A side-by-side comparison of the performance and time-and-motion data of VITEK MS.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry systems are designed for rapid and reliable microbial identification. VITEK MS PRIME is the bioMérieux's new generation instrument equipped with a continuous load-and-go sample loading system, urgent slide prioritization for critical patient samples and new internal components for faster identification. The aim of this study was to assess the performance of VITEK MS PRIME and to compare it to that of the VITEK MS system. In addition, at two sites, we performed a time-and-motion study to evaluate the efficiency of sample analysis from colony picking to slide removal from the instrument. We analyzed by VITEK MS and VITEK MS PRIME a total of 1413 isolates (1320 bacterial and 76 yeast) deriving from routine diagnostic samples that came into four laboratories in Canada, France, Italy, and Spain. VITEK MS PRIME and VITEK MS were concordant to the species and genus level for 1354/1413 (95.8%) and to the species level for 1341/1413 (94.9%). The identification and concordance rates in individual centers were largely homogenous. Overall, VITEK MS PRIME identified 1370/1413 (97.0%) of isolates compared to 1367/1413 (96.7%) identified by VITEK MS. Identification rates were consistently high for all microorganism categories. A time-and-motion study showed that the use of VITEK MS PRIME was associated with significant time saving. VITEK MS PRIME performs as well as VITEK MS and reduces the time necessary for pathogen identification. To fully optimize the laboratory process and obtain maximum efficiency, VITEK MS PRIME must be integrated into the laboratory workflow.

A persistently replicating SARS-CoV-2 variant derived from an asymptomatic individual.

BACKGROUND: Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. METHODS: Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. RESULTS: The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. CONCLUSIONS: Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.

Lymphomagenic properties of a HIV p17 variant derived from a splenic marginal zone lymphoma occurred in a HIV-infected patient.

Despite antiretroviral therapy, HIV+ individuals still have increased risk to develop lymphomas, including marginal zone lymphomas, suggesting that factors other than HIV-related immunosuppression are probably acting as lymphomagenic factors in the HIV setting. The possible pathogenic involvement of HIV p17 protein variants was investigated in a particularly informative case of HIV-related splenic marginal zone lymphoma, which was negative for oncogenic virus infections, thus allowing us to assess the possible direct contribution of these HIV-encoded proteins to lymphomagenesis. The presence of p17 protein was analyzed by immunohistochemistry in lymphoma tissue. Recombinant p17 protein derived from the dominant sequence detected in plasma and lymphoma biopsy was characterized for B-cell proliferation, clonogenicity in soft agar, in vitro tube formation and wound healing. Intracellular signaling was investigated by immunoblotting. HIV p17 protein was detected in reactive lymphoid follicles but not within lymphoma cells. An identical dominant variant p17 sequence, p17-Lyrm, carrying a 117 to 118 Ala-Ala insertion was detected in both plasma and lymphoma tissue. Recombinant p17-Lyrm enhanced B-cell proliferation and clonogenicity promoted the formation of capillary-like structures and enhanced endothelial cell migration. Unlike reference p17, the p17-Lyrm variant enhanced the activation of Akt and ERK, critical kinases in lymphomagenesis. p17-Lyrm clonogenic activity was dependent on the activation of Akt but not of ERK1/2. These results indicated that HIV p17 variants with distinct molecular signatures and functional properties may accumulate in lymphoid tissues of HIV-infected individuals where they may act as a local stimulus promoting the development of lymphomas.

Correction to: Acute epididymo-orchitis due to Salmonella Typhi in a young man from Bangladesh.

The original version of this article unfortunately contained a mistake. Arnaldo Caruso was not listed among the authors.

Acute epididymo-orchitis due to Salmonella Typhi in a young man from Bangladesh.

S. typhi infection rarely involves the genitourinary system. We report the first described case of acute epididymo-orchitis due to S. typhi in a 14-year-old boy from Bangladesh. A high index of suspicion should be maintained when evaluating patients coming from endemic countries also in case of unusual sites of infection.

Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.

Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117-118 (Ala-Ala) or 125-126 (Gly-Asn or Gly-Gln-Ala-Asn-Gln-Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117-118, and one with the Ala-Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala-Ala at position 117-118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly-Asn insertion at position 125-126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala-Ala insertion mutant is destabilized compared with refp17, whereas the Gly-Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1-related NHL.

In-depth analysis of compartmentalization of HIV-1 matrix protein p17 in PBMC and plasma.

HIV-1 p17 plays an important role in the virus life-cycle and disease pathogenesis. Recent studies indicated a high heterogeneity of p17. A high number of insertions in the p17 carboxy-terminal region have been more frequently detected in patients with non-Hodgkin lymphoma (NHL), suggesting a role of altered p17 in lymphomagenesis. Based on p17 heterogeneity, possible PBMC/plasma compartmentalization of p17 variants was explored by ultra-deep pyrosequencing in five NHL patients. The high variability of p17 with insertions at the carboxy-terminal region was confirmed in plasma and observed for the first time in proviral genomes. Quasispecies compartmentalization was evident in 4/5 patients. Further studies are needed to define the possible role of p17 quasispecies compartmentalization in lymphomagenesis.

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens.

The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.

A natural HIV p17 protein variant up-regulates the LMP-1 EBV oncoprotein and promotes the growth of EBV-infected B-lymphocytes: implications for EBV-driven lymphomagenesis in the HIV setting.

Human immunodeficiency virus p17 matrix protein is released by infected cells and may accumulate within lymphoid tissues where it may deregulate the biological activities of different cell populations by binding to CXCR1 and CXCR2 cellular receptors. S75X, a natural p17 variant, was recently shown to enhance the malignant properties of lymphoma cells. We investigated a reference p17 protein and the S75X variant for their ability to bind to Epstein-Barr virus (EBV)-infected primary and fully transformed B-lymphocytes and trigger downstream effects of potential pathogenic relevance. We demonstrate that EBV infection of primary B-lymphocytes or the ectopic expression of the latent membrane protein-1 viral oncoprotein in EBV-negative B-cells up-regulates CXCR2, but not CXCR1. Multispectral imaging flow cytometry showed that EBV-infected primary B-cells more efficiently bind and internalize p17 proteins as compared with activated B-lymphocytes. The S75X variant bound more efficiently to EBV-infected primary and fully transformed B-lymphocytes compared with reference p17, because of a higher affinity to CXCR2, and enhanced the proliferation of these cells, an effect associated with cyclin D2 and D3 up-regulation and increased interleukin-6 production. Notably, the S75X variant markedly up-regulated latent membrane protein-1 expression at both mRNA and protein levels and enhanced the activation of Akt, ERK1/2 and STAT3 signaling, thereby contributing to EBV(+) B-cell growth promotion. These results indicate that EBV infection sensitizes B-lymphocytes to CXCR2-mediated effects of p17 proteins and provide evidence supporting a possible contribution of natural p17 variants to EBV-driven lymphomagenesis in the human immunodeficiency virus setting.

Long-lasting humoral immune response induced in HIV-1-infected patients by a synthetic peptide (AT20) derived from the HIV-1 matrix protein p17 functional epitope.

OBJECTIVE: A therapeutic vaccination based on a synthetic peptide (AT20) representative of the HIV-1 matrix protein p17 (p17) functional region, coupled to keyhole limpet hemocyanin (KLH) AT20-KLH was capable of inducing the production of high-avidity antibodies (Abs) toward a previous untargeted p17 hotspot of functional activity in highly active antiretroviral therapy (HAART)-treated HIV-1-infected patients. Since avidity of Abs after immunization and the retention of antigens are important in sustaining the long-lasting production of specific humoral responses, we asked whether AT20-KLH vaccination would result in development of a long-lived immune response. METHODS: The long-term duration of Ab response to AT20-KLH has been evaluated in 10 patients previously enrolled for the AT20-KLH vaccination trial at day 898 post-immunization. Ab titer and their avidity was assessed using specifically designed ELISA assays, whereas their neutralizing capacity was estimated in vitro using a 'wound sealing assay'. RESULTS: Data obtained show that high titers of specific anti-AT20 Abs were maintained at more than 2 years after the last immunization. Furthermore, these Abs were capable to neutralize exogenous p17, as assessed by ability of sera derived from AT20-KLH-immunized patients to block the ability of p17 to promote cell migration in vitro. CONCLUSION: This finding attests for a successful AT20-KLH vaccine molecule formulation and for an effective HAART-dependent Ab persistence.

