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.

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.

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.

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.

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.

Application of multiple laboratory tests for Mycobacterium avium ssp. paratuberculosis detection in Crohn's disease patient specimens.

The difficulties involved in detecting and enumerating Mycobacterium avium subsp. paratuberculosis (MAP) as a pathogen potentially involved in Crohn's disease (CD) are well known. This study aimed to improve this situation through the application of multiple laboratory diagnostic tests to detect and isolate this bacterium from different specimens collected from CD-patients and non-CD subjects as controls. A total of 120 samples (terminal ileum and colon biopsies, blood and stool) were obtained from 19 CD-patients and from 11 individuals who did not have a clinicopathological diagnosis of CD (non-CD controls) attending for ileocolonoscopy. All samples were processed by staining techniques, culture on both solid and liquid media, and Insertion Sequence 900/F57 real-time PCR. The MAP frequency in CD-patients was found in a significantly greater proportion than in non-CD subjects; the most positive samples were biopsies from CD-patients tested by real-time PCR. MAP detection in biopsies, and in the other samples, by applying multiple and validated laboratory diagnostic tests, could be a marker of active infection, supporting MAP involvement in CD.

Detection of herpesviruses 1-6 and community-acquired respiratory viruses in patients with chronic rhinosinusitis with nasal polyposis.

OBJECTIVE: To evaluate the prevalence of human herpesviruses (HHV) 1-6 and community-acquired respiratory viruses (CARVs) in specimens from patients with nasal polyposis undergoing functional endoscopic sinus surgery (FESS) and investigate the potential clinical role. METHODS: Viral occurrence was evaluated by molecular methods in polyp, turbinate mucosa, and pre- and postoperative scraping specimens from 35 consecutive patients at different time points in relation to FESS. RESULTS: Overall, 21 patients (60%) were positive to at least one virus in at least one specimen; in particular, 12.1% of all specimens for HHV-6 (3/35 polyps, 11/31 turbinates, 1 presurgical scraping) and 10.5% for Epstein-Barr virus (EBV) (8/35 polyps, 3/31 turbinates, 1/29 pre- and 1/29 postsurgical scraping), followed by CMV and HSV-1 (both 1.6%; 1/35 polyps, 1/29 postsurgical scraping and 2/35 polyps, respectively). EBV positivity tended to be higher in polyps, as well as HHV-6 in adjacent healthy turbinate mucosa, although no significant association was found. Only one preoperative cytological specimen was positive to parainfluenza virus-1. CONCLUSION: No association between the development of nasal polyps, herpesviruses and CARVs seems to exist. However, the higher EBV frequency in polyps could suggest a causative role or persistence in the inflammatory lymphoid tissue.

Evaluation of polyomavirus BK cellular immune response by an ELISpot assay and relation to viral replication in kidney transplant recipients.

The failure of immune surveillance may be associated with polyomavirus BK reactivation, potentially leading to the development of nephropathy in kidney transplantation. BK-specific cellular immune response may be used to modulate immunosuppressive therapy, but few studies have investigated the topic. Herein, we serially evaluated BK-specific response in 149 kidney transplant recipients and found that only 14/149 (9.4%) were responders. Episodes of viral reactivation (viremia and/or viruria) occurred only in non-responder patients. The frequency of BK-specific immune response appears to be lower than that for other persistently infecting viruses such as cytomegalovirus.

QuantiFERON CMV Test and CMV Serostatus in Lung Transplant: Stratification Risk for Infection, Chronic and Acute Allograft Rejection.

