Inhibition of prostaglandin biosynthesis in human endotoxin-stimulated peripheral blood monocytes: effects of caffeine.

There is an ongoing debate about possible advantages of the coadministration of caffeine with cyclooxygenase (COX) inhibitors in the treatment of pain. There are results suggesting interference by caffeine with COX expression and activity in rat immune cells. In the present study, we have used, therefore, human endotoxin-stimulated monocytes to investigate a possible influence of caffeine on indometacin-induced inhibition of prostaglandin E(2) (PGE(2)) formation. Endotoxin caused a concentration- and time-dependent increase in immunoreactive PGE(2) that was dependent on CD14-mediated mechanisms. In order to investigate pharmacological inhibition of the COX activity, a submaximal concentration of 1 ng/ml endotoxin (4 h exposure time) was used. Indometacin caused a concentration-dependent inhibition of PGE(2) with an apparent IC(50) of 8.9 +/- 1.4 x 10(-9) mol/l and an I(max) of 1 x 10(-7) mol/l. Caffeine (5 x 10(-6) to 1.5 x 10(-4) mol/l) on its own produced no statistically significant effect on endotoxin-induced PGE(2) formation. In the presence of caffeine (5 x 10(-6) to 1.5 x 10(-4) mol/l), inhibition of PGE(2) biosynthesis by indometacin (1 x 10(-8) mol/l) was not significantly altered. These results show that, in human monocytes, caffeine, up to concentrations severalfold higher than those reached in patients, has no significant effect on endotoxin-induced PGE(2) formation nor on its inhibition by indometacin.

Determination of human immunodeficiency virus type 1 subtypes by a rapid method useful for the routine diagnostic laboratory.

The existence of human immunodeficiency virus type 1 (HIV-1) subtypes has many important implications for the global evolution of HIV and for the evaluation of pathogenicity, transmissibility, and candidate HIV vaccines. The aim of this study was to establish a rapid method for determination of HIV-1 subtypes useful for a routine diagnostic laboratory and to investigate the distribution of HIV-1 subtypes in Austrian patients. Samples were tested by a subtyping method based on a 1.3-kb sequence of the polymerase gene generated by a commercially available drug resistance assay. The generated sequence was subtyped by means of an HIV sequence database. Results of 74 routine samples revealed subtype B (71.6%) as the predominant subtype, followed by subtype A (13.5%) and subtype C (6.8%). Subtypes E, F, G, and AE (CM240) were also detected. This subtyping method was found to be very easy to handle, rapid, and inexpensive and has proved suitable for high-throughput routine diagnostic laboratories. The specific polymerase gene sequence, however, must be existent.

Effects of storage and type of blood collection tubes on hepatitis C virus level in whole blood samples.

In this study, we compared serum hepatitis C virus (HCV) RNA concentrations with HCV RNA concentrations in whole blood collection tubes, including two different types of EDTA tubes and nucleic acid stabilization tubes (NASTs). We also investigated the impact of a processing delay on HCV RNA concentration in these tubes. In NASTs, the mean HCV RNA concentration was comparable to the mean serum HCV RNA concentration at "date zero." In EDTA tubes, mean baseline HCV RNA concentrations were higher. Storage at room temperature up to 96 h did not result in a decline of HCV RNA concentration in any of the whole blood collection tubes. In NASTs, HCV RNA concentrations remained stable during the whole study period, whereas a significant increase of HCV RNA was observed in both types of EDTA tubes at 96 h compared to date zero. We concluded that HCV RNA remains stable in NASTs at room temperature for at least 96 h, allowing greater flexibility in sample collection and transport.

Cytomegalovirus diagnosis in renal and bone marrow transplant recipients: the impact of molecular assays.

