Cobas Amplicor Test Research Articles

A Systematic Review and Meta-Analysis of Different Diagnostic Procedures to Detect Gonococcus Infection in Resource-Limited Scenario

AbstractContext:Neisseria gonorrhoeae is a Gram-negative diplococcus, an obligate human pathogen, and the etiologic agent of the sexually transmitted infection (STI), gonorrhoea. culture is the standard procedure for diagnosis, which may be supported by nucleic acid tests and microscopy. Aims: To determine the best possible method of diagnosis for Gonococcus infection in resource-limited settings. Settings and Design: The meta-analyses were designed to determine the difference in diagnosis between Culture and nucleic acid amplification tests (NAATs) and also between the different Amplification Tests and widely available Roche COBAS AMPLICOR test. Subjects and Methods: Databases searched were Pubmed, Medline, Google Scholar and Cochrane reviews. Risk ratio (RR) with 95% confidence intervals was estimated for the dichotomous outcomes. The random-effect model was applied for all the studies in the analysis. Statistical Analysis Used: The meta-analysis was computed in RevMan Version 5.3, Copenhagen, Denmark. Results: In the first analysis, NAATs significantly improved the chances of detection in comparison to the standard culture and final RR was 1.24 (1.05–2.51), which put the diamond on the right of no-effect axis, indicating more positives by NAATs. In the second analysis, AMPLICOR had the more positive results, which may have indicated better detection rate, as well as less specificity and final RR was 0.809 (0.737–0.888), which put the diamond on the left of the non-effect axis, indicating more positives by AMPLICOR. Conclusions: In a resource-limited scenario like India, the syndromic management of STIs are considered to be the norm. A positive diagnosis is only given if the tests are confirmed by Culture, as it is still considered to be the gold standard of diagnosis. However, in many cases, due to suboptimal transportation and lack of proper handling, culture in unable to grow even if the patient is infected. In such cases, Nucleic Acid Tests should be able to detect an infection.

O24 Confirming gc naat results: is it always necessary?: Abstract O24 Table 1

<h3>Introduction</h3> Current guidance recommends that all specimens testing positive using a <i>N. gonorrhoeae</i> Nucleic Acid Amplification Test (GC NAAT) be confirmed using a second test with an alternative target, in order to achieve a positive predictive value above 90%. <h3>Aim</h3> To determine rates of GC NAAT confirmations by primary screening test and specimen site. <h3>Methods</h3> 994 specimens which were GC NAAT positive at local laboratories were sent for confirmation using an in-house multiplex PCR with <i>Por</i>A and <i>opa </i>gene targets. A correlation between the confirmatory real-time PCR results, specimen site and GC screening NAAT was undertaken. For the purposes of this analysis equivocal results were regarded as positive and inhibited results were excluded. <h3>Results</h3> Overall, 57% of specimens examined could be confirmed as GC positive using the in-house real-time PCR test (Table 1). <h3>Discussion</h3> High rates of confirmation can be achieved when examining genital, rectal and urine specimens irrespective of the GC screening NAAT. However &gt;90% confirmatory rates were only achieved when examining male urine specimens which had been screened using the Probetec and Cobas Amplicor tests, although caution should be applied if extrapolating this data to low prevalence settings. Poor confirmation rates from throat specimens is probably due to cross-reactivity with commensal Neisseria, and highlights confirmation is essential when testing these samples.

Human Papillomavirus Infection in Benign Tonsil Swabs and Tonsillectomy Specimens

Objectives: (1) Determine incidence of HPV (human papillomavirus) infection in routine tonsillectomy swabs and specimens. (2) Determine if vaccination impacts tonsillar HPV infection. Methods: This was a prospective study conducted from July 2012 to January 2014. Patients ages 1 to 100 years undergoing tonsillectomy for nonmalignant indications were enrolled. Demographic data and immunization status were collected. Specimens collected during surgery included a flocked swab and half of the left and right tonsil. All specimens were tested for HPV DNA using the polymerase chain reaction–based Roche Cobas Amplicor test and for the presence of E6/E7 mRNA using the Gen-Probe Aptima HPV test. Results: Patients were stratified into 2 groups, pediatric (ages 1-12 years) and adults (age 13 years and up) based on age of potential sexual maturity. In the pediatric group (n = 130) mean age was 5.2 years, 42% were female, 58% were male, and only 3 patients were fully vaccinated against HPV. In the adult group (n = 64) mean age was 24 years, 76% female, 24% male, and 47% were either fully or partially vaccinated. All specimens were negative for HPV infection via both testing methods. Internal controls confirmed the tests were functioning properly. Conclusions: This is the first study to examine fresh tonsil swabs and tonsillectomy specimens for HPV infection using a Food and Drug Administration–approved method. Interestingly, both adult and pediatric specimens were negative for HPV infection. The role of vaccination in oropharyngeal HPV infection remains unclear.

