Impact of nucleic acid extraction procedures on human papillomavirus (HPV) detection and genotyping.

Human papillomavirus (HPV) infections are often asymptomatic, but some of the >200 HPV genotypes confer a high risk for precancerous cervical lesions and cervical cancer. Current clinical management of HPV infections relies on reliable nucleic acid testing detection and genotyping. We prospectively compared nucleic acid extraction without and with prior centrifugation enrichment for detecting and genotyping HPV in cervical swabs with atypical squamous or glandular cells. Consecutive swabs were analyzed from 45 patients with atypical squamous or glandular cells. Nucleic acids were extracted in parallel using three procedures, Abbott-M2000, Roche-MagNA-Pure-96 Large-Volume Kit without (Roche-MP-large) and with prior centrifugation (Roche-MP-large/spin) and tested using Seegene-Anyplex-II HPV28. In total, 54 HPV-genotypes were detected in 45 samples, 51 by Roche-MP-large/spin, 48 by Abbott-M2000 and 42 by Roche-MP-large. The overall concordance was 80% for detecting any HPV and 74% for specific HPV-genotypes. Roche-MP-large/spin and Abbott-M2000 showed the highest concordance for HPV detection (88.9%; kappa 0.78), and genotyping (88.5%). Two and more HPV-genotypes were detected in 15 samples, often with one HPV being more abundant. Dilution series confirmed the specific detection of multiple HPV-genotypes and their relative abundance. In 285 consecutive follow-up samples extracted by Roche-MP-large/spin, the top three detected genotypes were the high-risk HPV16, HPV53, HPV56 and the low-risk HPV42, HPV54 and HPV61. Rate and breadth of HPV detection in cervical swabs depends on extraction protocols being highest after centrifugation/enrichment. As multivalent HPV-vaccine coverage increases, detecting the evolving HPV virome depends on improved extraction and broader genotype coverage.

Comparing cytomegalovirus diagnostics by cell culture and quantitative nucleic acid testing in broncho-alveolar lavage fluids.

Many clinical laboratories have replaced virus isolation in cell-culture (VIC) for cytomegalovirus (CMV) by quantitative-nucleic-acid testing (QNAT), rendering clinically relevant CMV-replication difficult to distinguish from CMV-shedding or latent infection. We compared direct VIC in 1109 consecutive bronchoalveolar lavage fluids (BALFs) and a well-validated CMV-QNAT (Basel-CMV-UL111a-77bp). In the retrospective Group 1 (N = 694) and Group 2 (N = 303), CMV-QNAT was performed within 48 h from 2-fold and 10-fold concentrated total nucleic acid (TNA) eluates, respectively. In Group 3 (N = 112), 2-fold and 10-fold concentrated TNA eluates were prospectively analyzed in parallel to VIC. CMV was detected by VIC in 79 of 694 (11%) and 26 of 303 (9%) of Groups 1 and 2, but in 114 of 694 (16%) and 57 of 303 (17%) by CMV-QNAT, respectively. Median CMV loads were significantly higher in VIC-positive than in VIC-negative BALF. The likelihood for CMV detection by VIC was 85% for BALF CMV- loads >4 log10 copies/ml. In the prospective Group 3, CMV was detected by VIC in 10 of 112 (9%), and in 14 of 112 (13%) and 20 of 112 (18%) by CMV-QNAT, when using 2-fold and 10-fold concentrated TNA eluates, respectively. Notably, CMV was undetectable by CMV-QNAT in 10 VIC-positive cases of Groups 1 and 2, but in none of Group 3. We conclude that CMV-QNAT can be adopted to BALF diagnostics but requires several careful steps in validation. CMV-QNAT loads >10 000 copies/ml in BALF may indicate significant CMV replication as defined by VIC, if short shipment and processing procedures can be guaranteed. Discordance of detecting CMV in time-matched plasma samples emphasises the role of local pulmonary CMV replication, for which histopathology remains the gold standard of proven CMV pneumonia.

Epidemiology and precision of SARS-CoV-2 detection following lockdown and relaxation measures.

