Comparison of three cartridge-based platforms for syndromic testing for respiratory viruses.

Syndromic testing, the simultaneous testing for multiple pathogens causing similar symptoms, has recently gained ground in clinical diagnostics. This approach can significantly shorten time to diagnosis and speed up decision-making, leading to an improved outcome for the patient. Here, we compared three automated multiplex PCR platforms for syndromic testing of respiratory samples in a retrospective study, and assessed their relative sensitivities. The PPA between BioFire and QIAstat compared to ePlex was 98.4 % and 93.8 %, respectively, and 6 discrepant results were observed. The BioFire was identified as the platform with the highest relative sensitivity. Overall, the platforms performed similarly and are all suitable for syndromic testing of respiratory samples.

Management of Suspected Cases of Feline Immunodeficiency Virus Infection in Eurasian Lynx (Lynx lynx) During an International Translocation Program.

The Eurasian lynx (Lynx lynx) population in Switzerland serves as a source for reintroductions in neighboring countries. In 2016-2017, three lynx from the same geographical area were found seropositive for feline immunodeficiency virus (FIV) in the framework of an international translocation program. This novel finding raised questions about the virus origin and pathogenicity to lynx, the emerging character of the infection, and the interpretation of serological results in other lynx caught for translocation. Archived serum samples from 84 lynx captured in 2001-2016 were retrospectively tested for FIV antibodies by Western blot. All archived samples were FIV-negative. The three seropositive lynx were monitored in quarantine enclosures prior to euthanasia and necropsy. They showed disease signs, pathological findings, and occurrence of co-infections reminding of those described in FIV-infected domestic cats. All attempts to isolate and characterize the virus failed but serological data and spatiotemporal proximity of the cases suggested emergence of a lentivirus with antigenic and pathogenic similarities to FIV in the Swiss lynx population. A decision scheme was developed to minimize potential health risks posed by FIV infection, both in the recipient and source lynx populations, considering conservation goals, animal welfare, and the limited action range resulting from local human conflicts. Development and implementation of a cautious decision scheme was particularly challenging because FIV pathogenic potential in lynx was unclear, negative FIV serological results obtained within the first weeks after infection are unpredictable, and neither euthanasia nor repatriation of multiple lynx was acceptable options. The proposed scheme distinguished between three scenarios: release at the capture site, translocation, or euthanasia. Until April 2021, none of the 40 lynx newly captured in Switzerland tested FIV-seropositive. Altogether, seropositivity to FIV was documented in none of 124 lynx tested at their first capture, but three of them seroconverted in 2016-2017. Diagnosis of FIV infection in the three seropositive lynx remains uncertain, but clinical observations and pathological findings confirmed that euthanasia was appropriate. Our experiences underline the necessity to include FIV in pathogen screenings of free-ranging European wild felids, the importance of lynx health monitoring, and the usefulness of health protocols in wildlife translocation.

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).

Bacterial but no SARS-CoV-2 contamination after terminal disinfection of tertiary care intensive care units treating COVID-19 patients.

BACKGROUND: In intensive care units (ICUs) treating patients with Coronavirus disease 2019 (COVID-19) invasive ventilation poses a high risk for aerosol and droplet formation. Surface contamination of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) or bacteria can result in nosocomial transmission. METHODS: Two tertiary care COVID-19 intensive care units treating 53 patients for 870 patient days were sampled after terminal cleaning and preparation for regular use to treat non-COVID-19 patients. RESULTS: A total of 176 swabs were sampled of defined locations covering both ICUs. No SARS-CoV-2 ribonucleic acid (RNA) was detected. Gram-negative bacterial contamination was mainly linked to sinks and siphons. Skin flora was isolated from most swabbed areas and Enterococcus faecium was detected on two keyboards. CONCLUSIONS: After basic cleaning with standard disinfection measures no remaining SARS-CoV-2 RNA was detected. Bacterial contamination was low and mainly localised in sinks and siphons.

Evaluation of the RIDA®GENE RT-PCR assays for detection of sapovirus, astrovirus, adenovirus, and rotavirus in stool samples of adults in Switzerland.

Sapovirus (SaV) and astrovirus (AstV) increasingly are recognized as cause of acute viral gastroenteritis (AGE). We evaluated the real-time RT-PCR assays RIDA®GENE SaV and viral stool panel II (RGN RT-PCR) for detection of SaV, AstV, adenovirus (AdV) F40/41 and rotavirus (RoV) in clinical stool samples (n = 69). Results were compared with reference singleplex RT-PCRs. The sensitivity for SaV, AstV and RoV are 100%, the specificity ranges from 98.1% to 100%. In 10 out of 11 AdV (all types) samples, the RGN RT-PCR for AdV F40/41 displayed negative results. Retrospectively, 196 stool specimens from adult patients previously tested negative for norovirus (NoV) were analyzed. In about 10% of NoV-negative stool samples, AdV (n = 9), RoV (n = 6), AstV (n = 3) or SaV (n = 3) were found. The RGN RT-PCR assays are useful for detection of enteric viruses other than NoV. This study emphasizes the need for further testing of NoV-negative stool samples in patients with AGE.