BACKGROUND: Cytomegalovirus (CMV) infections are a major threat in transplant recipients. In recent years, new assays for routine CMV diagnosis, based on molecular techniques, have become available. OBJECTIVE: The impact of molecular assays for CMV diagnosis in transplant recipient was evaluated. STUDY DESIGN: A total of 51 transplant recipients were screened for CMV infection. Serological (AxSYM CMV IgG and recombinant CMV IgM assays), antigenemia, CMV DNA (qualitative in house PCR and the quantitative COBAS AMPLICOR CMV MONITOR Test), and CMV mRNA (NucliSens CMV pp67 Test) tests were compared. RESULTS: In 11/20 bone marrow transplant (BMT) recipients and 10/31 renal transplant (RTX) recipients there was no evidence of active CMV infection. Ten RTX recipients and one BMT recipient were antigenemia positive, 21 RTX and seven BMT recipients were PCR positive (qualitative CMV PCR). There were more BMT recipients CMV DNA positive in serum (7/21) than antigenemia positive (1/21). CMV mRNA was found positive in two BMT recipients (one case with no other evidence of CMV infection, the other one CMV DNA positive and antigenemia negative). The only antigenemia positive BMT recipient was found negative for CMV mRNA, but positive in all other tests. Eight RTX recipients were found positive for CMV mRNA. Six of them were also antigenemia positive and five of those were also found positive for CMV IgM. One CMV mRNA positive RTX recipient was CMV IgM positive but antigenemia negative and the other one CMV mRNA positive RTX recipient was found negative in all other tests. Two antigenemia positive RTX recipients were found negative for mRNA and CMV IgM. CONCLUSION: Antigenemia was found to be a good screening test for CMV infection in RTX recipients. In BMT recipients, tests based on molecular techniques appeared to be superior compared to antigenemia.

Semiautomated quantification of hepatitis B virus DNA in a routine diagnostic laboratory.

The Cobas Amplicor HBV Monitor test for quantitative determination of hepatitis B virus (HBV) DNA in serum has recently been introduced. To evaluate the performance of this assay in a routine diagnostic laboratory, reproducibility of results was determined with the First European Union Concerted Action HBV Proficiency Panel and the Accurun 325 HBV DNA Positive Control, Series 300. Results for 270 routine serum samples were additionally evaluated. To avoid the retesting of a large number of samples due to titers exceeding the upper limit for the linear range of the assay, sera of patients with chronic hepatitis B (CHB) were diluted prior to the assay to 10(-4) in normal human plasma, which is included in the assay. The mean coefficient of variation was 22.9% for all input HBV DNAs. Of 270 routine serum samples, 182 (150 sera from transplant donors and 32 sera from patients who had recovered from CHB) tested negative. Eighty-six sera were found to be HBV DNA positive; in six sera, HBV DNA levels were found to exceed the upper limit for the linear range of the assay and had to be retested. In the remaining two sera, inhibition occurred. The semiautomated Cobas Amplicor HBV Monitor test showed sufficient reproducibility and helped in avoiding human error. The relatively narrow linear range of detection is a limitation of the new assay.

Detection of Herpes simplex virus DNA by real-time PCR.

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.

Identification of different states of hepatitis B virus infection with a quantitative PCR assay.

The level of hepatitis B virus (HBV) DNA in serum reflects the replicative activity of HBV. To compare serum HBV DNA levels in different states of hepatitis B, 47 sera of patients with HBeAg-positive chronic hepatitis B, 4 sera of patients with HBeAg-negative chronic hepatitis B, 40 samples of patients after HBeAg seroconversion during alpha interferon treatment, 57 sera of inactive HBsAg carriers, and 42 sera of patients who had recovered from chronic hepatitis B more than 12 months prior to blood collection were checked for the presence of HBV DNA with the Amplicor HBV Monitor Test. In patients with HBeAg-positive chronic hepatitis B, the median of serum HBV DNA levels (8.3 x 10(8) copies/ml) was significantly higher than that for patients after HBeAg seroconversion (6.2 x 10(3) copies/ml) and than that for inactive HBsAg carriers (5.6 x 10(3) copies/ml). None of the patients who had recovered from hepatitis B had detectable HBV DNA in serum. Quantitative PCR proved to be a valuable tool for identification of different states of HBV infection. This technique was found to be a good method for determination of serum HBV DNA levels both for patients with HBeAg seroconversion and for inactive carriers who showed low viremia not detectable by conventional hybridization assays.

