Aeromonas species associated with necrotizing enteritis and septicemia in an adult male ostrich (Struthio camelus).

A deceased 10-yr-old male ostrich was diagnosed with severe necrotizing enteritis and septicemia. The bird was inappetent for 3 wk and had neurologic signs 2 days prior to death. Macroscopically, no significant lesions were noted aside from congestion of the liver, kidneys, and spleen. Histopathology revealed severe fibrinonecrotic enteritis,associated with large numbers of gram-negative bacteria, multifocal fibrinoid necrosis in portal arteries, accumulation of fibrin in hepatic sinusoids, myocardial degeneration, and necrosis. There was also squamous metaplasia in the glands of the esophagus and external ears. A gram-negative rod was isolated in pure culture from intestine, liver, lungs, and trachea and identified as an Aeromonas species. The concentration of vitamin A in the liver was extremely low. The lesions seen in the intestine and liver and the isolation of an Aeromonas sp. from various tissues strongly suggest that this bacterium was the cause of the necrotizing enteritis, septicemia, and death of this ostrich. Vitamin A deficiency might have predisposed the bird to the Aeromonas infection.

Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response in patients with chronic hepatitis C virus infection.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are present in the peripheral blood and liver of chronically infected patients. The current study was performed to study the relationship between the strength of the CTL response, liver disease severity, and viral load. The results may be summarized as follows: first, using CTL precursor frequency (CTLpf) analysis to quantitate the peripheral blood CTL response, chronically infected patients were less strongly sensitized to a panel of well-defined HCV epitopes than they were to an epitope within the influenza matrix protein. Second, HCV-specific CTLpf did not correlate with disease activity or viral load in the majority of patients on a cross-sectional basis, although it did increase in three patients concomitant with sharp increases in liver disease. Finally, interferon therapy did not enhance the CTLpf against the HCV epitopes studied in these patients, indicating that its antiviral effect is independent of the CTL response. Since the HCV-specific CTLpf in the blood is actually quite low, the CTL may contribute to ongoing liver disease in these patients while being quantitatively inadequate to destroy all of the infected hepatocytes, thereby facilitating HCV persistence and contributing to chronic liver disease.

Listeriosis in a cockatiel (Nymphicus hollandicus).

Listeriosis was diagnosed in a 4-yr-old female cockatiel (Nymphicus hollandicus) that died after exhibiting clinical signs that included a fluffed-up appearance, weakness, and loss of weight of several days duration. Grossly, the bird was moderately emaciated, and the liver and spleen were enlarged. Microscopically, there was mild-to-moderate inflammation associated with rod-shaped, gram-positive bacteria in the liver, spleen, kidneys, adrenal glands, bone marrow, and esophagus. Listeria monocytogenes was isolated from the liver, trachea, and intestine. The isolate was identified as type 1 by agglutination with specific antisera, and it further identified as belonging to serovar group 1/2a, 3a by multiplex polymerase chain reaction assay. Listeria monocytogenes also was detected in affected tissues by immunohistochemistry.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

AIM: To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. METHOD: Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. RESULTS: In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. CONCLUSION: In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Clinical evaluation of branched DNA signal amplification for quantifying HIV type 1 in human plasma.

