Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification.

Viral culture isolation has been widely accepted as the "gold standard" for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at -70 degrees C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

Cutaneous fibropapilloma in a mountain lion (Felis concolor).

A 12-yr-old mountain lion (Felis concolor) developed a 0.5-cm3 raised nonpigmented and nonulcerated mass between the lip and the nasal planum. The tumor was surgically removed and diagnosed histologically as a fibropapilloma. The tumor recurred 1 yr later, at which time it was again excised, and the diagnosis was reconfirmed by biopsy. Frozen tissue from the second excision was submitted for polymerase chain reaction testing for papillomavirus. The 176-base pair polymerase chain reaction product recovered from the tumor was cloned and sequenced. The papillomavirus had 96% homology with a papillomavirus previously retrieved from a fibropapilloma in a domestic cat and is the next most closely related to bovine papillomavirus type 1. This is the first report of a virus-associated fibropapilloma in a mountain lion.

Camelid mucoutaneous fibropapillomas: clinicopathologic findings and association with papillomavirus.

Five camelid mucocutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by a dermal fibroblastic proliferation and overlying, often ulcerated hyperplastic epidermis with thin rete pegs extending down into the dermis. Two of the tumors came from llamas and three from alpacas. Four of the animals were 6-year-old females. The fifth was a 6-year-old castrated male. The fibropapillomas were located on the nose, lip, and cheeks. One of the llama tumors waxed and waned before surgery and recurred and spread after surgery. None of the other tumors recurred. All five tumors were positive for papillomavirus (PV) DNA by polymerase chain reaction testing. Nucleotide sequence analysis of the PCR product from one of the llama fibropapillomas confirmed a unique PV. This report provides the microscopic and clinical features of fibropapillomas in camelids as well as evidence for a PV etiology.

Applying molecular biological techniques to detecting biological agents.

Recent efforts in the civilian and military health care and public health communities are directed at strengthening surveillance systems and our national laboratory capabilities for early detection of infectious disease outbreaks. These new systems will address proper specimen collection, transport, nucleic acid processing, molecular assay diagnostic reagent and equipment development, and standardization for sensitive and rapid detection of bioagents in blood and other clinical samples. A greater understanding of the genetic diversity and virulence factors for each organism that could be used for bioterrorism would aid design of rapid molecular detection strategies. Combinations of appropriate diagnostic technologies (culture, immunoassay, and molecular assay) can provide rapid diagnostic response capabilities to microbial threats with antimicrobial resistant organisms, new emerging infectious disease agents, and possible agents of bioterrorism.

Primary effusion lymphoma with herpesvirus 8 DNA in patients coinfected with HIV and hepatitis C virus: a report of 2 cases.

The primary effusion lymphoma (PEL), commonly described in patients with AIDS, is a unique subset of diffuse large cell lymphoma in which the malignant lymphocytes proliferate exclusively in serous cavities. The cytologic, immunophenotypic, and molecular features of PEL are presented from findings of 2 patients coinfected with HIV and hepatitis C virus who presented with abdominal pain. Abdominal radiography in both patients displayed marked peritoneal effusions. Cytomorphologic examination of peritoneal fluid revealed a malignant lymphoma in both. Their immunophenotypic expression was CD30 (Ki-1) and epithelial membrane antigen. Molecular analysis demonstrated human herpesvirus 8 DNA in both patients and bcl-2 oncogene rearrangement within the major breakpoint region of t(14;18) chromosome translocation in Case B only. Clinical correlation supports the current concept that PEL represents a primary HIV/AIDS-related lymphoma in effusion. Cytomorphologic examination of body cavity fluid serves as a tool for the initial diagnosis of PEL.

Feline cutaneous fibropapillomas: clinicopathologic findings and association with papillomavirus infection.

