Serum zonulin levels in patients with liver cirrhosis: Prognostic implications.

BACKGROUND: Increased gut permeability and bacterial translocation play an important role in liver cirrhosis. Zonulin is a recently recognized protein involved in the disintegration of the intestinal barrier. AIM: To investigate possible differences in serum zonulin levels among patients with different cirrhosis stages and their potential prognostic implications. METHODS: Consecutive cirrhotic patients who attended our liver clinic were included in the study. Serum zonulin levels, clinical, radiological and biochemical data were collected at baseline. Patients who accepted participation in a regular surveillance program were followed-up for at least 12 mo. RESULTS: We enrolled 116 cirrhotics [mean Child-Turcotte-Pugh (CTP) score: 6.2 ± 1.6; model for end-stage liver disease score: 11 ± 3.9]. The causes of cirrhosis were viral hepatitis (39%), alcohol (30%), non-alcoholic fatty liver disease (17%), and other (14%). At baseline, 53% had decompensated cirrhosis, 48% had ascites, and 32% had history of hepatic encephalopathy. Mean zonulin levels were significantly higher in patients with CTP-B class than CTP-A class (4.2 ± 2.4 ng/dL vs 3.5 ± 0.9 ng/dL, P = 0.038), with than without ascites (P = 0.006), and with than without history of encephalopathy (P = 0.011). Baseline serum zonulin levels were independently associated with the probability of decompensation at 1 year (P = 0.039), with an area under the receiving operating characteristic of 0.723 for predicting hepatic decompensation. Higher CTP score (P = 0.021) and portal vein diameter (P = 0.022) were independent predictors of mortality. CONCLUSION: Serum zonulin levels are higher in patients with more advanced chronic liver disease and have significant prognostic value in identifying patients who will develop decompensation.

Serum HBsAg kinetics and usefulness of interferon-inducible protein 10 serum in HBeAg-negative chronic hepatitis B patients treated with tenofovir disoproxil fumarate.

The kinetics of serum HBsAg and interferon-inducible protein 10 (IP10) levels in patients with chronic hepatitis B infection treated with tenofovir are unclear. We evaluated the changes of HBsAg levels and the predictability of IP10 for HBsAg decline in 160 HBeAg-negative patients receiving tenofovir for ≥12 months. Serum samples taken before and at 6, 12, 24, 36 and 48 months after tenofovir were tested for HBsAg levels. In 104 patients, serum samples before tenofovir were tested for IP10 levels. Compared to before tenofovir, HBsAg levels decreased by a median of 0.08, 0.11, 0.24, 0.33 and 0.38 log10 IU/mL at 6, 12, 24, 36 and 48 months, respectively (P < 0.001). HBsAg kinetics did not differ between nucleos(t)ide analogue(s) naive and experienced patients. The 12-, 24-, 36- and 48-month cumulative rates of ≥0.5 log10 HBsAg decline were 8%, 16%, 24% and 41% and of HBsAg ≤100 IU/mL were 9%, 12%, 14% and 18%, respectively. The only factor associated with HBsAg ≤100 IU/mL was lower HBsAg levels before tenofovir (P < 0.001), while HBsAg decline ≥0.5 log10 was associated with higher IP10 levels (P = 0.002) and particularly with IP10 > 350 pg/mL (P < 0.001). In conclusion, tenofovir decreases serum HBsAg levels in both nucleos(t)ide analogue(s) naive and experienced patients with HBeAg-negative chronic hepatitis B infection. After 4 years of therapy, HBsAg ≤100 IU/mL can be achieved in approximately 20% of patients, particularly in those with low baseline HBsAg levels. HBsAg decline is slow (≥0.5 log10 in 40% of patients after 4 years) and is associated only with higher baseline serum IP10 levels.

Long-term safety of anti-TNF treatment in patients with rheumatic diseases and chronic or resolved hepatitis B virus infection.

