Performance of a Novel Real-Time PCR-Based Assay for Rapid Monkeypox Virus Detection in Human Samples.

The ongoing epidemic of mpox, namely human monkeypox virus (MPXV) infection, requires rapid and reliable laboratory diagnosis. We report on the QIAstat-Dx viral vesicular panel PCR assay that allows the detection of (within 75 min) six vesicular disease-causing viruses, including MPXV. We analyzed 168 clinical samples, known to be positive (51 samples) or negative (117 samples) for MPXV clade II, obtained from patients at their mpox diagnosis or follow-up. QIAstat assay results were compared to those of a MPXV-specific reference PCR assay. The QIAstat assay detected MPXV (clade II) in 51 (100%) of 51 samples and did not detect MPXV in 117 (100%) of 117 samples, resulting in a positive or negative agreement of 100% (95% CI, 93.0-100) and 100% (95% CI, 96.8-100), respectively. Of the 20 patients diagnosed with mpox, 18 (90.0%) had at least a vesicular swab and 1 (5.0%) had only an oropharyngeal swab positive for MPXV. At mpox follow-ups, 2 (10.0%) of 20 patients had first-time positive whole blood samples. Thirteen MPXV-negative samples were positive for mpox-mimicking viruses. Our findings show the excellent performance of the QIAstat-Dx assay for MPXV detection in clinical samples. Further studies are needed before considering a large-scale application of the QIAstat-Dx assay.

Prevalence, incidence and predictors of anal high-risk HPV infections and cytological abnormalities in HIV-infected individuals.

OBJECTIVES: Our purpose was to investigate prevalence, incidence and risk factors of anal high risk-HPV infections and cytological abnormalities in HIV-positive individuals. METHODS: A cohort of consecutively enrolled HIV-positive patients underwent, at baseline visit, a sexual behaviors questionnaire, anoscopy, HPV testing and cytological examination. Hybridization and multiplex-PCR were used for DNA detection and typing; HPV E6-E7 mRNA expression was analyzed in HR-HPV+ patients. Logistic regression was used to assess predictors of HR-HPV infection and anal dysplasia. RESULTS: 233 HIV-infected patients were enrolled (81% males, median age 44 years). HR-HPV was detected in 144 anal swabs and showed a positive association with CDC stage C and a negative association with a higher CD4 count and the use of a NNRTI-based antiretroviral regimen. HR-HPV DNA detection and anal warts at baseline were associated to cytological abnormalities; a detectable HIV-RNA independently predicted new onset anal dysplasia at follow-up (incidence 15.4 per 100 patients-year). Incidence of new HR-HPV infection was 44.2 per 100 patients-year. CONCLUSIONS: The relevance of screening for anal dysplasia in HIV+ patients is emphasized, especially in those with detectable plasma HIV-RNA, anal HR-HPV infection or compromised immunological status.

Porphyromonas gingivalis and the pathogenesis of rheumatoid arthritis: analysis of various compartments including the synovial tissue.

INTRODUCTION: We evaluated the presence of Porphyromonas gingivalis (Pg) DNA in the synovial tissue through synovial biopsy and in other compartments of rheumatoid arthritis (RA) patients in comparison with patients affected by other arthritides. Possible links with clinical, immunologic and genetic features were assessed. METHODS: Peripheral blood (PB), sub-gingival dental plaque, synovial fluid (SF) and synovial tissue samples were collected from 69 patients with active knee arthritis (32 with RA and 37 with other arthritides, of which 14 had undifferentiated peripheral inflammatory arthritis - UPIA). Demographic, clinical, laboratory and immunological data were recorded. The presence of Pg DNA was evaluated through PCR. The HLA-DR haplotype was assessed for 45 patients with RA and UPIA. RESULTS: No differences arose in the positivity for Pg DNA in the sub-gingival plaque, PB and SF samples between RA and the cohort of other arthritides. Full PB samples showed a higher positivity for Pg DNA than plasma samples (11.8% vs. 1.5%, P = 0.04). Patients with RA showed a higher positivity for Pg DNA in the synovial tissue compared to controls (33.3% vs. 5.9%, P <0.01). UPIA and RA patients carrying the HLA DRB1*04 allele showed a higher positivity for Pg DNA in the synovial tissue compared to patients negative for the allele (57.1% vs. 16.7%, P = 0.04). RA patients positive for Pg DNA in the sub-gingival plaque had a lower disease duration and a higher peripheral blood leucocyte and neutrophil count. The presence of Pg DNA did not influence disease activity, disease disability or positivity for autoantibodies. CONCLUSIONS: The presence of Pg DNA in the synovial tissue of RA patients suggests a pathogenic role of the bacterium. The higher positivity of Pg DNA in full peripheral blood and synovial tissue samples compared to plasma and synovial fluid suggests a possible intracellular localization of Pg, in particular in patients positive for HLA-DR4.

Comparison of real-time PCR and pp65 antigen assays for monitoring the development of Cytomegalovirus disease in recipients of solid organ and bone marrow transplants.

