Elements of informed consent for pharmacogenetic research; perspective of the pharmacogenetics working group.
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Clinical Trials as Topic↗
Biomedical subjects
S C Ray
Publications and source records attributed to S C Ray.
Determinants of viral clearance and persistence during acute hepatitis C virus infection.
The virological and immunological features of hepatitis C virus (HCV) infection were studied weekly for 6 months after accidental needlestick exposure in five health care workers, four of whom developed acute hepatitis that progressed to chronicity while one subject cleared the virus. In all subjects, viremia was first detectable within 1-2 weeks of inoculation, 1 month or more before the appearance of virus-specific T cells. The subject who cleared the virus experienced a prolonged episode of acute hepatitis that coincided with a CD38+ IFN-gamma- CD8+ T cell response to HCV and a small reduction in viremia. Subsequently, a strong CD4+ T cell response emerged and the CD8+ T cells became CD38- and started producing IFN-gamma in response to HCV, coinciding with a rapid 100,000-fold decrease in viremia that occurred without a corresponding surge of disease activity. Chronic infection developed in two subjects who failed to produce a significant T cell response and in two other subjects who initially mounted strong CD4+ T cell responses that ultimately waned. In all subjects, viremia was higher at the peak of acute hepatitis than it was when the disease began, and the disease improved during the viremia. These results provide the first insight into the host-virus relationship in humans during the incubation phase of acute HCV infection, and they provide the only insight to date into the virological and immunological characteristics of clinically asymptomatic acute HCV infection, the commonest manifestation of this disease. In addition, the results suggest that the vigor and quality of the antiviral T cell response determines the outcome of acute HCV infection, that the ability of HCV to outpace the T cell response may contribute to its tendency to persist; that the onset of hepatitis coincides with the onset of the CD8+ T cell response, that disease pathogenesis and viral clearance are mediated by different CD8+ T cell populations that control HCV by both cytolytic and noncytolytic mechanisms, and that there are different pathways to viral persistence in asymptomatic and symptomatic acute HCV infection.
Acute Disease↗
Dynamics of SEN virus infection among injection drug users.
SEN virus (SENV) is a recently discovered group of DNA viruses whose members (SENV-D and SENV-H) are linked to posttransfusion hepatitis. Of 397 injection drug users (IDUs) in Baltimore, Maryland, SENV-D infection was detected by polymerase chain reaction in serum samples from 130 (32.7%) and SENV-H infection in 149 (37.5%). Of 41 IDUs in whom SENV-D DNA was initially detected, retesting for viral persistence a median of 9.3 years later detected SENV-D in 25 (61.0%), whereas SENV-H was detected on retesting in only 14 (26.9%) of 52 IDUs in whom the virus was originally found. Reinfection was apparent (>5% nucleotide difference) in 77.8% of IDUs who repeatedly tested positive for SENV-D DNA and in 55.6% of those who repeatedly tested positive for SENV-H DNA. Among Baltimore IDUs, SENV-D and SENV-H infections are common and dynamic, including both viral clearance and reinfection. The clinical significance of SENV infection in this setting remains unknown.
Adult↗
HIV-1 drug resistance profiles in children and adults with viral load of <50 copies/ml receiving combination therapy.