Evaluation of AMPLILINK software for the COBAS AMPLICOR system.

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.

Automation of polymerase chain reaction-based systems for detection of hepatitis C virus RNA.

Polymerase chain reaction-based molecular assays are gaining increasing importance in the diagnosis and monitoring of infectious diseases. Over the past several years, the development and application of these techniques has initiated a revolution in the diagnosis and monitoring of hepatitis C infection. Presently, molecular assays are exclusively done in especially dedicated laboratories. Advances in automation will bring these technologies into routine diagnostic laboratories and will make them widely used.

Quantitative detection of hepatitis B virus DNA with a new PCR assay.

The Amplicor HBV Monitor Test for quantitative detection of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates. Evaluation in a routine diagnostic laboratory showed excellent sensitivity and adequate reproducibility; however, a more automated format would be desirable. The Amplicor HBV Monitor Test is useful for recognizing those patients who might benefit from antiviral treatment and for evaluation of the efficacy of anti-hepatitis B virus treatment.

Bell peppers (Capsicum annuum) express allergens (profilin, pathogenesis-related protein P23 and Bet v 1) depending on the horticultural strain.

BACKGROUND: Little is known about the role of bell peppers in food allergy. We collected sera from 11 patients with food allergy to bell peppers to analyze bell pepper extracts for allergen composition. METHODS: Proteins of mature fruits of eight horticultural strains of bell peppers were extracted and tested with patients' sera for IgE binding and with monoclonal and polyclonal antibodies in immunoblot. RESULTS: Profilin was detected in bell pepper extracts by an anti-celery profilin antibody. It showed high IgE binding activity in all extracts, which could be inhibited by recombinant birch pollen profilin. Anti-birch pollen monoclonal antibody BIP3, directed against birch pollen proteins between 30 and 69 kD, bound to bell pepper antigens of comparable molecular weights. A homologue of the major birch pollen allergen Bet v 1 was detected in four of eight horticultural strains of bell peppers, and was shown to bind IgE in 1 of the 11 patients. A 23-kD allergen of bell peppers was shown to correspond to the 23-kD major paprika allergen by IgE absorption experiments. Its N-terminal sequence showed 100% identity to P23 from tomatoes. CONCLUSION: The appearance of profilin in all and Bet v 1 in 50% of the tested horticultural strains indicates that bell peppers have to be considered potentially dangerous for Bet v 1- and profilin-sensitized patients. Moreover, in 4 of 8 horticultural strains of bell peppers a homologue of the osmotin-like protein P23 from tomatoes is responsible for substantial IgE binding. Contact with Bet v 1 and P23 homologues in bell peppers can therefore be minimized by avoidance of the respective horticultural strains.

Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B.

BACKGROUND: The Amplicor HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates. OBJECTIVE: The performance of the Amplicor HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor HBV Monoitor assay was compared to the Digene Hybrid Capture System HBV DNA assay for the quantitation of HBV in patient sera. STUDY DESIGN: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays. RESULTS: The detection limit was found to be 10(3) copies/ml with the Amplicor PCR assay compared to 10(6) to 10(7) copies/ml with the Digene hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10(3) and 10(7) copies/ml and all of them tested below the detection limit with the hybridization assay. CONCLUSION: The Amplicor HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.

Evaluation of molecular parameters for routine assessment of viremia in patients with chronic hepatitis C who are undergoing antiviral therapy.