The QuantiFERON CMV (QCMV) test evaluates specific adaptive immune system activity against CMV by measuring IFN-γ released by activated CD8+ T lymphocytes. We aimed to evaluate the QCMV test as a predictive tool for CMV manifestations and acute or chronic lung allograft rejection (AR and CLAD) in lung transplant (LTx) patients. A total of 73 patients were divided into four groups based on donor and recipient (D/R) serology for CMV and QCMV assay: group A low-risk for CMV infection and disease (D-/R-); group B and C at intermediate-risk (R+), group B with non-reactive QCMV and group C with reactive QCMV; group D at high-risk (D+/R-). Group D patients experienced higher viral replication; no differences were observed among R+ patients of groups B and C. D+/R- patients had a higher number of AR events and group C presented a lower incidence of AR. Prevalence of CLAD at 24 months was higher in group B with a higher risk of CLAD development (OR 6.33). The QCMV test allows us to identify R+ non-reactive QCMV population as the most exposed to onset of CLAD. This population had a higher, although non-significant, susceptibility to AR compared to the R+ population with reactive QCMV.

Maribavir treatment for resistant cytomegalovirus disseminated disease in kidney transplant recipients: A case-based scoping review of real life data in literature.

The treatment of refractory CMV is often associated with high toxicity. Maribavir (MBV) is a novel oral antiviral, known for its favourable safety profile in fragile patients. We describe a case of CMV disease with end organ damage following kidney transplantation at high risk, for recipient-donor serological mismatch. A 54-year-old female with history of obesity, hypertension, and chronic kidney disease, on prednisone and tacrolimus after kidney transplantation in November 2022, soon after developed primary CMV infection, treated with Valganciclovir and CMV Ig. In January 2023 the patient presented with fever and dyspnea. Pulmonary miliary opacities and right-upper lobe consolidation were found at CT-scan along with CMV-DNA positivity on BAL and serum. Lung biopsy confirmed CMV infection. Antiviral was switched to Ganciclovir. Despite initial benefit, fever and respiratory failure happened 8 days later, leading to intubation at day 15. Due to slow decrease serum CMV-DNA and detection of UL97 mutation, conferring resistance to valganciclovir and ganciclovir, the patient was started on foscarnet and letermovir. She was extubated after a gradual respiratory improvement and discharged from ICU to rehabilitation department with HFNC; reduction in serum CMV-DNA, but persistently elevated CMV-DNA on BAL were documented. At week 8, MBV was started and letermovir continued, for a 8 weeks course, without notable adverse effects. Respiratory function improved but soon after septic shock occurred. A bone marrow biopsy resulted in lymphoma, without indications for treatment: the patient developed coma and died 6 months after admission. MBV has recently been approved in Europe for treatment of R/R CMV in HSCT and SOT recipients. MBV showed superior rates of viraemia clearance after 8 weeks compared to SOC, demonstrating also a favourable safety profile with fewer patients discontinuing treatment and being affected by nephrotoxicity and neutropenia. Its main side effects are taste impairment, gastro-intestinal symptoms and asthenia. Based on actual promising perspectives regarding antiviral stewardship, more data are required to corroborate benefit of MBV in terms of toxicity and impact on mortality in highly fragile populations as SOT recipients. MBV received approval for the treatment of refractory or resistant CMV infections to other antiviral agents. Nevertheless, real-life data on efficacy and safety of MBV are still lacking. We conducted a narrative review of the current literature on MBV as treatment for CMV infection in kidney transplant recipients to understand clinical characteristics, safety and outcomes of MBV in this population. A search was run on the main scientific databases. 194 papers were identified, of which 188 were excluded by title and abstract evaluation. Subsequently, 6 papers were included. We performed descriptive statistics on the entire study population. The studies included in our analysis showed a higher prevalence of male subjects. The median age was 57 year. CKD was the most frequently reported comorbidity. Seven patients reported a donor/recipient mismatch (D+/R-). The case report and the cohort of patients collected from the literature show that MBV was used as an option in R/R CMV, notably for the presence or suspicion of CMV resistance to previous treatment. The clinical presentation of CMV in kidney SOT was heterogenous and varied from isolated reactivation of CMV-DNAemia, isolated fever or gastrointestinal involvement. For mild to moderate CMV disease, as with the cases reported in our review, or for proven ganciclovir, foscarnet or cidofovir resistance, MBV could be a valuable option. Outcomes of the patients treated with MBV were not reported in all the studies; however, where reported, 45.4% of the cases developed virological failure during MBV treatment with the development of specific resistance to MBV. MBV was generally well-tolerated, with low rates of toxicity, normally reversible. The introduction of new oral antivirals, such as MBV, could improve treatment, prophylaxis and preemptive treatment strategies, especially in anti-CMV treatment experienced patients.