CONTEXT: The continued release of human immunodeficiency virus type 1 (HIV-1) into plasma at very low levels during highly active antiretroviral therapy (HAART) can be detected using specialized techniques, but the nature and significance of this low-level viremia, especially as related to acquisition of drug resistance mutations, are unclear. OBJECTIVE: To determine genetic resistance profiles of low-level plasma HIV-1 in patients with prolonged viral suppression (<50 copies/mL of plasma HIV-1 RNA) while receiving HAART. DESIGN AND SETTING: Cross-sectional study conducted at a US academic hospital from November 1999 to February 2001 using a novel method for amplification of low levels of viral genomes in plasma. PATIENTS: Eighteen HIV-1-infected patients (7 children and 11 adults), enrolled in a longitudinal study of HIV-1 reservoirs, who had suppression of viral replication while receiving protease inhibitor-containing combination therapy. Two patients (1 adult and 1 child) with less optimal suppression of viral replication were included to assess virus predominating when plasma HIV-1 RNA levels are low but detectable (<1000 copies/mL). Follow-up analyses were conducted in 3 patients. MAIN OUTCOME MEASURE: Detection of drug resistance mutations in clones amplified from low-level plasma virus. RESULTS: Viral sequences were amplified from 8 of the 18 patients with simultaneous plasma HIV-1 measurements of less than 50 copies/mL and from 2 patients with 231 and 50 copies/mL. Clones from 3 treatment-naive patients with less than 50 copies/mL of plasma HIV-1 RNA showed continued release, for as long as 42 months, of wild-type drug-sensitive virus. The 7 patients with prior nonsuppressive therapy, with viral loads below 50 copies/mL and during "blips" to 231 and 64 copies/mL, had only resistance mutations consistent with pre-HAART therapy (although reverse transcriptase inhibitor mutations may have continued to occur). New HAART-related mutations were seen in a control patient with prior viral load levels of about 400 to 1000 copies/mL. For phylogenetic analysis, sequences were available for both resting CD4(+) T cells and plasma HIV for 7 of 10 patients and showed patient-specific clustering of sequences and a close relationship between virus in the plasma and the latent reservoir. CONCLUSIONS: Based on the samples that could be amplified, low-level viremia in children and adults receiving HAART with prolonged suppression of viremia to less than 50 copies/mL of HIV-1 RNA may result primarily from archival, pre-HAART virus, reflecting earlier treatment conditions, and does not appear to require development of new, HAART-selected mutations reflecting partial resistance to therapy. Low-level viremia below 50 copies/mL may represent less of a concern regarding impending drug failure of current HAART regimens. However, the archival drug-resistant virus may be relevant regarding future treatment strategies.
Adult↗
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.
Anaplasma↗
Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies.
When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.
Adult↗
Genetic epidemiology of hepatitis C virus throughout egypt.
Hepatitis C virus (HCV) infection is a major health problem in Egypt, where the seroprevalence is 10-20-fold higher than that in the United States. To characterize the HCV genotype distribution and concordance of genotype assessments on the basis of multiple genomic regions, specimens were obtained from blood donors in 15 geographically diverse governorates throughout Egypt. The 5' noncoding, core/E1, and NS5B regions were amplified by reverse transcription-polymerase chain reaction and analyzed by both restriction fragment length polymorphism (RFLP) and phylogenetic tree construction. For the 5' noncoding region, 122 (64%) of 190 specimens were amplified and analyzed by RFLP: 111 (91%) were genotype 4, 1 (1%) was genotype 1a, 1 (1%) was genotype 1b, and 9 (7%) could not be typed. Phylogenetic analyses of the core/E1 and NS5B regions confirmed the genotype 4 preponderance and revealed evidence of 3 new subtypes. Analysis of genetic distance between isolates was consistent with the introduction of multiple virus strains 75-140 years ago, and no clustering was detected within geographic regions, suggesting widespread dispersion at some time since then.
Blood Donors↗
Sex and the genetic diversity of HIV-1.
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Acquired Immunodeficiency Syndrome↗
Determinants of the quantity of hepatitis C virus RNA.
To test the hypothesis that person-to-person variability in blood levels of hepatitis C virus (HCV) RNA can be explained, the quantity of HCV RNA was assessed in 969 persons who acquired HCV infection in the context of injection drug use. Serum HCV RNA levels ranged from 200,000 to >120 million equivalents/mL (the linear range of the assay). The median log10 HCV RNA level was 0.46 higher in 468 human immunodeficiency virus (HIV)-positive persons than in 501 HIV-negative persons (P<.001). In addition, among HIV-negative persons, lower HCV RNA levels were independently associated with younger age (P<.001), ongoing hepatitis B infection (P=.005), and the absence of needle sharing (P=.02). However, >90% of the person-to-person HCV RNA level variability was not explained by these sociodemographic, environmental, and virologic factors. Additional research is necessary to ascertain what determines the level of HCV RNA in blood.
