Detection of SARS-CoV-2 Δ426 ORF8 Deletion Mutant Cluster in NGS Screening.

Next-generation sequencing (NGS) from SARS-CoV-2-positive swabs collected during the last months of 2022 revealed a large deletion spanning ORF7b and ORF8 (426 nt) in six patients infected with the BA.5.1 Omicron variant. This extensive genome loss removed a large part of these two genes, maintaining in frame the first 22 aminoacids of ORF7b and the last three aminoacids of ORF8. Interestingly, the deleted region was flanked by two small repeats, which were likely involved in the formation of a hairpin structure. Similar rearrangements, comparable in size and location to the deletion, were also identified in 15 sequences in the NCBI database. In this group, seven out of 15 cases from the USA and Switzerland presented both the BA.5.1 variant and the same 426 nucleotides deletion. It is noteworthy that three out of six cases were detected in patients with immunodeficiency, and it is conceivable that this clinical condition could promote the replication and selection of these mutations.

One-Year Surveillance of SARS-CoV-2 Exposure in Stray Cats and Kennel Dogs from Northeastern Italy.

Dogs and cats are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). During the pandemic, several studies have been performed on owned cats and dogs, whereas limited data are available on the exposure to stray animals. The objective of this study was to investigate the exposure to SARS-CoV-2 of feral cats and kennel dogs in northeastern Italy, through serological and molecular methods. From May 2021 to September 2022, public health veterinary services collected serum, oropharyngeal, and rectal swab samples from 257 free-roaming dogs newly introduced to shelters, and from 389 feral cats examined during the routinely trap-neutered-return programs. The swabs were analyzed for viral RNA through a real-time reverse transcriptase PCR (rRT-PCR), and sera were tested for the presence of the specific antibody against SARS-CoV-2 (enzyme-linked immunosorbent assay). Serology was positive in nine dogs (9/257) and three cats (3/389), while two asymptomatic cats tested positive to rRT-PCR. One cat turned out to be positive both for serology and molecular analysis. In addition, this study described the case of a possible human-to-animal SARS-CoV-2 transmission in a cat that travelled in close contact to a COVID-19-positive refugee from Ukraine. This study shows that SARS-CoV-2 can infect, in natural conditions, stray cats and kennel dogs in northeastern Italy, although with a low prevalence.

Corneal transplantation during the COVID-19 pandemic: An operational guide.

PURPOSE: To provide an operational guide for corneal transplantation during the COVID-19 pandemic aimed to maintain surgery and avoid spreading of SARS-CoV-2. METHODS: Prospective observational case series study in patients requiring corneal graft manage toward separate free and restricted pathways for those COVID-19 negative or positive, respectively. RESULTS: During the national lockdown, 30 consecutive patients underwent endothelial (n = 16), penetrating (n = 9), and anterior lamellar keratoplasty (n = 5). Two patients followed the COVID-19 restricted pathway, as they were considered positive while waiting for test results. Nine patients were hospitalized one night in the hospital. On admission to the hospital before surgery, at surgery, the day after surgery and at 7 and 30 days all patients and health-care personnel showed no symptoms and resulted negative at risks factors/exposure to the SARS-CoV-2 infection and occurrence of COVID-19. Nucleic acid testing resulted not detectable in all patients and SARS-CoV-2 antibodies quantification showed IgG and IgM below the positive predicted value in 29 patients. One patient showed IgM above the cut-off of significance (1.21 and 1.03 preoperative and 1-month postoperative, respectively) that were considered irrelevant because of the absence of symptoms and exposure risks. CONCLUSIONS: The concept of donor emergency (i.e. short-term availability of transplant tissues), makes corneal transplantation an always-urgent activity because it is related to the availability of the corneal tissue from a donor. Modest adjustments to ophthalmic clinic and eye surgery organization are required to maintain surgery and care of eye patients in a safe environment.

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR.

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

Significance of anti-HBc only in blood donors: a serological and virological study after hepatitis B vaccination.

BACKGROUND: Blood donors positive only for anti-HBc may have a resolved hepatitis B virus (HBV) infection, low grade chronic infection or infection with variant strains of HBV. We aimed to assess the significance of this serological pattern after hepatitis B vaccination in such cases. MATERIALS AND METHODS: Twenty-four anti-HBc only blood donors were vaccinated with the Engerix HBV vaccine and a serological and virological evaluation was performed before HBV vaccination and 7-10 days after each dose. Subjects were classified as non-responders if their anti-HBs levels stayed below 10 IU/L after full vaccination, while the response was considered secondary (anamnestic) if anti-HBs levels rose over 10 IU/L after the first vaccine dose, and primary if anti-HBs levels rose over 10 IU/L only after the second or third vaccine dose. RESULTS: Of the 21 fully evaluable donors, six had no response, eight showed a primary response and seven had an anamnestic response. One non-responder had transient positivity for HBV-DNA at low levels (12 IU/mL) with persistent negativity for HBsAg. DISCUSSION: Anti-HBc-only positive blood donors are a heterogeneous population including HBV naïve subjects with a likely false-positive anti-HBc reactivity, subjects with a resolved HBV infection, and subjects with persistent low-level HBV replication. The analysis of the anti-HBs response after a dose of HBV vaccine may help to distinguish among the different causes of the isolated anti-HBc positivity, thereby enabling proper counselling and potential readmission to blood donation.

A multicenter evaluation of the Abbott RealTime HCV Genotype II assay.

Genotype determination is recommended before starting anti-HCV therapy to determine the duration of treatment (PEG-Interferon+ribavirin). The Versant HCV Genotype 2.0 assay, based on the reverse hybridization of the 5'UTR segment and core region of hepatitis C virus (HCV), has been one of the assays used most widely for HCV genotyping. A multicenter evaluation of the more automated Abbott RealTime HCV Genotype II assay was carried out on 124 HCV positive sera tested previously with the Versant HCV Genotype 2.0 assay. There was good agreement between the two assays. Type concordance was 95.9% (117/122) while concordance at the subtype level for genotype 1 was 95.6% (43/45). The Abbott RealTime HCV Genotype II assay is automated, allowing a substantial reduction of time-to results and hands-on time. The combined features of full automation, objective interpretation and digital archiving make this assay useful in a diagnostic setting.