition of one or more antigens recognized during acute infection, and the median reduction in the magnitude of responses was 85%. Most significantly, despite ongoing viremia, those who had persistent infection did not develop new epitope specificities after the first 6 months of infection. In conclusion, in most individuals, the CD8+ T cell responses generated early in HCV infection decline in peripheral blood and are not replaced with new responses. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:104–112.)

Divergent and convergent evolution after a common-source outbreak of hepatitis C virus

The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune-driven selection and, in the absence of immune pressure, reversion toward an ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18–22 yr after infection to sequences present in the shared inoculum and found that HCV evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). These data suggest that vaccines for genetically diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.

Genotypic Analysis of HIV-1 Drug Resistance at the Limit of Detection: Virus Production without Evolution in Treated Adults with Undetectable HIV Loads

Human immunodeficiency virus (HIV) production continues in patients receiving highly active antiretroviral therapy (HAART) with undetectable (<50 copies/mL) virus loads. Our initial cross-sectional study showed that this viremia is composed of viruses that lack new resistance mutations to the HAART regimen. Here we describe a longitudinal, clonal genotypic analysis of plasma virus loads in treated adults who had undetectable virus loads. We document a continuous production of virus in 8 HIV-1-infected adults who maintained suppression of viremia for up to 15 months. Using analytical approaches for distinguishing selected resistance mutations from nonselected mutations and polymerase chain reaction errors, we detected no evolution of resistance in the reverse-transcriptase and protease genes. Sporadic resistance mutations were detected in some viral clones that were not selected for subsequently. Thus, in some patients, HAART suppresses replication to a level that does not allow the evolution of drug resistance over a time frame of years.

Oral antibiotic treatment of right-sided staphylococcal endocarditis in injection drug users: Prospective randomized comparison with parenteral therapy

PURPOSE To compare the efficacy and safety of inpatient oral antibiotic treatment (oral) versus standard parenteral antibiotic treatment (intravenous) for right-sided staphylococcal endocarditis in injection drug users. PATIENTS AND METHODS In a prospective, randomized, non-blinded trial, febrile injection drug users were assigned to begin oral or intravenous (IV) treatment on admission, before blood culture results were available. Oral therapy consisted of ciprofloxacin and rifampin. Parenteral therapy was oxacillin or vancomycin, plus gentamicin for the first 5 days. Antibiotic dosing was adjusted for renal dysfunction. Administration of other antibacterial drugs was not permitted during the treatment or follow-up periods. Bacteremic subjects having right-sided staphylococcal endocarditis received 28 days of inpatient therapy with the assigned antibiotics. Test-of-cure blood cultures were obtained during inpatient observation 6 and 7 days after the completion of antibiotic therapy, and again at outpatient follow-up 1 month later. Criteria for treatment failure and for drug toxicity were prospectively defined. RESULTS Of 573 injection drug users who were hospitalized because of a febrile illness and suspected right-sided staphylococcal endocarditis, 93 subjects (16.2%) had two or more sets of blood cultures positive for staphylococci; 85 of these bacteremic subjects (14.8%) satisfied diagnostic criteria for at least possible right-sided staphylococcal endocarditis (no other source of bacteremia was apparent) and entered the trial. Forty-four (oral, 19; IV, 25) of these 85 subjects completed inpatient treatment and evaluation including test-of-cure blood cultures. There were four treatment failures (oral, 1 [5.2%]; IV, 3 [12.0%]; not significant, Fishers exact test). Drug toxicity was significantly more common in the parenterally treated group (oral, 3%; IV, 62%; P < 0.0001), consisting largely of oxacillin-associated increases in liver enzymes. CONCLUSIONS For selected patients with right-sided staphylococcal endocarditis, oral ciprofloxacin plus rifampin is effective and is associated with less drug toxicity than is intravenous therapy.