Multisite Performance Evaluation of the cobas 5800 System and Comparison to the cobas 6800/8800 Systems for Quantitative Measurement of HBV, HCV, and HIV-1 Viral Load.

The cobas 5800 System ("cobas 5800") is a new low- to mid-throughput PCR-based nucleic acid testing system which performs both qualitative and quantitative testing, including viral load (VL) determination. cobas 5800 shares numerous design elements and technical characteristics with the existing cobas 6800/8800 Systems. We compared HBV, HCV, and HIV-1 VL results from cobas 5800 in three different laboratories to those from the same specimens tested on a cobas 6800 system. We also assessed cobas 5800 assay reproducibility by repetitive testing of specimens with VL close to values used as thresholds for patient management or classification. The correlation between VL measurements generated using cobas 5800 versus 6800 was extremely high, with r2 correlation coefficients between 0.990 and 0.999 for the three targets at the different sites. The slope of the Deming regression line ranged from 0.994 (HBV, site 3) to 1.025 (HIV-1, site 1). The standard deviation values ranged from 0.04 to 0.19 log10 IU/mL for HBV, 0.06 to 0.33 log10 IU/mL for HCV, and 0.05 to 0.34 log10 copies/mL for HIV-1. In general, variability was higher at lower VL. Between 98.6% and 100% of results fell within the allowable total difference zone that defines expected variability on the existing 6800/8800 system. This multisite comparison study demonstrates equivalent performance of the new cobas 5800 system compared with cobas 6800. This establishes cobas 5800 as a new option for low- to mid-throughout laboratories seeking to optimize efficiency of their viral molecular testing. IMPORTANCE These are the first published data that demonstrate equivalent performance of the new cobas 5800 system compared with cobas 6800. This fulfills an unmet need for low- to mid-throughout laboratories seeking to optimize efficiency of their viral molecular testing.

The importance of effect sizes when comparing cycle threshold values of SARS-CoV-2 variants.

PURPOSE: We aimed to elaborate whether cycle threshold (Ct) values differ significantly between wild type SARS-CoV-2 (wtV) and certain viral variants and how strong or weak a potential significant effect might be. METHODS: In a retrospective study, we investigated 1873 SARS-CoV-2 positive samples for the occurrence of viral marker mutations. Age, gender, clinical setting, days after onset of symptoms, and Ct values were recorded. Statistical analysis was carried out with special consideration of effect sizes. RESULTS: During the study period wtV was detected in 1013 samples (54%), while 845 (45%) patients carried the Alpha variant of concern (VOC), and 15 (1%) the Beta VOC. For further analysis, only wtV and the Alpha VOC were included. In a multi-factor ANOVA and post-hoc test with Bonferroni-correction for the age groups we found significant main-effects for Ct values of the viral variant (wtV mean 26.4 (SD 4.27); Alpha VOC mean 25.0 (SD 3.84); F (1,1850) = 55.841; p < .001) and the clinical setting (outpatients: mean 25.7 (SD 4.1); inpatients: mean 27.0 (SD 4.2); F (1,1850) = 8.520, p = .004). However, since the effect sizes were very small (eta squared for the Alpha VOC = .029 and the clinical setting = .004), there was only a slight trend towards higher viral loads of the Alpha VOC compared to wtV. CONCLUSIONS: In order to compare different variants of SARS-CoV-2 the calculation of effect sizes seems to be necessary. A combination of p-values as estimates of the existance of an effect and effect sizes as estimates of the magnitude of a potential effect may allow a better insight into transmission mechanisms of SARS-CoV-2.

Where have the enteric viruses gone? - Differential effects on frequent causes of infectious diarrhoea by SARS-CoV-2 pandemic lockdown measures.