OBJECTIVES: The aim of this prospective study was to examine the safety of anti-tumour necrosis factor (TNF) therapy in patients with rheumatic disease and hepatitis B virus (HBV) infection. METHODS: 14 patients with chronic HBV infection, 19 HBV-vaccinated patients and 19 patients with resolved HBV infection were included in the study. All HBV-infected patients received combination therapy with oral antivirals and anti-TNF agents. During treatment the levels of hepatitis B surface antibodies (anti-HBs) in HBV-vaccinated patients and of serum HBV DNA in patients with chronic or resolved HBV infection were monitored. RESULTS: No viral reactivation was observed in patients with resolved HBV infection while anti-HBs titres decreased during anti-TNF treatment in vaccinated patients, similarly to patients treated with methotrexate alone. None of the HBV-infected patients developed liver decompensation or a significant increase in alanine aminotransferase levels. One patient (7%) treated with lamivudine and etanercept showed viral reactivation due to the emergence of a lamivudine-resistant mutant strain. CONCLUSIONS: Anti-TNF agents represent a safe option for patients with chronic HBV infection when combined with antiviral therapy, as well as in patients previously exposed to HBV receiving no HBV prophylaxis. Resistant HBV strains may arise in patients with chronic hepatitis B, necessitating the initial use of anti-HBV agents with a low risk of resistance.

Serum hepcidin levels are related to the severity of liver histological lesions in chronic hepatitis C.

Hepcidin is synthesized in the liver and has a crucial role in iron homoeostasis. Its synthesis is up-regulated in chronic inflammation and iron excess. We examined the determinants of serum hepcidin and liver hepcidin mRNA levels and their association with histological lesions in patients with chronic hepatitis C (CHC) and healthy controls. We studied 96 patients with CHC and 30 controls. Serum hepcidin levels were measured by an in-house competitive ELISA. Hepcidin mRNA levels were determined by a one-step qRT-PCR in total RNA extracted from liver biopsy specimens of 27 patients with CHC and six disease controls. Histological lesions were evaluated according to Ishak's classification. Serum hepcidin was significantly lower in patients with CHC than healthy controls (14.6 ± 7.3 vs 34.6 ± 17.3 ng/mL, P < 0.001). In patients with CHC, serum hepcidin correlated positively with aspartate aminotransferase (r = 0.334, P = 0.001) and insulin resistance (r = 0.27, P = 0.016) and had a trend for correlation with alanine aminotransferase (r = 0.197, P = 0.057) and serum haemoglobin (r = 0.188, P = 0.067) but not with ferritin. A significant positive correlation was also found between serum hepcidin levels and both necroinflammation (r = 0.259, P = 0.011) and fibrosis (r = 0.214, P = 0.036). Serum hepcidin was among others an independent predictor of cirrhosis (odds ratio: 1.145, P = 0.039). Liver hepcidin mRNA levels did not differ between patients and controls and were relatively lower in patients with than without cirrhosis (19.3 ± 21.7 vs 38.3 ± 26.0, P = 0.067). Patients with CHC have reduced serum hepcidin levels, which correlate with worse necroinflammation and fibrosis. The previously mentioned observations suggest a viral effect on hepatic hepcidin production, but might also support its involvement in the inflammatory process.

Serum HCV RNA levels and HCV genotype do not affect insulin resistance in nondiabetic patients with chronic hepatitis C: a multicentre study.

