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Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of COVID-19 infection. Due to pre-analytical and technical limitations, samples with low viral load are often misdiagnosed as false-negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT-qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID-19 were analyzed by droplet digital PCR (ddPCR) and RT-qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)-approved probe for the SARS-CoV-2 N gene were used. SYBR-Green RT-qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT-qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT-qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10-fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT-qPCR for the diagnosis of COVID-19 infection in false-negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID-19 and the follow-up of positive patients until complete remission.

The advent of disease modifying therapies in spinal muscular atrophy (SMA) has increased life expectancy but also raising new challenges. We aimed to explore the neurobehavioral profile in SMA type I subjects and in those identified by newborn screening (NBS). Behavioral assessment included screening questionnaires (strengths and difficulties questionnaire (SDQ), social communication questionnaire (SCQ), and sensory profile 2 (SP2)), neurobehavioral observation, CARS2 and DSM-5 criteria. The cohort included thirty-one children (25 type I and 6 NBS) aged 2–10 years. On SDQ prosocial scale, 14/31 showed borderline or abnormal results. 6/14 had borderline scores at the SCQ questionnaire, while none had abnormal scores. Neurobehavioral observation suggested the presence of ASD in 3/31, confirmed by CARS2 and DSM-5 criteria. 5/31 showed other behavioral disorders. Our findings suggest that autism is present in SMA infants in a percentage slightly higher than in the general population. Other neurobehavioral difficulties are less frequent. Our study highlighted the challenges to select appropriate tools in infants with limited mobility and the need for a clear diagnostic pathway, starting with screening questionnaires followed by more appropriate diagnostic tools to reduce the number of false positive results.

Objective The aim of this paper was to report the 2‐year follow‐up in type I patients treated with Nusinersen and to assess whether possible changes in motor function are related to the subtype, age, or SMN2 copy number. Methods Sixty‐eight patients, with ages ranging from 0.20 to 15.92 years (mean: 3.96; standard deviation: +3.90) were enrolled in the study. All patients were assessed using the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) and the developmental section of the Hammersmith Infant Neurological Examination (HINE‐2) at the time they started treatment and 12 and 24 months after that. Results For both CHOP and HINE‐2 repeated measures analysis of variance showed a significant difference (P < 0.001) between baseline and 12 months, 12 months and 24 months, and baseline and 24‐month scores for the whole group. When age subgroups (<210 days, <2 years, 2–4 years, 5–11 years, 12–18 years) were considered, on the CHOP INTEND the difference was significant between baseline and 24 months in all age subgroups. On the HINE‐2, the difference between baseline and 24 months was significant in all the subgroups before the age of 4 years. Age was predictive of changes on both scales (P < 0.05), whereas SMN2 copy number and decimal classification were not. Interpretation Our results suggest that some improvement of motor function can be observed even after the first year of treatment. This is more obvious in the infants treated in the first 2 years but some improvement can also be found in older children.

Invasive pulmonary aspergillosis (IPA) reports significant mortality rates among critically ill patients. A prompt microbiological diagnosis is essential to establish a coherent antifungal treatment. Despite its low sensitivity and prolonged turn-around time, culture represents the conventional diagnostic technique. Additionally, galactomannan detection may support the diagnostic process. Ultimate generation methods, such as the real-time polymerase chain reaction (Real-Time PCR), integrated the diagnostic procedure to improve the overall laboratory effectiveness, especially regarding a quantitative Aspergillus spp. DNA detection. Herein, we propose a retrospective analysis where a quantitative real-time PCR was performed on respiratory samples belonging to patients with or without probable pulmonary aspergillosis. The study enrolled 62 samples, whose PCR results were compared to culture and galactomannan indexes. Additionally, clinical and general data were collected for all the patients. The qPCR assay reported 100% sensitivity and negative predictive value, while specificity reached 59.2% and the positive predictive value was 76.1%. Moreover, IPA patients reported fungal DNA loads higher than 103 in a logarithmic scale, while non-aspergillosis episodes reported a maximum level of 103. We hypothesized a future possibility to define a specific cut-off in distinguishing colonization from infection cases, requiring further investigations and speculations about IPA patients and respiratory samples.

The study reports real world data in type 2 and 3 SMA patients treated for at least 2 years with nusinersen. Increase in motor function was observed after 12 months and during the second year. The magnitude of change was variable across age and functional subgroup, with the largest changes observed in young patients with higher function at baseline. When compared to natural history data, the difference between study cohort and untreated patients swas significant on both Hammersmith Functional Motor Scale and Revised Upper Limb Module both at 12 months and at 24 months.