Twenty-three feline cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by dermal fibroblastic proliferation with overlying, often ulcerated hyperplastic epidermis. Electron microscopic findings supported the fibroblastic nature of the neoplastic cells. The 23 tumors came from 20 cats and were submitted from veterinary clinics in Wisconsin and Minnesota. These tumors occurred most commonly in young cats and were found primarily on the head, neck, and digits. Fifteen of the 17 cats for which breed was reported were domestic shorthair cats. In 11/20 cases, there was confirmed exposure to cattle. Local recurrence of the tumor following surgical excision was reported in 7 of the 18 cats for which follow-up information was available. Metastasis was not documented in any of the cases. Two of the 19 tumors tested by polymerase chain reaction (PCR) had no amplifiable DNA. The remaining 17 were positive for papillomavirus by PCR. No papillomavirus DNA was detected in three other feline skin tumors (cutaneous mast cell tumor, malignant lymphoma, and fibrosarcoma) that served as controls. This is the first report of detection of papillomavirus in feline tumors that have clinicopathologic features similar to equine sarcoids.

Common metastatic carcinoma of California sea lions (Zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus.

Tissues from 10 adult California sea lions (Zalophus californianus, seven females and three males) that had metastatic carcinoma in sublumbar area lymph nodes were examined histologically. A distinctive epithelial proliferative lesion interpreted as intraepithelial neoplasia was found in genital tracts of all ten animals; in vagina (5/7), cervix (7/7), uterus (3/7), penis (3/3) and prepuce (3/3). Intraepithelial neoplasia closely resembled metastatic carcinomas and was directly contiguous with invasive carcinoma in one animal. Rare eosinophilic intranuclear inclusion bodies were found in penile and preputial intraepithelial neoplasia (one animal), cervical intraepithelial neoplasia (one animal), invasive cervical carcinoma (one animal) and metastatic carcinoma (two animals). Electron microscopic examination of tissues from two sea lions (one with intraepithelial neoplasia and one with metastatic carcinoma) demonstrated viral particles consistent with a herpesvirus. An immunohistochemical stain for the latent membrane protein of Epstein-Barr virus was positive in intraepithelial neoplasia in one sea lion. Herpesvirus DNA sequences were detected by consensus primer polymerase chain reaction (PCR) in metastatic carcinomas from all four sea lions from which unfixed tumor samples were available. Results of sequencing were consistent with a novel gammaherpesvirus in the genus Rhadinovirus. DNA extracted from the four metastatic carcinomas also was tested for papillomavirus by Southern blot and PCR with consensus papillomavirus primers; all samples were negative by both methods. These findings support the genital origin of the sea lion carcinoma and implicate a novel gammaherpesvirus as a possible cause.

Molecular genetic evidence of a novel morbillivirus in a long-finned pilot whale (Globicephalus melas).

A long-finned pilot whale with morbilliviral disease was stranded in New Jersey. An immunohistochemical stain demonstrated morbilliviral antigen. Reverse transcriptase-polymerase chain reaction for morbillivirus P and N genes was positive. Novel sequences most closely related to, but distinct from, those of dolphin and porpoise morbilliviruses suggest that this virus may represent a third member of the cetacean morbillivirus group.

Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.

BACKGROUND: Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. METHODS AND RESULTS: Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. CONCLUSIONS: Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.

Differential expression of cyclin D1 in mantle cell lymphoma and other non-Hodgkin's lymphomas.

Mantle-cell lymphomas are associated with a characteristic chromosomal translocation, t(11;14)(q13;q32). This translocation involves rearrangement of the bcl-1 proto-oncogene from chromosome 11 to the immunoglobulin heavy chain gene on chromosome 14, resulting in an overexpression of cyclin D1 mRNA (also known as bcl-1 and PRAD1). In the current study performed on paraffin-embedded tissue, cyclin D1 mRNA could be detected in 23 of 24 mantle-cell lymphomas by reverse transcription polymerase chain reaction (RT-PCR) whereas only 9 of 24 demonstrated a t(11;14) by PCR. However, we also found that cyclin D1 mRNA could be detected in the majority (11 of 17, 65%) of non-mantle-cell lymphomas and in a minority of atypical lymphoid hyperplasias (3 of 7, 43%). Cyclin D1 mRNA expression was not observed in floridly reactive lymph nodes (0 of 9) or in unstimulated lymph nodes (0 of 20), suggesting that it is a sensitive adjunct marker for malignant lymphoproliferative processes, but not specific for mantle-cell lymphoma. A semiquantitative RT-PCR assay was developed that compared the ratio of cyclin D1 to the constitutively expressed gene beta2-microglobulin. Using this assay on a limited number of our specimens, cyclin D1 overexpression in mantle-cell lymphoma could be reliably distinguished from its expression in other non-Hodgkin's lymphomas. This assay for cyclin D1 expression, designed for formalin-fixed, paraffin-embedded tissue, was a very sensitive and specific marker for mantle-cell lymphoma.

