[Real-time PCR assay for rapid detection of clarithromycin-resistant helicobacter pylori in gastric biopsy specimens]. / Schneller Nachweis von clarithromycinresistenten Helicobacter pylori in der Magenbiopsie mittels Real-Time-PCR.

The main cause for failure of Helicobacter pylori eradication therapy is resistance to clarithromycin which is due to point mutations. The use of real-time PCR allows the detection of these mutations directly on biopsy specimens within a few hours. In our routine laboratory, we compared LightCycler PCR to conventional detection and susceptibility testing of H. pylori by culture. PCR showed a positive result for H. pylori in 74 specimens. PCR was confirmed by culture in 69 specimens. In five specimens which were positive by PCR but negative on culture the (13)C urea breath test confirmed the PCR results. Sensitivity and specificity of our LightCycler assay for the detection of H. pylori in biopsy specimens were both 100 %. In 26 out of 68 specimens conventional susceptibility testing yielded resistance to clarithromycin. Corresponding point mutations were found in 24 of these specimens. Compared to culture, PCR gave a false-resistant, respectively, a false sensitive result in one specimen each. In another specimen, culture yielded a resistant strain whereas PCR detected both a resistant mutant and the wild-type strain. From two other specimens clarithromycin-sensitive strains were cultured but both a wild-type strain and a mutant were detected by PCR. Sensitivity and specificity of LightCycler PCR for resistance to clarithromycin were 96.2 % and 97.6 %, respectively. This assay had an accuracy comparable to culture and could be performed within 3 hours, allowing it to be used before the administration of H. pylori eradication therapy.

Prenatal diagnosis of congenital cytomegalovirus infection in 189 pregnancies with known outcome.

Prenatal diagnosis (PD) of fetal cytomegalovirus (CMV) infection was performed in 242 pregnancies, with known outcome in 189 cases. In 141/189 pregnancies, PD was carried out on account of suspicious maternal CMV serology up to gestational week (WG) 23, and in 48 cases on account of abnormal ultrasonic findings detected between WG 18 and 39. Chorionic villus samples (n = 6), amniotic fluid (AF, n = 176) and/or fetal blood specimens (n = 80) were investigated for detection of virus by cell culture, shell vial assay, PCR and/or CMV-specific IgM antibodies. Of 189 fetuses correctly evaluated by CMV detection either in fetal tissue following therapeutic abortion/stillbirth (n = 24) or in urine of neonates within the first 2 weeks of life (n = 33), 57 were congenitally infected. In women with proven or suspected primary infection, the intrauterine transmission rates were 20.6% (7/34) and 24.4% (10/41), respectively. Of the congenitally infected live-born infants, 57.6% (19/33) had symptoms of varying degree. The overall sensitivity of PD in the serologic and ultrasound risk groups was 89.5% (51/57). A sensitivity of 100% was achieved by combining detection of CMV-DNA and CMV-specific IgM in fetal blood or by combined testing of AF and fetal blood for CMV-DNA or IgM antibodies. There was no instance of intrauterine death following the invasive procedure. The predictive value of PD for fetal infection was 95.7% (132/138) for negative results and 100% (51/51) for positive results. Correct results for congenital CMV infection by testing AF samples can be expected with samples obtained after WG 21 and after a time interval of at least 6 weeks between first diagnosis of maternal infection and PD. In case of negative findings in AF or fetal blood and the absence of ultrasound abnormalities at WG 22-23, fetal infection and neonatal disease could be excluded with high confidence. Positive findings for CMV infection in AF and/or fetal blood in combination with CMV suspicious ultrasound abnormalities predicted a high risk of cytomegalic inclusion disease (CID). Furthermore, detection of specific IgM antibodies in fetal blood was significantly correlated with severe outcome for the fetus or the newborn (p = 0.0224). However, normal ultrasound of infected fetuses at WG 22-23 can neither completely exclude an abnormal ultrasound at a later WG and the birth of a severely damaged child nor the birth of neonates which are afflicted by single manifestations at birth or later and of the kind which are not detectable by currently available ultrasonographic techniques.

Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system.

