Immune System Deficiencies Do Not Alter SARS-CoV-2 Evolutionary Rate but Favour the Emergence of Mutations by Extending Viral Persistence.

During the COVID-19 pandemic, immunosuppressed patients showed prolonged SARS-CoV-2 infections, with several studies reporting the accumulation of mutations in the viral genome. The weakened immune system present in these individuals, along with the effect of antiviral therapies, are thought to create a favourable environment for intra-host viral evolution and have been linked to the emergence of new viral variants which strongly challenged containment measures and some therapeutic treatments. To assess whether impaired immunity could lead to the increased instability of viral genomes, longitudinal nasopharyngeal swabs were collected from eight immunocompromised patients and fourteen non-immunocompromised subjects, all undergoing SARS-CoV-2 infection. Intra-host viral evolution was compared between the two groups through deep sequencing, exploiting a probe-based enrichment method to minimise the possibility of artefactual mutations commonly generated in amplicon-based methods, which heavily rely on PCR amplification. Although, as expected, immunocompromised patients experienced significantly longer infections, the acquisition of novel intra-host viral mutations was similar between the two groups. Moreover, a thorough analysis of viral quasispecies showed that the variability of viral populations in the two groups is comparable not only at the consensus level, but also when considering low-frequency mutations. This study suggests that a compromised immune system alone does not affect SARS-CoV-2 within-host genomic variability.

Predictors of urinary tract infection after lower gastrointestinal surgery.

BACKGROUND: Urinary tract infections (UTIs) cause postoperative morbidity in patients undergoing lower gastrointestinal (GI) surgery that can prolong postoperative hospital stays. In patients with a fever of unknown origin (FUO), clinicians ignore what to do while waiting for the results of the urine culture test. This study aimed to develop a nomogram predicting UTI in the case of postoperative FUO. METHODS: This observational, retrospective study included all consecutive patients from 1 November 2020 to 1 November 2021 undergoing lower-GI surgery at the Chirurgia Generale 3, University Hospital of Padua, Italy. A nomogram was created and externally validated in 90 consecutive patients undergoing urine culture tests for FUO at the Chirurgia Oncologica Unit, Veneto Institute of Oncology. RESULTS: In the development cohort, 109 (N = 109) patients performed a urine culture test for FUO, and 39 were diagnosed with UTI. In a multivariate analysis of patients who underwent urine culture tests for FUO, UTI was associated with female sex, older age, and duration of catheterization at the date of the urine culture test. We developed a nomogram to predict UTI in surgical patients with a C-index of 0.76. In the validation cohort, 90 consecutive patients, who had lower-GI surgery, underwent a urine culture test for FUO and were tested with this nomogram. In the validation cohort, the C-index of the nomogram for predicting a positive urine culture test was 0.71. CONCLUSIONS AND RELEVANCE: UTIs are a common problem in patients undergoing lower-GI surgery. A nomogram including the major risk factors may help to reduce the inappropriate use of antibiotics during the period awaiting the result of the urine culture test.

Reconstruction of the Maxillary Sinus After Previous Major Sinus Elevation Failure: A Study with a 3-Year Follow-up.

Fourteen maxillary sinuses were reconstructed in 12 patients who presented with a previous sinus elevation failure. In all cases, large perforations of the sinus membrane occurred during removal of the failed graft from the sinus; the perforations were sealed with fibrin glue, then the site was grafted using homologous fibrin glue (HFG) mixed with a calcium phosphate scaffold (CPS). Histologic analyses revealed that the CPS-HFG graft was followed by an ossification process, with the formation of spongy bone similar to that of the normal skeleton. Twenty-four endosseous implants were successfully placed into the newly regenerated bone. All implants were successfully restored with ceramic crowns 6 months after placement. At the 3-year follow-up, no infections or implant failures were reported. The described technique offers several clinical advantages, as the removal of the failed graft, the sinus perforation repair, and the sinus elevation can be achieved in the same surgery without needing to postpone the regenerative surgery phase.

Neutralising reactivity against SARS-CoV-2 Delta and Omicron variants by vaccination and infection history.

