Cristina Mazzi

Background: Whipple disease (WD) is a rare, potentially fatal infection caused by Tropheryma whipplei (TW). Central Nervous System involvement (CNS‐WD) is a severe complication of WD with a challenging diagnosis, due to limited data integrating neurological features, imaging, cerebrospinal fluid analysis, PCR, and intestinal histopathology. Prompt recognition and treatment are essential to improve patient outcomes. Methods: A scoping review of the literature from 2000 to 2025 was conducted, including all CNS‐WD case reports and series, to outline its clinical spectrum and diagnostic workup. Results: Included studies were 89, accounting for 111 CNS‐WD cases. Most patients were male (79.8%), with a mean age of 53.6 years. Notably, 13.2% presented with CNS involvement in the absence of systemic WD. Pathognomonic signs were rare (13/111, 11.7%). Most patients (82.9%, 92/111) presented with two or more neurological symptoms, the most frequently reported being altered mentation, followed by ataxia, eye movement abnormalities, and other movement disorders. CSF was abnormal in 74.7% (56/75) of patients and TW PCR was positive in 75% (48/64); 64% (55/86) of cases showed intestinal histopathology consistent with WD. TW PCR on gastrointestinal biopsies was positive in 67.9% (19/28) cases. CNS imaging revealed abnormalities in 85.4% (82/96) of patients, with no distinctive pattern. Conclusions: CNS‐WD diagnosis remains challenging due to nonspecific symptoms, variable imaging, and occasional discordance among CSF, TW PCR or biopsy findings. Our review suggests that CNS‐WD should be considered in the differential diagnosis of unexplained neurological syndromes, even in patients with atypical clinical or radiological profiles, supporting broader indications for TW testing.

Background: Data on tick-borne diseases (TBDs) in Italy are fragmented. This study aimed to describe tick-borne pathogens (TBPs) and TBDs observed in a hospital in Italy and to assess the correlation between TBPs and human disease. Methods: A prospective observational study was conducted in a single hospital between March 2024 and January 2025. Inclusion criteria were: individuals ≥ 8 years of age and consent to study participation. Data and blood samples were collected at baseline (T0), and 12–18 weeks later (T1). Ticks collected at T0 were pooled and analyzed by real-time PCR. Serological and molecular tests were used for TBD diagnosis. Results: Ninety-three individuals (median age 39 years; 52.7% female) were enrolled. The pathogens most frequently detected in the tick pools were Rickettsia spp. (15.1%) and Borrelia spp. (7.5%). Sixteen participants (17.2%) developed TBD-compatible symptoms, but TBD was confirmed in only four cases: Lyme borreliosis (n = 2), scalp eschar and neck lymphadenopathy after tick bite (SENLAT, n = 1), and tick-borne encephalitis (TBE, n = 1). Concordance between TBP detection and the corresponding TBD development in humans was observed in only two cases (2.1%). Conclusions: The low concordance between pathogens detected in participant-level tick pools and confirmed human tick-borne disease supports the limited clinical value of tick testing in this setting.

This study aimed primarily to evaluate the performance of two Conformité Européenne—In Vitro Diagnostic (CE-IVD) multiplex real-time PCR (rt-PCR) assays for the molecular identification of tick-borne pathogens (TBPs) of human interest on ticks removed from human skin and collected through a citizen science-based approach. As a secondary objective, the aggregated results were used to describe tick species distribution, developmental stages, and seasonal TBP circulation in 2024 in the considered area. The comparison was conducted on 116 tick samples collected in 2024 voluntarily delivered to a hospital in northeastern Italy. Detected TBPs were further confirmed with in-house-validated PCR methods and, where applicable, resolved to the species level. Clinically relevant pathogen species were identified as single infections or coinfections. Overall, 33.6% of tick samples tested positive for at least one TBP, and 6.9% showed coinfections. Kit B exhibited a higher detection rate for Borrelia spp. and Rickettsia spp. targets, partly reflecting its broader diagnostic specificity, while statistically significant differences in cycle threshold values were observed for Anaplasma phagocytophilum detection. The most frequently involved ticks were Ixodes ricinus nymphs, and the most represented area was Verona province. Late spring and early summer were identified as the periods with the highest tick conferment and pathogen diversity. Overall, the results support the use of multiplex real-time PCR commercial kits combined with citizen science-based tick collection as an effective approach for both diagnostic screening and regional surveillance of circulating ticks and TBPs.