BACKGROUND: Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products. OBJECTIVES: to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines. STUDY DESIGN: 73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay). RESULTS: with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24-48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load. CONCLUSIONS: LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.

Evaluation of enterovirus serological tests IgM-EIA and complement fixation in patients with meningitis, confirmed by detection of enteroviral RNA by RT-PCR in cerebrospinal fluid.

An enzyme immunoassay (EIA) for detection of anti-enterovirus IgM antibodies was compared with complement fixation test in 43 patients with confirmed enterovirus meningitis by RT-PCR of cerebrospinal fluids (CSF). In 34% of patients with enterovirus meningitis, IgM antibodies could be found, whereas complement fixation tests were positive in only 20%. The specificity was determined with sera of 105 patients with non-enterovirus meningitis. Specificity of IgM EIA and of complement fixation was 94% and 85%, respectively. In four patients with meningitis but without enterovirus detection in CSF, RT-PCR and virus isolation from stools were positive. In three of these patients, IgM antibodies were detected, giving a strong indication of an enterovirus-associated disease. Because of the high specificity of IgM EIA, diagnosis of enterovirus-associated diseases can be carried out in a single serum sample, whereas by complement fixation tests, only fourfold increases in antibody titres in paired sera indicate an acute infection. The application of IgM EIA is especially important in cases of meningitis when CSF samples are not available and for diagnosis of enterovirus diseases with other clinical symptoms such as fever, enteritis, and hand-foot-and-mouth disease.

Fast and type-specific analysis of herpes simplex virus types 1 and 2 by rapid PCR and fluorescence melting-curve-analysis.

Diagnosis of central nervous system (CNS) infection with herpes simplex virus (HSV) requires sensitive and rapid techniques. PCR therefore is considered to be the diagnostic gold standard in these cases. However, current PCR protocols are time-consuming and labor-intensive. In addition, the need for post-amplification manipulations increases the risk of laboratory contaminations with amplified products. In order to improve conventional PCR techniques we compared our current semiautomated HSV-PCR-ELISA assay with a new micro-volume rapid-cycle PCR system that combines real-time monitoring and fluorescence melting-curve analysis without the need for post-amplification sample manipulations. Spiking experiments with supernatants of tissue culture-grown HSV type 1 (HSV-1) and type 2 (HSV-2) in HSV-negative control cerebrospinal fluid (CSF) and sterile water revealed that the new rapid cycle PCR protocol is as sensitive and specific as the PCR-ELISA. Furthermore, a mismatch (G:T) within the probe-targeted region of the HSV-2 glycoprotein B gene decreases the probe/product melting temperature (Tm) from 69 degrees C for HSV-1 to 64 degrees C for HSV-2, enabling the simultaneous identification of the two HSV genotypes by melting-curve analysis within one run. This type specificity of the system was confirmed with 30 genital swabs previously analyzed for the presence of HSV-1/2 in cell culture. While our current PCR-ELISA method needs up to 1 day from sample preparation to result generation, the new procedure takes only 1 h. We consider this system as a promising new tool for the analysis of HSV DNA in CSF and in other human body fluids as well as for the diagnosis of other infectious agents where rapid diagnosis, high sensitivity and specificity are required.

A new enzyme immunoassay for the detection of enteroviruses in faecal specimens.

A new enzyme immunoassay (EIA) for direct detection of enteroviruses based on a group-specific monoclonal antibody was evaluated using stool samples from patients with suspected enteroviral infection. The EIA was compared with polymerase chain reaction (PCR) and virus isolation in cell culture. Of 204 samples tested, 20 were positive by EIA, 34 by PCR, and 18 by cell culture. Compared with PCR, the most sensitive method, the sensitivity of EIA was 58% (20/34); the sensitivity of cell culture isolation was 52% (18/34). The results of both assays correlated in only 60% of cases. The combination of EIA and cell culture isolation detected 76% of PCR-positive stool samples. Enterovirus EIA provides results within 3-4 hr and requires only standard EIA equipment. It represents a rapid, reliable, and cost-effective diagnostic tool for enterovirus diagnosis from faecal samples. Negative results must be confirmed by other techniques, such as PCR or virus isolation in cell culture.