Molecular techniques recently integrated the candidiasis diagnostic workflow, avoiding the culture-based prolonged turn-around time and lack of sensitivity. The present retrospective study evaluated the OLM CandID Real-Time PCR on serum samples in the early and rapid candidaemia diagnosis among ICU patients. The final purpose of the protocol was to demonstrate the effectiveness of a PCR assay in the invasive candidiasis diagnostic workflow due to the high sensitivity rates and species identification possibility. The evaluation screened 60 suitable patients, accounting for 10 probable and 7 proven candidiasis cases. Patients with at least a positive (1→3)-β-D-glucan (BDG) value underwent molecular procedures. A sensitivity of 83.3%, a specificity of 94.3%, a positive predictive value of 87.5%, and a negative predictive value of 91.7% emerged for the PCR assay. As a conclusion, Candida PCR assays may represent useful diagnostic assistance tools when applied together with serological markers and culture-based assays.

Objective This study investigated myostatin levels in SMA patients receiving disease‐modifying therapies (DMTs) to understand their relationship with treatment duration and functional status. Methods Our study includes both cross‐sectional and longitudinal analyses of myostatin levels in treated SMA patients. The longitudinal cohort included 46 treatment‐naive patients assessed at baseline and 12 months post‐treatment. Myostatin levels were measured using ELISA. Age‐matched controls (n = 89) were included for comparison. The cross‐sectional study included 128 patients with variable durations of treatment (from 0.4 to 7.2 years). In both cohorts, myostatin levels were correlated with SMA type, functional status, and clinical outcomes. Results Baseline myostatin levels were significantly lower than controls (p < 0.001), except during the neonatal period in presymptomatic patients. After 12 months of treatment, there were no significant changes compared to baseline levels (p = 0.1652). The only substantial changes were observed in presymptomatic neonates, who showed a reduction of myostatin despite treatment intervention. There was a significant correlation between myostatin levels, functional status, and SMA type both in the cross‐sectional and longitudinal groups. Interpretation This study demonstrates lower myostatin levels in SMA patients compared to controls. The association between myostatin levels, functional status, and SMA type suggests its possible role as a disease severity biomarker. The utility of myostatin as a biomarker for DMT response remains controversial; while we observed no significant increase in myostatin levels following treatment, we also did not observe the progressive reduction previously reported in untreated patients.

Isavuconazole is a new broad-spectrum triazole, with significant in vitro activity against yeasts. Isavuconazole in vitro susceptibility can be evaluated through broth microdilution as a reference method. Considering difficulties in equipping such methods in a laboratory routine, a commercial MIC Strip test has been designed. This study aims to implement data about isavuconazole in vitro activity and compare EUCAST broth microdilution and MIC Strip test in defining yeast isavuconazole susceptibility. The study involved 629 isolates from positive blood cultures (January 2017–December 2021). The identified species were C. albicans (283), C. glabrata (53), C. krusei (23), C. tropicalis (68), C. parapsilosis complex (151), C. guilliermondii (12), C. famata (6), S. cerevisiae (12), C. neoformans (5), S. capitata (12), and Rhodotorula species (4). All the isolates were tested with EUCAST microdilution and MIC Strip methods. The total essential agreement between these two methods was 99.3%. As a result, we can consider that both methods are useful in testing isavuconazole susceptibility. Proposed cut-off values (P-ECOFF) were calculated using ECOFFinder software. Further studies could lead to either definitive E-COFF or clinical breakpoints, which represent the most important categorization tool of the laboratory data, allowing a better insertion of an antimicrobial drug in clinical practice.

Parvovirus B19 (B19V) re-emerged across Europe in 2024, raising concerns about vertical transmission and neonatal morbidity. We undertook a prospective, single-centre cohort study to characterise the early clinical course of congenitally infected neonates born between April and December 2024. Seventy-one pregnancies with serologically or PCR-confirmed maternal infection were enrolled; seven neonates met laboratory criteria for congenital B19V infection and were followed with serial clinical, biochemical and imaging assessments through the first year of life. Troponin I and CK-MB were measured on days 1, 3, 7 and 15; electrocardiogram (ECG) and echocardiography were repeated in parallel, and cranial ultrasound (US), ophthalmologic and audiologic screening were scheduled prospectively. Mean troponin rose from 50.7 ng L−1 on day 1 to a peak of 120.7 ng L−1 on day 7 (p < 0.01), normalising by one month, while echocardiograms remained structurally normal, and only one transient arrhythmia was recorded. CK-MB exceeded the reference range in 29% of infants but showed no clinical sequelae. Multiple periventricular hyperechogenicities were identified in 8/70 neonates (11%), and moderate anaemia (Hb ≤ 9.8 g/dL) occurred in 2 cases. Serum PCR detected high-level viraemia (>108 genome equivalents mL−1) in 40% of those tested; saliva and urine were consistently negative. No instances of myocarditis or hydrops were observed. Our findings indicate that congenital B19V infection during the current outbreak is marked by transient biochemical myocardial stress and subtle neurosonographic changes rather than overt cardiac disease, supporting an outpatient-focused follow-up strategy incorporating serial biomarkers and targeted neuroimaging.

Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.