Molecular diagnosis of Cytomegalovirus infection: clinical performance of the Aptima transcription-mediated amplification assay toward conventional qPCR chemistry on whole blood samples.

Human Cytomegalovirus (HCMV) infection is life-threatening for immunocompromised patients. Quantitative molecular assays on whole blood or plasma are the gold standard for the diagnosis of invasive HCMV infection and for monitoring antiviral treatment in individuals at risk of HCMV disease. For these reasons, an accurate standardization toward the WHO 1st International Standard among different centers and diagnostic kits represents an effort for better clinical management of HCMV-positive patients. Herein, we evaluate, for the first time, the performance of a new transcription-mediated amplification (TMA) assay versus quantitative polymerase chain reaction (qPCR) chemistry, used as a routine method, on whole blood samples. A total of 755 clinical whole blood specimens were collected and tested simultaneously with TMA and qPCR assays. The data showed a qualitative agreement of 99.27% for positive quantified samples and 89.39% for those undetected between the two tested methods. Evaluation of viremia in positive samples highlighted a good correlation between TMA and qPCR chemistries in terms of International Units (ΔLog10 IU/mL: -0.29 ± 0.40). The TMA assay showed a significant correlation with qPCR in patients monitored for up to 3 months, thus allowing an accurate assessment of viremia in transplant patients. Therefore, TMA chemistry showed good agreement with qPCR testing, used as a current diagnostic routine. It also offers important advantages, such as FDA approval on plasma and In Vitro Diagnostic (IVD) on both plasma and whole blood, automated workflow with minimal hands-on time, and random access loading, thus enabling a rapid and reliable diagnostic in HCMV-infected patients. IMPORTANCE: In this paper, we describe the clinical performance of a novel transcription-mediated amplification (TMA) assay for the detection and quantification of human Cytomegalovirus (HCMV) DNA from whole blood samples. This is a pivotal analysis in immunocompromised patients [transplanted, HIV-positive, and Hematopoietic Stem Cell (HSC) recipients], and molecular tests with high sensitivity and specificity are necessary to evaluate the HCMV viral load in these patients. To our knowledge, this is the first in-depth evaluation of TMA chemistry for HCMV diagnosis on whole blood samples. Moreover, also technical aspects of this assay make it suitable for clinical diagnostics.

Herpes Virus Infection in Lung Transplantation: Diagnosis, Treatment and Prevention Strategies.

Lung transplantation is an ultimate treatment option for some end-stage lung diseases; due to the intense immunosuppression needed to reduce the risk of developing acute and chronic allograft failure, infectious complications are highly incident. Viral infections represent nearly 30% of all infectious complications, with herpes viruses playing an important role in the development of acute and chronic diseases. Among them, cytomegalovirus (CMV) is a major cause of morbidity and mortality, being associated with an increased risk of chronic lung allograft failure. Epstein-Barr virus (EBV) is associated with transformation of infected B cells with the development of post-transplantation lymphoproliferative disorders (PTLDs). Similarly, herpes simplex virus (HSV), varicella zoster virus and human herpesviruses 6 and 7 can also be responsible for acute manifestations in lung transplant patients. During these last years, new, highly sensitive and specific diagnostic tests have been developed, and preventive and prophylactic strategies have been studied aiming to reduce and prevent the incidence of these viral infections. In this narrative review, we explore epidemiology, diagnosis and treatment options for more frequent herpes virus infections in lung transplant patients.

Antiviral Approach to Cytomegalovirus Infection: An Overview of Conventional and Novel Strategies.

Human cytomegalovirus (HCMV) is a herpesvirus capable of establishing a lifelong persistence in the host through a chronic state of infection and remains an essential global concern due to its distinct life cycle, mutations, and latency. It represents a life-threatening pathogen for immunocompromised patients, such as solid organ transplanted patients, HIV-positive individuals, and hematopoietic stem cell recipients. Multiple antiviral approaches are currently available and administered in order to prevent or manage viral infections in the early stages. However, limitations due to side effects and the onset of antidrug resistance are a hurdle to their efficacy, especially for long-term therapies. Novel antiviral molecules, together with innovative approaches (e.g., genetic editing and RNA interference) are currently in study, with promising results performed in vitro and in vivo. Since HCMV is a virus able to establish latent infection, with a consequential risk of reactivation, infection management could benefit from preventive treatment for critical patients, such as immunocompromised individuals and seronegative pregnant women. This review will provide an overview of conventional antiviral clinical approaches and their mechanisms of action. Additionally, an overview of proposed and developing new molecules is provided, including nucleic-acid-based therapies and immune-mediated approaches.

