Comparative evaluation of three commercial systems for detection of high-risk human papillomavirus in cervical and vaginal ThinPrep PreservCyt samples and correlation with biopsy results.

Genital human papillomavirus (HPV) is the etiologic agent of more than 99% of all cervical cancers worldwide, with 14 genotypes being considered oncogenic or "high risk" because of their association with severe dysplasia and cervical carcinoma. Among these 14 high-risk types, HPV-16 and -18 account for approximately 70% of cervical cancers. The aim of this study was to evaluate three FDA-approved HPV nucleic acid-based tests for the ability to predict high-grade cervical intraepithelial neoplasias (CIN2 or worse) in corresponding tissue biopsy specimens. Residual specimens (total n = 793, cervical n = 743, vaginal n = 50) collected in ThinPrep PreservCyt medium with a cytologic result of ≥ atypical squamous cells of undetermined significance were tested by the Hybrid Capture 2 (HC2) assay (Qiagen, Gaithersburg, MD), the cobas HPV test (Roche Diagnostics, Indianapolis, IN), and the APTIMA HPV assay (Hologic, San Diego, CA). Genotyping for HPV-16 and HPV-18 was simultaneously performed by the cobas HPV test. Results were compared to cervical or vaginal biopsy findings, when they were available (n = 350). Among the 350 patients with corresponding biopsy results, 81 (23.1%) showed ≥ CIN2 by histopathology. The ≥ CIN2 detection sensitivity was 91.4% by the cobas and APTIMA assays and 97.5% by HC2 assay. The specificities of the cobas, APTIMA, and HC2 assays were 31.2, 42.0, and 27.1%, respectively. When considering only positive HPV-16 and/or HPV-18 genotype results, the cobas test showed a sensitivity and a specificity of 51.9 and 86.6%, respectively. While the HC2, cobas, and APTIMA assays showed similar sensitivities for the detection of ≥ CIN2 lesions, the specificities of the three tests varied, with the greatest specificity (86.6%) observed when the HPV-16 and/or HPV-18 genotypes were detected.

Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices?

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Detection and differentiation of norovirus genogroups I and II from clinical stool specimens using real-time PCR.

A real-time RT-PCR assay was designed to detect and differentiate norovirus genogroups I (GI) and II (GII), with primers and probes targeting the nonstructural polyprotein gene. Stool samples (n = 100) submitted for routine testing by the BioFire FilmArray® GI panel were also tested by the norovirus GI/GII real-time PCR assays. When compared to the FilmArray GI panel, the norovirus real-time PCR assay demonstrated a sensitivity of 77.5% (62/80) and specificity of 95% (19/20). Specimens yielding discordant results (n = 19) were tested at two outside laboratories for adjudication. Following discordant resolution, the adjusted sensitivity and specificity of the norovirus real-time PCR assays were 96.9% (63/65) and 100% (35/35), respectively. These results suggest that the real-time PCR assays are able to accurately detect and differentiate norovirus GI/GII from clinical stool specimens. Furthermore, our report highlights a potential issue with the specificity of the BioFire FilmArray® norovirus assay, which warrants additional investigation.

Relationship of HHV8 replication and Kaposi's sarcoma after solid organ transplantation.

HHV8 DNA sequences have recently been isolated from all types of Kaposi's sarcomas, and its association in the etiopathogenesis of this tumor has been established. However, little is known about the regulation of HHV8 replication in immunocompromised patients seropositive for this virus, and its impact on the development of Kaposi's sarcoma (KS). Through the study of a heart transplant patient who developed KS and in whom peripheral blood lymphocytes (PBLs) had been prospectively collected before and after transplantation, we have investigated the pathogenesis of HHV8. Our results indicate that (i) HHV8 can reactivate soon after transplantation; (ii) viral replication, as determined by quantification of HHV8 DNA load of PBLs, increases significantly after transplantation; and (iii) increased HHV8 DNA levels in PBLs are associated with the development of KS.