Resolution of plasma sample mix-ups through comparison of patient antibody patterns to E. coli.

BACKGROUND: Accidental sample mix-ups and the need for their swift resolution is a challenge faced by every analytical laboratory. To this end, we developed a simple immunoblot-based method, making use of a patient's characteristic plasma antibody profile to Escherichia coli (E. coli) proteins. METHODS: Nitrocellulose strips of size-separated proteins from E. coli whole-cell lysates were incubated with patient plasma and visualised with an enzyme-coupled secondary antibody and substrate. Plasma samples of 20 random patients as well as five longitudinal samples of three patients were analysed for antibody band patterns, to evaluate uniqueness and consistency over time, respectively. For sample mix-ups, antibody band patterns of questionable samples were compared with samples of known identity. RESULTS: Comparison of anti-E. coli antibody patterns of 20 random patients showed a unique antibody profile for each patient. Antibody profiles remained consistent over time, as shown for three patients over several years. Three example cases demonstrate the use of this methodology in mis-labelling or -pipetting incidences. CONCLUSION: Our simple method for resolving plasma sample mix-ups between non-related individuals can be performed with basic laboratory equipment and thus can easily be adopted by analytical laboratories.

Unbiased metagenomic sequencing complements specific routine diagnostic methods and increases chances to detect rare viral strains.

Multiplex PCR assays for respiratory viruses are widely used in routine diagnostics, as they are highly sensitive, rapid, and cost effective. However, depending on the assay system, cross-reactivity between viruses that share a high sequence homology as well as detection of rare virus isolates with sequence variations can be problematic. Virus sequence-independent metagenomic high-throughput sequencing allows for accurate detection of all virus species in a given sample, as we demonstrate here for human Enterovirus and Rhinovirus in a lung transplant patient. While early in infection a commercial PCR assay recorded Rhinovirus, high-throughput sequencing correctly identified human Enterovirus C104 as the source of infection, highlighting the potential of the technology and the benefit of applying open assay formats in complex diagnostic situations.

Detection of antibodies to the feline leukemia Virus (FeLV) transmembrane protein p15E: an alternative approach for serological FeLV detection based on antibodies to p15E.

The aim of this report was to investigate whether the diagnosis of feline leukemia virus (FeLV) infection by serology might be feasible and useful. Among the various viral proteins, the FeLV env-gene product (SU) and the envelope transmembrane protein p15E were considered promising candidates for the serological diagnosis of FeLV infection. Thus, we evaluated p15E and three other FeLV antigens, namely, a recombinant env-gene product, whole FeLV, and a short peptide from the FeLV transmembrane protein, for their potential to detect FeLV infection. To evaluate possible exposure of cats to FeLV, we tested serum and plasma samples from experimentally and naturally infected and vaccinated cats for the presence of antibodies to these antigens by enzyme-linked immunosorbent assays (ELISAs). The serological results were compared with the p27 and proviral real-time PCR results. We found that p15E displayed a diagnostic sensitivity of 95.7% and a specificity of 100% in experimentally infected cats. In naturally infected cats, p15E showed a diagnostic sensitivity of 77.1% and a specificity of 85.6%. Vaccinated cats displayed minimal antibody levels to p15E, suggesting that anti-p15E antibodies indicate infection rather than vaccination. The other antigens turned out to be too unspecific. The lower specificity in cats exposed to FeLV under field conditions may be explained by the fact that some cats become infected and seroconvert in the absence of detectable viral nucleic acids in plasma. We conclude that p15E serology may become a valuable tool for diagnosing FeLV infection; in some cases, it may replace PCR.

Use of reverse-transcriptase-based HIV-1 viral load assessment to confirm low viral loads in newly diagnosed patients in Switzerland.