HIV-1 matrix protein p17 promotes lymphangiogenesis and activates the endothelin-1/endothelin B receptor axis.

OBJECTIVE: AIDS-related lymphomas are high grade and aggressively metastatic with poor prognosis. Lymphangiogenesis is essential in supporting proliferation and survival of lymphoma, as well as tumor dissemination. Data suggest that aberrant lymphangiogenesis relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. HIV-1 matrix protein p17 was found to accumulate and persist in lymph nodes of patients even under highly active antiretroviral therapy. Because p17 was recently found to exert a potent proangiogenic activity by interacting with chemokine (C-X-C motif) receptors 1 and 2, we tested the prolymphangiogenic activity of the viral protein. APPROACH AND RESULTS: Human primary lymph node-derived lymphatic endothelial cells were used to perform capillary-like structure formation, wound healing, spheroids, and Western blot assays after stimulation with or without p17. Here, we show that p17 promotes lymphangiogenesis by binding to chemokine (C-X-C motif) receptor-1 and chemokine (C-X-C motif) receptor-2 expressed on lymph node-derived lymphatic endothelial cells and activating the Akt/extracellular signal-regulated kinase signaling pathway. In particular, it was found to induce capillary-like structure formation, sprout formation from spheroids, and increase lymph node-derived lymphatic endothelial cells motility. The p17 lymphangiogenic activity was, in part, sustained by activation of the endothelin-1/endothelin receptor B axis. A Matrigel plug assay showed that p17 was able to promote the outgrowth of lymphatic vessels in vivo, demonstrating that p17 directly regulates lymphatic vessel formation. CONCLUSIONS: Our results suggest that p17 may generate a prolymphangiogenic microenvironment and plays a role in predisposing the lymph node to lymphoma growth and metastasis. This finding offers new opportunities to identify treatment strategies in combating AIDS-related lymphomas.

HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2.

Vascular diseases supported by aberrant angiogenesis have increased incidence in HIV-1-infected patients. Several data suggest that endothelium dysfunction relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. The HIV-1 matrix protein p17 is known to deregulate the biological activity of different immune cells. Recently, p17 was found to mimic IL-8 chemokine activity by binding to the IL-8 receptor CXCR1. Here we show that p17 binds with high affinity to CXCR2, a CXCR1-related receptor, and promotes the formation of capillary-like structures on human endothelial cells (ECs) by interacting with both CXCR1 and CXCR2 expressed on the EC surface. ERK signaling via Akt was defined as the pathway responsible for p17-induced tube formation. Ex vivo and in vivo experimental models confirmed the provasculogenic activity of p17, which was comparable to that induced by VEGF-A. The hypothesis of a major role for p17 in HIV-1-induced aberrant angiogenesis is enforced by the finding that p17 is detected, as a single protein, in blood vessels of HIV-1-patients and in particular in the nucleus of ECs. Localization of p17 in the nucleus of ECs was evidenced also in in vitro experiments, suggesting the internalization of exogenous p17 in ECs by mechanisms of receptor-mediated endocytosis. Recognizing p17 interaction with CXCR1 and CXCR2 as the key event in sustaining EC aberrant angiogenesis could help us to identify new treatment strategies in combating AIDS-related vascular diseases.

Molecular interaction studies of HIV-1 matrix protein p17 and heparin: identification of the heparin-binding motif of p17 as a target for the development of multitarget antagonists.

Once released by HIV(+) cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (K(d) = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.

HIV-1 matrix protein p17 binds to the IL-8 receptor CXCR1 and shows IL-8-like chemokine activity on monocytes through Rho/ROCK activation.