Novel mutation in the CMV UL97 gene associated with resistance to ganciclovir therapy.

Cytomegalovirus (CMV) strains resistant to ganciclovir have been associated with specific mutations in the UL97 and UL54 genes. The UL97 gene of a CMV strain isolated from a renal transplant recipient before and after 438 days of ganciclovir treatment was amplified by polymerase chain reaction and sequenced. A novel mutation resulting in deletion of codons 595 to 603 was identified in the viral DNA from specimens obtained after, but not before, prolonged ganciclovir therapy. Clinical and virological resolution of CMV disease occurred after switching to foscarnet therapy. Although many ganciclovir resistance mutations have been mapped to the UL97 codon range 591-607, this one is unusual in that it involves deletion of half these codons. Because UL97 seems to be necessary for effective CMV replication, this deletion suggests that much of codons 591-607 can be removed without destroying the biological function of UL97, and that this codon range can be altered in various ways to affect ganciclovir susceptibility. Rapid, flexible genotypic assays directed at this part of UL97 may facilitate the early recognition of ganciclovir resistance.

Granulomatous vasculitis associated with herpes virus: a persistent, painful, postherpetic papular eruption.

Cutaneous granulomatous vasculitis manifesting as a postherpetic reaction pattern is uncommon hand has previously been reported as a delayed complication of varicella-zoster virus infection. We describe three patients who had persistent, painful, postherpetic papules in a zosteriform distribution that histologically demonstrated a small vessel granulomatous vasculitis. Herpes simplex virus DNA detected by the polymerase chain reaction technique was demonstrated in two cases.

The effect of centrifugation on the rapid detection of adenovirus in shell vials.

Monoclonal antibody, specific for the adenovirus group-reactive hexon antigen, was used for the detection of this agent by immunofluorescence 24 and 48 hours after inoculation of HEp-2 cell monolayers in 1-dram shell vials after low-speed centrifugation (700 X g, 30 minutes). Of 31 adenovirus isolates, 16 (sensitivity, 52%) and 30 (sensitivity, 97%) were detected after incubation for 24 hours and 48 hours, respectively. All adenovirus strains were detected in conventional tube cell cultures but required an average of four days incubation. The shell vial assay is rapid, sensitive, and specific and can be easily implemented in laboratories with cell culture experience.

New developments in the diagnosis of viral diseases.

Major technical advances have occurred, especially in the last 5 years, in the laboratory diagnosis of viral infections. Immunologic detection of immediate early antigens in specimens such as bronchoalveolar lavage fluid and blood inoculated into shell vial cell cultures, particularly for herpesvirus (cytomegalovirus, herpes simplex virus, varicella-zoster virus), has provided results 16 to 48 hours after inoculation rather than the several days required for recognition of cytopathic effects in conventional tube cell cultures. Similarly, cytomegalovirus viremia can be detected directly by immunostaining of peripheral blood leukocytes with commercially available reagents the same day the specimen is submitted to the laboratory. Single-test membrane immunoassays have provided rapid (15 minutes) detection of viral antigens (respiratory syncytial virus, rotavirus, influenza virus type A). In the near future, diagnostic virology laboratories will be expected to monitor viral strains for susceptibility to the growing list of antiviral drugs. Amplification of nucleic acid sequences of viruses from cerebrospinal fluid or tissue, which generally does not yield isolates by conventional diagnostic techniques, has added a new dimension to the laboratory diagnosis of viral infection.

Diagnosis of cytomegalovirus hepatitis by histopathology and in situ hybridization in liver transplantation.

Hematoxylin-eosin staining (H&E) of cytomegalovirus (CMV) inclusion bodies was compared with in situ hybridization using a biotinylated DNA probe for the detection of CMV. Of 29 biopsy specimens selected from 23 patients with clinical CMV hepatitis and typical CMV inclusions on histopathologic examination, 23 (79%) were positive by DNA probe and 17 (59%) were detected in cell cultures. The mean number of CMV foci per tissue section was higher by DNA probe (12) compared with H&E (5). We do not recommend in situ hybridization in microbiology laboratories as a replacement for histopathology for the diagnosis of CMV in tissue specimens.