BACKGROUND: Measures of distancing, wearing face/medical masks and lockdown introduced in many countries to meet the challenges of the SARS-CoV-2 pandemic have led to gross changes in the epidemiology of important infections. The observation of decline of positive norovirus tests after introduction of lockdown in Germany led us to investigate changes in the detection of major causes of diarrhoea by comparing pre-pandemic quarters (PPQ: 1Q/17 through 1Q/20) since 2017 and pandemic quarters (PQ: 2Q/20 through 1Q/21). METHODS AND SETTING: Bioscientia Laboratory Ingelheim is a large regional clinical pathology laboratory serving > 50 hospitals and > 5000 general practitioners and specialist outpatient practices located in the federal states Hesse, Rhineland-Palatinate and North Rhine-Westphalia, Germany. Antigen detection assays were used for detection of astrovirus, adenovirus, rotavirus, and Campylobacter antigen and Clostridium difficile Toxin A/B, while norovirus was detected by qualitative RT-PCR. FINDINGS: The mean positivity-ratios of norovirus, adenovirus and astrovirus assays were 3-20 fold lower in periods PQ (2Q/20 through 1Q/21) compared to PPQ (1Q/17 through 1Q/20) (p<.01). The mean positivity-ratio was lower in PQ compared to PPQ for rotavirus (p=.31), but failed to reach statistical significance, while for campylobacter antigen (p=.91) and C. difficile Toxin A/B (p=.17) the mean positivity-ratio was even higher in PQ compared to PPQ. CONCLUSIONS: Apparently, hygienic measures used to contain the SARS-CoV-2 pandemic have differential effects on incidence of diarrhoea viruses as compared to bacterial gastrointestinal agents, particularly C. difficile, which may lead to re-evaluate measures implemented against this important cause of nosocomial diarrhoea.

Early Diagnosis of HIV-1 and HIV-2 Using Cobas HIV-1/HIV-2 Qualitative Test: A Novel Qualitative Nucleic Acid Amplification Test for Plasma, Serum, and Dried Blood Spot Specimens.

BACKGROUND: Nucleic acid amplification tests (NATs) minimize the time from HIV infection to diagnosis, reducing transmission during acute HIV. NATs are especially useful for diagnosing HIV in children younger than 18 months and discriminating between HIV-1 and HIV-2. METHODS: We evaluated the performance of the cobas HIV-1/HIV-2 qualitative (cobas HIV-1/2 Qual) test for use on cobas 6800/8800 Systems. The results of adult plasma and serum samples and pediatric dried blood spots were compared with those of the recomLine HIV-1 & HIV-2 Immunoglobulin G serological test and COBAS AmpliPrep/COBAS TaqMan HIV-1 qualitative test, v2.0. Genotype inclusivity and limits of detection were determined, and sensitivity on seroconversion panels was compared with that in the Bio-Rad Geenius HIV 1/2 Confirmatory Assay, Abbott ARCHITECT HIV Ag/Ab Combo serological test, and cobas TaqScreen MPX, v2.0. RESULTS: Concordance of cobas HIV-1/2 Qual test with the comparator serological test and COBAS AmpliPrep/COBAS TaqMan test was ≥99.6% with all sample types. Reactivity with all HIV genotypes was 100%. LOD in plasma samples was 14.8, 12.6, and 27.9 copies/mL for HIV-1 group M, HIV-1 group O, and HIV-2, respectively, with similar results for serum samples. LOD in dried blood spots was 255 copies/mL for HIV-1 and 984 copies/mL for HIV-2. HIV infection was detected 18.9 days and 8.5 days earlier than the confirmatory and serological assays, respectively, and at a similar time to the NAT. CONCLUSIONS: The cobas HIV-1/2 Qual test enables early and accurate diagnoses of HIV-1 and HIV-2 in adults and children across sample types. The assay could help avert transmission during acute HIV, simplify HIV diagnostic algorithms, and promote the survival of HIV-infected children.

Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by Mass Spectrometry.

BACKGROUND: Amplification of viral ribonucleic acid (RNA) by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is the gold standard to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the initial outbreak, strategies to detect and isolate patients have been important to avoid uncontrolled viral spread. Although testing capacities have been upscaled, there is still a need for reliable high throughput test systems, specifically those that require alternative consumables. Therefore, we tested and compared two different methods for the detection of viral PCR products: rRT-PCR and mass spectrometry (MS). METHODS: Viral RNA was isolated and amplified from oro- or nasopharyngeal swabs. A total of 22 samples that tested positive and 22 samples that tested negative for SARS-CoV-2 by rRT-PCR were analyzed by MS. Results of the rRT-PCR and the MS protocol were compared. RESULTS: Results of rRT-PCR and the MS test system were in concordance in all samples. Time-to-results was faster for rRT-PCR. Hands-on-time was comparable in both assays. CONCLUSIONS: MS is a fast, reliable and cost-effective alternative for the detection of SARS-CoV-2 from oral and nasopharyngeal swabs.