OBJECTIVES: Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to the clinical and epidemiological assessment of CoVID-19. We cross-validated manual and automated high-throughput testing for SARS-CoV-2-RNA, evaluated SARS-CoV-2 loads in nasopharyngeal-oropharyngeal swabs (NOPS), lower respiratory fluids, and plasma, and analyzed detection rates after lockdown and relaxation measures. METHODS: Basel-S-gene, Roche-E-gene, and Roche-cobas®6800-Target1 and Target2 were prospectively validated in 1344 NOPS submitted during the first pandemic peak (Week 13). Follow-up cohort (FUP) 1, 2, and 3 comprised 10,999, 10,147, and 19,389 NOPS submitted during a 10-week period until Weeks 23, 33, and 43, respectively. RESULTS: Concordant results were obtained in 1308 cases (97%), including 97 (9%) SARS-CoV-2-positives showing high quantitative correlations (Spearman's r > .95; p < .001) for all assays and high precision by Bland-Altman analysis. Discordant samples (N = 36, 3%) had significantly lower SARS-CoV-2 loads (p < .001). Following lockdown, detection rates declined to <1% in FUP-1, reducing single-test positive predictive values from 99.3% to 85.1%. Following relaxation, rates flared up to 4% and 12% in FUP-2 and -3, but infected patients were younger than during lockdown (34 vs. 52 years, p < .001). In 261 patients providing 936 NOPS, SARS-CoV-2 loads declined by three orders of magnitude within 10 days postdiagnosis (p < .001). SARS-CoV-2 loads in NOPS correlated with those in time-matched lower respiratory fluids or in plasma but remained detectable in some cases with negative follow-up NOPS, respectively. CONCLUSION: Manual and automated assays significantly correlated qualitatively and quantitatively. Following a successful lockdown, declining positive predictive values require independent dual-target confirmation for reliable assessment. Confirmatory and quantitative follow-up testing should be obtained within <5 days and consider lower respiratory fluids in symptomatic patients with SARS-CoV-2-negative NOPS.

Hunting coronavirus by transmission electron microscopy - a guide to SARS-CoV-2-associated ultrastructural pathology in COVID-19 tissues.

Transmission electron microscopy has become a valuable tool to investigate tissues of COVID-19 patients because it allows visualisation of SARS-CoV-2, but the 'virus-like particles' described in several organs have been highly contested. Because most electron microscopists in pathology are not accustomed to analysing viral particles and subcellular structures, our review aims to discuss the ultrastructural changes associated with SARS-CoV-2 infection and COVID-19 with respect to pathology, virology and electron microscopy. Using micrographs from infected cell cultures and autopsy tissues, we show how coronavirus replication affects ultrastructure and put the morphological findings in the context of viral replication, which induces extensive remodelling of the intracellular membrane systems. Virions assemble by budding into the endoplasmic reticulum-Golgi intermediate complex and are characterised by electron-dense dots of cross-sections of the nucleocapsid inside the viral particles. Physiological mimickers such as multivesicular bodies or coated vesicles serve as perfect decoys. Compared to other in-situ techniques, transmission electron microscopy is the only method to visualise assembled virions in tissues, and will be required to prove SARS-CoV-2 replication outside the respiratory tract. In practice, documenting in tissues the characteristic features seen in infected cell cultures seems to be much more difficult than anticipated. In our view, the hunt for coronavirus by transmission electron microscopy is still on.

Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2 Emergence Amidst Community-Acquired Respiratory Viruses.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China as the cause of coronavirus disease 2019 in December 2019 and reached Europe by late January 2020, when community-acquired respiratory viruses (CARVs) are at their annual peak. We validated the World Health Organization (WHO)-recommended SARS-CoV-2 assay and analyzed the epidemiology of SARS-CoV-2 and CARVs. METHODS: Nasopharyngeal/oropharyngeal swabs (NOPS) from 7663 patients were prospectively tested by the Basel S-gene and WHO-based E-gene (Roche) assays in parallel using the Basel N-gene assay for confirmation. CARVs were prospectively tested in 2394 NOPS by multiplex nucleic acid testing, including 1816 (75%) simultaneously for SARS-CoV-2. RESULTS: The Basel S-gene and Roche E-gene assays were concordant in 7475 cases (97.5%) including 825 (11%) SARS-CoV-2 positives. In 188 (2.5%) discordant cases, SARS-CoV-2 loads were significantly lower than in concordant positive ones and confirmed in 105 (1.4%). Adults were more frequently SARS-CoV-2 positive, whereas children tested more frequently CARV positive. CARV coinfections with SARS-CoV-2 occurred in 1.8%. SARS-CoV-2 replaced CARVs within 3 weeks, reaching 48% of all detected respiratory viruses followed by rhinovirus/enterovirus (13%), influenza virus (12%), coronavirus (9%), respiratory syncytial virus (6%), and metapneumovirus (6%). CONCLUSIONS: Winter CARVs were dominant during the early SARS-CoV-2 pandemic, impacting infection control and treatment decisions, but were rapidly replaced, suggesting competitive infection. We hypothesize that preexisting immune memory and innate immune interference contribute to the different SARS-CoV-2 epidemiology among adults and children.

Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements.

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r, 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific.IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

Comparing the Cobas Liat Influenza A/B and respiratory syncytial virus assay with multiplex nucleic acid testing.

Influenza virus and respiratory syncytial virus (RSV) detection with short turn-around-time (TAT) is pivotal for rapid decisions regarding treatment and infection control. However, negative rapid testing results may come from poor assay sensitivity or from influenza-like illnesses caused by other community-acquired respiratory viruses (CARVs). We prospectively compared the performance of Cobas Liat Influenza A/B and RSV assay (LIAT) with our routine multiplexNAT-1 (xTAG Respiratory Pathogen Panel; Luminex) and multiplexNAT-2 (ePlex-RPP; GenMark Diagnostics) using 194 consecutive nasopharyngeal swabs from patients with influenza-like illness during winter 2017/2018. Discordant results were reanalyzed by specific in-house quantitative nucleic acid amplification testing (NAT). LIAT was positive for influenza virus-A, -B, and RSV in 18 (9.3%), 13 (6.7%), and 55 (28.4%) samples, and negative in 108 samples. Other CARVs were detected by multiplexNAT in 66 (34.0%) samples. Concordant results for influenza and RSV were seen in 190 (97.9%), discordant results in 4 (2.1%), which showed low-level RSV (<40 000 copies/mL). Sensitivity and specificity of LIAT for influenza-A, -B, and RSV were 100%, 100% and 100%, and 100%, 99.5% and 100%, respectively. The average TAT of LIAT was 20 minutes compared to 6 hours and 2 hours for the multiplexNAT-1 and -2, respectively. Thus, LIAT demonstrated excellent sensitivity and specificity for influenza and RSV, which together with the simple sample processing and short TAT renders this assay suitable for near-patient testing.

Donor-derived, metastatic urothelial cancer after kidney transplantation associated with a potentially oncogenic BK polyomavirus.

BK polyomavirus has been linked to urothelial carcinoma in immunosuppressed patients. Here, we performed comprehensive genomic analysis of a BK polyomavirus-associated, metachronous, multifocal and metastatic micropapillary urothelial cancer in a kidney transplant recipient. Dissecting cancer heterogeneity by sorting technologies prior to array-comparative genomic hybridization followed by short tandem repeat analysis revealed that the metastatic urothelial cancer was of donor origin (4-year-old male). The top 50 cancer-associated genes showed no key driver mutations as assessed by next-generation sequencing. Whole genome sequencing and BK polyomavirus-specific amplification provided evidence for episomal and subgenomic chromosomally integrated BK polyomavirus genomes, which carried the same unique 17-bp deletion signature in the viral non-coding control region (NCCR). Whereas no role in oncogenesis could be attributed to the host gene integration in chromosome 1, the 17-bp deletion in the NCCR increased early viral gene expression, but decreased viral replication capacity. Consequently, urothelial cells were exposed to high levels of the transforming BK polyomavirus early proteins large tumour antigen and small tumour antigen from episomal and integrated gene expression. Surgery combined with discontinuation of immunosuppression resulted in complete remission, but sacrificed the renal transplant. Thus, this report links, for the first time, BK polyomavirus NCCR rearrangements with oncogenic transformation in urothelial cancer in immunosuppressed patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Enterovirus detection in patients with acute gastroenteritis in Switzerland.