HIV-coinfection leads to a modest increase in plasma HCV-RNA load in patients with chronic HCV infection

The influence of HIV coinfection on plasma hepatitis C virus (HCV) RNA load has not been reliably evaluated. We analyzed plasma HCV RNA load in 396 HCV-monoinfected and 467 HIV/HCV-coinfected patients. Median HCV RNA concentrations (interquartile range) in HCV-monoinfected patients were 5.88 (5.3–6.2) log10IU/mL versus 5.96 (5.6–6.5) log10IU/mL in HIV/HCV-coinfected individuals (p=0.033) as determined with the Cobas Amplicor Test and 6.06 (5.4–5.7) log10IU/mL versus 6.3 (5.5–6.9) log10IU/mL (p=0.026) using the Cobas TaqMan System. The plasma HCV RNA load in patients with HIV infection and undetectable plasmatic HIV RNA was similar to that observed in HCV-monoinfected individuals [6.02 (5.45–6.61) log10IU/mL versus 6.01 (5.36–6.59) log10IU/mL, respectively (p=1.0)]. In conclusion, HIV coinfection tends to be associated with higher plasma HCV RNA load, however, the magnitude of the differences is small and this effect can be counterbalanced with antiviral therapy.

Chlamydia trachomatis ompA genotypes in male patients with urethritis in Greece – Conservation of the serovar distribution and evidence for mixed infections with Chlamydophila abortus

PCR amplification and nucleotide sequencing of the ompA gene of Chlamydia trachomatis were used to determine the prevalence and distribution of genotypes in 51 urine and urethral specimens from Greek male patients with urethritis, that were positive by the COBAS Amplicor test. A single C. trachomatis serovar was identified in 43 of the 51 amplified samples. Serovars F and E were the most prevalent (both 12, 28%), followed by D (9, 21%), G (4, 9%), B and K (both 2, 5%) and H and J (both 1, 2%). Over one third of the samples bared a variant ompA genotype that had been previously identified in other areas worldwide. Two results in this study, both observed for the first time, were of particular interest. First, the emergence of the unique variant genotype D/Ep6 ( X77364.2) identified in 3 urethral samples. Second, the ompA genotype OCLH196 of the animal pathogen Chlamydophila abortus as well as a 23S rRNA gene fragment of this species detected by the assay ArrayTube™ was found in 7 urethral samples. The implications resulting from this observation for the health of the general population are discussed.

Therapy of chronic hepatitis C: Virologic response monitoring

Virological testing is considered to be essential in the management of hepatitis C virus (HCV) infection in order to diagnose infection, and, most importantly, as a guide for treatment decisions and assess the virological response to antiviral therapy. The aim of this study was to determine the rate of a sustained virological response (SVR) and various factors associated with response rates in chronic hepatitis C infected patients treated with peg interferon alpha (PEG-INF) and ribavirin (RBV) combination therapy. A total of 34 patients, treated with PEG-IFN and RBV were studied. Serum HCV-RNA was measured before the treatment, 12 weeks following the start of the therapy and 6 weeks after the treatment cessation. SVR was defined as undetectable serum HCV-RNA 6 months of post-treatment follow-up, virologic relapse (VR) as relapse of HCV-RNA during the posttreatment follow-up. Serum HCV-RNA was measured with the Cobas Amplicor test. At the end of post-treatment follow-up 19 (55.8%) patients demonstrated a SVR. The majority of the patients were genotype 1 (27), and the other were genotype 3 (5 patients) and genotype 4 (2 patients). There was VR in 6 patients 6 months after the therapy. In 9 patients HCV-RNA was positive after 12 weeks. We demonstrated that patients with chronic HCV infection can be successfully treated with combination of PEG-INF and RBV. This result emphasizes also that post-treatment follow-up to identify patients with SVR or VR could be important.

