qpcr-is-sensitive-rapid-method-for-detection-cytomegaloviral-dna

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Indiana University School of Medicine

Equine influenza virus (EIV) is the leading cause of acute respiratory infection in horses worldwide. In recent years, the precise mechanism by which influenza infection kills host cells is being re-evaluated. In this report, we examined whether caspases, a group of intracellular proteases, are activated following EIV infection and contribute to EIV-mediated cell death. Western blotting analysis indicated that a nuclear target of caspase-3, poly(ADP-ribose) polymerase (PARP) was proteolytically cleaved in EIV-infected MDCK cells, but not in mock-infected cells. In comparison with caspase-3 specific inhibitor Ac-DEVD-CHO, a general caspase inhibitor Boc-D-FMK provided much stronger inhibition of EIV-induced cytopathic effect and apoptosis. Our results suggest that EIV may activate more than one caspase. Caspase activation and cleavage of its cellular targets may play a critical role in EIV-mediated cytotoxicity.

Caspase activation in equine influenza virus induced apoptotic cell death.

Resulting from its subjective nature, cytopathic effect (CPE) due to virus infection in cell culture has long been difficult to quantify. This report illustrates the use of electric cell-substrate impedance sensing (ECIS) for monitoring the progression of CPE due to influenza A virus infection. ECIS monitors the impedance of a non-invasive ac current flowing through cell culture medium by gold film electrodes placed on the surface of the culture dish. As cultured cells attach and spread onto the electrodes, the current is impeded proportional to the number of attached cells, the number of tight junctions between cells and the shortness in distance between the cells and the substratum. In the case at hand, a healthy monolayer of cells was insulted with influenza A virus infection and exhibited a characteristic rounded cell morphology and cell detachment. These effects resulted in reduced impedance, which was monitored with ECIS. Since data obtained through ECIS are both quantitative and in real-time, it was possible to monitor continuously cell behavior during infection. This, in turn, allowed for a more detailed and comprehensive data set to analyze. More importantly, through ammonium chloride treatment of cells, it was also shown that ECIS may be exploited to examine a treatment's effect on the reduction of resistance because of its antiviral activity. Thus, ECIS may be a powerful approach for screening antiviral compounds quantitatively in a real-time fashion.

Use of electric cell-substrate impedance sensing as a tool for quantifying cytopathic effect in influenza A virus infected MDCK cells in real-time.

An abortion storm occurred in a goat herd, resulting in 75 aborted kids and 1 neonatal death from December 2004 to February 2005.

Serologic and reproductive findings after a herpesvirus-1 abortion storm in goats.

Serological evidence for influenza A, subtype H1 and H3 virus infections of bovines, associated with respiratory disease and decreased milk production, has been reported. Equine H3N8 influenza virus circulates widely and was responsible for the introduction of H3N8 influenza into canines.

Infectivity of equine H3N8 influenza virus in bovine cells and calves.

A 62-year-old woman with a 6-month history of jaundice.

Histopathologic diagnosis of gastrointestinal (GI) tract cytomegaloviral (CMV) infection relies on hematoxylin and eosin (H&E)-stained tissue, along with the aid of immunohistochemistry (IHC). However, non-classic appearing inclusions or atypical IHC staining patterns remain an ongoing concern for pathologists. We reported the use of real-time polymerase chain reaction (qPCR) on nucleic acid extracted from paraffin-embedded, formalin-fixed tissue of GI biopsies from cases of CMV infection (n = 91) diagnosed by H&E and IHC. Seventy-nine biopsies, including normal colon biopsies (n = 35), active colitis (n = 25), and active duodenitis (n = 19), were used as negative controls. Of 91 CMV-positive biopsies diagnosed by histology, 88 tested positive by qPCR, with a sensitivity of 96.7%. Of 79 negative controls, 78 were negative and 1 positive by qPCR, resulting in a specificity of 98.7%. Of the cases that were positive for CMV by histopathology, there were an additional 40 biopsies taken from these patients either during the same or previous procedures, some taken just days prior, which were negative for CMV by histology. Interestingly, 22 (55%) of these biopsies tested positive by qPCR, which correlated well with additional clinical CMV results. By defining qPCR as the "gold standard" for a CMV result, histology (H&E and/or IHC) had a sensitivity and specificity of 79% and 97%, respectively. Eighteen biopsies were found negative by H&E and equivocal by IHC. Among them, 14 (78%) tested positive for CMV by qPCR, which also correlated well with additional clinical results. qPCR is a sensitive, specific, and rapid molecular tool that may be helpful to aid in early diagnosis of CMV infection on equivocal or clinically highly suspicious small GI biopsies.

qPCR increases sensitivity to detect cytomegalovirus in formalin-fixed, paraffin-embedded tissue of gastrointestinal biopsies.

Listeriosis is characterized by bacteremia or meningitis. We searched for listeriosis case series and outbreak investigations published in English by 2013, and assessed the strength of evidence for foodborne acquisition among patients who ate hospital food. We identified 30 reports from 13 countries. Among the case series, the median proportion of cases considered to be hospital-acquired was 25% (range, 9%-67%). The median number of outbreak-related illnesses considered to be hospital-acquired was 4.0 (range, 2-16). All patients were immunosuppressed in 18 of 24 (75%) reports with available data. Eight outbreak reports with strong evidence for foodborne acquisition in a hospital implicated sandwiches (3 reports), butter, precut celery, Camembert cheese, sausage, and tuna salad (1 report each). Foodborne acquisition of listeriosis among hospitalized patients is well documented internationally. The number of listeriosis cases could be reduced substantially by establishing hospital policies for safe food preparation for immunocompromised patients and by not serving them higher-risk foods.

Morgan H. McCoy

Department of Pathology and Laboratory Medicine

Indiana University School of Medicine

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Morgan H. McCoy

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Indiana University School of Medicine

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Department of Pathology and Laboratory Medicine