Acute gastroenteritis (GE) has a major impact on morbidity and mortality worldwide, yet comprehensive data regarding infectious agents including enteroviruses are scarce. We hypothesized that enteroviruses constitute a significant cause of acute GE. We analyzed 677 stool samples from 504 patients, which had been submitted for suspected infectious GE. 0.2 mL of stool suspension was extracted using the Abbott m2000sp robot and analysed by multiplex nucleic acid testing (NAT) using the Luminex xTAG gastrointestinal pathogen panel (GPP) as well as by specific NATs detecting enteroviruses and polioviruses. Median age of the patients was 6.6 years (IQR 1.1-50.6; pediatric <18 years). 292 of 677 (43%) samples were positive for at least one pathogen. Enterovirus was detected in 5.3% (36/677) as sole pathogen (67%), and more frequently in children (P = 0.0054). Only rotavirus (18.6%) and norovirus (12.1%) were more frequent. Clostridium difficile and Campylobacter jejuni were detected in 5.5% and 2.2% of stools, respectively. Adenovirus, E. coli O157, Salmonella, Shiga toxin-producing E. coli (STEC), Shigella, Giardia lamblia, Cryptosporidium, and Entamoeba histolytica were rare (<1% of samples). Vibrio cholerae, Yersinia enterocolitica, enterotoxigenic E. coli (ETEC) and poliovirus were not detected. Thus, non-polio enteroviruses are the third most frequent pathogen in acute GE suggesting that enteroviruses may play an important role in GE even in developed, industrial health care settings.

Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression.

UNLABELLED: In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged noncoding control regions (rr-NCCRs) that increase viral early gene expression, replicative fitness, and cytopathology. rr-NCCRs result from various deletions and duplications of archetype NCCR (ww-NCCR) sequences, which alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear. We inactivated 28 TFBS in the archetype NCCR by selective point mutations and examined viral gene expression in bidirectional reporter constructs. Compared to the archetype, group 1 mutations increased viral early gene expression similar to rr-NCCR and resulted from inactivating one Sp1 or one Ets1 TFBS near the late transcription start site (TSS). Group 2 mutations conferred intermediate early gene activation and affected NF1, YY1, and p53 sites between early and late TSS. Group 3 mutations decreased early and late gene expression and included two other Sp1 sites near the early TSS. Recombinant viruses bearing group 1 NCCRs showed increased replication in human renal epithelial cells similar to clinical rr-NCCR variants. Group 2 and 3 viruses showed intermediate or no replication, respectively. A literature search revealed unnoticed group 1 mutations in BKPyV nephropathy, hemorrhagic cystitis, and disseminated disease. IMPORTANCE: The NCCRs of polyomaviruses mediate silent persistence of the viral genome as well as the appropriately timed (re)activation of the viral life cycle. This study indicates that the basal BKPyV NCCR is critically controlled by a hierarchy of single TFBS in the archetype NCCR that direct, modulate, and execute the bidirectional early and late viral gene expression. The results provide new insights into how BKPyV NCCR functions as a viral sensor of host cell signals and shed new light on how transcription factors like Sp1 control bidirectional viral gene expression and contribute to replication and pathology.

CMX001 (1-O-hexadecyloxypropyl-cidofovir) inhibits polyomavirus JC replication in human brain progenitor-derived astrocytes.

Polyomavirus JC (JCV) replication causes progressive multifocal leukoencephalopathy (PML), a frequently fatal brain disease in immunodeficient patients, yet antiviral drugs are lacking. We characterized the lipid conjugate 1-O-hexadecyloxypropyl-cidofovir (CMX001) regarding JCV (Mad-4) replication in human brain progenitor-derived astrocytes (PDA) and the simian virus 40 (SV40) large-T-antigen-expressing COS-7 cells up to 7 days postinfection (dpi). We examined JCV loads by PCR, the infection rate by immunofluorescence, and host cell toxicity by WST-1 and BrdU incorporation assays. Supernatants from CMX001-treated PDA demonstrated a drug concentration-dependent decrease in JCV loads and infectivity. CMX001 had only a modest effect on host cell metabolism but reduced overall BrdU incorporation. In PDA at 7 dpi, the CMX001 50% effective concentration (EC50) was 5.55 nM, the 50% cytotoxic concentration (CC50) was 184.6 nM, and the 50% selectivity index (SI50) was 33.3. The EC90 was 19.7 nM, the CC90 was 5,054 nM, and the SI90 was 256.1. In COS-7 cells, JCV replication was faster and the EC50 and EC90 were 18- and 37-fold higher than those in PDA, i.e., 0.1 µM and 0.74 µM (CC50, 0.67 µM; SI50, 6.7; CC90, 12.2 µM; SI90, 16.5) at 5 dpi. We conclude that CMX001 inhibits JCV replication at concentrations in vitro that can be attained by oral administration without significant side effects in clinical studies.