Extraction Bottleneck in the Diagnosis of SARS-CoV-2: Evaluation of an Alternative Protocol Derived from Veterinary Use.

The COVID-19 pandemic represented a challenge for health-care systems, and a major bottleneck in SARS-CoV-2 diagnosis was the unavailability of extraction reagents. To overcome this limitation, we performed a comparative analysis to evaluate the performance of an alternative extraction protocol derived from veterinary use adapted to an open robotic platform (Testing method). A total of 73 nasopharyngeal swabs collected for diagnosis of SARS-CoV-2 infection were simultaneously extracted with the Testing protocol and the laboratory Standard of Care in order to assess the performance of the first one. The Cohen's coefficient between both procedures was excellent (K Value = 0.955). Analysis of cycle threshold and linear regression showed a significant correlation between the two methods for each tested genetic target. Although validated for veterinary applications, the Testing method showed excellent performances in RNA extraction, with several advantages: lower sample input volume, the possibility to overcome the lack of deep-well plates and adaptability to robotic liquid handlers.

Clinical features of respiratory syncytial virus bronchiolitis in an infant: rapid and fatal brain involvement.

BACKGROUND: Respiratory Syncytial Virus (RSV) infection is a significant cause of bronchiolitis and pneumonia, mostly responsible for hospitalization and infant death worldwide. However, in recent years the importance of extrapulmonary RSV manifestations, especially at neurological level, have become evident. Seizures, lethargy, ataxia and status epilepticus are suggestive of brain involvement, but also in their absence a direct neurological damage RSV-related need to be evaluated. CASE PRESENTATION: A 40-day old male infant was admitted to the Emergency Department with severe bronchiolitis and dyspnea. The patient was reported to be coughing for a week with a vomiting episode in the previous two days. The nasopharyngeal swab confirmed the diagnosis of RSV infection and blood gas test showed hypoxemia and respiratory acidosis. For these reasons, the patient was provided with oxygen therapy. A few hours later, after an initial improvement in clinical parameters, a worsening of respiratory dynamics occurred and the patient was prepared for endotracheal intubation, but in the meantime death occurred. During all the observation period in the Emergency Room, no signs of neuropathological damage were evident. Post mortem examination showed lungs congestion with alveolar atelectasis and white matter degradation with severe edema at brain level. Microbiological analysis performed on autoptic samples confirmed the presence of RSV genome in tracheobronchial aspirate, meningeal swabs, pericardic and abdominal fluids, lung and brain biopsies. CONCLUSIONS: RSV is usually associated with respiratory diseases, however, as reported by an increasingly number of studies, the systemic dissemination of virus during severe disease can lead to a sudden infant death. The clinical picture herein reported showed a severe bronchiolitis resulting in a fatal and underestimated cerebral involvement due to RSV neurotropic behaviour and underline the need for clinicians to pay more attention to neurological involvement of RSV infection, even in absence of cerebral damage evidence.

Detection of Carbapenemase and CTX-M Encoding Genes Directly from Bronchoalveolar Lavage Using the CRE and ESBL ELITe MGB Assays: Toward Early and Optimal Antibiotic Therapy Management of Critically Ill Patients with Pneumonia.

The detection of carbapenemase extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales (EB) has become a major issue among critically ill patients, especially due to their impact on appropriate antimicrobial therapy. This study aimed at evaluating the potential contribution of molecular assays to early optimization of empirical antibiotic therapy among critically ill patients with carbapenemase- and/or CTX-M-producing EB pneumonia. The CRE and ESBL ELITe MGB® assays were evaluated directly on 197 bronchoalveolar lavage (BAL) samples obtained from 120 patients. Molecular results were then compared to routine culture-based diagnostic results, and a retrospective analysis of the therapeutic antimicrobial management was performed. Among the 197 clinical specimens, blaKPC-like and blaCTX-M-like were detected in 20 (10.2%) and 12 (6.1%) specimens belonging to 15 and 11 patients, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of the CRE ELITe MGB Kit were 85% [95% confidence interval [CI]: 64.9-94.6] and 100%, respectively. PPV and NPV of the ESBL ELITe MGB Kit were 75% [95% CI: 49.4-90.2] and 100%, respectively. Retrospective analysis of the therapeutic antimicrobial management at the time of BAL collection showed that in ∼50% of patients with carbapenemase- and CTX-M-producing EB pneumonia empirical antibiotic therapy could have been optimized at least 48-72 hr earlier if positive molecular data had been used. The CRE and ESBL ELITe MGB assays might be an interesting tool for expediting optimization of empirical antibiotic therapy in critically ill patients with pneumonia, depending on local epidemiology of antibiotic resistance, patient risk stratification for EB infection, and availability of an antimicrobial stewardship team.