Background: Chemoimmunotherapy combinations represent the standard first-line treatment for non-oncogene addicted metastatic NSCLC (mNSCLC). However, evidence in elderly patients remains limited and conflicting. We conducted an analysis of the efficacy and safety of chemoimmunotherapy in patients aged ≥75 years enrolled in the Real-Combo Lung study, an observational study including patients with non-oncogene-addicted mNSCLC and PD-L1 expression < 50%. Patients and Methods: The primary objective of the study was to compare progression-free survival (PFS) and overall survival (OS) between patients aged ≥75 (elderly cohort) and those aged p = 0.29). The median OS was 17.5 months (95% CI: 14.7–NR) versus 21.4 months (95% CI: 17–NR), respectively (unadjusted HR 1.09, 95% CI: 0.76–1.56, p = 0.63). In multivariable analysis, ECOG PS ≥ 2 and baseline use of steroids were significantly associated with a worse outcome in the elderly cohort for both PFS and OS. Safety data did not differ significantly between cohorts. Conclusions: In this real-world study, elderly patients with mNSCLC derived outcomes comparable to those of younger patients, with similar efficacy and a manageable safety profile when treated with chemoimmunotherapy combinations.

Extracellular vesicles (EVs) from human body fluids are valuable tools for biomarker discovery and for exploring the mechanisms underlying various pathologies, including infectious diseases. The translation of EV research into clinical practice is however hindered by the variability in EV pre-clinical investigations. Therefore, standardisation of analytical procedures and reporting policies is essential. Human serum is a key biological matrix for biomarker discovery and is commonly stored within biobanks. Here, we investigated different strategies for EV enrichment from small volumes of biobanked serum and evaluated their impact on EVs’ downstream analyses. EVs were obtained from 250 μL of biobanked serum using ultracentrifugation (UC), size-exclusion chromatography-based methods (ExoSpin-ES, qEV1-35 nm, and qEV1-70 nm), or ExoRNeasy (ER). The resulting EVs were subsequently characterised for morphology, concentration, surface phenotype, and multi-omics profiles. All methods successfully enriched small EVs expressing tetraspanins on their surface, although at different concentrations. The most efficient method for proteomics analyses was qEV1-70 nm, followed by ES, which was more susceptible to contamination by serum proteins. EV-miRNA cargo was effectively profiled in UC-, ES-, and ER-EVs, with the latter providing the broadest miRNA coverage. Our results support the feasibility of using biobanked serum for EV-based research and further highlight the importance of selecting appropriate EV enrichment methods, since they influence both miRNA and protein cargo characterisation.

Background: Efficient nucleic acid extraction is essential for reliable viral load testing, yet performance can differ widely depending on the extraction system and sample type. We compared three automated platforms, QIAcube, EZ1 Advanced, and Maxwell RSC, for their ability to recover cytomegalovirus (CMV) DNA and West Nile virus (WNV) RNA from common clinical matrices. Methods: Mock specimens were prepared using whole blood, plasma, serum, and urine collected from two donors. Samples were spiked with CMV or WNV culture material and extracted in triplicate on each platform according to the manufacturers’ protocols. Viral loads were measured using ELITech ELITE MGB assays on the InGenius system. Whole blood samples were also tested after a 1:4 dilution. Matrix-specific standard curves were applied, and viral loads were compared using Wilcoxon rank-sum tests with false-discovery rate adjustment. Results: Extraction efficiency differed substantially by platform and specimen type. For CMV, QIAcube consistently produced the highest DNA recovery across all matrices, with particularly large differences in plasma and serum, where EZ1 and Maxwell RSC yielded significantly lower loads. The WNV results varied by matrix: EZ1 and QIAcube performed similarly in plasma, while Maxwell RSC achieved the highest RNA recovery in whole blood. While the QIAcube exhibited reduced WNV recovery in blood, it achieved the best performance in serum, as specified by the kit. No significant platform differences were observed for urine. Diluting whole blood reduced variability between platforms. Conclusions: Both sample matrix and extraction system strongly influence nucleic acid recovery. These results highlight the need for matrix-specific validation and standardized extraction approaches in molecular diagnostics.

