Histopathologic, immunohistochemical, and polymerase chain reaction assays in the study of cases with fatal sporadic myocarditis.

Paraffin tissue blocks from 27 cases with sporadic myocarditis were collected during a 12-year period at a single medical examiner's office. Blocks were studied by using histopathology; immunohistochemistry for viruses (adenovirus, enterovirus, influenza A and B, and human herpes types 4 and 5), bacteria (Neisseria meningitidis, Ehrlichia sp, spotted fever group Rickettsia) and parasites (Toxoplasma gondii and Trypanosoma cruzi); and polymerase chain reaction (PCR)/RT-PCR for adenovirus and enterovirus. We identified enterovirus in 5 (18.5%) cases and Sarcocystis in a 36-year-old woman who had focal inflammation and myocyte necrosis. Immunohistochemical evidence of enteroviruses was found in the myocytes of 2 patients less than 6 months old who had diffuse mononuclear myocardial inflammation, interstitial pneumonitis; one also had encephalitis. In these 2 patients, the presence of enterovirus was confirmed by RT-PCR targeting the 5' nontranslated region and was serotyped as coxsackievirus B2 by sequencing the VP1 capsid region. In another 3 cases (ages 12, 47, and 54), enterovirus was detected by the 5' nontranslated region region; VP1 sequencing identified these as echoviruses 6, 13, and 7, respectively. Accurately identifying an infectious agent is the foundation for clinical and public health interventions. Despite using multiple diagnostic methods, an organism could only be detected in a small proportion of sporadic myocarditis cases.

Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses.

The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3'-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68.3 % of isolates, while the HEV-A and HEV-C primers accounted for 9.7 and 11.3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6.5 %) were amplified by more than one primer set and eight isolates (4.3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3'-non-translated region sequences.