BACKGROUND: Chronic hepatitis C (CHC) induces insulin resistance (IR) and subsequently diabetes. AIM: To examine viral, metabolic and histological predictors of IR in 275 CHC patients to test the hypothesis that IR differs among HCV genotypes and that viral replication directly affects IR. METHODS: We studied 275 nondiabetic treatment-naïve CHC patients. Histological lesions were evaluated according to Ishak. IR was assessed using homeostasis model assessment for insulin resistance (HOMA-IR). RESULTS: HOMA > 3.0 was found in 37% of patients, independently associated with higher BMI and GGT. In genotype non-3 patients, HOMA > 3.0 was associated with higher BMI and GGT values, while no significant association was noted in genotype 3 patients. In non-obese patients with minimal fibrosis, HOMA > 3.0 was found in 20% of cases without significant differences among genotypes. No association between HOMA > 3.0 and HCV-RNA levels was found. Severe fibrosis (stage 5-6) related to older age (OR:1.048), HOMA-IR (OR:1.177), necroinflammation (OR: 2.990) and higher ALT (OR: 1.009) and GGT (OR:1.006). CONCLUSIONS: IR develops at early stages of CHC without significant differences among genotypes. It is more frequent in obese patients with steatosis and contributes to fibrosis progression. However, IR does not seem to be associated with viraemia and therefore its exact pathogenetic mechanism in CHC remains elusive.

Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B.

The immunopathogenesis of hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-gamma (IFN-gamma) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with overlapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182-191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-gamma producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-gamma assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained.

Longitudinal changes in serum HBV DNA levels and predictors of progression during the natural course of HBeAg-negative chronic hepatitis B virus infection.

We evaluated the longitudinal changes of viraemia and predictors of progression in a prospectively followed cohort of 150 untreated patients with HBeAg-negative chronic hepatitis B virus (HBV) infection. According to the first year of follow-up, 85 patients were classified into inactive carrier state and 65 into chronic hepatitis B (CHB). Serum HBV DNA levels were determined at baseline in all patients, at year-1 in carriers or last pretherapy visit in CHB patients and during alanine aminotransferase (ALT) elevations in carriers progressing to CHB. HBV DNA levels at any occasion were > or =80, > or =2000 or > or =20 000 IU/mL in 81%, 23% or 0% of carriers and 100%, 95% or 83% of CHB patients. The cumulative progression rate from carrier to CHB was 11%, 16%, 24% at 2-, 3-, 4 years and was independently associated with higher baseline ALT (always within traditional normal range) and baseline HBV DNA > or =2000 or > or =5000 IU/mL. In 12 carriers progressed to CHB, HBV DNA increased by >1 log(10) IU/mL. During 7.5 months of median follow-up, HBV DNA change > or =1 log(10) IU/mL was observed in 49% of CHB patients. In conclusion, serum HBV DNA levels are detectable in the majority of inactive HBV carriers exceeding 2000 IU/mL in only 23% and 20 000 IU/mL in none of them. Carriers have approximately 15% 3-year risk of progression to CHB, which is associated with higher baseline ALT and viraemia > or =2000-5000 IU/mL, and thus should be closely followed. Approximately 20% of HBeAg-negative CHB patients have HBV DNA <20 000 IU/mL with fluctuations >1 log(10) occurring in many of them.

Serum apoptotic caspase activity as a marker of severity in HBeAg-negative chronic hepatitis B virus infection.

