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H H Kessler

Publications and source records attributed to H H Kessler.

32 records · Page 2Linked to original sources
PubMed1997

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.

Adolescent↗
PubMed1997

Prevalence and significance of hepatitis C virus (HCV) genotypes in anti-HCV positive patients in northwest Croatia.

Hepatitis C virus (HCV) transmission is related to blood transfusion and transmission in hemodialysis (HD) units. The aim of the study was to investigate the distribution of HCV genotypes and routes of HCV transmission in three groups of anti-HCV positive patients in north-west Croatia. A total of 111 patients were studied. Patients were classified into three groups: 45 HD patients with a low percentage of anti-HCV positivity (group 1), 60 HD patients treated at another HD unit and with high percentage of anti-HCV positivity (group 2), and six anti-HCV positive patients with chronic hepatitis (group 3). Most of the HD patients were treated during the sama shift, but with separate equipment. Serum HCV RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Genotyping of HCV isolates was performed with a line-probe assay. Patients of groups 1 and 2 did not show significant differences with regard to the clinical profile. In group 1, all anti-HCV positive patients were RT-PCR positive and all of them were infected with the same genotype (genotype 3). In group 2, 89% of anti-HCV positive patients were RT-PCR positive and all infected with the same genotype (genotype 1 b). In group 3, 50% anti-HCV positive chronic hepatitis patients were RT-PCR positive. Two of them were infected with genotype 1 b and one with genotype 4. The homogeneity of HCV genotypes in the patients from both HD units (groups 1 and 2), seemed to indicate nosocomial transmission of HCV, whereas viremia was found to be related to blood transfusion in all group 3 patients. The exact mechanism involved in the transmission of HCV in HD units remains to be discovered.

Croatia↗
PubMed1996

Evaluation of two nonculture antigen tests and three serotests for detection of anti-chlamydial antibodies in the diagnosis of ocular chlamydial infections.

BACKGROUND: Diagnosis of chlamydial conjuctivitis is difficult in chronic diseases because chlamydial elementary bodies are mostly undetectable in conjunctival scrapings by cell culture. We therefore compared two nonculture antigen tests and three different serotests for anti-chlamydial antibodies with McCoy cell culture, the "gold standard" of chlamydial diagnosis. Conjunctival scrapings and serum samples of 93 patients attending the outpatient eye clinic in Graz because of chronic follicular conjunctivitis were tested. METHODS: A total of 558 conjunctival scrapings and 93 serum samples were investigated. Chlamydial antigen detection was done by McCoy cell culture, polymerase chain reaction (PCR; Amplicor, Roche), and direct immunofluorescence assay (DFA; Microtrak, Syva). Antichlamydial IgA and IgG antibodies in the sera were detected by an immunoperoxidase assay (IPAzyme, Savyon) and two different enzyme-linked immunosorbent assays (SeroELISA, Savyon and rELISA, medac). RESULTS: Cell culture and PCR yielded identical results. The positivity rate for chlamydial conjunctivitis was 8.6% (8 of 93 patients). PCR proved most sensitive and most specific. IPAzyme was 75% sensitive for IgA and 100% for IgG; SeroELISA and rELISA were less sensitive. IPAzyme was 81% specific for IgA and 47.3% for IgG. SeroELISA and rELISA were less specific for IgA, but more specific for IgG. Post-test likelihood of disease was greatest in IPAzyme. CONCLUSIONS: PCR proved to be a good alternative to cell culture; DFA is useful for quick diagnosis. Genus-specific serotests cannot compete with chlamydial antigen detection. They differ in sensitivity and specificity because of the antigen type they present. They are still of only supportive value in cases where chlamydial antigen detection is not possible. Recently introduced species-specific antibody tests should be of greater value.

Adolescent↗
PubMed1996

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.

Journal Article↗
PubMed1995

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.

Journal Article↗
PubMed1994

Comparison of two hybridization assays for the rapid detection of PCR amplified HSV genome sequences from cerebrospinal fluid.

