Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity.

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection.

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 developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

Development of a highly sensitive and specific blastomycosis antibody enzyme immunoassay using Blastomyces dermatitidis surface protein BAD-1.

Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis.

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

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.

Genotype prevalence and risk factors for severe clinical adenovirus infection, United States 2004-2006.

BACKGROUND: Recently, epidemiological and clinical data have revealed important changes with regard to clinical adenovirus infection, including alterations in antigenic presentation, geographical distribution, and virulence of the virus. METHODS: In an effort to better understand the epidemiology of clinical adenovirus infection in the United States, we adopted a new molecular adenovirus typing technique to study clinical adenovirus isolates collected from 22 medical facilities over a 25-month period during 2004-2006. A hexon gene sequence typing method was used to characterize 2237 clinical adenovirus-positive specimens, comparing their sequences with those of the 51 currently recognized prototype human adenovirus strains. In a blinded comparison, this method performed well and was much faster than the classic serologic typing method. RESULTS: Among civilians, the most prevalent adenovirus types were types 3 (prevalence, 34.6%), 2 (24.3%), 1 (17.7%), and 5 (5.3%). Among military trainees, the most prevalent types were types 4 (prevalence, 92.8%), 3 (2.6%), and 21 (2.4%). CONCLUSIONS: For both populations, we observed a statistically significant increasing trend of adenovirus type 21 detection over time. Among adenovirus isolates recovered from specimens from civilians, 50% were associated with hospitalization, 19.6% with a chronic disease condition, 11% with a bone marrow or solid organ transplantation, 7.4% with intensive care unit stay, and 4.2% with a cancer diagnosis. Multivariable risk factor modeling for adenovirus disease severity found that age <7 years (odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4-7.4), chronic disease (OR, 3.6; 95% CI, 2.6-5.1), recent transplantation (OR, 2.7; 95% CI, 1.3-5.2), and adenovirus type 5 (OR, 2.7; 95% CI, 1.5-4.7) or type 21 infection (OR, 7.6; 95% CI, 2.6-22.3) increased the risk of severe disease.

Role of cell culture for virus detection in the age of technology.

Viral disease diagnosis has traditionally relied on the isolation of viral pathogens in cell cultures. Although this approach is often slow and requires considerable technical expertise, it has been regarded for decades as the "gold standard" for the laboratory diagnosis of viral disease. With the development of nonculture methods for the rapid detection of viral antigens and/or nucleic acids, the usefulness of viral culture has been questioned. This review describes advances in cell culture-based viral diagnostic products and techniques, including the use of newer cell culture formats, cryopreserved cell cultures, centrifugation-enhanced inoculation, precytopathogenic effect detection, cocultivated cell cultures, and transgenic cell lines. All of these contribute to more efficient and less technically demanding viral detection in cell culture. Although most laboratories combine various culture and nonculture approaches to optimize viral disease diagnosis, virus isolation in cell culture remains a useful approach, especially when a viable isolate is needed, if viable and nonviable virus must be differentiated, when infection is not characteristic of any single virus (i.e., when testing for only one virus is not sufficient), and when available culture-based methods can provide a result in a more timely fashion than molecular methods.