BACKGROUND: The continuous emergence of SARS-CoV-2 variants of concern (VOC) with immune escape properties, such as Delta (B.1.617.2) and Omicron (B.1.1.529), questions the extent of the antibody-mediated protection against the virus. Here we investigated the long-term antibody persistence in previously infected subjects and the extent of the antibody-mediated protection against B.1, B.1.617.2 and BA.1 variants in unvaccinated subjects previously infected, vaccinated naïve and vaccinated previously infected subjects. METHODS: Blood samples collected 15 months post-infection from unvaccinated (n=35) and vaccinated (n=41) previously infected subjects (Vo' cohort) were tested for the presence of antibodies against the SARS-CoV-2 spike (S) and nucleocapsid (N) antigens using the Abbott, DiaSorin, and Roche immunoassays. The serum neutralising reactivity was assessed against B.1, B.1.617.2 (Delta), and BA.1 (Omicron) SARS-CoV-2 strains through micro-neutralisation. The antibody titres were compared to those from previous timepoints, performed at 2- and 9-months post-infection on the same individuals. Two groups of naïve subjects were used as controls, one from the same cohort (unvaccinated n=29 and vaccinated n=20) and a group of vaccinated naïve healthcare workers (n=61). RESULTS: We report on the results of the third serosurvey run in the Vo' cohort. With respect to the 9-month time point, antibodies against the S antigen significantly decreased (P=0.0063) among unvaccinated subjects and increased (P<0.0001) in vaccinated individuals, whereas those against the N antigen decreased in the whole cohort. When compared with control groups (naïve Vo' inhabitants and naïve healthcare workers), vaccinated subjects that were previously infected had higher antibody levels (P<0.0001) than vaccinated naïve subjects. Two doses of vaccine elicited stronger anti-S antibody response than natural infection (P<0.0001). Finally, the neutralising reactivity of sera against B.1.617.2 and BA.1 was 4-fold and 16-fold lower than the reactivity observed against the original B.1 strain. CONCLUSIONS: These results confirm that vaccination induces strong antibody response in most individuals, and even stronger in previously infected subjects. Neutralising reactivity elicited by natural infection followed by vaccination is increasingly weakened by the recent emergence of VOCs. While immunity is not completely compromised, a change in vaccine development may be required going forward, to generate cross-protective pan-coronavirus immunity in the global population.

Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study.

Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available. Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells. Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R 2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550-8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease. Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.

Frontline Screening for SARS-CoV-2 Infection at Emergency Department Admission by Third Generation Rapid Antigen Test: Can We Spare RT-qPCR?

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96-98), and a sensitivity of 85% (95% CI: 82-89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86-95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657-41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.

SARS-CoV-2 RNA in plasma samples of COVID-19 affected individuals: a cross-sectional proof-of-concept study.

BACKGROUND: Recent studies showed that plasma SARS-CoV-2 RNA seems to be associated with worse COVID-19 outcome. However, whether specific population can be at higher risk of viremia are to date unexplored. METHODS: This cross-sectional proof-of-concept study included 41 SARS-CoV-2-positive adult individuals (six affected by haematological malignancies) hospitalized at two major hospital in Milan, for those demographic, clinical and laboratory data were available. SARS-CoV-2 load was quantified by ddPCR in paired plasma and respiratory samples. To assess significant differences between patients with and patients without viremia, Fisher exact test and Wilcoxon test were used for categorical and continuous variables, respectively. RESULTS: Plasma SARS-CoV-2 RNA was found in 8 patients (19.5%), with a median (IQR) value of 694 (209-1023) copies/mL. Viremic patients were characterized by an higher mortality rate (50.0% vs 9.1%; p = 0.018) respect to patients without viremia. Viremic patients were more frequently affected by haematological malignancies (62.5% vs. 3.0%; p < 0.001), and had higher viral load in respiratory samples (9,404,000 [586,060-10,000,000] vs 1560 [312-25,160] copies/mL; p = 0.002). CONCLUSIONS: Even if based on a small sample population, this proof-of-concept study poses the basis for an early identification of patients at higher risk of SARS-CoV-2 viremia, and therefore likely to develop severe COVID-19, and supports the need of a quantitative viral load determination in blood and respiratory samples of haematologic patients with COVID-19 in order to predict prognosis and consequently to help their further management.

Genomic epidemiology of SARS-CoV-2 reveals multiple lineages and early spread of SARS-CoV-2 infections in Lombardy, Italy.

From February to April 2020, Lombardy (Italy) reported the highest numbers of SARS-CoV-2 cases worldwide. By analyzing 346 whole SARS-CoV-2 genomes, we demonstrate the presence of seven viral lineages in Lombardy, frequently sustained by local transmission chains and at least two likely to have originated in Italy. Six single nucleotide polymorphisms (five of them non-synonymous) characterized the SARS-CoV-2 sequences, none of them affecting N-glycosylation sites. The seven lineages, and the presence of local transmission clusters within three of them, revealed that sustained community transmission was underway before the first COVID-19 case had been detected in Lombardy.

Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients.

Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.

A Step-by-Step Procedure for Bone Regeneration Using Calcium Phosphate Scaffolds: From Site Preparation to Graft Placement.

Hydroxyapatite (HA) is the main mineral component of bone and teeth. HA is often used as a bone substitute and especially in its granular form it is osteoconductive and osseointegrating as shown by many investigations in animals and humans. HA granules are used for filling bone defects, but they have poor handling qualities and retention at the surgical site, leading to graft voids between the granules and bone tissue and resulting in mechanical instability. Furthermore HA implantation is not constantly followed by favorable results, especially when it is carried out for augmentation of the alveolar ridge. This article offers a protocol for bone reconstruction and predictable implant treatment outcomes. We provide a step-by-step description of both the recipient site and composite graft preparations using coralline HA granules (CHAG) and homologous fibrin glue (HFG). In the present study, 20 randomly selected patients (12 women and 8 men) underwent bone regeneration using CHAG-HFG before a dental implant procedure. Radiographic imaging, physical examination, and histological analysis were performed during a 2-year period. Biopsies were obtained at second-stage surgery before implant insertion using a 2.8-mm trephine bur. A morphological study of 20 bioptic human specimens was performed. Our results demonstrate that this surgical protocol for the preparation of the recipient site associated with a mixture of coralline HA granules with homologous fibrin glue provides reliable bone regeneration, thus reducing failures and minimizing risks of postoperative morbidity.