BACKGROUND AND AIMS: In chronic hepatitis C and non-alcoholic fatty liver disease, apoptotic caspases are activated in liver, and serum caspase activity has been suggested as a sensitive marker of early liver injury. An investigation was carried out into whether the serum levels of caspase-generated fragments of cytokeratin-18 (CK-18) are associated with the severity of liver lesions in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Patients/ METHODS: CK-18 fragment serum levels were determined in 115 treatment-naive, consecutive HBV patients and 30 healthy controls. Hepatic-expression of CK-18 fragments was evaluated by immunocytochemistry in chronic hepatitis B patients. RESULTS: CK-18 fragment levels (U/l) were significantly lower in healthy controls (mean (SD), 154 (31)) than in 53 inactive carriers (172 (24), p = 0.003) and in 62 chronic hepatitis B patients (474 (488), p<0.001). The receiver operating characteristic curve showed excellent diagnostic accuracy (c-statistic: 0.87) for differentiating inactive carriers from chronic hepatitis B patients. A CK-18 fragment cut-off level of 240 U/l gave a sensitivity of 60%, and a specificity and positive predictive value of 100% for chronic hepatitis B diagnosis. CK-18 fragment levels were also lower in inactive carriers than in 16 chronic hepatitis B patients with transiently normal alanine aminotransferase (ALT; 327 (256), p = 0.001), offering good accuracy for such a differentiation (c-statistic: 0.78). In chronic hepatitis B patients, serum CK-18 fragments correlated positively with ALT/aspartate aminotransferase (AST), viraemia, grading score and their immunohistochemical hepatic expression, and negatively with platelet counts, but not with fibrosis or steatosis severity. CONCLUSIONS: Serum apoptotic caspase activity is strongly associated with the presence of liver injury in patients with HBeAg-negative chronic HBV infection. CK-18 fragment levels seem to be a very useful marker for differentiation between the inactive HBV carrier state and HBeAg-negative chronic hepatitis B, but not for estimation of the severity of liver histological lesions among HBeAg-negative chronic hepatitis B patients.

Severe hepatitis C virus-related cryoglobulinaemic sensory-motor polyneuropathy treated with pegylated interferon-a2b and ribavirin: clinical, laboratory and neurophysiological study.

BACKGROUND/AIMS: Severe involvement of central and/or peripheral nervous system is a rare complication of hepatitis C virus (HCV)-related cryoglobulinaemia. METHOD: Four patients with HCV-related type II/III cryoglobulinaemia (three males with genotype 1, one female with genotype 3) who presented with severe sensory-motor polyneuropathy, one with central nervous system involvement as well, were treated with pegylated IFNa-2b 1.5 microg/kg/week and ribavirin 10.6 mg/kg/daily for 48 weeks. Neurological evaluation involved detailed clinical motor and sensory scores/scales and neurophysiological studies before and after treatment. RESULTS/CONCLUSION: Three out of four patients had undetectable serum HCV-RNA, normal levels of aminotransferases and substantially lower or undetectable levels of cryoglobulins at the end of treatment and at 24 weeks follow-up period. Treatment was well tolerated and all patients exhibited significant improvement of neuropathy based on solid clinical and laboratory criteria that was associated with the virological response.

Study of host and virological factors of patients with chronic HCV infection and associated laboratory or clinical autoimmune manifestations.

OBJECTIVE: Chronic hepatitis C virus (HCV) infection is associated with an array of autoimmune laboratory and clinical manifestations. The goals of our study were to identify host and/or virological factors that are implicated in the pathogenesis of these manifestations. METHODS: We performed a detailed prospective study of various demographic, virological, biochemical, immunological (including lymphocyte subsets, Fc gamma-receptor and HLA class-II genotyping), histological and host genetic parameters in 3 well defined subgroups of HCV patients (n = 40): patients with liver disease only (group I, n = 11) or with laboratory (group II, n = 20) and clinical (group III, n = 9) autoimmune manifestations. RESULTS: Group III patients, mainly with features of mixed cryoglobulinemia, were older, with higher levels of rheumatoid factor and circulating cryoglobulins while they tended to have a longer estimated disease duration compared to the other two groups of patients. We did not identify any specific immunological features that could differentiate symptomatic versus asymptomatic patients, except from the elevated soluble interleukin-2 receptor levels. An increased frequency of the R/R131 FcR gamma IIIA and the NA1/NA1 Fc gamma RIIIB genotypes was observed in our total HCV population, regardless of autoimmune manifestations, compared to historical controls. No statistically significant differences in HLA class II allele frequencies was detected between patient subgroups or in comparison to healthy controls. CONCLUSIONS: Chronically infected HCV patients with symptomatic mixed cryoglobulinemia display a number of unique characteristics that differentiate them from asymptomatic patients with chronic hepatitis C.

Hepatitis C virus RNA assays: a comparison of SuperQuant and Monitor.