Renal Allograft Biopsies with Polyomavirus BK Nephropathy: Turin Transplant Center, 2015-19.

BACKGROUND: In kidney transplant patients, polyomavirus-associated nephropathy (PVAN) represents a serious complication; the key factor for the development of PVAN is immunosuppression level and modulation of anti-rejection treatment represents the first line of intervention. Allograft biopsy and histology remain the criterion standard for diagnosing PVAN. METHODS: All consecutive renal biopsies with the diagnosis of PVAN carried out at the University Hospital City of Health and Science of Turin over a five-years period were studied. Renal allograft biopsy was performed due to renal function alterations associated to medium-high polyomavirus BK (BKV)-DNA levels on plasma specimen. RESULTS: A total of 21 patients underwent a first biopsy to diagnose a possible BKV nephropathy, in 18, a second biopsy was made, in eight, a third biopsy, and finally, three underwent the fourth renal biopsy; following the results of each biopsies, immunosuppressant agents dosages were modified in order to reduce the effect of PVAN. CONCLUSIONS: In this study, the clinical and histological features of 21 kidney transplant recipients with BKV reactivation and development of PVAN are described. To date, the only treatment for PVAN consists in the reduction of immunosuppressive agents, constantly monitoring viral load.

Kinetics of cytomegalovirus and Epstein-Barr virus DNA in whole blood and plasma of kidney transplant recipients: Implications on management strategies.

This retrospective multicenter cohort study investigated the kinetics (ascending and descending phases) of cytomegalovirus (CMV) and Epstein-Barr virus (EBV)-DNA in whole blood (WB) and plasma samples collected from adult kidney transplant (KT) recipients. CMV-DNA kinetics according to antiviral therapy were investigated. Three hundred twenty-eight paired samples from 42 episodes of CMV infection and 157 paired samples from 26 episodes of EBV infection were analyzed by a single commercial molecular method approved by regulatory agencies for both matrices. CMV-DNAemia followed different kinetics in WB and plasma. In the descending phase of infection, a slower decay of viral load and a higher percentage of CMV-DNA positive samples were observed in plasma versus WB. In the 72.4% of patients receiving antiviral therapy, monitoring with plasma CMV-DNAemia versus WB CMV-DNAemia could delay treatment interruption by 7-14 days. Discontinuation of therapy based on WB monitoring did not result in relapsed infection in any patients. Highly different EBV-DNA kinetics in WB and plasma were observed due to lower positivity in plasma; EBV positive samples with a quantitative result in both blood compartments were observed in only 11.5% of cases. Our results emphasize the potential role of WB as specimen type for post-KT surveillance of both infections for disease prevention and management.

Controlled ovarian stimulation and progesterone supplementation affect vaginal and endometrial microbiota in IVF cycles: a pilot study.

PURPOSE: Does controlled ovarian stimulation (COS) and progesterone (P) luteal supplementation modify the vaginal and endometrial microbiota of women undergoing in vitro fertilization? METHODS: Fifteen women underwent microbiota analysis at two time points: during a mock transfer performed in the luteal phase of the cycle preceding COS, and at the time of fresh embryo transfer (ET). A vaginal swab and the distal extremity of the ET catheter tip were analyzed using next-generation 16SrRNA gene sequencing. Heterogeneity of the bacterial microbiota was assessed according to both the Bray-Curtis similarity index and the Shannon diversity index. RESULTS: Lactobacillus was the most prevalent genus in the vaginal samples, although its relative proportion was reduced by COS plus P supplementation (71.5 ± 40.6% vs. 61.1 ± 44.2%). In the vagina, an increase in pathogenic species was observed, involving Prevotella (3.5 ± 8.9% vs. 12.0 ± 19.4%), and Escherichia coli-Shigella spp. (1.4 ± 5.6% vs. 2.0 ± 7.8%). In the endometrium, the proportion of Lactobacilli slightly decreased (27.4 ± 34.5% vs. 25.0 ± 29.9%); differently, both Prevotella and Atopobium increased (3.4 ± 9.5% vs. 4.7 ± 7.4% and 0.7 ± 1.5% vs. 5.8 ± 12.0%). In both sites, biodiversity was greater after COS (p < 0.05), particularly in the endometrial microbiota, as confirmed by Bray-Curtis analysis of the phylogenetic distance among bacteria genera. Bray-Curtis analysis confirmed significant differences also for the paired endometrium-vagina samples at each time point. CONCLUSIONS: Our findings suggest that COS and P supplementation significantly change the composition of vaginal and endometrial microbiota. The greater instability could affect both endometrial receptivity and placentation. If our findings are confirmed, they may provide a further reason to encourage the freeze-all strategy.