Trichomonas vaginalis Detection in Female Specimens with cobas® TV/MG for use on the cobas® 6800/8800 Systems.

Trichomoniasis, a common curable sexually transmitted infection caused by the protozoan Trichomonas vaginalis (TV), is usually asymptomatic. However, symptomatic women may experience vaginal discharge and/or vulvar irritation. This study evaluated cobas® TV/ Mycoplasma genitalium (MG) (Conformité Européene marking for in vitro diagnostic medical devices [CE-IVD]) against other nucleic acid amplification tests (NAATs) for detecting TV in female urogenital specimens. Matched de-identified specimens from 412 females were collected. cobas® TV/MG results were compared against a composite reference (CR) of 3 different NAATs for TV (Aptima TV, modified S-DiaMGTV™, and a laboratory-developed test). The overall TV prevalence rate was 6.2%, based on cobas® TV/MG results. Relative to the CR, cobas® TV/MG sensitivity/specificity for the specimen types were endocervical swabs (ES) 100%/99.2%, vaginal swabs (VS) 100%/99.7%, urine (U) 100%/99.7%, and cervical specimens in PreservCyt® solution (PC) 100%/99.5%. There was no significant statistical difference between clinician-collected and self-collected VS (p = 0.28). Correlation of cobas® TV/MG vs. Aptima TV demonstrated the following positive, negative, and overall percent agreements, respectively: ES 69.0%, 98.7%, and 96.6%; VS 88.9%, 99.5%, and 98.8%; U 100%, 100%, and 100%; and PC 95.5%, 99.0%, and 98.8%. Detection of TV with cobas® TV/MG for use on the cobas® 6800/8800 systems demonstrated excellent performance in female urogenital specimens (overall sensitivity/specificity of 100%≥99.2%).

Frequency of hepatitis B surface antigen variants (HBsAg) in hepatitis B virus genotype B and C infected East- and Southeast Asian patients: Detection by the Elecsys® HBsAg II assay.

BACKGROUND: To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant 'a' determinant region, which vary by ethnographic region. OBJECTIVE: We evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys® HBsAg II Qualitative assay. STUDY DESIGN: We analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the 'a' determinant region using the Elecsys® HBsAg II Qualitative assay. RESULTS: Overall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct mutations, the majority of the amino acid mutations were found in HBV genotype C (64.8%). We identified 25 previously unknown distinct mutations, mostly prevalent in genotype C-infected Korean patients (n = 18) followed by Chinese (n = 12) patients. All 898 samples were correctly identified by the Elecsys® HBsAg II Qualitative assay. CONCLUSIONS: We observed 237 distinct (including 25 novel) mutations, demonstrating the complexity of HBsAg variants in HBV-infected East- and Southeast Asian patients. The Elecsys® HBsAg II Qualitative assay can reliably detect HBV-positive samples and is suitable for routine diagnostic use in East and Southeast Asia.

Substantial variation in the hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-positive patients from South Africa: Reliable detection of HBV by the Elecsys HBsAg II assay.

BACKGROUND: It is essential that hepatitis B surface antigen (HBsAg) diagnostic assays reliably detect genetic diversity in the major hydrophilic region (MHR) of HBsAg to avoid false-negative results. Mutations in this domain display marked ethno-geographic variation and may lead to failure to diagnose hepatitis B virus (HBV) infection. OBJECTIVES: Evaluate diagnostic performance of the Elecsys® HBsAg II Qualitative assay in a cohort of South African HBV-positive blood donors. STUDY DESIGN: A total of 179 South African HBsAg- and HBV DNA > 100 IU/mL-positive blood donor samples were included. Samples were sequenced for genetic variation in HBsAg MHR using next-generation ultra-deep sequencing. HBsAg seropositivity was determined using the Roche Elecsys HBsAg II Qualitative assay. Mutation rates were compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the immunodominant MHR 'a' determinant region. Frequency of occult HBV infection-associated Y100C mutations was also determined. RESULTS: We observed a total of 279 MHR mutations (117 variants) in 102 (57%) samples, of which 91 were located in the 'a' determinant region. The major vaccine-induced escape mutation G145R was observed in two samples. All occult HBV infection-associated Y100C and common diagnostic and vaccine-escape-associated P120T, G145R, K122R, M133L, M133T, Q129H, G130N, and T126S mutations were reliably detected by the assay, which consistently detected the presence of HBsAg in all 179 samples including samples with 11 novel mutations. CONCLUSIONS: Despite substantial variation in HBsAg MHR, the Elecsys HBsAg II Qualitative assay robustly detects HBV infection in this South African cohort.