Development of antibodies to the nonstructural protein NS1 of parvovirus B19 during acute symptomatic and subclinical infection in pregnancy: implications for pathogenesis doubtful.

At present little is known about the mechanisms influencing the course and severity of parvovirus B19 infection. Antibodies to the parvovirus nonstructural protein NS1 were reported in patients with parvovirus-associated arthritis and those with persisting infection but not in those without complications, suggesting a potential involvement of NS1 or anti-NS1 antibodies in pathogenesis. The immune response to NS1 was examined retrospectively in 33 pregnant women with acute parvovirus B19 infection, 14 of whom experienced symptomatic infection and 19 in whom the infection was subclinical. Antibodies to NS1 were found in 15 (45%) of the women, seven with symptomatic and eight with subclinical infection. No association was found between the development of anti-NS1 antibodies and the occurrence of fetal complications. Of the seven cases in which fetal complications were observed, anti-NS1 antibodies were detected in only three. The finding that an immune response to NS1 can also be demonstrated in patients with asymptomatic infection suggests that anti-NS1 antibodies do not appear to represent a marker for an altered or severe course of infection in pregnant women or to contribute significantly to pathogenesis. Since anti-NS1 antibodies first become detectable at least six weeks postinfection, their presence can be used to exclude acute infection in patients with unclear serology or be used to aid differential diagnosis of rashlike illnesses.

Acute parvovirus B19 infection in pregnant women--an analysis of serial samples by serological and semi-quantitative PCR techniques.

The serological and virological course of parvovirus B19 infection was followed in 14 women who suffered symptomatic or subclinical acute infection during pregnancy. Serial serum samples from the patients were tested for IgG and IgM antibodies and the levels of parvovirus B19 DNA were monitored using a semi-quantitative PCR assay. In addition, the outcome of the pregnancies was documented by clinical information and by testing cord blood for parvovirus B19 specific antibodies as well as for parvovirus B19 DNA by PCR. Levels of IgG antibodies rose steadily within 2 months of infection and in some cases began to decline at the end of pregnancy. IgM antibodies were usually detected for at least 2 months and persisted for as long as 9 months in one case. Viral DNA was detectable for at least 8 weeks following infection and semi-quantitative analysis revealed a gradual reduction in virus load during the viraemic phase of infection. There were no apparent differences in the course of antibody development and duration of viraemia in symptomatic versus subclinical infections.

Life-threatening parvovirus B19-associated myocarditis and cardiac transplantation as possible therapy: two case reports.

Parvovirus B19 infection can cause a wide spectrum of disease syndromes. Two cases of parvovirus B19 infection were identified that resulted in life-threatening myocarditis shortly after acute infection in immunocompetent individuals. The diagnosis was made with serological and polymerase chain reaction techniques. One patient was successfully treated by heart transplantation. Sequence analysis showed that the parvovirus B19 cloned from the patients' sera had 99% homology with the prototype sequence. Clinicians should be alerted to the possible role of parvovirus B19 in myocarditis presenting in immunocompetent patients.

[Cat scratch disease. Bartonella henselae antibodies and DNA detection in regional lymphadenopathy]. / Katzenkratzkrankheit. Bartonella-henselae-Antikörper und Nukleinsäurenachweis bei regionaler Lymphadenopathie.

HISTORY AND CLINICAL FINDINGS: Two hard, pressure-sensitive nodules developed in the lower jaw of a 22-year-old woman. After a dental cause had been excluded, she was treated for suspected tonsillitis with Ceftibuten. Erythrocyte sedimentation rate was increased to 18 mm in the first hour. There were no other significant biochemical findings and fine-needle biopsy of one of the nodules showed nonspecific inflammatory reaction. INVESTIGATIONS: Sonography revealed two lymph nodes, 7 and 22 mm in diameter. Suspected cat scratch disease was confirmed by immunofluorescence with Bartonella (Rochalimaea) henselae and quintana antigens. TREATMENT AND COURSE: After a course of Clarithromycin (250 mg twice daily) for 6 weeks the lymph nodes had shrunk and the overlying skin was thin and discoloured brown. One node was incised and drained and the material examined. Microbiology was negative, but DNA sequencing confirmed Bartonella henselae. As a consequence, Rifampicin was given for 2 months (600 mg daily). Wound healing was very slow and the scar had regressed little after 9 months.