Evaluation of CMV DNA in dried blood spot.

Cytomegalovirus is the primary viral cause of congenital infection. However, diagnosis may be difficult for clinical and technical reasons. Currently, evaluation of CMV DNA on dried blood spot (DBS) is an important instrument to define a congenital infection. The aim of this study was to identify a clinically and technically suitable diagnostic work-flow for CMV DNA evaluation on DBS. Sensitivity was not significantly influenced by storage time of up to 12 months and extraction technique; however, analysis in triplicate was crucial to obtain reliable results. Considering viral load in an infected foetus at risk of developing disease, a threshold value of approximately 104 copies/mL was characterized by high operating characteristics for detection of positivity at 12 months on DBS.

Tailored combined cytomegalovirus management in lung transplantation: a retrospective analysis.

BACKGROUND: There is no univocal prophylactic regimen to prevent cytomegalovirus (CMV) infection/disease in lung transplantation (LT) recipients. The aim of this study is to evaluate short-term clinical outcomes of a tailored combined CMV management approach. METHODS: After 1-year follow up, 43 LT patients receiving combined CMV prophylaxis with antiviral agents and CMV-specific IgG were evaluated in a retrospective observational study. Systemic and lung viral infections were investigated by molecular methods on a total of 1134 whole blood and 167 bronchoalveolar lavage (BAL) and biopsy specimens. CMV immunity was assessed by ELISPOT assay. Clinical and therapeutic data were also evaluated. RESULTS: We found 2/167 cases of CMV pneumonia (1.2%), both in the donor-positive/recipient-positive (D+/R+) population, and 51/167 cases of CMV pulmonary infection (BAL positivity 30.5%). However, only 32/167 patients (19.1%) were treated due to their weak immunological response at CMV ELISPOT assay. Viremia ⩾100,000 copies/mL occurred in 33/1134 specimens (2.9%). Regarding CMV-serological matching (D/R), the D+/R- population had more CMV viremia episodes (p < 0.05) and fewer viremia-free days (p < 0.001). CONCLUSIONS: Compared to previous findings, our study shows a lower incidence of CMV pneumonia and viremia despite the presence of a substantial CMV load. In addition, our findings further confirm the D+/R- group to be a high-risk population for CMV viremia. Overall, a good immunological response seems to protect patients from CMV viremia and pneumonia but not from CMV alveolar replication. The reviews of this paper are available via the supplemental material section.

Collaborative national multicenter for the identification of conversion factors from copies/mL to international units/mL for the normalization of HCMV DNA load.

The present multicentric (n = 11 laboratories) study aimed to identify conversion factors from copies/mL to international units (IU)/mL for the normalization of HCMV DNA load using the first WHO International Standard for HCMV nucleic acid amplification techniques and to enhance interlaboratory agreement of HCMV DNA quantification methods. Study protocols for whole blood and plasma (extraction and amplification) were performed to calculate conversion factors from HCMV DNA copy number to IU. The greatest variability was observed in samples with lower HCMV concentrations (3.0 Log10) in both biological matrices. Overall, 73.1% (206/282) of whole blood and 82.2% (324/394) of plasma samples analyzed fell within an acceptable variation range (±0.5 Log10 difference). An average of 0.64 (range 0.21-1.17) was the conversion factor calculated for the HCMV whole blood panel and 0.82 (range 0.39-2.2) for the HCMV plasma panel.

Serological and molecular detection of Bartonella henselae in specimens from patients with suspected cat scratch disease in Italy: A comparative study.

