Morphologic, immunologic, and molecular methods to detect bacillus anthracis in formalin-fixed tissues.

Due to the importance of Bacillus anthracis as a cause of naturally occurring infection among humans and as an agent of bioterrorism, there is a vital need for rapid and specific assays, including immunohistochemistry (IHC) and polymerase chain reaction (PCR) assays, to detect the bacterium in formalin-fixed tissues. Colorimetric IHC assays were developed using a multistep indirect immunoalkaline phosphatase method with anti-B. anthracis cell wall (EAII-6G6-2-3) and anti-B. anthracis capsule (FDF-1B9) mAbs to detect B. anthracis antigens in formalin-fixed, paraffin-embedded bacterial cultures and tissues. B. anthracis antigens were localized, using both antibodies, in samples from B. anthracis-infected animals and humans. The colorimetric IHC assay with both antibodies was expedient in diagnosing the presence of B. anthracis in formalin-fixed, paraffin-embedded tissue from bioterrorism-associated cases of inhalational and cutaneous anthrax and from a case of naturally occurring cutaneous anthrax. Using the same antibodies, confocal microscopy demonstrated the structure of replicating B. anthracis in tissues. B. anthracis-specific primers were successfully used with PCR to amplify and detect B. anthracis sequences derived from formalin-fixed tissues of anthrax cases. In this study, morphologic, immunologic, and molecular assays were used to study and diagnose 22 veterinary and human anthrax cases.

Intestinal intussusception associated with adenovirus infection in Mexican children.

Formalin-fixed intestinal tissue specimens from 12 Mexican pediatric patients with intussusception were examined for the presence of adenovirus. Four patients (33%) had detectable adenovirus antigen in epithelial cells as determined by using immunohistochemical analysis. Two of the patients with positive immunohistochemical results had antigens in dendritic and mononuclear inflammatory cells, and 3 patients had positive results for species C adenovirus by in situ hybridization using adenovirus species-specific probes (A-F). A real-time polymerase chain reaction assay specific for species C (nonenteric) adenoviruses was used to confirm immunohistochemical results and to amplify adenovirus DNA for sequencing. A sequence similar to that for adenovirus serotype 1 was found in 1 patient, serotype 2 in another, and serotype 6 in a third; in the fourth patient, the sequence was indeterminate between serotypes 2 and 6. The assays used in this study proved useful for the identification of species C adenoviruses in formalin-fixed specimens from Mexican pediatric patients with intussusception.

The pathology of rotavirus-associated deaths, using new molecular diagnostics.

Rotavirus, the most common cause of severe, dehydrating gastroenteritis among children worldwide, annually causes approximately 500,000 deaths among children aged <5 years. The primary site of rotavirus infection is the small intestine. Pathologic investigations of patients who died of rotavirus infection are limited to data from a few reported autopsies, and dehydration with electrolyte imbalance is believed to be the major cause of death. Several recent reports suggest that children who died during a rotavirus illness were viremic before death, because rotavirus was detected at several extraintestinal sites. We report 3 rotavirus-associated deaths among children, 2 of whom had evidence of rotavirus genome in extraintestinal tissues detected by use of novel molecular diagnostic methods. The part played by rotavirus in fatal cases is unclear and requires additional investigation of diarrhea-associated deaths, because a better understanding might alter the approach to treatment and the need for antiviral therapy.

Molecular and immunological methods to detect rotavirus in formalin-fixed tissue.

In 1999, a tetravalent rhesus-based rotavirus vaccine was withdrawn from the market after reports of intussusception cases among vaccinated infants. Methods to detect rotavirus in formalin-fixed pathology specimens from such patients will be important in examining the possible associations between the vaccine and intussusception, in investigating fatalities caused by natural rotavirus infection, and in furthering our understanding of the pathogenesis of rotavirus disease. Three different methods, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and in situ hybridization (ISH), were developed to detect rotavirus in infected cell lines that were fixed in formalin and embedded in paraffin. Using specific primer pairs to identify the VP4 gene with a one-step RT-PCR method, we detected simian rotavirus strains RRV and YK-1 in the liver of an RRV-infected SCID mouse and in the small intestine of an YK-1 infected macaque, respectively. Using a two-step indirect immunoalkaline phosphatase technique, we found RRV antigens in the liver of an infected SCID mouse with a rabbit polyclonal anti-group A rotavirus antibody and a murine monoclonal anti-rotavirus VP2 antibody. Using riboprobes designed to detect RRV genes, VP4 and NSP4, we obtained a positive hybridization signal in the same area of the infected SCID mouse liver as the area in which rotavirus antigens were localized. These techniques should prove valuable to detect rotavirus antigens and nucleic acids in tissues from patients infected naturally with rotavirus or with intussususception associated with rotavirus vaccine.