Diagnostic value of an enzyme-linked immunosorbent assay using the recombinant CT694 species-specific protein ofChlamydia trachomatis

To study the performance of the CT694 protein in relation to the microimmunofluorescence (MIF) and the pELISA tests for the serodiagnosis of Chlamydia trachomatis infections. The CT694 protein was produced as recombinant protein and was used as antigen in ELISA test for the detection of C. trachomatis IgG antibodies. The performance of the developed ELISA test was compared to the MIF test at two cut-off values of 16 and 64, and to the specific pELISA test using a panel of 342 sera. These sera were from children MIF C. trachomatis and Chlamydophila pneumoniae negative, patients MIF C. pneumoniae positive, patients MIF C. trachomatis positive, patients suspected to have chlamydial infections diagnosed by the Cobas Amplicor test, healthy blood donors and prostitutes. Our results indicate that the developed ELISA test has performed better compared with the MIF and the pELISA tests. The highest performance was obtained when comparing the developed ELISA test in relation to the pELISA, yielding an overall sensitivity and specificity of 85% and 87% respectively. The CT694 ELISA showed the best performance when compared to the species-specific pELISA test and may be used for the serodiagnosis of C. trachomatis infections. The CT694 ELISA test responds to the criteria of both sensitivity and specificity according to the MIF and pELISA tests and may be used for serodiagnosis of C. trachomatis infections.

Evaluation of replacing the existing diagnostic strategy for Neisseria gonorrhoeae and Chlamydia trachomatis infections with sole molecular testing of urine specimens in a sexually transmitted infection clinic setting

Objectives: To evaluate nucleic acid testing of urine specimens against conventional Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) tests in genital swab specimens in a sexually transmitted infection (STI) clinic...

Rapid viral response and treatment outcome in genotype 2 and 3 chronic hepatitis C: comparison between two HCV RNA quantitation methods

Fifty consecutive patients with genotype 2 or 3 chronic hepatitis C were treated with peg-IFN alfa-2a 135 microg weekly and ribavirin (11 mg/kg body weight) daily during 24 weeks. Rapid viral response treatment week 4, end-of-treatment response, and sustained viral response were analyzed by two different HCV RNA quantitation methods, the Cobas Amplicor Monitor test and the TaqMan test with a sensitivity of 600 and 15 IU/ml, respectively. The TaqMan test differentiated patients with rapid viral response finally achieving sustained viral response better. Hence, patients with and without rapid viral response as tested by the TaqMan test finally achieved sustained viral response in 97% (32/33) versus in 75% (12/16), P < 0.017. The corresponding figures for the Cobas Amplicor test was 91% (41/45) versus (80%) 4/5 a non-significant difference. In conclusion, the more sensitive TaqMan test yielded a lower number of patients with rapid viral response than the less sensitive Amplicor Monitor test, but predicted sustained viral response in a higher percentage of patients with rapid viral response than the Amplicor Monitor test. A rapid viral response meaning HCV RNA levels <15 IU/ml predicted a sustained viral response in 97% of patients with genotype 2 or 3.

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

Specific andSensitive Detection of Neisseria gonorrhoeae in ClinicalSpecimens by Real-Time PCR

Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients' health and infectivity. We report the development of a specific and sensitive TaqMan assay for the detection of N. gonorrhoeae in clinical samples. The target sequence is a 76-bp fragment of the 5' untranslated region of the opa genes that encode opacity proteins. A panel of 448 well-defined N. gonorrhoeae isolates was used to evaluate and optimize the assay. The method employs two minor-groove binding probes, one of them recognizing a newly identified sequence in the opa genes. Testing a large panel of related and unrelated microorganisms revealed that other Neisseria strains and other microorganisms tested negative in the opa test. With a lower detection limit of one genome per reaction, the opa test appeared more sensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV, Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a panel of 122 COBAS AMPLICOR-positive samples revealed that 68% were negative in both the 16S rRNA test and the opa assay (confirming that the COBAS AMPLICOR test produces false positives), while 30% were positive in both assays. Three samples were opa positive and 16S rRNA negative, which may be due to the higher sensitivity of the opa assay. We conclude that the opa gene-based real-time amplification assay offers a sensitive, specific, semiquantitative, and reliable assay suitable for the detection of N. gonorrhoeae in clinical specimens and/or for confirmation of less specific tests.

Use of Pooled Urine Samples and Automated DNA Isolation To Achieve Improved Sensitivity and Cost-Effectiveness of Large-Scale Testing for Chlamydia trachomatis in Pregnant Women

The success of large-scale screening for Chlamydia trachomatis depends on the availability of noninvasive samples, low costs, and high-quality testing. To evaluate C. trachomatis testing with pregnant women, first-void urine specimens from 750 consecutive asymptomatic pregnant women from the Rotterdam area (The Netherlands) were collected. Initially, we investigated the performance of three different DNA isolation methods with 350 of these urines and 70 pools of 5 of the same subset of urine samples. The routinely used COBAS AMPLICOR test was compared to the COBAS AMPLICOR test with prior DNA isolation by use of the MagNA Pure large-volume kit and the MagNA Pure bacterial DNA isolation kit. The latter combination provided the best DNA test for pooled urines, with a sensitivity twice that of the other methods. Next, using all 750 urines, the COBAS AMPLICOR performance for individual testing was compared to pooled testing with the standard COBAS AMPLICOR procedure and subsequently to pooled testing with COBAS AMPLICOR in combination with the MagNA Pure bacterial DNA isolation kit. The sensitivity of COBAS AMPLICOR was 65% on individual and 42% on pooled urines but improved to 92% on pooled urines with the MagNA Pure bacterial DNA isolation kit, making this combination the best screening method. The C. trachomatis prevalence in this population appeared to be 6.4%. Additionally, the cost of the combined MagNA Pure bacterial DNA isolation kit and COBAS AMPLICOR method on pooled urines was only 56% of the cost of the standard COBAS AMPLICOR test applied to individual urines. Costs per positive case detected in the combined method were 39% of standard costs.