Hepatitis C RNA testing has been used extensively to assess the efficacy of antiviral therapy and has increasingly become an integral part of clinical management of patients with chronic hepatitis C. A variety of commercially available hepatitis C virus (HCV) RNA tests are used to detect HCV RNA qualitatively or quantitatively. These commercial tests have fundamental differences that are reflected on the values they generate. We compared two widely used assays, HCV SuperQuant (SQ) and Amplicor HCV Monitor (M1 and M2), in sera of patients with chronic hepatitis C. A total of 506 sera from 79 patients were tested with both assays. The data were logarithmically transformed and analyzed by linear regression and measurement of agreement. Two hundred thirty-eight sera had HCV RNA values within the dynamic range of both assays. The correlation between the assays was fair, with a correlation coefficient (r) of 0.699. Overall, SQ generated higher values than M1 with a mean difference of 0.558 log (SD = 0.624). One hundred ninety-four (38%) and 121 (24%) of the sera were below the dynamic range of M1 and SQ, respectively. Seventy-three sera, undetectable by M1, were positive by SQ. The Amplicor HCV Monitor 2.0 (M2) was performed in 66 sera. All were positive by SQ and M2, but only 38 were within the dynamic range of M1. The correlations between these tests were good (r = 0.68-0.78), but the agreement was rather poor. In conclusion, this study confirms that both SQ and M2 are more sensitive than M1. Additionally, our results show rather poor agreements between these assays. The recent attempts in standardizing the reporting of these assays should make their results more easily interchangeable.

Ciliocytophthoria in clinical virology.

Direct immunofluorescence assays (DFAs) are used in the clinical virology laboratory for the rapid detection of viruses. An assessment of the cellularity of specimens submitted for DFA is necessary for the most effective use of this assay. This assessment ensures that an adequate number of the appropriate cells are present for examination. During this assessment, clinical virologists may encounter unfamiliar cellular elements or cellular fragments. One of these elements, ciliocytophthoria, has been misinterpreted as a parasite in specimens submitted for cytologic testing. We describe a similar case in which a technologist thought that ciliocytophthoria possibly represented a ciliated parasite in a nasopharyngeal specimen sent for respiratory syncytial virus DFA. After a thorough morphologic examination, the staff dismissed the possibility of a ciliated parasite. We confirmed this entity as ciliocytophthoria using morphologic criteria and the Diff-Quik stain. This near misidentification of ciliocytophthoria as a ciliated parasite affords us the opportunity to raise the awareness of clinical virologists about ciliocytophthoria. Additionally, we briefly review useful features for differentiating ciliocytophthoria from the only ciliate parasitic for humans, Balantidium coli. Finally, we present the utility of a commonly used cytologic stain, the Diff-Quik stain, for the confirmation of ciliocytophthoria.

Screening and confirmatory testing for extended spectrum beta-lactamases (ESBL) in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca clinical isolates.

Escherichia coli and Klebsiella spp. were screened for ESBL based on routine susceptibility testing results. Isolates with intermediate or resistant susceptibilities for extended spectrum cephalosporins or aztreonam were reported as probable ESBL producers. By using the NCCLS proposed ESBL confirmatory method, we tested 61 screen-positive isolates from 42 patients, 30 randomly selected susceptible isolates, and 12 isolates with previously characterized beta-lactamases. Ceftazidime contributed to 97% of screen-positive isolates, whereas aztreonam added a single patient isolate. An ESBL was confirmed in 86% of K. pneumoniae, 100% of K. oxytoca, and 20% of E. coli screen-positive single patient isolates. None of the susceptible isolates were shown to produce ESBL. Based on these findings a comment regarding the presence of ESBL seems sufficient for Klebsiella spp. but confirmatory testing is indicated for E. coli. 0.25 microg/mL was used to indicate the presence of ESBL, the specificity of the assay increased to 100%. The NCCLS ESBL phenotypic confirmatory method was reproducible and accurate enough to be used in the clinical laboratory.