Cat scratch disease (CSD) is an infectious disease caused by Bartonella henselae, usually characterized by self-limiting regional lymphadenopathy and fever. Given the low clinical diagnostic sensitivity and specificity of conventional anti-B. henselae indirect immunofluorescence assays (IFAs), real-time polymerase chain reaction (PCR)-based detection of B. henselae is now being proposed as a more sensitive tool to diagnose CSD. Thus, here we have assessed the efficacy of real-time PCR in detecting B. henselae in different specimens from patients with suspected CSD and compared it to that of IFA. From March 2011 to May 2016, at the Microbiology and Virology Unit, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy, 115 clinical specimens (56 aspirated pus, 39 fresh lymph node biopsies, and 20 whole blood samples) and 99 sera from 115 patients with suspected CSD (62 females and 53 males between the ages of 3 months and 68 years) were analyzed by both real-time PCR, used in a qualitative way, and IFA (IgM and IgG) for the presence of B. henselae. For 16 patients, serological results were not available due to a clinical decision not to request the test. B. henselae DNA positivity was detected by real-time PCR in 37.39% of patients, while 62.61% of them were negative. Thus, patients were divided into two groups: real-time PCR+ (n = 43) and real-time PCR- (n = 72). Real-time PCR screening of whole blood, biopsies, and aspirated pus revealed B. henselae positivity in 40%, 38.46%, and 35.71% of patients, respectively. When we analyzed samples by IFA, we found the presence of B. henselae in 28 out of 99 (28.28%) patients, of which 11 (11.11%) belonged to the real-time PCR+ group and 17 (17.17%) to the real-time PCR- group. Among the 71 seronegative subjects, 16 (16.16%) were found positive for B. henselae by real-time PCR. Thus, by combining the results of both assays, we were able to increase the percentage of B. henselae positive specimens from 27.27% (real-time PCR) or 28.28% (IFA) to 44.44% (real-time PCR+IFA). Altogether, these findings indicate that the early detection of B. henselae in patients with suspicious CSD through combined real-time PCR and serological analyses can lead to a more accurate diagnosis of CSD, thereby allowing prompt and appropriate disease management.

Recurrent herpes labialis and Herpes simplex virus-1 genitalis: what is the link?

BACKGROUND: Recently, Herpes simplex virus (HSV)-1 seroprevalence declined among adolescents, rendering young people lacking HSV-1 antibodies more susceptible to genital HSV-1 acquisition, if sexually exposed. The aim of the present study was to identify the possible risk factors for the development of HSV-1 related Herpes genitalis (HG). METHODS: From January 2012 to December 2015, patients with HG attending three Sexually Transmitted Infections Units in Northern Italy were recruited. A genital swab on the lesions for the search of HSV-1/2 DNA through real time polymerase chain reaction (PCR) and a serum sample for HSV-1/2 specific serology were performed. Moreover, patients were asked whether they had personal history of herpes labialis (HL). Patients with PCR proved HSV-1 HG were included as cases; asymptomatic subjects attending STI Units for a blood check were recruited as controls and were checked for HSV-1/2 serology. RESULTS: The study included 141 cases and 70 controls. Specific HSV-1 antibodies were found in 34.7% of the cases and 67% of the controls. History of recurrent herpes labialis (RHL) was found in 4% of the cases and 31% of the controls. The occurrence of RHL in HSV-1 seropositive patients resulted lower in the case group compared to the control group. CONCLUSIONS: We can speculate about a protective role for RHL against the clinical appearance of HSV-1 HG. The clinical usefulness of our study involved especially the counselling in serodiscordant couples. The presence of HSV-1 antibodies in asymptomatic sexual partners does appear protective for HG manifestation only in presence of RHL history.