Histologic, immunohistochemical, and polymerase chain reaction studies of bottlenose dolphins from the 1987-1988 United States Atlantic coast epizootic.

Tissues from 95 bottlenose dolphins (Tursiops truncatus) that died during the 1987-1988 US Atlantic coast epizootic and 11 bottlenose dolphins that died along the Atlantic coast prior to 1987 were examined histologically and immunohistochemically. Polymerase chain reaction (PCR) testing was performed on 36 of the epizootic and all of the pre-1987 cases. Epizootic cases had syncytia and rare intranuclear and intracytoplasmic inclusion bodies within lung, lymph node, and spleen. Lymphoid depletion was present in lymph node, spleen, and gut-associated lymphoid tissue of epizootic cases. Pre-1987 cases did not have these pulmonary and lymphoid lesions. A larger percentage of epizootic than pre-1987 cases had bacterial and/or fungal infections (primarily pneumonias), pulmonary and lymphoid tissue histiocytosis, mucocutaneous ulcers, and evidence of negative energy balance. Immunohistochemically, 49/95 (52%) epizootic dolphins were positive for morbilliviral antigen. Morbilliviral antigen was detected in lung, lymph node, spleen, thymus, skin, tongue, esophagus, liver, pancreas, gastrointestinal tract, urinary bladder, oviduct, and mammary gland by immunohistochemistry. PCR testing identified morbilliviral RNA in 35/36 (97%) epizootic cases tested. Neither morbilliviral antigen nor morbilliviral RNA were detected in pre-1987 cases. Histologic, immunohistochemical, and PCR results provide strong evidence that morbillivirus infection was the primary cause of the 1987-1988 bottlenose dolphin epizootic.

Herpes simplex virus in primary graft failure.

OBJECTIVE: To determine the frequency of herpes simplex virus type 1 (HSV-1) in primary corneal graft failures. METHODS: The clinical data submitted about all cases of corneal graft failure on file at the Armed Forces Institute of Pathology, Washington, DC, from the last 25 years (1970-1995) were evaluated. Cases that met the definition of primary graft failure were examined microscopically and analyzed using polymerase chain reaction (PCR) for the DNA of HSV-1. RESULTS: Three (2.8%) of the 106 cases of graft failure were primary graft failures. The DNA from 2 of the 3 corneal buttons was amplifiable by PCR analysis and results of the PCR analysis and Southern blotting were positive for HSV-1. None of the results of the PCR analysis and Southern blotting of the corneal buttons from the 3 graft failures occurring later than 30 days were positive for HSV-1. Results of the PCR analysis and Southern blotting indicated that 2 of 3 corneal buttons in the control group of clinically suspected herpetic keratitis were positive for HSV-1. The cornea from the first case of primary graft failure showed acute inflammation with stromal necrosis. The cornea in the second case had loss of endothelium without inflammation. CONCLUSIONS: The finding of DNA from HSV-1 in corneal buttons from 2 cases of primary graft failure supports similar observations by Cleator et al and suggests that HSV-1 may be pathogenic in some cases of primary graft failure. A larger study is needed to determine if HSV-1 is a causative factor in primary graft failure.

Initial genetic characterization of the 1918 "Spanish" influenza virus.