High-Throughput Testing of Urogenital and Extragenital Specimens for Detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae with Cobas® CT/NG.

We compared the analytical and clinical performance of cobas® CT/NG for use on the Cobas® 6800/8800 Systems with the Cobas® 4800 CT/NG Test from urogenital and extragenital specimens in over 12,000 specimens from both male and female subjects in Germany and the United States. The analytical sensitivity was ≤40 EB/ml for Chlamydia trachomatis (CT) and ≤1 CFU/ml for Neisseria gonorrhoeae (NG). Using clinical specimens, the overall percent agreement with the Cobas® 4800 CT/NG Test was >98.5%. Across urogenital specimens, there were 93 discrepant specimens; 76 (93.8%) of 81 CT discrepant specimens were 6800+/4800- and 10 (83.3%) of 12 NG discrepant specimens were 6800+/4800-. Sequencing verified CT results for 45 (61.6%) of 73 samples positive by 6800 and 1 (20%) of 5 positive by 4800. Similarly, 7 (70.0%) of 10 NG samples positive by 6800 and 1 of 2 positive by 4800 were confirmed by sequencing. Among discrepant extragenital specimens (all 6800+/4800-), 7 (50%) of 14 oropharyngeal and 23 (76.7%) of 30 anorectal CT discordant samples were confirmed as CT positive by sequencing; all 8 anorectal and 20 (90.9%) of 22 oropharyngeal NG discordant results were also confirmed as NG positive. In conclusion, Cobas® CT/NG for use on the Cobas® 6800/8800 Systems provides high-throughput automated solutions for sexually transmitted infection (STI) screening programs.

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

Development of a second version of the Cobas AmpliPrep/Cobas TaqMan hepatitis C virus quantitative test with improved genotype inclusivity.

Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of real-time PCR-based assays with low limits of detection and broad dynamic ranges for quantification. In the present study, the performance of two second-version prototypes of the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with decreased sample input volume and improved genotype inclusivity was investigated. A total of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype 1 [GT1], n = 108; GT2, n = 8; GT3, n = 24; GT4, n = 87; GT5, n = 3; and GT6, n = 2) were processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM), Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4 transcripts containing rare variant sequences were tested. The mean log(10) HCV RNA differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to CAM were -0.05, 0.05, -0.12, -0.10, -0.44, and -0.29 for patients with GT1, GT2, GT3, GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including isolates with known variants within the 5' untranslated region (G145A, A165T) that were underquantified with CAP/CTM were correctly quantified with the second-version prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA across all HCV genotypes, including specimens with rare polymorphisms previously associated with underquantification.

Detection and quantitation of HBV DNA in miniaturized samples: multi centre study to evaluate the performance of the COBAS ® AmpliPrep/COBAS ® TaqMan ® hepatitis B virus (HBV) test v2.0 by the use of plasma or serum specimens.

Laboratory analysis of blood specimens is an increasingly important tool for rapid diagnosis and control of therapy. So, miniaturization of test systems is needed, but reduced specimens might impair test quality. For rapid detection and quantitation of HBV DNA, the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV test has proved a robust instrument in routine diagnostic services. The test system has been modified recently for application of reduced samples of blood plasma and for blood serum, too. The performance of this modified COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV v2.0 (HBV v2.0 (this test is currently not available in the USA)) test was evaluated by comparison with the former COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV v1.0 (HBV v1.0) test. In this study a platform correlation of both assay versions was done including 275 HBV DNA positive EDTA plasma samples. Comparable results were obtained (R(2)=0.97, mean difference -0.03 log(10)IU/ml). The verification of equivalency of the sample matrix (plasma vs. serum samples tested in HBV v2.0 in the same run) showed comparable results for all 278 samples with a R(2)=0.99 and a mean difference of 0.06 log(10)IU/ml. In conclusion, the new test version HBV v2.0 is highly specific and reproducible and quantifies accurately HBV DNA in EDTA plasma and serum samples from patients with chronic HBV infection.