Benign Paroxysmal Positional Vertigo After Piezosurgical Removal of Osseointegrated Implants.

With the increasing popularity of dental implants, the explantation of fractured implants has become a major challenge for clinicians. Several tools can be used for the removal of osseointegrated implants; however, few of these have the characteristics of easy control, selective cutting, and rapid healing. We report a case of benign paroxysmal positional vertigo (BPPV) developed immediately after piezosurgical removal of osteointegrated implants.The BPPV may be an unpleasant complication of piezosurgical removal of osseointegrated implants and may cause considerable stress if not identified correctly and managed properly.

A Step-By-Step Technique for the Piezosurgical Removal of Fractured Implants.

With the increasing popularity of dental implants, the removal of fractured implants has become a major challenge for clinicians. Several tools can be used for the removal of osseointegrated implants; however, few of these have the characteristics of easy control, selective cutting, and rapid healing. In this study, the authors describe a step-by-step technique for the removal of osseointegrated fractured implants via multiple peri-implant osteotomies performed using a piezoelectric device and appropriate inserts. All patients ended with primary wound closure without any soft tissue dehiscence and no healing problems during the postoperative period. As the use of ultrasonic inserts enables precise and selective cuts, piezosurgical implant removal is an attractive alternative to trephine burs or rotary drills.

A literature review and hypothesis for the etiologies of cervical and root caries.

The presence of endogenous acids from bacteria acting on a suitable substrate combined with sources of exogenous biocorrosives such as exogenous acids and proteolytic enzymes in areas of stress concentration are hypothesized to lead to the development and progression of cervical and root caries (RC). Quantifying the effects of each of the mechanisms (stress and biocorrosion) is a daunting task to investigate since so many factors are involved at various times in the etiology of noncarious cervical lesions (NCCLs), cervical caries (CC), and RC. Frictional action of the tongue has a cleansing effect and lingual serous saliva, which has a high flow rate buffering capacity from bicarbonates seem to account for the paucity of lingual NCCLs, cervical, and RC in these areas of teeth. Future studies are indicated to determine the effects of stress and biocorrosion and their factors in the etiology of CC and RC. CLINICAL SIGNIFICANCE: This manuscript presents hypothetical and literary information that the combined effects of stress concentration and biocorrosion contribute to the formation as well as progression of cervical and root caries.

Metformin associated lactic acidosis (MALA): clinical profiling and management.

Metformin (MF) accumulation during acute kidney injury is associated with high anion gap lactic acidosis type B (MF-associated lactic acidosis, MALA), a serious medical condition leading to high mortality. Despite dose adjustment for renal failure, diabetic patients with chronic kidney disease (CKD) stage III-IV are at risk for rapid decline in renal function by whatever reason, so that MF toxicity might arise if the drug is not timely withdrawn. Sixteen consecutive patients were admitted to our Hospital's Emergency Department with clinical findings consistent with MALA. Fifteen had prior history of CKD, 60 % of them with GFR between 30 and 60 ml/min. Of these, 5 required mechanical ventilation and cardiovascular support; 3 promptly recovered renal function after rehydration, whereas 10 (62 %) required continuous veno-venous renal replacement treatment. SOFA and SAPS II scores were significantly related to the degree of lactic acidosis. In addition, lactate levels were relevant to therapeutic choices, since they were higher in dialyzed patients than in those on conservative treatment (11.92 mmol/l vs 5.7 mmol/l, p = 0.03). The overall death rate has been 31 %, with poorer prognosis for worse acidemia, as serum pH was significantly lower in non-survivors (pH 6.96 vs 7.16, p > 0.04). Our own data and a review of the literature suggest that aged, hemodynamically frail patients, with several comorbidities and CKD, are at greater risk of MALA, despite MF dosage adjustment. Moreover, renal replacement therapy rather than simple acidosis correction by administration of alkali seems the treatment of choice, based on eventual renal recovery and overall outcome.

The removal of a malpositioned implant in the anterior mandible using piezosurgery.

In oral, cranio, and maxillofacial surgery, a close relationship among the bone, nerves, and blood vessels can be regularly observed. Surgical procedures for the removal of dental implants have the potential to cause vascular injury and bleeding in the floor of the mouth and internal anterior region of the mandible. Furthermore, conventional osteotomy techniques always require extensive protection of adjacent soft tissue because cutting is not limited to bone and could easily affect other tissues when applied improperly. We report the removal by means of piezosurgery of a malpositioned osseointegrated implant that had previously caused a sublingual hematoma during its insertion. The postoperative course was uneventful, no bleeding, infection, or hematoma formation was noted and the patient reported 100% resolution of all symptoms.