The "Spanish" influenza pandemic killed at least 20 million people in 1918-1919, making it the worst infectious pandemic in history. Understanding the origins of the 1918 virus and the basis for its exceptional virulence may aid in the prediction of future influenza pandemics. RNA from a victim of the 1918 pandemic was isolated from a formalin-fixed, paraffin-embedded, lung tissue sample. Nine fragments of viral RNA were sequenced from the coding regions of hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1, and matrix protein 2. The sequences are consistent with a novel H1N1 influenza A virus that belongs to the subgroup of strains that infect humans and swine, not the avian subgroup.

Transcription analysis and nucleotide sequence of tox promoter/operator mutants of corynebacteriophage beta.

The production of diphtheria toxin (DT) by Corynebacterium diphtheriae C7 (beta) is transcriptionally regulated by the iron-dependent diphtheria toxin repressor, DtxR. Transcription of the tox gene was studied in wild-type C. diphtheriae C7 (beta) and in lysogens carrying mutants of beta that determine insensitivity to inhibition of DT production by iron. Under low iron conditions in all strains, tox-specific mRNA appeared and DT production began during late-log phase, and they increased to maximal levels at stationary phase. Under high iron conditions, tox-specific mRNA and DT production were strongly repressed in C7 (beta) but only partially repressed in C7 (beta tox-202) and C7 (beta tox-201). Under high and low iron conditions, DT production and tox-specific mRNA levels were greater in C7 (beta tox-201) and C7 (beta tox-202) than in wild-type C7 (beta). Addition of iron or rifampicin to low iron cultures of C. diphtheriae C7 (beta) repressed tox-mRNA production promptly and with a similar time course. In contrast, repression of tox-mRNA synthesis in C. diphtheriae C7 (beta tox-201) occurred promptly after addition of rifampicin but more slowly after addition of iron. Nucleotide sequence analysis revealed single G to A mutations at positions -47 and -48, within the preferred '-10' sequence of the tox promoter, in beta tox-201 and beta tox-202, respectively. The single nucleotide substitutions in the tox-201 and tox-202 regulatory alleles, therefore, have pleiotropic effects, causing increased activity of the promoter and partial resistance of the operator to iron-dependent repression.

Coordinate regulation of siderophore and diphtheria toxin production by iron in Corynebacterium diphtheriae.

Iron is an environmental signal which regulates the coordinate expression of genes associated with virulence in many pathogenic bacteria. In response to iron-deprivation, lysogenic Corynebacterium diphtheriae C7 (beta) synthesizes and secretes diphtheria toxin and siderophore and induces a high-affinity iron uptake system. Diphtheria toxin is encoded by beta phage, but genes for siderophore production are encoded on the bacterial chromosome. Diphtheria toxin and siderophore production were shown to be coordinately induced during late logarithmic phase growth of wild-type C7(beta) in iron-limited medium. C. diphtheriae mutant C7hm723 produced siderophore and toxin constitutively under low-iron and high-iron conditions, but in mutants HC1, HC3, HC4, and HC5 their synthesis was partially repressed under high-iron conditions. The phenotypes of HC1, HC3, HC4, and HC5 are consistent with their severe defects in iron uptake, but the phenotype of C7hm723 is more likely to be explained by inactivation of the repressor for the iron regulon of C. diphtheriae.

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.

Cofactor requirements of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities in cell extracts of Clostridium scindens.

Two neutral steroid-transforming activities were demonstrated in cell extracts of Clostridium scindens. Steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase were found to be inducible in cells cultured in the presence of cortisol. Both activities required manganese ions and NAD+ or NADH for activity. Cortisol, cortisone and 11-desoxycortisol were substrates as well as inducers of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. 17 alpha-Hydroxyprogesterone was an effective inducer but did not serve as a substrate for either enzyme activity. C. scindens is the first bacterial species of the normal human intestinal flora reported to elaborate inducible steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. The results of cofactor, substrate specificity and induction studies suggest that these two activities may reside in the same enzyme complex.