The misdiagnosis of acute febrile illness (AFI) aetiology is associated with antibiotic overuse and improper patient management. To develop novel strategies, a more comprehensive understanding of the pathophysiological mechanisms underlying the early response to different causes of fever is essential. Here we examined the host response to AFI, through the retrospective investigation of serum samples from febrile patients with bacterial infection (n = 52) or malaria (n = 73). The systemic levels of CXCL10, IFNγ, IL-2, IL-4, IL-6, IL-8, procalcitonin (PCT), TRAIL, ICAM-1, VCAM-1, osteopontin, TNFα, MMP-2 and MMP-9 were measured by multiplex immunoassays, while the relative abundance of 37 different surface markers of circulating extracellular vesicles (EVs) was determined using the MACSplex kit (Miltenyi Biotec). Markers of endothelial activation (CD29⁺, CD62P⁺, CD9⁺ EVs) and immune cell migration (MMP-2 and MMP-9) appeared elevated in bacterial AFI (q-value < 0.05). Mediators associated with antigen recognition (CD8⁺, CD81⁺, HLA-DR/DP/DQ⁺ EVs) and cytokine storm (IFNγ, CXCL10, TNFα) were instead prominent in malaria infection (q-value < 0.005). Interestingly, HLA-DR/DP/DQ⁺ EVs distinguished between bacterial and malaria AFI with area under the ROC curve (AUC) of 0.87, not significantly different from platelets’ AUC (0.92), which was the best individual discriminator. Our results showed different biological processes as associated with bacterial and malaria AFI, paving the way for the identification of novel potential markers for a timely and reliable AFI diagnosis. The study also provides novel insights on the potential role of EVs as mediators of pro-inflammatory signalling during acute fever, which should be investigated more comprehensively in vitro.

Background/Objectives: The timely diagnosis of schistosomiasis and strongyloidiasis is important because of their potentially severe, even lethal, consequences. European diagnostic laboratories must comply with the European In Vitro Diagnostic (IVD) Regulation, which requires justifying the use of in-house assays when CE-IVD-marked kits are available. We aimed to compare the performance of the Biosynex Helminths AMPLIQUICK^® RT-PCR and the multiplex in-house RT-PCR for the diagnosis of Schistosoma mansoni and Strongyloides stercoralis currently used in our department, an Italian reference centre for tropical diseases. Methods: We conducted a performance comparison study on biobanked frozen stool samples classified as cases or controls according to PCR and/or copromicroscopy at diagnosis. Both RT-PCRs were performed on DNA re-extracted from the same stool aliquot. Sensitivity and specificity were compared using McNemar’s Chi-squared test, while agreement was assessed using Gwet’s AC1 and Cohen’s K coefficients, and Bland–Altman analysis. Results: A total of 45 S. mansoni cases with 52 controls and 29 S. stercoralis cases with 54 controls were analyzed. For both S. mansoni and S. stercoralis, sensitivity and specificity were not significantly different between RT-PCRs (p = 1). Concordance was perfect for controls (AC1 = 1) in both cohorts, but was poor for S. mansoni cases (AC1 = 0.38) and good for S. stercoralis cases (AC1 = 0.78). Conclusions: Performance was not significantly different between in-house and Biosynex RT-PCRs. Nevertheless, careful assessment of the specific molecular targets included in the panels and prospective evaluation of any newly introduced tests should be implemented to minimize the impact of clinically significant discrepancies.

The WHO recently issued guidelines for public health control of Strongyloides stercoralis in endemic areas. We aimed to evaluate the feasibility of implementing the WHO recommendations in Rwanda, and our secondary objective was to estimate S. stercoralis prevalence. We conducted a community-based cross-sectional study in two Rwandan districts (Gisagara and Rubavu) including a training session focused on diagnostics for S. stercoralis: parasitological assays (Baermann and agar plate culture, APC) and a novel rapid diagnostic test (RDT). Technicians' perceptions of each assay were evaluated via a questionnaire; 1,415 individuals were screened. A critical aspect of the parasitological assays was the length of training, but there was no issues with RDT implementation. Based on the combination of Baermann and APC diagnostics, prevalence was 1.1% (95% CI 0.5-2.3) in Gisagara, and 3.9% (95% CI 2.6-5.7) in Rubavu, which was similar for the RDT. Overall, we found the implementation of S. stercoralis-specific tests was feasible, though intense training was crucial.