Rearranged JC virus noncoding control regions found in progressive multifocal leukoencephalopathy patient samples increase virus early gene expression and replication rate.

Polyomavirus JC (JCV) infects ∼ 60% of the general population, followed by asymptomatic urinary shedding in ∼ 20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs). Although JCV NCCR rearrangements are deemed pathognomonic for PML, their role as a viral determinant is unclear. We sequenced JCV NCCRs found in CSF of eight HIV/AIDS patients newly diagnosed with PML and analyzed their effect on early and late gene expression using a bidirectional reporter vector recapitulating the circular polyomavirus early and late gene organization. The rr-NCCR sequences were highly diverse, but all increased viral early reporter gene expression in progenitor-derived astrocytes, glia-derived cells, and human kidney compared to the expression levels with the at-NCCR. The expression of simian virus 40 (SV40) large T antigen or HIV Tat expression in trans was associated with a strong increase of at-NCCR-controlled early gene expression, while rr-NCCRs were less responsive. The insertion of rr-NCCRs into the JCV genome backbone revealed higher viral replication rates for rr-NCCR compared to those of the at-NCCR JCV in human progenitor-derived astrocytes or glia cells, which was abrogated in SV40 large T-expressing COS-7 cells. We conclude that naturally occurring JCV rr-NCCR variants from PML patients confer increased early gene expression and higher replication rates compared to those of at-NCCR JCV and thereby increase cytopathology.

Usefulness of the GenMark ePlex RPP assay for the detection of respiratory viruses compared to the FTD21 multiplex RT-PCR.

Cartridge-based multiplex panels covering numerous pathogens offer an advantage of minimal hands-on-time and short time to result to commercial RT-PCR assays. In this study, we evaluated the performance of the ePlex respiratory pathogen panel (RPP) compared to the Fast Track Diagnostics Respiratory pathogens 21 multiplex RT-PCR assay (FTD21) using 400 clinical respiratory samples. Discrepant results were further analysed by a reference nucleic acid amplification testing (NAT) and a composite reference approach was used for final interpretation. Discordant results were observed in 56 targets corresponding to 54 samples. Sensitivities and specificities were 85.5% and 99.9% for the ePlex RPP and 95.8% and 99.7% for the FTD21 system, respectively. Altogether, the ePlex RPP is a valuable tool for the rapid detection of a number of different respiratory viruses with the exception of the coronavirus family (low sensitivity ranging from 50-80%) and samples with a low pathogen load (Ct values >33).

1-O-hexadecyloxypropyl cidofovir (CMX001) effectively inhibits polyomavirus BK replication in primary human renal tubular epithelial cells.

Antiviral drugs for treating polyomavirus BK (BKV) replication in polyomavirus-associated nephropathy or hemorrhagic cystitis are of considerable clinical interest. Unlike cidofovir, the lipid conjugate 1-O-hexadecyloxypropyl cidofovir (CMX001) is orally available and has not caused detectable nephrotoxicity in rodent models or human studies to date. Primary human renal proximal tubular epithelial cells were infected with BKV-Dunlop, and CMX001 was added 2 h postinfection (hpi). The intracellular and extracellular BKV DNA load was determined by quantitative PCR. Viral gene expression was examined by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence microscopy. We also examined host cell viability, proliferation, metabolic activity, and DNA replication. The titration of CMX001 identified 0.31 µM as the 90% effective concentration (EC(90)) for reducing the extracellular BKV load at 72 hpi. BKV large T antigen mRNA and protein expression was unaffected at 24 hpi, but the intracellular BKV genome was reduced by 90% at 48 hpi. Late gene expression was reduced by 70 and 90% at 48 and 72 hpi, respectively. Comparisons of CMX001 and cidofovir EC(90)s from 24 to 96 hpi demonstrated that CMX001 had a more rapid and enduring effect on BKV DNA and infectious progeny at 96 hpi than cidofovir. CMX001 at 0.31 µM had little effect on overall cell metabolism but reduced bromodeoxyuridine incorporation and host cell proliferation by 20 to 30%, while BKV infection increased cell proliferation in both rapidly dividing and near-confluent cultures. We conclude that CMX001 inhibits BKV replication with a longer-lasting effect than cidofovir at 400× lower levels, with fewer side effects on relevant host cells in vitro.