Quantification of HIV-1 RNA in human plasma has provided unique insight into AIDS pathogenesis and promises to hasten progress in antiretroviral therapy and vaccine research. However, no generally available HIV-1 RNA assay has yet been subjected to rigorous clinical testing or to comparative evaluation with research-based RNA assays using large numbers of well-characterized clinical specimens. In this study, the Chiron Quantiplex branched DNA (bDNA) signal amplification assay was used to measure viral RNA in the plasma of 152 HIV-1-positive individuals at all stages of infection and in 12 patients before and after initiating zidovudine therapy. Eighty-six percent of patients had bDNA assay results above the 10,000-RNA Eq/ml sensitivity cutoff. Branched DNA values were significantly correlated with plasma viral RNA levels determined by quantitative competitive polymerase chain reaction (QC-PCR) assay (Spearman rank correlation, r = 0.89), infectious plasma virus titers (r = 0.72), p24 antigen levels (r = 0.51), immune complex dissociated p24 antigen levels (r = 0.56), and CD4+ lymphocyte counts (r = -0.72; p < 0.0001 for all comparisons). Plasma viral RNA determinations by bDNA and QC-PCR assays were quantitatively similar in the range of 10(4) to 10(7) RNA molecules/ml [log bDNA = 0.93 + 0.80 (log QC-PCR); R2 = 0.81, p < 0.0001] and declined identically following the institution of zidovudine therapy (68-73% decrease from baseline). The close quantitative correlation between bDNA and QC-PCR results, and their significant association with other viral markers and CD4+ counts, support the use of plasma viral RNA measurement in HIV-1 clinical trials.

The pathogenicity of Aeromonas strains relative to genospecies and phenospecies identification.

The relative pathogenicity of 80 Aeromonas strains typed by biochemical (phenospecies) and genetic (genospecies) methods was assessed by determining the 50% lethal dose for each isolate in Swiss-Webster mice by intraperitoneal injection. Overall, the maximum difference in virulence potential observed between the least and most pathogenic strains was a four log (10,000-fold) difference. Results according to phenospecies designation supported previous investigations indicating that both A. hydrophila and A. sobria are inherently more pathogenic for mice than A. caviae. According to genospecies designation, the relative virulence of individual groups in decreasing order was as follows: HG 9 (A. jandaei) greater than HG 1 (A. hydrophila) and HG 12 (A. schubertii) greater than HG 10 (A. veronii biotype veronii) greater than HG 8 (A. veronii biotype sobria) greater than HG 11 (unnamed) greater than HG 2 (unnamed) greater than HG 3 (A. salmonicida), HG 4 (A. caviae) and HG 6 (A. eucrenophila) greater than HG 5 (A. media) greater than HG 7 (A. sobria).

Isolation and identification of autoagglutinating serogroup O:11 Aeromonas strains in the clinical laboratory.

We evaluated the extent to which serogroup O:11 Aeromonas strains could be recovered from both clinical and environmental specimens and the cultural parameters that affected the phenotypic marker (autoagglutination) associated with this group. Of over 200 Aeromonas strains screened, serogroup O:11 was identified only among the phenospecies A. hydrophila and A. sobria and was associated with clinical isolates more frequently than with environmental strains. Blood and wound isolates accounted for almost 50% of all O:11 strains identified. The autoagglutination phenotype associated with O:11 strains could be detected in most commercial liquid media, under a wide range of growth temperatures, and within 15 min of incubation at 100 degrees C. The results suggest that clinical laboratories can recognize this important group of Aeromonas strains by two simple tests.

Laboratory investigations on the low pathogenic potential of Plesiomonas shigelloides.

The pathogenic properties of 16 Plesiomonas shigelloides strains recovered from humans with extraintestinal and intestinal illnesses, infected animals, and environmental sources were investigated. Most strains possessed a high cell charge and low surface hydrophobicity analogous to those of Shigella spp.; additionally, serogroup O:17 strains reacted with Shigella group D antisera. However, unlike the shigellae, P. shigelloides strains did not universally bind Congo red, were noninvasive in HEp-2 cell assays, and did not produce a Shiga-like toxin on Vero cells. On HEp-2, Y1, and possibly Vero cells, a low-level cytolysin was consistently produced by all 16 P. shigelloides strains when grown in either Evan Casamino Acids-yeast extract or Penassay broth. The median 50% lethal dose for all 16 P. shigelloides strains in outbred Swiss Webster mice was 3.5 x 10(8) CFU (range, 3.2 x 10(7) to greater than 1 x 10(9) CFU). Animal pathogenicity did not correlate with cytolysin expression, possession of a greater than or equal to 120-MDa plasmid, protein profile, or resistance to complement-mediated lysis. No strain analyzed produced siderophores or a heat-stable enterotoxin. The results suggest that members of the genus Plesiomonas have an overall low pathogenic potential, irrespective of the site of isolation or phenotypic, serologic, or surface properties shared with other traditional enteropathogens.