Rapid diagnosis of herpes simplex encephalitis (HSE) can only be achieved by the polymerase chain reaction (PCR). In order to carry out PCR under routine conditions, it is of great importance to establish an easy DNA extraction protocol and especially a rapid and sensitive DNA detection method. In the present study, two different solid phase hybridization assays (Gen-Eti-K-DNA Enzyme Immunoassay (DEIA), Sorin Biomedica, Italy and Enzymun-Test DNA detection, Boehringer Mannheim, Germany) were compared for detection of PCR amplified HSV DNA polymerase genome region, using standard primers, from cerebrospinal fluid (CSF) samples. 122 CSF samples obtained from patients suffering from encephalitis and hospitalized at the University Clinics of Frankfurt and Graz during the period January 1992 to July 1993 were tested. To ascertain the sensitivity of the hybridization assays, dilution series of a plasmid, encoding the amplified region of the polymerase gene, were investigated. The detection limit of the DEIA assay was one copy of the plasmid/microliter, and the lowest amount of DNA which could be detected by the Enzymun assay as well as Southern blot was 10 copies/microliter. 15 CSF samples obtained from patients with HSE were found positive by the three assays. Concordant results were also obtained with CSF samples from non-HSE patients. The results of this study show that new hybridization systems guarantee a fast and high-sensitive detection of amplified HSV DNA. HSV PCR in CSF can be carried out routinely by the combined use of rapid hybridization and a simple extraction procedure.

Base Sequence↗
PubMed1994

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique.

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.

Artifacts↗
PubMed1993

Rapid detection of Legionella species in bronchoalveolar lavage fluids with the EnviroAmp Legionella PCR amplification and detection kit.

A molecular assay based on a rapid DNA extraction protocol and the EnviroAmp Legionella Kits was used to detect Legionella species in bronchoalveolar fluid specimens. All Legionella strains isolated from tap water in hospitals could be detected distinctly. Both sensitivity and specificity were tested. In a prospective study, bronchoalveolar lavage fluids obtained from patients with atypical pneumonia were investigated. Three positive samples were detected with the molecular techniques and were subsequently confirmed by culture. Application of the system described may lead to safe and early diagnosis of Legionnaires' disease in patients with atypical pneumonia.

Bacteriological Techniques↗
PubMed1989

[Which immunologic mechanisms facilitate a successful pregnancy].

Three possibilities are discussed for the immunological mechanisms which may be operative in establishing and maintaining a successful grafting of a semiallogenic embryo: (1) The lack of maternal allogenic recognition due to either the absence or a decreased alloantigenicity of paternal alloantigens (HLA). (2) The local suppression of maternal immune reactions against the embryo by hormonally induced decidual suppressor cells and by molecules released from the placenta. (3) The placenta may serve as an immunological barrier filtering potentially cytotoxic cells and molecules out of the maternal circulation before they reach the fetus.

Embryo, Mammalian↗
PubMed1989

[Expression of histocompatibility antigens in human pre-implantation embryos].

The expression of HLA antigens on polyploid human preimplantation embryos, obtained in an in vitro fertilization program, was investigated by indirect immunofluorescence test using a panel of well-defined monoclonal antibodies. Neither HLA class I antigens and beta 2-microglobulin, nor HLA class II molecules could be detected on blastomeres and the zona pellucida. It is supposed that the absence of HLA antigens on human preimplantation embryos may be one of the mechanisms important for the acceptance of the embryo by the immunocompetent mother.

Embryo Transfer↗
PubMed1988

The importance of an early diagnosis of fertilization disorders.

Typical possibilities of abnormal development in human oocytes cultured and fertilized in vitro were studied using phase-contrast microscopy. All oocytes were checked three times for maturity, fertilization and embryo development. A total of 180 non-cleaved oocytes and 30 embryos originating from polypronuclear oocytes are described. It is concluded that an exact inspection at the fertilization check is essential to avoid the transfer of abnormally developing oocytes and embryos.

Chorionic Gonadotropin↗
PubMedDate not supplied

Rapid detection of Chlamydia trachomatis in conjunctival, pharyngeal, and urethral specimens with a new polymerase chain reaction assay.

GOAL OF THIS STUDY: The Amplicor polymerase chain reaction (PCR) assay for the detection of Chlamydia trachomatis (Roche Molecular Systems, Branchburg, NJ) was evaluated on conjunctival, pharyngeal, and urethral swabs. STUDY DESIGN: A total of 515 conjunctival, pharyngeal, and urethral swabs. The reference system was culture with McCoy cells in shell vials with fluorescent immunostaining. One swab was used for both cell culture and the molecular assay. Initial storage took place in 2-SP medium. After transfer to Amplicor specimen transport medium the molecular assay was done using the Amplicor Chlamydia trachomatis amplification and detection kits. RESULTS: The total positive rate was 6.6%. Specificity of culture was 100%. The evaluated molecular assay gave a specificity of 99.8%. Sensitivities of PCR and culture were 100% and 85.3%, respectively. CONCLUSIONS: Because of the high sensitivity, specificity, and ease of use, the molecular assay was found to be a good alternative to culture for detection of C. trachomatis in conjunctival, pharyngeal, and urethral specimens.

Adolescent↗
PubMedDate not supplied

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.

Adult↗