Comparison of VIDAS with direct immunofluorescence for the detection of respiratory syncytial virus in clinical specimens.

BACKGROUND: requirements for infection control measures and the decision for treatment with antiviral agents make the rapid detection of respiratory syncytial virus (RSV) essential for hospitalized, pediatric and immunocompromised patients. Immunofluorescence is considered to be the most rapid and sensitive method for direct detection of RSV in clinical specimens, but several enzyme-linked immunoassays have also been commercially available. OBJECTIVES: to compare the performance of VIDAS RSV assay (Vitek ImmunoDiagnostic Assay System, BioMerieux Vitek), which is an automated enzyme-linked fluorescent immunoassay (ELFA) to direct immunofluorescence (DFA), in rapid detection of RSV in respiratory specimens. STUDY DESIGN: respiratory specimens collected during the 'RSV season', between the months of November 1997 and February 1998, were tested by both methods. DFA was performed upon receipt of the sample and an aliquot of the original specimen was stored in -70 degrees C for batch testing with VIDAS. RESULTS: from 238 samples that were tested, 231 could be evaluated by both methods. The two assays were in agreement for 213 specimens (92%), or 32 positive and 181 negative results. Eighteen discrepant results were generated; seven specimens were VIDAS-/DFA+ and 11 specimens were VIDAS+/DFA-. In addition, seven specimens had an inadequate number of cells for evaluation with DFA. One of these samples tested positive with VIDAS. VIDAS relative sensitivity and specificity were 82% and 94%, respectively, when compared to DFA. CONCLUSIONS: VIDAS is simple to perform, does not require expertise in interpretation and appears to be an acceptable method for the rapid detection of RSV.

Amplicor enterovirus polymerase chain reaction in patients with aseptic meningitis: a sensitive test limited by amplification inhibitors.

OBJECTIVE: To evaluate the sensitivity and specificity of reverse-transcription polymerase chain reaction (RT-PCR) and routine cell culture for the detection of enterovirus in cerebrospinal fluid. METHODS: Thirty-eight cerebrospinal fluid specimens were included. Cell culture was inoculated immediately and incubated for 14 days. An aliquot was kept frozen for Amplicor RT-PCR. Chart review was performed to determine the validity of the results. RESULTS: Nine of 38 specimens were positive for enterovirus by culture, and 14 were positive by RT-PCR. There were 7 discrepancies between the 2 methods. Six specimens were positive by RT-PCR and negative by the culture method. The 1 culture-positive but RT-PCR--negative specimen was determined to contain PCR inhibitors. All discrepant results were confirmed as true positives by chart review. Patients whose cerebrospinal fluid was negative by both methods had a final diagnosis other than enterovirus infection. CONCLUSION: Amplicor PCR is more sensitive than cell culture (93.3% vs. 60%) and is very specific. With the incorporation of appropriate controls for the detection of amplification inhibitors, RT-PCR could be a valuable tool in the diagnosis of enteroviral meningitis.

Evaluation of Two Methods for Quantitation of Hepatitis C Virus RNA.