The susceptibility of S-layer-positive and S-layer-negative Aeromonas strains to complement-mediated lysis.

Forty strains of Aeromonas hydrophila and Aeromonas veronii recovered from invasive and non-invasive infections were tested for their susceptibility to complement-mediated lysis by 65% pooled human serum (PHS). Based upon the results of this assay, two major populations could be defined. The first group (n = 20) consisted of serogroup O:11 strains, all of which possessed a paracrystalline surface layer (S layer); all of these strains were refractory to the bactericidal activity of 65% PHS with the exception of A. hydrophila strain AH-121, which was composed of mixed subpopulations of serum-susceptible and serum-resistant clones. A second collection of isolates (n = 20), all of which were S-layer-negative, contained a subgroup of strains (n = 7) that were highly susceptible to complement-mediated lysis, showing a greater than 100-fold reduction of viable progeny within 30 min of exposure to 65% PHS. Serum-resistant strains from both groups could not be lysed by exposure of bacterial cells to polyclonal somatic or whole cell antisera or to 30 micrograms ml-1 of polymyxin B nonapeptide prior to challenge with 65% PHS. Analysis of selected serum-resistant and serum-susceptible strains from both groups showed that all isolates activated the complement pathway and most bound C3b to the cell surface, indicating that the inability of complement to lyse serum-resistant strains was related to a defect in the terminal portions of the complement pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoretic analysis of the surface components of autoagglutinating surface array protein-positive and surface array protein-negative Aeromonas hydrophila and Aeromonas sobria.

The protein and lipopolysaccharide (LPS) compositions of 10 autoagglutinating Aeromonas hydrophila and Aeromonas sobria strains were studied; one group consisted of five serogroup O:11 strains that contained an S layer, while a second group was composed of diverse serogroups that were S layer negative by transmission electron microscopy. All serogroup O:11 strains were found to contain a predominant 52,000- to 54,000-molecular-weight protein that was present on both whole-cell and outer membrane protein profiles; this protein was found to be glycine extractable under low-pH (pH 4) conditions and was identified as the surface array protein. LPS analysis revealed that all O:11 strains exhibited homogeneous-length O-polysaccharide side chains characterized primarily by two or three major bands. In contrast, S-layer-negative autoagglutinating strains of other serogroups lacked this predominant surface array protein, and silver stain analysis of LPS indicated that such profiles mainly consisted of core antigens and were deficient in or devoid of O-polysaccharide side chains. These collective results offer potential explanations for observed differences between these two groups in virulence, disease spectrum, and pathogenic properties.

Characterization of classic and atypical serogroup O:11 Aeromonas: evidence that the surface array protein is not directly involved in mouse pathogenicity.

A number of different techniques were used to analyse classic and atypical serogroup O:11 Aeromonas isolates. Five of seven atypical O:11, S layer-negative strains lacking a homogeneous LPS side-chain pattern exhibited varying degrees of mouse pathogenecity. One virulent atypical strain (AH-77) synthesized a surface array protein (SAP) but was unable to anchor it to the cell surface in an intact form, presumably due to a defect in its LPS architecture. Proteinase K digestion to remove the SAP, or growth at elevated temperatures (42 degrees C) to reduce the proportion of SAP synthesized from classic O:11 S layer-positive strains, did not alter their LD50 values in outbred mice. In addition, a spontaneous mutant, AS-180-1, that was S layer-negative was as virulent as the parental S layer-positive strain in the mouse model. These results suggest neither the SAP nor the characteristic serogroup O:11 homogeneous LPS side-chain pattern are directly involved in mouse pathogenicity.

Immunochemical analysis and possible biological role of an Aeromonas hydrophila surface array protein in septicaemia.