OBJECTIVE: To define the usefulness of molecular parameters in patients with chronic hepatitis C who are undergoing antiviral therapy. Anti-hepatitis C virus (HCV) treatment was monitored by determination of serum HCV load and by presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). STUDY DESIGN/METHODS: Fifty-one patients with chronic hepatitis C undergoing antiviral therapy with interferon-alpha plus ribavirin were studied. Serum HCV RNA load was tested with a quantitative assay (Amplicor HCV Monitor Test) before, during, and up to 12 months after end of treatment. If HCV RNA was not detectable, serum samples were subsequently tested with a qualitative assay (Cobas Amplicor HCV Test) and corresponding ethylenediaminetetraacetic acid (EDTA)-treated blood was checked for presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). Sustained virologic response was defined by loss of HCV RNA 12 months after the end of treatment. RESULTS: Four patients (7.8%) were found to be sustained virologic responders, 17 (33.3%) were transient virologic responders, and 30 (58.8%) were virologic nonresponders. No significant difference was found in the median pretreatment serum HCV RNA load between sustained virologic responders, transient virologic responders, and virologic nonresponders. At 1 month after start of therapy, HCV RNA was not detectable with both the serum and the PBMC assay in 12 (23.5%) of 51 patients. Four remained HCV RNA-negative until 12 months after the end of treatment. In 14 of 17 transient virologic responders, reappearance of HCV RNA was detected earlier in PBMCs than in serum. CONCLUSIONS: Based on these results in 51 patients, quantitation of baseline serum HCV RNA does not appear to be a decisive factor to the management of the individual patient. Early assessment of serum HCV RNA level after start of anti-viral treatment seems to be of major importance to identify virologic nonresponders. Reappearance of HCV RNA may be demonstrated earlier in PBMCs than in serum.

Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format.

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.

Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay.

The AMPLICOR Enterovirus Test was evaluated with 103 cerebrospinal fluid (CSF) specimens. Twenty-seven CSF specimens were culture positive. With the AMPLICOR test, enterovirus RNA was detected in 34 specimens. Compared with culture, the AMPLICOR test gave a sensitivity of 96.3% and a specificity of 100%. The sensitivity of culture was 79.4% in comparison with the AMPLICOR test.

Performance of the automated COBAS AMPLICOR system for the detection of hepatitis C virus RNA.

BACKGROUND: The COBAS AMPLICOR (CA) instrument for the amplification and detection steps of the AMPLICOR molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator. OBJECTIVE: The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test). STUDY DESIGN: Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR Test. RESULTS: There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number. CONCLUSION: The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.

Patterns of hepatitis C viremia in patients receiving hemodialysis.

OBJECTIVES: Chronic hepatitis C virus (HCV) infection is common in patients who receive hemodialysis (HD). The aim of this study was to determine the natural history of hepatitis C viremia and the clinical utility of quantitation and genotyping of HCV in this population of patients. METHODS: Consecutive sera from two groups of HD patients who were HCV RNA positive, a group of 33 patients treated with interferon alfa (5 MU, three times a week for 4 months) and a group of 31 untreated patients, were analyzed by qualitative polymerase chain reaction, quantitative polymerase chain reaction, and a line probe assay for genotyping. RESULTS: Serum HCV RNA was detected continuously in 20 of 31 untreated patients (65%), and 11 patients (35%) showed a fluctuating pattern of viremia with virus-free intervals of up to 4 wk. Twenty-five of 33 patients (76%) treated with interferon alfa became HCV RNA negative during therapy; eight of these 25 patients had a breakthrough, which was transient in seven patients and persistent in one. Of the remaining 24 end-of-treatment responders, 17 relapsed after completion of therapy, and seven (21%) had a sustained response with undetectable serum HCV RNA for 1 yr of follow-up. Initial serum HCV RNA levels in HD patients were generally low (median, 1 x 10(5) genome eq/ml). Sustained responders had significantly lower median levels of viremia (4 x 10(4) eq/ml) than relapsers and nonresponders (9 x 10(4) and 1.8 x 10(5) eq/ml, respectively). Genotyping revealed a predominance of genotype 1a (33%) and 1b (48%). CONCLUSIONS: This study documents that fluctuating hepatitis C viremia with periods of undetectable HCV RNA is common and that low viral load predicts a sustained response to interferon therapy in HD patients. Diagnosis of chronic hepatitis C and monitoring of interferon therapy in HD patients should include initial HCV RNA quantitation and repeated qualitative measurements of HCV RNA.