Background: Accurate quantitation of hepatitis C virus (HCV) RNA in serum may provide a means to predict disease course and response to interferon-alpha therapy. Several quantitative assays are commercially available, but none have been accepted as the gold standard. Methods and Results: The branched DNA quantitative hybridization assay (Quantiplex HCV 1.0, Chiron, Emeryville, CA) and a quantitative reverse transcription polymerase chain reaction (Amplicor HCV MONITOR, Roche Diagnostic Systems, Branchburg, NJ) were compared using a panel of 53 sera from patients with chronic hepatitis C. All sera contained HCV RNA of known genotype. Overall, there was a positive correlation between the results for the 41 sera that gave discrete values in both tests (r =.81, linear regression; P <.01, Kendall's rank test); however, the mean number of HCV copies per milliliter was 13.5-fold higher with Quantiplex (P <.01). A plot of the difference between methods against their means showed poor agreement between the methods. No correlation between the results of the two tests was observed for sera with MONITOR values greater than 5.0 x 10(5) copies/mL. Discrete MONITOR values were obtained for all 12 sera that were below the lower limit of quantitation of Quantiplex (mean, 1.78 x 10(5)). Parallel testing of serial dilutions of two sera showed that each method gave linear responses over the stated dynamic ranges; however, the proportional systematic error was greater with MONITOR. The mean coefficient of variation for replicate determinations was 23% for Quantiplex and 45% for MONITOR (P =.13). Conclusions: Despite a positive correlation, systematic differences exist between the two methods for quantitation of HCV RNA and they cannot be used interchangeably.

Analysis of human immunodeficiency virus type 1 mRNA splicing patterns during disease progression in peripheral blood mononuclear cells from infected individuals.

HIV-1 produces more than 20 mRNAs encoding the viral proteins. We have used a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) approach to determine HIV-1 transcriptional patterns during the course of viral infection in unstimulated peripheral blood mononuclear cells (PBMCs) from different patients. Several sets of PCR primers, used in parallel reactions, allowed the amplification and specific detection of almost all individual HIV-1 transcripts. We investigated the transcriptional profile in two individuals during primary acute and early chronic infection. In these individuals, HIV-1 mRNA expression was elevated at the first time points examined and declined over time. In addition, we performed a detailed study of HIV-1 expression in several individuals over a minimum of 7 years following seroconversion. We found that long-term asymptomatic individuals had undetectable or low levels of the three classes of HIV-1 transcripts (unspliced, singly spliced, and multiply spliced). Individuals who demonstrated disease progression showed either a general increase in the amount of expression of all transcripts or elevated levels of unspliced transcripts in late-stage disease. The splicing pattern in each patient was conserved over the years and differed among the different individuals. No evidence of major changes in the splicing pattern was found during disease progression within the same individual. Thus, HIV-1 transcriptional patterns are viral strain specific rather than disease stage specific. These results indicate that high-level expression of any class of HIV-1 transcripts is associated with clinical progression. Our analysis also demonstrates the importance of using more than one set of primers to evaluate HIV-1 RNA expression, since virus in patient PBMCs showed sequence heterogeneity in conserved regions.

Splicing variability in HIV type 1 revealed by quantitative RNA polymerase chain reaction.

A quantitative RNA-polymerase chain reaction (PCR) method able to detect the majority of mRNAs produced by human immunodeficiency virus type 1 (HIV-1) was developed and used to study expression of different HIV-1 clones in human cells. Amplified mRNAs were compared to known cDNA standards. This comparison permitted the optimization of PCR conditions and eliminated the generation of artifactual PCR bands. The use of RNA and cDNA standards demonstrated that the RNA amplification is linear within the tested range and suggested that it can be used to quantitate individual mRNAs. The results demonstrate the overall conservation of splicing in different HIV-1 clones. Although, in general, splicing was conserved, extensive qualitative and quantitative variability was observed in different HIV-1 clones. This variability is likely one determinant of the biological characteristics of the different HIV-1 clones, and demonstrates a great plasticity of the HIV-1 genome. The described RNA-PCR methodology was used for the study of HIV-1 expression in unstimulated peripheral blood mononuclear cells (PBMCs) of infected individuals. In general, the same mRNAs were identified in HIV-infected cultured cell lines and in unstimulated PBMCs. Analysis of a variant band found after amplification of PBMC RNA from an HIV-infected individual revealed a new splice site for the generation of Rev/Nef-encoding mRNAs. The availability of a sensitive, rapid, and essentially quantitative method to examine the major HIV-1 mRNAs will facilitate the detailed analysis of HIV-1 expression in human cells.