Exogenous HIV-1 matrix protein p17 was found to deregulate biologic activities of many different immune cells that are directly or indirectly involved in AIDS pathogenesis after binding to unknown cellular receptor(s). In particular, p17 was found to induce a functional program in monocytes related to activation and inflammation. In the present study, we demonstrate that CXCR1 is the receptor molecule responsible for p17 chemokine-like activity on monocytes. After CXCR1 binding, p17 was capable of triggering rapid adhesion and chemotaxis of monocytes through a pathway that involved Rho/ROCK. Moreover, CXCR1-silenced primary monocytes lost responsiveness to p17 chemoattraction, whereas CXCR1-transfected Jurkat cells acquired responsiveness. Surface plasmon resonance studies confirmed the capacity of p17 to bind CXCR1 and showed that the p17/CXCR1 interaction occurred with a low affinity compared with that measured for IL-8, the physiologic CXCR1 ligand. In all of its activities, p17 mimicked IL-8, the natural high-affinity ligand of CXCR1. Recent studies have highlighted the role of IL-8 and CXCR1 in HIV-1 replication and AIDS pathogenesis. Our findings herein call for an exploration of the therapeutic potential of blocking the p17/IL-8/CXCR1 axis in HIV-1 infection.

Emergence of carbapenem-resistant Klebsiella Pneumoniae strains producing KPC-3 in Brescia Hospital, Italy.

Carbapenem-resistant K. pneumoniae has recently been reported as a new multidrug-resistant nosocomial pathogen. This study reports the emergence of carbapenem-resistant K. pneumoniae strains in Brescia Civic Hospital, Italy. Different samples, collected from April 2012 to February 2013, showed that 29 patients presented infections from multidrug-resistant K. pneumoniae and three of these patients were intestinal carriers. In total, 40 carbapenem-resistant K. pneumoniae strains were isolated from multiple specimens of these patients. In 39 out of 40 samples, we identified the bla(KPC-3) carbapenemase gene variant responsible for bacterial carbapenem resistance. The DiversiLab analysis showed four different genetic patterns within multidrug-resistant K. pneumoniae isolates, with pattern 1 and 2 including 95% of the bacterial strains. Carbapenem-resistant K. pneumoniae strains belonging to patterns 1 and 2 were also detected in the intestinal tract of the three asymptomatic carriers. Moreover, isolation of the same strains in other body sites of the same patients and in bronchial fluid of a non-colonized patient in the same ward indicates an initial dissemination of this pathogen. Our results highlight the emergence of carbapenemase-producing K. pneumoniae strains in different hospital wards and the urgent need for infection control, antibiotic stewardship programmes and utilization of a surveillance and prevention system.

For timing of HAART is less more? CD4+/CD8+ ratio and CD4+ percentage as surrogate markers for more complex immunological features.

There is disagreement on the optimal timing of HAART initiation based on absolute CD4+ T-cell count (CD4+ count). We investigated if na�ve patients with CD4+ T-cell percentage (%CD4+) <29% or CD4+/CD8+ ratio <1 display signs of immune deterioration notwithstanding CD4+ count ?500 cells/?l. We found that these patients show B-cell aberrations and an impaired control of Torque Teno Virus replication. By contrast, patients with CD4+?500/?l, %CD4+?29% and CD4+/CD8+?1 displayed features of healthy subjects. Results obtained suggest that a combination of these parameters could be an adequate surrogate marker of immunological competence. This will be helpful in deciding when to start HAART.

Chronic Hepatitis C Cascade of Care in Prisoners-Is There Still Some Work to Do? Analysis of Two Large Penitentiaries in Northern Italy.