Quantitation and genotyping of hepatitis C virus RNA in sera of hemodialysis and AIDS patients.

BACKGROUND: Hepatitis C virus (HCV) infection is highly prevalent in hemodialysis and AIDS patients. Little information exists about the viral load in those patients. OBJECTIVE: To characterize HCV infection in hemodialysis and AIDS patients, the viral load in the sera was measured. Results were compared with genotypes, gender of the patients, and biochemical markers of active hepatitis. STUDY DESIGN: Sera from a total of 442 patients were screened with a third-generation EIA, and anti-HCV immunoreactivity was confirmed with the Wellcozyme HCV Western Blot. After qualitative PCR with the Amplicor PCR Test, positives were genotyped using a reverse hybridization test. Determination of HCV levels was done with the Amplicor HCV Monitor assay. RESULTS: HCV RNA was detected in the sera of 95 (74.8%) EIA-positive patients. HCV RNA levels ranged from 1 x 10(4) to 1.4 x 10(6) molecules of HCV RNA/ml. Median HCV RNA levels of AIDS patients were slightly higher than those of hemodialysis patients. Male patients had higher median HCV RNA levels compared with female patients. No association between HCV RNA levels and both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was found. The most common genotypes were type 1b and type 1a, followed by type 3, type 4, and type 2a. There were no significant differences in HCV RNA levels among patients with genotypes 1a, 1b, and 2a. Patients infected with types 3 and 4, respectively, had significantly lower HCV RNA levels compared with other genotypes. CONCLUSION: Because the Amplicor HCV Monitor assay allows quantitation of low-titer viremic patients, HCV RNA levels were distinctly lower compared with previous reports. HCV RNA levels of males did not differ significantly from those of females. ALT and AST are very poor indicators of ongoing HCV infection. Patients with chronic type 3 or type 4 HCV infection tended to have lower HCV RNA levels.

Detection of hepatitis C viral sequences in serum by 'nested' polymerase chain reaction (PCR) and a commercial single-round PCR assay.

BACKGROUND: Demonstration of the hepatitis C virus (HCV) genome is usually done with combined reverse transcription and polymerase chain reaction (RT-PCR) employing nested primer sets. Recently, a commercial PCR assay (Amplicor PCR assay), based on a simplified sample preparation procedure, a single, combined reverse transcription and polymerase chain reaction (RT-PCR), and a microwell plate capture and detection, has been developed. OBJECTIVE: The aim of the present study was to compare the new Amplicor assay with an 'in-house' PCR. Additional testing included a third-generation enzyme immunoassay for anti-HCV antibodies, the Wellcozyme HCV Western Blot, which is equivalent to a third-generation recombinant immunoblot assay. Furthermore, HCV genotypes were classified. STUDY DESIGN: Sera from a total of 127 patients were studied. After screening with a third-generation enzyme immunoassay (EIA), the Wellcozyme HCV Western Blot, was performed as well as the conventional RT-PCR and the Amplicor PCR. Specimens, which were found positive by testing with the Amplicor kit, were subjected to storage at room temperature for 96 h. RESULTS: A total of 52 patients were found to be positive for anti-HCV by the third-generation EIA. With the Amplicor assay, the HCV genome was detected in 38 patients. In comparison with the 'in-house' assay, two discrepant results were found. Resolution of discrepant samples increased the total number of true positives to 39. A good correlation was found between a positive anti-HCV test result and the presence of HCV-RNA by RT-PCR. No significant reduction in the amount of amplification product was observed by retesting of suboptimally stored samples with the Amplicor assay. CONCLUSION: Because of the rapidity and the improved ease of handling, the Amplicor assay was found to be a good contribution for detection of HCV in serum.