Comparison of SARS-CoV-2 detection with the Cobas® 6800/8800 system on gargle samples using two sample processing methods with combined oropharyngeal/nasopharyngeal swab.

BACKGROUND: Gargle samples have been proposed as a noninvasive method for detection of SARS-CoV-2 RNA. The clinical performance of gargle specimens diluted in Cobas® PCR Media and in Cobas® Omni Lysis Reagent was compared to oropharyngeal/nasopharyngeal swab (ONPS) for the detection of SARS-CoV-2 RNA. STUDY DESIGN: Participants were recruited prospectively in two COVID-19 screening clinics. In addition to the ONPS, participants gargled with 5 ml of natural spring water split in the laboratory as follows: 1 ml was added to 4.3 ml of polymerase chain reaction (PCR) media and 400 µl was added to 200 µl of lysis buffer. Testing was performed with the Cobas® SARS-CoV-2 test on the Cobas® 6800 or 8800 platforms. RESULTS: Overall, 134/647 (20.7%) participants were considered infected because the ONPS or at least one gargle test was positive. ONPS had, respectively, a sensitivity of 96.3% (95% confidence interval [CI]: 91.3-98.5); both gargle processing methods were slightly less but equally sensitive (90.3% [95% CI: 83.9-94.3]). When ONPS and gargle specimens were both positive, the mean cycle threshold (Ct ) was significantly higher for gargles, suggesting lower viral loads. CONCLUSION: Gargle specimens directly added in PCR Media provide a similar clinical sensitivity to chemical lysis, both having a slightly, not significantly, lower sensitivity to ONPS.

Comparison of saliva with oral and nasopharyngeal swabs for SARS-CoV-2 detection on various commercial and laboratory-developed assays.

The accurate laboratory detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial element in the fight against coronavirus disease 2019 (COVID-19). Reverse transcription-polymerase chain reaction testing on combined oral and nasopharyngeal swab (ONPS) suffers from several limitations, including the need for qualified personnel, the discomfort caused by invasive nasopharyngeal sample collection, and the possibility of swab and transport media shortage. Testing on saliva would represent an advancement. The aim of this study was to compare the concordance between saliva samples and ONPS for the detection of SARS-CoV-2 on various commercial and laboratory-developed tests (LDT). Individuals were recruited from eight institutions in Quebec, Canada, if they had SARS-CoV-2 RNA detected on a recently collected ONPS, and accepted to provide another ONPS, paired with saliva. Assays available in the different laboratories (Abbott RealTime SARS-CoV-2, Cobas® SARS-CoV-2, Simplexa™ COVID-19 Direct, Allplex™ 2019-nCoV, RIDA®GENE SARS-CoV-2, and an LDT preceded by three different extraction methods) were used to determine the concordance between saliva and ONPS results. Overall, 320 tests were run from a total of 125 saliva and ONPS sample pairs. All assays yielded similar sensitivity when saliva was compared to ONPS, with the exception of one LDT (67% vs. 93%). The mean difference in cycle threshold (∆C t ) was generally (but not significantly) in favor of the ONPS for all nucleic acid amplification tests. The maximum mean ∆​​​​​C t was 2.0, while individual ∆C t varied importantly from -17.5 to 12.4. Saliva seems to be associated with sensitivity similar to ONPS for the detection of SARS-CoV-2 by various assays.

The diamond mesh, a phase-error- and loss-tolerant field-programmable MZI-based optical processor for optical neural networks.

This paper presents the performance analysis of a phase error- and loss-tolerant multiport field-programmable MZI-based structure for optical neural networks (ONNs). Compared to the triangular (Reck) mesh, our proposed diamond mesh makes use of a larger number of MZIs, leading to a symmetric topology and adding additional degrees of freedom for the weight matrix optimization in the backpropagation process. Furthermore, the additional MZIs enable the diamond mesh to optimally eliminate the excess light intensity that degrades the performance of the ONNs through the tapered out waveguides. Our results show that the diamond topology is more robust to the inevitable imperfections in practice, i.e., insertion loss of the constituent MZIs and the phase errors. This robustness allows for better classification accuracy in the presence of experimental imperfections. The practical performance and the scalability of the two structures implementing different sizes of optical neural networks are analytically compared. The obtained results confirm that the diamond mesh is more error- and loss-tolerant in classifying the data samples in different sizes of ONNs.