Cytomegalovirus and Epstein-Barr Virus DNA Kinetics in Whole Blood and Plasma of Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Currently, no consensus has been reached on the optimal blood compartment to be used for surveillance of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNAemia. Although several comparative studies have been performed correlating CMV and EBV DNA loads in whole blood (WB) versus plasma, to our knowledge, no studies to date have analyzed the kinetics of both viruses in the 2 blood compartments. In this retrospective noninterventional multicenter cohort study, the kinetics of CMV and EBV DNA in 121 hematopoietic stem cell transplantation (HSCT) recipients were investigated by analyzing in parallel 569 and 351 paired samples from 80 and 58 sequential episodes of CMV and EBV DNAemia, respectively. Unlike previous studies, this study used a single automated molecular method that was CE-marked and Food and Drug Administration-approved for use in quantifying CMV and EBV DNA in both plasma and WB. Furthermore, the complete viral replication kinetics of all episodes (including both the ascending and the descending phases of the active infection) was examined in each patient. The previously observed overall correlation between CMV DNA levels in WB and plasma was confirmed (Spearman's ρ = .85; P < .001). However, although WB and plasma CMV DNAemia reached peak levels simultaneously, in the ascending phase, the median CMV DNA levels in plasma were approximately 1 log10 lower than WB. Furthermore, in patients who received preemptive therapy, CMV DNA showed a delayed decrease in plasma compared with WB. A lower correlation between EBV DNA levels in plasma versus WB was found (Spearman's ρ = .61; P < .001). EBV DNA kinetics was not consistent in the 2 blood compartments, mostly due to the lower positivity in plasma. Indeed, in 19% of episodes, EBV DNA was negative at the time of the EBV DNA peak in WB. Our results suggest a preferential use of WB for surveillance of CMV and EBV infection in HSCT recipients.

Development of an EliSPOT assay for HSV-1 and clinical validation in lung transplant patients.

Cellular immunity plays a major role in the control of HSV-1 infection/reactivation with a potential impact on the clinical-therapeutic management of immunocompromised patients, such as transplant recipients. Herein, we quantitatively evaluated T-cell response directed at HSV-1 by a newly developed IFN-γ EliSPOT assay in 53 patients (including 45 lung transplant recipients and eight subjects in waiting list). Overall, 62.2% of transplant patients and 62.5% of subjects on the waiting list showed a response to HSV-1 with no significant difference in the level of virus-specific cellular immunity. Response tended to be lower in the first three months posttransplantation with a progressive recovery of pretransplantation status by the second year and in the presence of HSV-1 DNA positivity in bronchoalveolar lavage. As expected, no response was found in seronegative patients. No significant difference in the level of response according to IgM and IgG status was found. Further studies are required to define the role of HSV-1 specific immune response for the clinical-therapeutic management of lung transplant patients and in other clinical settings and to define cut-off levels discriminating between absence/low and strong response to be related to the risk of viral infection/reactivation.

Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection.

Diagnosis of viral gastroenteritis: limits and potential of currently available procedures.

The diagnostic approaches to viral gastroenteritis have evolved substantially over the past decades because of the advances in detection methods, the emergence of new pathogens, and the increase in diarrhea hospitalizations attributed to viruses, especially in young children in non-industrialized countries. Overall, these factors have lead to a relevant improvement of types and operating characteristics of diagnostic methods (including sensitivity and specificity), as well as turnaround time. In this review, clinical and laboratory approaches to the diagnosis of viruses causing gastroenteritis are presented; in particular, specimen collection and detection methods are reviewed and discussed, taking into account performance and limitations.

Pre-transplant assessment of CMV-specific immune response by Elispot assay in kidney transplant recipients.

Cytomegalovirus (CMV) primary infection or re-activation in solid organ transplant (SOT) recipients is associated with increased morbidity and mortality, with patients with IgG-CMV D+/R- sero-matching at greater risk. The impact of pre-transplant CMV-specific host cellular immunity on the long-term risk of CMV replication in kidney transplants (KT) was prospectively evaluated in eighty patients by CMV-EliSpot assay. The study population included 54 male and 26 female recipients, with CMV-IgG distribution: 60 D+/R+, 11 D-/R+, 7 D+/R-, 2 D-/R-. At pre-transplantation, 49 KT (61.3%) were CMV-responders by EliSpot. At 3-month follow up, 16 (32.7%) out of 49 CMV-responders showed CMV blood infection, compared to 8 (25.8%) out of 31 non-responders. No further episode of CMV viraemia was reported in the responder group, in comparison to 15 out 31 non-responders (48.4%) showing at least one episode of CMV-DNAemia at 12-month follow-up. Baseline CMV-IgG serology showed a strong correlation with EliSpot determinations; KT recipients exhibiting at least one episode of CMV viraemia at 12-month follow-up showed lower baseline CMV-EliSpot values than those without signs of CMV replication. The study suggests that monitoring CMV-specific T-cell responses at pre-transplantation by EliSpot assay may be useful for predicting the post-transplantation risk of CMV infection and reactivation.