BACKGROUND: Treatment-naïve patients newly diagnosed with HIV occasionally present with low viral RNA of ≤1'000 copies/ml, raising concerns about viral load underestimation. Because falsely low or undetectable viral loads might lead to inadvertent virus transmission or treatment delays, confirmation of such cases by a sequence-independent viral load test is recommended in Switzerland. METHODS: HIV-1 RNA ≤1'000 cp/ml by Roche's or Abbott's tests in patients newly diagnosed from 2010 to 2012 in Switzerland were subjected to viral load testing by the product-enhanced-reverse transcriptase (PERT) assay. These investigations were complemented with repeat and/or alternative viral RNA measurements. RESULTS: HIV-1 RNA ≤1'000 cp/ml was observed in 71 of 1814 newly diagnosed patients. The PERT assay suggested clinically relevant viral load underestimation in 7 of 32 cases that could be investigated. In four patients, the PERT viral load was 10-1'000-fold higher; this was confirmed by alternative HIV-1 RNA tests. Six of the 7 underestimates had been obtained with meanwhile outdated versions of Roche's HIV-1 RNA test. In the seventh patient, follow-up revealed similar results for RNA and PERT based viral loads. CONCLUSION: PERT assay revealed occasional severe viral load underestimation by versions of HIV-1 RNA tests meanwhile outdated. Underestimation by contemporary tests appears rare, however.

Comparative performances of HIV-1 RNA load assays at low viral load levels: results of an international collaboration.

Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ∼100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

Investigations on the diagnosis and retroviral aetiology of renal neoplasia in budgerigars (Melopsittacus undulatus).

The high susceptibility of budgerigars (Melopsittacus undulatus) to neoplasia, and specifically renal neoplasia, has often been reported. Further investigations led to a suspicion of a retrovirus as the causative agent for renal neoplasia in budgerigars, but definitive proof has yet to be found. In the present study, 32 budgerigars suspected of having renal neoplasia (based on the clinical presentation) were examined. The objectives were to investigate the use of different diagnostic methods for the ante-mortem diagnosis of this condition and to find more supporting evidence of a retroviral aetiology. The predominant clinical signs observed in budgerigars with renal neoplasia were lameness and absence of deep pain sensation of one leg. Alterations in haematology, plasma chemistry, and urine analyses could not pinpoint the cases of renal neoplasia. Contrast radiography of the intestinal tract proved to be diagnostically more useful compared with plain radiographic studies. Histology confirmed the renal neoplasia as adenocarcinoma. Investigations for virus identification included product-enhanced reverse transcriptase assay and enzyme-linked immunosorbent assay for the detection of avian leucosis virus group-specific antigen. Cell cultures and electron microscopy were performed on a limited number of patients. These investigations could find no presence of an exogenous, replicating retrovirus, neither could viral particles be detected by electron microscopy. Based on the current findings, it can be concluded that there is no evidence of retroviral involvement in the occurrence of renal neoplasia in budgerigars.

Detection of reverse transcriptase activity in human melanoma cell lines and identification of a murine leukemia virus contaminant.

BACKGROUND: Stimulated by earlier reports on the presence of retroviruses in mouse and hamster melanoma cell lines, we addressed the question as to whether human melanoma cell lines might also harbour a retrovirus. METHODS AND RESULTS: The melanoma cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO, MML-I, MeWo, A-375, Colo-38, BS-780 were confirmed to be human by human leucocyte antigen (HLA) typing, and supernatants were tested by the product-enhanced reverse transcriptase (PERT) assay for reverse transcriptase (RT) activity. Cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO and MML-I were positive, whereas cell lines MeWo, A-375, Colo-38 and BS-780 were negative. The RT activity peaked at a buoyant density in sucrose typical for retroviruses. From this peak fraction an R-U5 sequence indistinguishable from murine leukemia virus (MLV) was identified by particle-associated retrovirus RNA amplification (PARRA). Semiquantitative MLV-specific RNA-PCR demonstrated colocalization of the MLV-like RNA and RT activity on the sucrose gradient of SK-Mel-25. MLV RNA and DNA were also detectable in culture supernatants of SK-MEL-28, MEL-JUSO and MML-I, but not of MeWo, A-375, Colo-38 and BS-780 by semiquantitative polymerase chain reaction (PCR). Sequence comparison revealed highest homology with the RET sequence previously identified in mouse myeloma SP2/0-AG14 cells. Taken together, our data strongly suggest that certain human melanoma cell lines are productively infected by a MLV which was probably introduced during tumour passage in mice or by laboratory contamination many years ago and subsequently spread to other lines. CONCLUSION: We recommend mandatory testing of melanoma and other human cell lines for contamination with infectious MLV or other animal retroviruses, similar to mycoplasma screening, in order to avoid artificial experimental data.

Direct evidence for natural transmission of small-ruminant lentiviruses of subtype A4 from goats to sheep and vice versa.