Brief validation of the novel GeneXpert Xpress SARS-CoV-2 PCR assay.

The clinical and epidemiologic management of the SARS-CoV-2 pandemic is critically dependent on molecular assays with short turn-around time. We validated the novel Xpert Xpress SARS-CoV-2 assay using a commercial nucleic acid testing (Roche Cobas 6800). We found an excellent concordance over a range of SARS-CoV-2 loads and across established human coronaviruses.

Antibody responses to recombinant polyomavirus BK large T and VP1 proteins in young kidney transplant patients.

BK virus (BKV)-specific immunity is critical for polyomavirus-associated nephropathy, but antibody responses are incompletely defined. We compared the hemagglutination inhibition assay (HIA) with immunoglobulin G enzyme immunoassays (EIA) to BKV proteins expressed in baculovirus-infected insect cells. N-terminal, internal, and C-terminal domains of the BKV large T antigen (BKLT) were fused to glutathione S-transferase (GST), yielding GST-BKLTD1, GST-BKLTD2, and GST-BKLTD3, respectively. The BKV capsid VP1 was expressed as a GST fusion (BKVP1) or as a native VP1 assembled into viruslike particles (BKVLP). We tested 422 sera from 28 healthy donors (HD), 99 dialysis patients (DP; median age, 15 years; range, 3 to 32 years), and 46 age-matched kidney transplant patients (KTP; median age, 15 years; range, 2 to 33 years). In HD, HIA and BKVLP EIA both yielded a 91.7% seroreactivity, whereas all other EIA responses were lower (BKVP1, 83.3%; BKLTD1, 25%; BKLTD2, 29%; BKLTD3, 40%). HIA titers significantly correlated with EIA levels for BKVLP, BKVP1, and BKLTD1 but not for BKLTD2 or BKLTD3, which were barely above the cutoff. In DP, the seroreactivities of HIA, BKVLP, and BKLTD1 were lower than that in HD (63.6%, 86.9%, and 10.1%, respectively) and they had lower titers (P < 0.001). In KTP, seropositivities for BKVLP, BKVP1, and BKLTD1 were 78%, 50%, and 17%, respectively, but anti-BKVLP levels increased significantly in KTP with viruria and viremia, whereas anti-BKLTD1 levels increased after clearing sustained BKV viremia. In conclusion, anti-BKVLP is equivalent to HIA in HD but is more sensitive to determine the BKV serostatus in DP and KTP. In KTP, anti-BKVLP responds to recent BKV viruria and viremia, whereas anti-BKLTD1 may indicate emerging BKV-specific immune control.

Investigation of the hepatitis C virus replication complex.

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors, is a hallmark of all positive-strand RNA viruses. In the case of HCV, RNA replication takes place in a likely endoplasmic reticulum-derived membrane alteration referred to as the "membranous web." In vitro transcription-translation, membrane extraction and flotation analyses, immunofluorescence microscopy, fluorescent in situ hybridization, and RNA metabolic labeling followed by confocal laser scanning microscopy have yielded insights into the structure and function of the HCV replication complex. We describe these techniques and highlight selected results.

Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results.

BACKGROUND: Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTM-CMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014). OBJECTIVES: To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT. STUDY DESIGN: The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction. RESULTS: Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by >90% indicating the presence of unprotected CMV genomic DNA. CONCLUSIONS: Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons.

Viral dynamics in transplant patients: implications for disease.

Viral infections cause substantial morbidity and mortality in transplant patients. Quantifying viral loads is widely appreciated as a direct means to diagnose and monitor the course of viral infections. Recent studies indicate that the kinetics of viral load changes rather than single viral load measurements better correlate with organ involvement. In this Review, we will summarise the current knowledge regarding the kinetics of viruses relevant to transplantation including cytomegalovirus, Epstein-Barr virus, the herpes viruses 6 and 7, hepatitis C virus, GB virus C, adenovirus, and the emerging human polyomavirus type BK. We discuss the implications of viral kinetics for organ pathology as well as for the evaluation of antiviral interventions in transplant patients.