Adult↗
Prospective characterization of full-length hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy.
The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.
Amino Acid Sequence↗
Hypervariable region 1 sequence stability during hepatitis C virus replication in chimpanzees.
The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex-single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.
Amino Acid Sequence↗
Report from ICAAC 2000: HIV & viral co-infections.
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AIDS-Related Opportunistic Infections↗
PCR detection of adenovirus in a bone marrow transplant recipient: hemorrhagic cystitis as a presenting manifestation of disseminated disease.
Adenoviruses (AdV), causing fatal disseminated infections in bone marrow transplant (BMT) recipients, are associated not only with hemorrhagic cystitis (HC) but also with hepatitis, conjunctivitis, and viral interstitial pneumonia. The importance of this virus as a cause of disseminated disease, however, has remained underappreciated. AdV infection has been diagnosed primarily through the use of cell culture. The fact that cell culture is insensitive for detecting this virus has hindered recognition of the role that AdV may play in morbidity and mortality in BMT recipients. To emphasize these points, we describe a patient who presented with HC due to AdV serotype 11, genotype c, and died with disseminated infection. In addition to cell culture, this study used a newly developed PCR-based method, capable of detecting all AdV serotypes tested, including different genotypes of serotype 11. The PCR result was positive in all culture-positive samples, including samples of urine, conjunctiva, and bronchoalveolar lavage (BAL). Importantly, the PCR method provided evidence of urinary shedding of AdV in a pretransplant, culture-negative specimen and showed dissemination in a subset of culture-negative specimens, including BAL, blood, and bone marrow samples. The lack of widespread awareness of the fact that localized infections may presage dissemination, and the previous associated lack of rapid, sensitive diagnostic assays, has impaired recognition of AdV infections in patients undergoing BMT. Early detection may contribute to therapy modification and avoidance of unwarranted diagnostic procedures. It may also assist in epidemiologic control of this highly infectious pathogen and lead to a renewed interest in preventive and therapeutic approaches.
Adenovirus Infections, Human↗
Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination.
The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is likely to depend on knowledge of circulating variants of genes other than the commonly sequenced gag and env genes. In addition, full-genome data are particularly limited for HIV-1 subtype C, currently the most commonly transmitted subtype in India and worldwide. Likewise, little is known about sequence variation of HIV-1 in India, the country facing the largest burden of HIV worldwide. Therefore, the objective of this study was to clone and characterize the complete genome of HIV-1 from seroconverters infected with subtype C variants in India. Cocultured HIV-1 isolates were obtained from six seroincident individuals from Pune, India, and virtually full-length HIV-1 genomes were amplified, cloned, and sequenced from each. Sequence analysis revealed that five of the six genomes were of subtype C, while one was a mosaic of subtypes A and C, with multiple breakpoints in env, nef, and the 3' long terminal repeat as determined by both maximal chi2 analysis and phylogenetic bootstrapping. Sequences were compared for preservation of known cytotoxic T lymphocyte (CTL) epitopes. Compared with those of the HIV-1LAI sequence, 38% of well-defined CTL epitopes were identical. The proportion of nonconservative substitutions for Env, at 61%, was higher (P < 0.001) than those for Gag (24%), Pol (18%), and Nef (32%). Therefore, characterized CTL epitopes demonstrated substantial differences from subtype B laboratory strains, which were most pronounced in Env. Because these clones were obtained from Indian seroconverters, they are likely to facilitate vaccine-related efforts in India by providing potential antigens for vaccine candidates as well as for assays of vaccine responsiveness.
Adult↗
Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy.
We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.
Acute Disease↗
Recrudescence of treated neurosyphilis in a patient with human immunodeficiency virus.