The biochemical, immunological, and biological properties of an S layer purified from an Aeromonas hydrophila strain (AH-342) involved in a case of bacteraemia were investigated. The S layer selectively removed from the cell surface was composed of a single acidic (pI 4.56) protein subunit (surface array protein, SAP) with a molecular mass of approximately 52 kDa. Amino acid analysis of this 52 kDa protein indicated a molecule composed of 498 amino acids with 46% hydrophobic residues. No cysteine residues were detected. The first 35 residues of the N-terminus were sequenced by Edman degradation; only 4-24% homology was noted between this sequence and those previously published for SAPs of Aeromonas salmonicida (A450) and a strain of A. hydrophila (TF7) originally isolated from a moribund fish. Polyclonal antibodies raised against AH-342 SAP were genospecific, reacting only against S layers produced by A. hydrophila strains and not those from Aeromonas veronii. Acute serum from the bacteraemic patient from whom AH-342 was isolated reacted strongly with the SAP of AH-342 in immunoblot studies. Purified SAP, when intraperitoneally co-inoculated with SAP- strains of A. hydrophila into Swiss-Webster mice, could reduce the 50% lethal dose by approximately 30-70 fold. The results suggest that the SAP of A. hydrophila strains may play an important role in systemic dissemination after invasion through the gastrointestinal mucosa.

Aeromonas species in septicemia: laboratory characteristics and clinical observations.

We retrospectively analyzed clinical and epidemiological data on and laboratory characteristics of 53 cases of aeromonas septicemia. Only four Aeromonas genomospecies (species defined by DNA relatedness) were associated with the 53 cases, with Aeromonas hydrophila (sensu stricto) predominating (47%). Nearly 60% of all Aeromonas isolates from blood fell into one of four somatic groups: serogroups O:11, O:16, O:18, and O:34. Unlike Aeromonas-associated gastroenteritis, septicemia did not peak in frequency during the warmer months but rather was most common in January through March, when approximately 40% of cases occurred. In vitro tests of the pathogenicity of 20 selected blood isolates of Aeromonas indicated that resistance to complement-mediated lysis, elevated levels of protease and hemolysin activity, and the ability to elaborate siderophores correlated with higher virulence. Species and serogroup designations also correlated with the degree of virulence. Susceptibility studies of 50 strains indicated that A. hydrophila was the most drug-resistant species and that Aeromonas veronii was the most susceptible. Susceptibility to first- and second-generation cephalosporins and carbenicillin was species-associated.

Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays.

A considerable proportion of patients with chronic hepatitis C who achieve a virologic end-of-treatment response relapse after discontinuation of therapy. It is conceivable that polymerase chain reaction (PCR)-based assays with a lower detection limit of 100 to 1, 000 hepatitic C virus (HCV) RNA copies/mL are still too insensitive to detect residual viremia. End-of-treatment serum samples of 47 patients with a virologic relapse according to results of qualitative PCR assays (Amplicor HCV; Roche Molecular Systems, Mannheim, Germany) were tested by transcription-mediated amplification (TMA), an isothermal, autocatalytic target amplification method that has the potential to detect less than 50 HCV RNA copies/mL. Virologic sustained responders (n = 59) and nonresponders (n = 49) served as controls. In end-of-treatment serum samples of virologic sustained responders and nonresponders an almost complete concordance between PCR and TMA results was observed (98%). However, HCV RNA was detectable by TMA in end-of-treatment serum samples from 16 of 25 relapse patients (64%) who were HCV-RNA-negative according to Amplicor HCV version 1.0 (lower detection limit 1,000 copies/mL) and in 8 of 22 patients (36%) who were HCV-RNA-negative according to Amplicor HCV version 2.0 (lower detection limit 100 copies/mL). End-of-treatment alanine transaminase (ALT) levels of sustained virologic responders and TMA-negative relapsers were similar, whereas a trend toward higher ALT values was observed in TMA-positive relapsers compared with sustained virologic responders (P = 0.09). In conclusion, HCV RNA can be detected at the end of treatment by TMA in a considerable proportion of patients who were classified as virologic end-of-treatment responders with a subsequent virologic relapse according to PCR-based methods.