Cytomegalovirus (CMV) infection is a frequent complication in transplant recipients. This retrospective study compared real-time PCR (rt-PCR) and a pp65 antigen assay as tools for monitoring CMV infection in solid organ (SOT) and bone marrow (SCT) transplant patients. The study tested 2662 samples by rt-PCR, and 1284 specimens with a pp65 antigen assay. 24.3% of the rt-PCR samples and 4.1% of the pp65 antigen samples were positive. 793 specimens, from 230 patients, were tested with both assays. In 6.7% of samples, both tests were positive; in 72.7% both were negative; in the remaining 20.6% of cases, the results were discordant. CMV disease was diagnosed in 50 patients. Results from the two methods were poorly correlated (r=0.460). The sensitivity of rt-PCR (94%) was higher than that of the pp65 antigen assay (27%). Both assays showed high specificity (92% and 99%, respectively). ROC curve analysis, performed separately for SOT and SCT patients, confirmed that rt-PCR outperformed the pp65 assay in the detection of CMV. These findings provide evidence that rt-PCR is a reliable diagnostic tool, and that it can be more effective than pp65 based assays in monitoring CMV infection progression and in guiding therapy in immunocompromised patients.

Cytomegalovirus DNA retrieval in the inner ear fluids of a congenitally deaf child one month after primary infection: a case report.

In the present article we report cytomegalovirus (CMV) DNA localization in the inner ear of a 15-month-old deaf boy 1 month after a virologically documented primary infection. CMV DNA retrieval was possible thanks to polymerase chain reaction analysis of the perilymph collected at cochlear implant surgery. To the authors' knowledge this is the first demonstration of CMV persistence in the cochlea of an immunocompetent subject after an acquired infection.

Clinical performance of human papillomavirus E6 and E7 mRNA testing for high-grade lesions of the cervix.

Infection with high-risk (HR) human papillomavirus (HPV) is the major cause of cervical cancer. However, relatively few infections progress to malignant disease. Progression to malignancy requires the overexpression of the E6 and E7 genes in the integrated HPV genome. It follows that the E6 and E7 transcripts could be useful markers of disease progression. The study presented here tests this possibility, using data from colposcopy and from cytological and histological tests to compare RNA assays for the E6 and E7 genes with DNA testing. A total of 180 women underwent colposcopy, cytology, and biopsy of suspected lesions (143 cases). Cervical brush specimens were analyzed for HPV DNA and for E6 and E7 mRNA. DNA from HR HPV was found in 57.8% of the specimens; E6 and E7 transcripts were found in 45%. The rates of detection of HPV DNA and of E6 and E7 transcripts were 33.3% and 25%, respectively, for specimens with normal findings; 51.4% and 31.9%, respectively, for specimens with cervical intraepithelial neoplasia grade 1 (CIN1); and 61.1% and 44.2% for specimens with CIN2, respectively. All specimens with CIN3 and 95.5% of specimens from patients with squamous cell carcinoma were positive by both assays. Thirty-seven patients with normal colposcopy findings did not undergo biopsy. HPV DNA and mRNA transcripts were found in 32.4% and 18.9% of these cases, respectively. Comparisons with cytological tests produced similar results. Overall, the mRNA tests showed a higher specificity than the DNA tests for high-grade lesions (72.7% and 56.2%, respectively) and a higher positive predictive value (59.3% and 49.0%, respectively). These findings suggest that mRNA assays could be more powerful than DNA testing for predicting the risk of progression and offer a strong potential as a tool for triage and patient follow-up.

RNA (E6 and E7) assays versus DNA (E6 and E7) assays for risk evaluation for women infected with human papillomavirus.

In the majority of cases, high-risk human papillomavirus (HR HPV) infections regress spontaneously, with only a small percentage progressing to high-grade lesions. Current screening methods are based on DNA detection. An alternative would be to monitor expression of the E6 and E7 viral oncogenes continuously expressed by malignant phenotypes. In the work reported in this paper, we compared the two methods for a group of women with high-risk HPV infections. Cervical specimens from 400 women, previously found to be HPV DNA positive, were analyzed for HPV DNA by a liquid hybridization assay and typed by multiplex PCR (for types 16, 18, 31, and 33). Identification of HR HPV E6 and E7 RNA transcripts was performed using real-time reverse transcription-PCR and nucleic acid sequence-based amplification assays. Results were compared with concurrent cytological data. HR HPVs were found in 61.2% of patients. The most common genotype was HPV type 16 (HPV-16) (47.1%), followed by HPV-18, HPV-31, and HPV-33. Nine percent of cases involved other genotypes. Among 223 HPV DNA-positive samples, only 118 were positive in the RNA test. Among HPV DNA-positive patients with normal cytology, we detected E6 and E7 RNA transcripts in two cases (18.2%). The rate of detection increased gradually with the grade of the observed lesions, rising from 20% for patients with atypical squamous cells of undetermined significance to 48.1% for women with low-grade squamous intraepithelial lesions and 86.3% for those with high-grade squamous intraepithelial lesions. These results suggest that testing for HPV E6 and E7 transcripts could be a useful tool for screening and patient management, providing more accurate predictions of risk than those obtained by DNA testing.