Assessment of Chlamydia trachomatis infection in asymptomatic male partners of infertile couples.

Three specimens from 111 asymptomatic male partners of infertile couples attending the Department of Urology in Amiens, France, were examined by the PCR COBAS AMPLICOR test (Roche Molecular Diagnostics) for the presence of Chlamydia trachomatis. The specimens analysed were: first void urine (FVU), urine obtained after prostatic massage (UPM) and semen specimens. Serum from each patient was also obtained and analysed for the presence of IgG and IgA chlamydial antibodies by in-house microimmunofluorescence (MIF) and pELISA. C. trachomatis was detected by PCR in 5.4% of FVU samples, 2.7% of semen specimens and in 0.9% of UPM samples. Two treatments for processing the samples (storage at -70 degrees C and heating to 95 degrees C) were routinely used before initial testing to reduce the effects of inhibitors of PCR. Despite these precautions, the PCR method revealed the presence of inhibitors in 7.3% of semen specimens and 3.6% of FVU samples. C. trachomatis was detected by PCR COBAS AMPLICOR in seven of 111 patients (6.3%) and by serology in five of 111 patients (4.5%). The detection of C. trachomatis in FVU, UPM and semen specimens can serve as a marker for the presence of this organism in the genital tract, and can be used as a reliable way of detecting asymptomatic carriers of infection.

Reliability of nucleic acid amplification methods for detection of Chlamydia trachomatis in urine: results of the first international collaborative quality control study among 96 laboratories.

The first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of C. trachomatis by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the performance of these assays.

Multicenter evaluation of the fully automated COBAS AMPLICOR PCR test for detection of Chlamydia trachomatis in urogenital specimens.

The fully automated COBAS AMPLICOR CT/NG test for the detection of Chlamydia trachomatis was evaluated in a multicenter trial. Test performance was evaluated for 2,014 endocervical swab and 1,278 urine specimens obtained from women and for 373 urethral swab and 254 urine specimens obtained from men. Culture served as the reference test. Culture-negative, COBAS AMPLICOR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the C. trachomatis major outer membrane protein gene were resolved as true positives. The overall prevalence of chlamydia was 4.3% in cervical swabs and 11.0% in urethral swabs from men. When the results for each specimen type were considered separately, the resolved sensitivities were 96.5% (83 of 86) for endocervical swab specimens, 95.1% (39 of 41) for urine specimens from women, 100.0% (41 of 41) for urethral swab specimens from men, and 94.4% (17 of 18) for urine specimens from men; the resolved specificities were 99.4% (1,912 of 1,924) for endocervical swab specimens, 99.8% (1,204 of 1,207) for urine specimens from women, 98. 5% (325 of 330) for urethral swab specimens from men, and 100.0% (236 of 236) for urine specimens from men. For the subset of patients from whom both swab and urine specimens were collected, the combined results for both specimen types were used to identify all infected patients. Using these combined reslts as criteria, the resolved sensitivities for the COBAS AMPLICOR test were 82.6% (38 of 46) for endocervical swab specimens, 84.4% (38 of 45) for urine specimens from women, 84.2% (16 of 19) for urethral swab specimens from men, and 89.5% (17 of 19) for urine specimens from men. In comparison, the sensitivity of culture was only 56.5% (26 of 46) for endocervical specimens and 63.2% (12 of 19) for urethral specimens from men. The internal control provided in the COBAS AMPLICOR test revealed that 2.9% of specimens were inhibitory when they were initially tested. Nevertheless, valid results were obtained for 99. 1% of specimens because 68.7% of the inhibitory specimens were not inhibitory when a second aliquot of the original sample was tested. Two additional COBAS AMPLICOR-positive specimens were detected by retesting inhibitory specimens. The COBAS AMPLICOR CT/NG test for the detection of C. trachomatis exhibited equally high sensitivities and specificities with both urogenital swab and urine specimens and, thus, is well-suited for use in screening.