Salmonella serogroups C2 and C3 identified by agglutination using an immunoglobulin G3(kappa) monoclonal antibody (32-1-E3) reactive with a somatic factor 8-like polysaccharide antigen.

An immunoglobulin G3(kappa) monoclonal antibody (MAb), MAb 32-1-E3, which was prepared in BALB/c mice by using a heated, alcohol-acetone-extracted Salmonella newport CDC 50 antigen, reacted with protein-free lipopolysaccharides from Salmonella groups C2 (O:6,8) and C3 (O:-,8) but not with those from any other serogroup tested. Sodium periodate did not inhibit antigen reactivity, which was consistent with its identity as the abequose-containing disaccharide O:8 antigen. Reactivity was inhibited by competition with serogroup C2 (O:6,8) and C3 (O:-,8) antigens but not with non-O:8 antigens. Reactivity was also inhibited by preincubation of the antigen with polyclonal rabbit antiserogroup C2 or C3 antibodies but not with antisera to serogroup C1 or other Salmonella serogroups. The MAb agglutinated with all strains of Salmonella serogroups C2 and C3 tested but not with other bacteria. Agglutination was inhibited by preabsorbing the MAb with either of two serogroup C3 Salmonella strains, S. virginia CDC 189 or S. haardt MDL 83A4545, which contain only O:8, but not by preabsortion with O:8-negative S. cholerasuis MDL 81A7623 (group C1; O:6,7), S. paratyphi type B CDC 157 (group B; O:1,[4],5,12), or Escherichia coli (O:157) (which contains no Salmonella serogroup antigens). The MAb reacted strongly (4+ agglutination) with all 140 wild-type strains of group C2 and C3 Salmonella spp. tested and showed no reaction with any of 1,324 wild-type strains of non-C2 or non-C3 Salmonella spp. tested. The MAb is useful as a replacement for absorbed, polyclonal, single-factor O:8 antiserum to discriminate Salmonella serogroups C2 and C3 from serogroup C1.

Structural and pathogenic properties of Aeromonas schubertii.

We investigated the phenotypic, structural, and pathogenic properties of 11 Aeromonas schubertii strains recovered from extraintestinal sites. Most A. schubertii strains were autoagglutination positive, possessed a high surface charge but low hydrophobicity, and fell into one or two biogroups on the basis of carbon substrate utilization patterns. Fatty acid methyl ester analysis of A. schubertii revealed this species to contain a relatively high percentage of branched fatty acids (i-13:0, i-15:0, i-17:1, i-17:0) compared with A. hydrophila. Immunologic and biochemical analysis of the lipopolysaccharides of A. schubertii strains allowed for two groups to be distinguished, namely, (i) a collection of six strains belonging to serogroup O:11 that possessed a characteristic homogeneous O polysaccharide side chain profile by silver staining and immunoblotting techniques and (ii) a second antigenically diverse group (five strains) that either exhibited a heterogeneous side chain profile or were side chain deficient. A, schubertii O:11 strains were all found to contain a 55-kDa major protein associated with the outer membrane fraction which was glycine-hydrochloride extractable; non-O:11 strains did not harbor a similar protein molecule. Screening of A. schubertii strains for reputed virulence factors indicated (i) that slightly more than half of the isolates produce an apparent contact-dependent hemolysin that is not cell associated or released extracellularly, (ii) a potent cytotoxin active against HEp-2 cells that is devoid of hemolytic activity, and (iii) lack of enterotoxigeniclike activity as determined by suckling mouse assays. All A. schubertii strains were pathogenic for mice as determined by 50% lethal dose assays, although no single factor correlated with mouse pathogenicity.

Performance characteristics of the QUANTIPLEX HIV-1 RNA 3.0 assay for detection and quantitation of human immunodeficiency virus type 1 RNA in plasma.