Detection of human cytomegalovirus in urine samples by cell culture, early antigen assay and polymerase chain reaction.

With the advent of effective therapy rapid, sensitive and reliable assays for diagnosis of human cytomegalovirus (HCMV) infections are required. In a total of 1,928 urine samples, detection of HCMV-immediate early antigen in a spin amplified microplate culture by a monoclonal antibody and immunoperoxidase staining (EA-assay) was compared with virus isolation in cell culture. Sensitivity of the EA assay was 85.5% and specificity was 99.5% compared with virus isolation. Overall agreement of both assays was 97.8%. In addition, in 235/1,928 urine samples amplification of HCMV-DNA was performed by means of polymerase chain reaction (PCR) using primers from the immediate early (IE1) gene region and 141/1,928 using primers from the late region (LA). The sensitivity of PCR compared with virus isolation was 67.8% for IE1 primers and 94.1% for LA primers (statistical significance: p < 0.01, Chi-square-test). Overall agreement between virus isolation and PCR was 88.5% for IE1-PCR and 84.4% for LA-PCR. Discordant results were more often found in adults with acute infection and immunocompromised patients than in infants.

Binding of NF-kB to the HIV-1 LTR is not sufficient to induce HIV-1 LTR activity.

Human immunodeficiency virus type 1 (HIV-1) spends a significant part of its life cycle as latent provirus in nonactivated cells. It induction requires mitogen stimulation. TPA treatment induces HIV-1 transcription by protein kinase C (PKC)-mediated activation of the cellular transcription factor NF-kB. PKC activation induces the dissociation of NF-kB from its inhibitor protein (IkB). The liberated NF-kB then binds to its proviral recognition sequence in the HIV-1 long terminal repeat (LTR) sequence. This step, however, is not sufficient to augment transcription. We demonstrate that NF-kB-mediated HIV-1 LTR activation is regulated by an additional event that is not dependent on IkB. A further phosphorylation event is proposed, since this step could be blocked by an inhibitor of a phospholipase C (PLC) type reaction. This inhibitor precludes the formation of diacylglycerols, which are required for activation of PKC isoenzymes. As an alternative pathway that is not dependent on PLC reactions, high-level transcription from the HIV-1 LTR is shown to require binding of both NF-kB and TAT.

Inhibition of c-fos transcription and phosphorylation of the serum response factor by an inhibitor of phospholipase C-type reactions.

Phospholipase C activity is necessary for transcriptional c-fos activation by providing diacylglycerol as an activator of protein kinase C. We found that transcriptional activation of c-fos and the phosphorylation of its major transcription factor were inhibited by tricyclodecan-9-yl xanthogenate, which blocks phospholipase C-type reactions. Transcription of the c-ras and beta-actin genes in the same cells remained unaffected.

Synergistic antiviral effect of xanthates and ionic detergents.

Xanthate compounds have been shown to exhibit antiviral activity against various DNA and RNA viruses under acidic pH conditions. It is now possible to utilize the unique broad range antiviral spectrum of these compounds under physiological pH conditions (pH 7.4) by simultaneous administration of certain ionic detergents. When used in conjunction with tricyclodecan-9-yl-xanthate (D609), sodium deoxycholate, sodium dodecylsulfate and certain fatty acids, which have no antiviral activity of their own, inhibit the replication of various DNA and RNA viruses (such as herpes simplex, vesicular stomatitis and Coxsackie B 4) in vitro at pH 7.4. Among saturated fatty acids of various chain lengths there was a marked size restriction in that the efficiency of undecanoic acid (11 C atoms) was three orders of magnitude greater than that of shorter (6 C atoms) or longer (18 C atoms) monocarbonic acids. Dose-response kinetics revealed a synergistic interaction between the xanthate and the monocarbonic acid. A dose that inhibited the replication of herpesvirus by a factor of 1000 still permitted mitotic activity in uninfected growing control cultures.