Laboratory diagnosis of Pneumocystis carinii infections by PCR directed to genes encoding for mitochondrial 5S and 28S ribosomal RNA.

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis carinii in clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage 1(BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.

Human papillomavirus infection and intraepithelial, in situ, and invasive carcinoma of penis.

The role of human papillomavirus (HPV) infection and other reported cofactors in the genesis, evolution, and clinical manifestations of precancerous and cancerous squamous cell lesions of the penis were studied in 34 men. Clinically, all lesions demonstrated aceto-whitening. Histologic changes of HPV infection formed a field-of-change that involved the components of the preputial cavity in all patients. These changes were associated with minor grades of penile intraepithelial neoplasia (PIN I and II) in 19 patients, major grades of PIN/carcinoma in situ (PIN III/Tis) in 7, and invasive squamous cell carcinoma (SCCa, Stages T2 and T3) in 8. Most of the patients (79.4%) were heavy smokers; 52.9 percent had a history of HPV infection, PIN, or invasive penile SCCa; and 60 percent of 30 patients had female sexual partners who had HPV-related genital neoplasia. A pilot virologic study of specimens obtained from 20 representative patients utilizing polymerase chain reaction amplification detected HPV DNA in 80 percent. Laser therapy was aimed at the entire field-of-change in 30 patients; recurrent minor-grade PIN or SCCa developed in 2 of 23 patients (8.7%) followed for up to three years. Of the 4 remaining patients treated with local excision or partial penectomy, 3 (75%) had development of recurrent minor-grade PIN when followed for up to four years. The combination of the host of carcinogenic factors and currently rampant immunologic disorders will likely lead to an increase in the historically low incidence of SCCa of the penis in the United States.

Detection of human papillomavirus DNA in primary squamous cell carcinoma of the male urethra.

OBJECTIVES: The prevalence of human papillomavirus deoxyribonucleic acid (DNA) in primary squamous cell carcinoma of the male urethra and corresponding control tissue was studied. METHODS: The technique of polymerase chain reaction DNA amplification was used to detect specific human papillomavirus DNA sequences in archival pathologic and control tissue. We analyzed 16 cases of primary squamous cell carcinoma of the male urethra and 22 specimens of normal male urethra stratified by location within the urethra. RESULTS: Primary squamous cell carcinoma of the pendulous urethra was significantly associated with human papillomavirus type 16 DNA (6 of 6 cases), but the control tissue from the pendulous urethra was not (0 of 12, P < 0.001). Primary squamous cell carcinoma of the bulbous and posterior urethra was not associated with human papillomavirus infection. CONCLUSIONS: Infection of the male urethra with human papillomavirus type 16 may have a role in the pathogenesis of primary squamous cell carcinoma of the pendulous urethra, for which it has a strong predilection, vis-à-vis the bulbous and posterior urethra.

Polymerase chain reaction (PCR) identification of human papillomavirus (HPV) DNA in verrucous carcinoma of the larynx.

The incidence of human papillomavirus (HPV) DNA in archival formalin-fixed, paraffin-embedded tissue sections from verrucous carcinoma of the larynx was determined using polymerase chain reaction (PCR) with consensus primers and by in situ hybridization designed to detect HPV types 6/11, 16/18, 31/33/35. HPV DNA was detected in 17 (85%) of 20 tissue samples by PCR; none of the 20 samples were positive for the seven genotype types tested by in situ hybridization. PCR is a valuable tool to detect HPV and therefore will significantly clarify the importance of HPV in squamous mucosal disorders.

Real-time PCR in clinical microbiology: applications for routine laboratory testing.

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Comparison of specimen processing and nucleic acid extraction by the swab extraction tube system versus the MagNA Pure LC system for laboratory diagnosis of herpes simplex virus infections by LightCycler PCR.