The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log(10) estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log(10) in most comparisons. Interlaboratory differences across runs were /=50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant results had higher HIV-1 RNA levels than the samples with concordant results (median, 34 versus 17 copies/ml; P = 0.0051 by the Wilcoxon two-sample test). The excellent reproducibility, broad linear range, and good sensitivity of the bDNA 3.0 assay make it a very attractive method for quantitation of HIV-1 RNA levels in plasma.

Biochemical and genetic characterization of autoagglutinating phenotypes of Aeromonas species associated with invasive and noninvasive disease.

The genetic characteristics and biochemical and structural properties of a number of autoagglutinating (AA) strains of Aeromonas associated with invasive and noninvasive disease in humans and infections in animals and from environmental sources were investigated. Of 27 strains analyzed by multilocus enzyme typing and DNA hybridization studies, 25 (93%) were confirmed to belong to either hybridization group 1 (phenospecies and genospecies Aeromonas hydrophila) or 8 (phenospecies Aeromonas sobria; genospecies Aeromonas veronii). Further analysis of 19 of these strains indicated that four major groups could be identified on the basis of serologic and surface characteristics, protein and lipopolysaccharide composition, and virulence properties; these groupings held true regardless of the site of isolation or disease process involved. The major AA+ group identified was serogroup O:11, whose strains possessed an S layer, were resistant to the bactericidal activity of normal serum, and were pathogenic in mice. The results suggest a set of useful phenotypic and structural markers for identification of specific subsets of mesophilic Aeromonas involved in a wide range of infections in the animal kingdom.

Increased transmission of vertical hepatitis C virus (HCV) infection to human immunodeficiency virus (HIV)-infected infants of HIV- and HCV-coinfected women.

The transmission of perinatal hepatitis C virus (HCV) infection was studied retrospectively in 62 infants born to 54 HCV- and human immunodeficiency virus (HIV)-coinfected women enrolled in a prospective natural history study of HIV transmission. Infant HCV infection was assessed by nested RNA polymerase chain reaction. The overall rate of vertical HCV transmission was 16.4% (9/62). Most HCV-infected children did not develop antibodies to HCV. The rate of HCV infection was higher among HIV-infected infants (40%) than among HIV-uninfected infants (7.5%; odds ratio, 8.2; P = .009). This difference in transmission was not related to differences in maternal HCV load, as measured by branched DNA assay, or mode of delivery. Why HIV-infected infants of HCV- and HIV-coinfected women have significantly higher rates of perinatal HCV transmission remains to be elucidated. The rate of HCV transmission in HIV-uninfected infants of HCV- and HIV-coinfected women is similar to that reported for infants born to HIV-seronegative mothers.

Differential cytotoxic T-lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients.

Cytotoxic T lymphocytes (CTL) are thought to control hepatitis B virus (HBV) infection, since they are readily detectable in patients who clear the virus whereas they are hard to detect during chronic HBV infection. In chronic hepatitis C virus (HCV) infection, however, the virus persists in the face of a CTL response. Indeed, most infected patients respond to one or more HCV-1 (genotype 1a)-derived CTL epitopes in the core, NS3, and NS4 proteins, and the CTL response is equally strong in patients infected by different HCV genotypes, suggesting broad cross-reactivity. To examine the effect of the HCV-specific CTL response in patients with chronic hepatitis C on viral load and disease activity, we quantitated the strength of the multispecific CTL response against 10 independent epitopes within the HCV polyprotein. We could not detect a linear correlation between the CTL response and viral load or disease activity in these patients. However, the CTL response was stronger in the subgroup of patients whose HCV RNA was below the detection threshold of the HCV branched- chain DNA assay than in branched-chain-DNA-positive patients. These results suggest that the HCV-specific CTL response may be able to control viral load to some extent in chronically infected patients, and they indicate that prospective studies in acutely infected patients who successfully clear HCV should be performed to more precisely define the relationship between CTL responsiveness, viral clearance, and disease severity in this infection.