The natural history of syphilis has been altered by the human immunodeficiency virus (HIV) epidemic. Treatment of patients coinfected with syphilis and HIV is currently controversial; progression and relapse of neurosyphilis have been reported. This case report documents failure of primary treatment and neurosyphilitic recrudescence. In a 32-year-old HIV-positive woman with syphilis who had no additional sexual contacts, the disease progressed to neurosyphilis despite three intramuscular doses of penicillin. After extended intravenous penicillin treatment, neurosyphilis later recurred and re-treatment was necessary. Because many urban centers are affected by high rates of sexually transmitted diseases, including common coinfections of syphilis and HIV, further efforts should be made to identify subsets of patients who may be at high risk of syphilitic recrudescence.
Adult↗
Autologous strain-specific cytolytic T lymphocyte responses directed against human immunodeficiency virus type 1 Env.
The Env glycoprotein of human immunodeficiency virus is critical for the pathogenesis of the acquired immunodeficiency syndrome, and has been the prime target for candidate HIV-1 vaccines. Cytolytic T lymphocytes (CTLs) may be important for the immunologic control of HIV infection and HIV-1 Env-specific cytolytic T cells have been isolated from infected individuals and seronegative recipients of HIV-1 vaccines. Most prior studies have used assays that detect Env-specific CTLs directed against standard laboratory viral variants. These studies may be limited because the Env proteins of these laboratory strains (for example, LAI and MN) may differ significantly from the Env proteins from primary HIV-1 strains, and a single amino acid change can abrogate the recognition of HIV-1 Env by some CTL clones. Therefore, this study measured CTL activity directed against HIV-1 Env representing the infected individual's (autologous) HIV-1 viral variants. For two HIV-1-infected individuals, recombinant vaccina viruses expressing cloned HIV-1 env genes were constructed. Using an in vitro stimulation method, strain-specific CTL activity directed against autologous HIV-1 Env was detected in both individuals. From one subject, strain-specific CTL clones directed against autologous and HIV-1LAI Env were characterized. Therefore, some infected individuals have Env-specific CTLs directed against autologous strains of HIV-1. Detection and characterization of autologous Env-specific CTL activity may have important implications relative to the current HIV-1 vaccine development strategies focusing on Env derived from laboratory strains of HIV-1.
Acquired Immunodeficiency Syndrome↗
Assessment of hepatitis C virus sequence complexity by electrophoretic mobilities of both single-and double-stranded DNAs.
To assess genetic variation in hepatitis C virus (HCV) sequences accurately, we optimized a method for identifying distinct viral clones without determining the nucleotide sequence of each clone. Twelve serum samples were obtained from seven individuals soon after they acquired HCV during a prospective study, and a 452-bp fragment from the E2 region was amplified by reverse transcriptase PCR and cloned. Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined heteroduplex analysis (HDA) and a single-stranded conformational polymorphism (SSCP) method to determine the number of clonotypes (electrophoretically indistinguishable cloned cDNAs) as a measure of genetic complexity (this combined method is referred to herein as the HDA+SSCP method). We calculated Shannon entropy, incorporating the number and distribution of clonotypes into a single quantifier of complexity. These measures were evaluated for their correlation with nucleotide sequence diversity. Blinded analysis revealed that the sensitivity (ability to detect variants) and specificity (avoidance of false detection) of the HDA+SSCP method were very high. The genetic distance (mean +/- standard deviation) between indistinguishable cloned cDNAs (intraclonotype diversity) was 0.6% +/- 0.9%, and 98.7% of cDNAs differed by <2%, while the mean distance between cloned cDNAs with different patterns was 4.0% +/- 3.2%. The sensitivity of the HDA+SSCP method compared favorably with either HDA or the SSCP method alone, which resulted in intraclonotype diversities of 1.6% +/- 1.8% and 3.5% +/- 3.4%, respectively. The number of clonotypes correlated strongly with genetic diversity (R2, 0.93), but this correlation fell off sharply when fewer clones were assessed. This HDA+SSCP method accurately reflected nucleotide sequence diversity among a large number of viral cDNA clones, which should enhance analyses to determine the effects of viral diversity on HCV-associated disease. If sequence diversity becomes recognized as an important parameter for staging or monitoring of HCV infection, this method should be practical enough for use in laboratories that perform nucleic acid testing.
Cloning, Molecular↗