Penitentiaries have a higher burden of communicable diseases compared to the general population. Prisoners should be tested for hepatitis C virus (HCV) and have direct access to treatment. We analysed the HCV cascade of care in two penitentiaries in Brescia, Northern Italy. At admission, prisoners are offered a voluntary screening for HCV, while patients with known infections are tested with an HCVRNA measurement. We performed an observational retrospective study including all the subjects admitted to the penitentiaries from 1 January 2015 to 31 October 2021. We conducted a descriptive analysis. During the study period, 5378 admissions were registered, and 2932 (54.5%) screenings were performed. Hepatitis C virus antibody positivity was found in 269 tests (9.2%). Hepatitis C virus RNA was detectable in 169 people. During the study period, 77 treatments with direct-acting antivirals (DAAs) were administered. Follow-up was available in 45 patients, and sustained virological response (SVR) was documented in 44 of them. Retention in care occurred in less than half of the prisoners after release. Our data demonstrate poor screening adherence that could benefit from educational programs. Treatment rates could be improved with test-and-treat programs. More efforts are needed to eliminate HCV as a public threat by 2030. Dedicated local networks, including infectious diseases (ID) departments, substance abuse services and prisons, could mitigate these issues.

Nutritional Strategies To Improve VRE Control.

The rise of Vancomycin-resistant enterococci (VRE) strains among cellular therapy recipients raises concerns due to increased morbidity, mortality, and hospitalization costs, particularly impacting transplanted patients with diminished survival expectations. Recent research linking lactose to Enterococcus growth and graft-versus-host disease (GVHD) emphasizes the need for data on reducing lactose in the diets of VRE-carrying patients, especially in cellular therapy contexts like CAR-T or allogeneic hematopoietic stem cell transplantation. Responding to elevated VRE positivity rates in rectal swabs among patients in our BMT Unit, a unique nutritional strategy was implemented, introducing lactose-free milk and strictly enforcing lactose-free diets. This approach resulted in a significant reduction in VRE carriers, with a 16% positivity rate in the Lactose Group versus 3.6% in the Lactose-Free Group, as of June 2023. These results indicate the potential efficacy of this innovative nutritional strategy in high-risk departments, such as BMT Units and Intensive Care Units, with implications for reducing isolation strategies and inappropriate antibiotic use in cases of VRE colonization.

Profile of toll-like receptors on peripheral blood cells in relation to acute graft-versus-host disease after allogeneic stem cell transplantation.

Toll-like receptors (TLRs) play a key role in the cross-talk between the innate and adaptive immune systems. Previous studies investigating associations between certain TLRs and acute graft-versus-host disease (aGVHD) have reported contrasting results, and no studies relating aGVHD to the expression and function of all human TLRs together have been published to date. We prospectively evaluated the expression of 9 TLRs on T lymphocytes and monocytes by flow cytometry in relation to aGVHD in 34 patients. Induction of TNF-α, IL-4, IFN-γ, and monocyte chemotactic protein 1 on TLR activation was assessed by ELISA on cell supernatants. Nineteen patients developed aGVHD, at a median time of 28 days (range, 20-50 days) after transplantation. A 2-step multivariate analysis was performed using principal component analysis and multifactor analysis of variance. The levels of TLR-5 expression on monocytes and T lymphocytes were positively correlated to aGVHD (P = .01), whereas levels of TLR-1 and -9 were negative predictors (P = .03 and .01, respectively). This profile of TLR-1, -5, and -9 can promote an overall immunostimulatory/proinflammatory response. If our findings are confirmed by further studies, this TLR profile could be a useful biomarker of aGVHD.

Human metapneumovirus-associated hospital admissions over five consecutive epidemic seasons: evidence for alternating circulation of different genotypes.

Human metapneumovirus (hMPV) is a pathogen of the respiratory tract with a worldwide distribution. The purpose of this study was to identify hMPV as the cause of acute respiratory diseases in children admitted at Spedali Civili, a public hospital in Brescia, Italy. Eight hundred forty-six nasopharyngeal aspirate samples negative for the presence of other common respiratory viruses were tested for the presence of hMPV RNA by reverse transcription-polymerase chain reaction. Of the 846 samples, 79 (9.3%) were positive for hMPV. Polymerase chain reaction products, obtained by amplification of the partial nucleotide sequence of gene F, were sequenced and compared with sequences deposited in GenBank. All four hMPV subtypes were identified, including the proposed subtype A2 sublineages "A" and "B". In successive epidemic seasons, large outbreaks of hMPV alternated with small outbreaks in a biannual pattern. This local study provides further evidence that hMPV infection should be considered as a reason for hospital admission for acute respiratory disease in children.