Detection of human cytomegalovirus in transbronchial biopsies from lung transplant recipients.

The role of human cytomegalovirus (HCMV) in lung transplantation (LT) and drawbacks related to viral quantification in bronchoalveolar lavage (BAL) underline the potential usefulness of investigating other specimens. Thirty-three LT recipients were prospectively studied by HCMV quantitative real time PCR on matched transbronchial biopsy (TBB), BAL, and whole blood specimens. Overall, 27/33 patients turned out HCMV-positive in at least one specimen: 7.1 %, 37.1 %, and 13.5 % of TBB, BAL, and blood samples, respectively. No significant association between HCMV on all types of specimens and acute rejection, lymphocytic bronchiolitis, bronchiolitis obliterans and bronchiolitis obliterans syndrome was found. HCMV pneumonia was associated to HCMV detection on TBB (p = 0.003) and whole blood (p = 0.008), not on BAL (p = 0.47). The highest mean viral load was detected in TBB from cases with HCMV pneumonia in comparison to all other cases, suggesting the potential use of HCMV investigation in TBB for evaluating posttransplant complications.

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.

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.

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.

Detection of Mimivirus in bronchoalveolar lavage of ventilated and nonventilated patients.

BACKGROUND/AIMS: We investigated the prevalence of Mimivirus in bronchoalveolar lavage (BAL) specimens from ventilated versus nonventilated patients. METHODS: The occurrence of Mimivirus DNA was evaluated by two previously developed real-time PCR assays in 69 BAL specimens: 30 from patients on mechanical ventilation for at least 48 h and 39 from nonventilated patients from different clinical settings, including lung transplant recipients. RESULTS: None of the BAL specimens from ventilated and nonventilated patients resulted positive for Mimivirus. CONCLUSION: This study, similarly to other studies that used molecular assays to detect Mimivirus, found no occurrence of the virus in the lower respiratory tract, thus being in contrast to serological investigations which reported a significant association between Mimivirus and the development of pneumonia. Gene polymorphism could explain these results or, alternatively, it could be hypothesized that Mimivirus does not represent a common cause of lower respiratory tract infection in either ventilated or nonventilated patients. Further studies on a larger population of patients from a different clinical setting evaluating both serology and DNA detection in lower respiratory tract specimens, including BAL and possibly tissue samples, could allow a better definition of the epidemiological and pathological role of Mimivirus in the development of pneumonia.

Detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients.

The occurrence of human rhinoviruses (HRV) and its relationship to clinical and histopathological findings were investigated in 127 bronchoalveolar lavage specimens from 36 lung transplant recipients by real-time RT-PCR. In addition, 286 samples from 235 other immunocompromised and immunocompetent patients were also studied. HRV was detected in 41.7% of lung transplant recipients vs 14.5% of other patients (p < 0.0001), and no differences in viral load were observed. Acute respiratory insufficiency was found in 15 cases, three of which were HRV positive (viral load, 6.3 x 10(6) RNA copies/ml in one patient with chronic graft dysfunction). A diagnosis of pneumonia was made in 10 out of 127 cases, two of which were HRV positive (viral load, 10(3)-10(4) in cases of co-infection). Acute rejection was diagnosed in 12 cases, three of which were HRV positive (viral load, 10(3) in two cases of co-infection and 10(5) in a single infection). HRV infection may involve the lower respiratory tract, particularly in the presence of an impaired pulmonary background, such as a transplanted lung. Clinical evaluation should take into account the viral load, with a load of >10(5) possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.

Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.