A total of 563 specimens (234 dermal and 329 genital swabs) from patients suspected of having herpes simplex virus (HSV) infections were processed using two different extraction methods (the MagNA Pure LC system and the swab extraction tube system [SETS]); HSV DNA was amplified by LightCycler PCR. HSV DNA was detected in 157 of 563 specimens (27.9%) processed by the MagNA Pure LC system and in 179 of 563 specimens (31.8%) processed by SETS (P < 0.0001). There was no specimen processed by the MagNA Pure LC extraction method that was positive only for HSV DNA. Of 157 specimens positive by both methods, HSV DNA copy levels were higher (using cycle crossover points [cycle threshold {C(T)}]) with SETS (mean C(T), 25.9 cycles) than with the MagNA Pure LC system (mean C(T), 32.0 cycles) (P < 0.0001). The time to process 32 samples was longer with the MagNA Pure LC extraction system (90 min) than with SETS (35 min). HSV DNA extraction using SETS is faster, less expensive, and more sensitive than the MagNA Pure LC system and could replace the latter for the laboratory diagnosis of HSV infections using LightCycler PCR.

Detection of herpes simplex virus in conventional tube cell cultures and in shell vials with a DNA probe kit and monoclonal antibodies.

Specimens submitted for diagnosis of herpes simplex virus (HSV) infection were inoculated into shell vials and reacted with a commercial DNA probe kit (Pathogene; Enzo Biochem, Inc., New York, N.Y.) and an immunofluorescence assay at 16 h postinoculation. The results were compared with isolation of the virus in conventional tube cell cultures. Of 504 specimens, 105 (20.8%) were positive for HSV. Of the 105, 93 HSV-positive specimens (89%) were detected by all three assay systems. Maximum detection of HSV (100 of 105 [95%]) was obtained by probe or monoclonal antibody assay in shell vials, which had sensitivities of 98 and 97%, respectively, compared with viral recovery in conventional tube cell cultures (mean time for recognition of cytopathic effects, 2 days). Both shell vial assays were 99% specific. The DNA probe kit may be used as an alternative to a monoclonal antibody and fluorescence assay in shell vials as a diagnostic method for rapid laboratory detection of HSV infection.

Simultaneous seeding and infecting of shell vials for rapid detection of cytomegalovirus infection.

Fifty cytomegalovirus isolates were used to infect shell vials containing confluent MRC-5 cell monolayers and shell vials which were seeded with MRC-5 cells at the time of inoculation. All 50 of the isolates were detected by immunofluorescence in shell vials containing confluent monolayers, whereas 39 (78%) of the isolates were detected in shell vials that had been seeded and inoculated simultaneously (P less than 0.001). Preformed monolayers of cells in shell vials provide the most sensitive system for the detection of cytomegalovirus infection.

Comparison of SHARP signal system and Southern blot hybridization analysis for detection of cytomegalovirus in clinical specimens by PCR.

Thirty cytomegalovirus cell culture-positive samples were tested by the SHARP Signal System. Twenty-seven specimens (100% agreement) were identified by both methods. The SHARP Signal System is rapid (4 h), easy to perform, and potentially adaptable to automation.

Profile of phospholipids in amniotic fluid to assess fetal lung maturity in high-risk pregnancy.

In assessing fetal lung maturity, an amniotic fluid profile, consisting of a total phospholipid (as phosphorus) (TPP) and total lecithin (LT) concentration, has been shown to yield a predictive accuracy of 97.5% with a false-negative rate of 2.4%. The LT had a range of specificity of 93% to 99%, whereas the one for the TPP was 88% to 96%. The cut-off values were 0.14 mg P/dl and 3.5 mg lecithin/dl of amniotic fluid. The analytical variables for laboratory tests for measuring amniotic fluid phospholipids led to the development of the quantitative tests used for this study. The proposed profile appears to offer the clinician an excellent means of judging how to manage the high-risk pregnancy.