Small-ruminant lentiviruses (SRLV), which include the caprine arthritis-encephalitis and the maedi-visna virus, cause persistent inflammatory infections in goats and sheep. SRLV are mainly transmitted from mother to offspring through milk. Transmission after prolonged contact between adult animals has also been observed. The observation that certain SRLV subtypes are found in both goats and sheep suggests that interspecies transmission has occurred on several occasions in the past. We investigated seropositive goats and sheep that were kept together in small mixed herds. Phylogenetic analysis of long proviral sequences in gag and pol, combined with epidemiologic information, demonstrated natural sheep-to-goat transmission of the recently identified SRLV subtype A4 in two instances and goat-to-sheep transmission of the same subtype in one instance. In a further mixed cluster, the direction of the interspecies transmission could not be determined. These findings present for the first time direct evidence that natural interspecies transmission of SRLV is ongoing in both directions. The findings are of relevance to virus eradication programs in both species.

Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade.

We performed a phylogenetic analysis of caprine and ovine lentiviruses using long sequences in gag and pol of 104 new Swiss isolates and six available corresponding database sequences. Forty-five isolates, forming five sequence clusters, were unclassifiable by the present classification. Pairwise DNA distance analysis indicated different categories of relatedness, requiring a new classification system. We propose four principal sequence groups, A-D, which differ by 25-37%. Groups A and B are further divided into subtypes which differ by 15-27%. Group D and four of the seven group A subtypes, A3, A4, A5 and A7, are formed by new Swiss isolates. Molecular epidemiology revealed that Swiss B1 strains differed no more from French, Brazilian or US strains than from each other, suggesting virus propagation through international livestock trade. Furthermore, infection of goats by subtypes A3 or A4 was significantly associated with documented contact with sheep, which also harbor these subtypes, thus indicating regularly occurring sheep-to-goat transmission.

Identification and characterization of two closely related unclassifiable endogenous retroviruses in pythons (Python molurus and Python curtus).

Boid inclusion body disease (BIBD) is a fatal disorder of boid snakes that is suspected to be caused by a retrovirus. In order to identify this agent, leukocyte cultures (established from Python molurus specimens with symptoms of BIBD or kept together with such diseased animals) were assessed for reverse transcriptase (RT) activity. Virus from cultures exhibiting high RT activity was banded on sucrose density gradients, and the RT peak fraction was subjected to highly efficient procedures for the identification of unknown particle-associated retroviral RNA. A 7-kb full retroviral sequence was identified, cloned, and sequenced. This virus contained intact open reading frames (ORFs) for gag, pro, pol, and env, as well as another ORF of unknown function within pol. Phylogenetic analysis showed that the virus is distantly related to viruses from both the B and D types and the mammalian C type but cannot be classified. It is present as a highly expressed endogenous retrovirus in all P. molurus individuals; a closely related, but much less expressed virus was found in all tested Python curtus individuals. All other boid snakes tested, including Python regius, Python reticulatus, Boa constrictor, Eunectes notaeus, and Morelia spilota, were virus negative, independent of whether they had BIBD or not. Virus isolated from P. molurus could not be transmitted to the peripheral blood mononuclear cells of B. constrictor or P. regius. Thus, there is no indication that this novel virus, which we propose to name python endogenous retrovirus (PyERV), is causally linked with BIBD.

Feline leukaemia provirus load during the course of experimental infection and in naturally infected cats.

Feline leukaemia virus (FeLV) infection in domestic cats can vary in its outcome (persistent, transient, no infection) for reasons that are not entirely known. It was hypothesized that the initial virus and provirus load could significantly influence the course of retrovirus infection. To determine the role of provirus loads, two methods of PCR, a nested PCR and a fluorogenic probe-based (TaqMan) real-time quantitative PCR, which were specific to the U3 region of FeLV-A were established. FeLV provirus in naturally and experimentally infected cats was then measured. Only 3 weeks after experimental FeLV-A infection, persistently infected cats demonstrated higher provirus loads and lower humoral immune responses than cats that had overcome antigenaemia. Lower initial provirus loads were associated with successful humoral immune responses. Unexpectedly, provirus in the buffy-coat cells of two cats that tested negative for the p27 antigen (a marker for viraemia) was also detected. In 597 Swiss cats, comparison of p27 antigen levels with PCR results revealed broad agreement. However, similar to the experimental situation, a significant number of animals (10%) was negative for the p27 antigen and FeLV-positive by PCR. These cats had a mean provirus load 300-fold lower than that of animals testing positive for the p27 antigen. In conclusion, an association between the provirus load and the outcome of FeLV infection was found. Detection of provirus carriers should contribute to further the control of FeLV. In addition, quantification of provirus loads will lead to a better understanding of FeLV pathogenesis and anti-retrovirus protective mechanisms.