All-optical retro-modulation for free-space optical communication.

This work presents device and system architectures for free-space optical and optical wireless communication at high data rates over multidirectional links. This is particularly important for all-optical networks, with high data rates, low latencies, and network protocol transparency, and for asymmetrical networks, with multidirectional links from one transceiver to multiple distributed transceivers. These two goals can be met by implementing a passive uplink via all-optical retro-modulation (AORM), which harnesses the optical power from an active downlink to form a passive uplink through retroreflection. The retroreflected optical power is modulated all-optically to ideally achieve terabit-per-second data rates. The proposed AORM architecture, for passive uplinks, uses high-refractive-index S-LAH79 hemispheres to realize effective retroreflection and an interior semiconductor thin film of CuO nanocrystals to realize ultrafast all-optical modulation on a timescale of approximately 770 fs. The AORM architecture is fabricated and tested, and ultimately shown to be capable of enabling multidirectional free-space optical communication with terabit-per-second aggregate data rates.

Ultrafast all-optical technologies for bidirectional optical wireless communications.

In this Letter, a spherical retro-modulator architecture is introduced for operation as a bidirectional transceiver in passive optical wireless communication links. The architecture uses spherical retroreflection to enable retroreflection with broad directionality (2π steradians), and it uses all-optical beam interaction to enable modulation on ultrafast timescales (120 fs duration). The spherical retro-modulator is investigated from a theoretical standpoint and is fabricated for testing with three glasses, N-BK7, N-LASF9, and S-LAH79. It is found that the S-LAH79 structure provides the optimal refraction and nonlinearity for the desired retroreflection and modulation capabilities.

Clinical prediction and diagnosis of neurosyphilis in HIV-infected patients with early Syphilis.

The diagnosis of neurosyphilis (NS) is a challenge, especially in HIV-infected patients, and the criteria for deciding when to perform a lumbar puncture (LP) in HIV-infected patients with syphilis are controversial. We retrospectively reviewed demographic, clinical, and laboratory data from 122 cases of HIV-infected patients with documented early syphilis who underwent an LP to rule out NS, and we evaluated 3 laboratory-developed validated real-time PCR assays, the Treponema pallidum particle agglutination (TPPA) assay, the fluorescent treponemal antibody absorption (FTA-ABS) assay, and the line immunoassay INNO-LIA Syphilis, for the diagnosis of NS from cerebrospinal fluid (CSF) samples of these patients. NS was defined by a reactive CSF-VDRL test result and/or a CSF white blood cell (WBC) count of >20 cells/µl. Thirty of the 122 patients (24.6%) had early NS. Headache, visual symptoms, a CD4 cell count of <500 cells/µl, and viremia, as defined by an HIV-1 RNA count of ≥50 copies/ml, were associated with NS in multivariate analysis (P = <0.001 for each factor). Blood serum rapid plasma reagin (RPR) titers were not associated with early NS (P = 0.575). For the diagnosis of NS, the PCR, FTA-ABS, TPPA, and INNO-LIA assays had sensitivities of 58%, 100%, 68%, and 100%, specificities of 67%, 12%, 49%, and 13%, and negative predictive values of 85%, 100%, 84%, and 100%, respectively. Visual disturbances, headache, uncontrolled HIV-1 viremia, and a CD4 cell count of <500 cells/µl were predictors of NS in HIV-infected patients with early syphilis, while blood serum RPR titers were not; therefore, RPR titers should not be used as the sole criterion for deciding whether to perform an LP in early syphilis. When applied to CSF samples, the INNO-LIA Syphilis assay easily helped rule out NS.

On-demand, hospital-based, severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomic epidemiology to support nosocomial outbreak investigations: A prospective molecular epidemiology study.

Objectives: We evaluated the added value of infection control-guided, on demand, and locally performed severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomic sequencing to support outbreak investigation and control in acute-care settings. Design and setting: This 18-month prospective molecular epidemiology study was conducted at a tertiary-care hospital in Montreal, Canada. When nosocomial transmission was suspected by local infection control, viral genomic sequencing was performed locally for all putative outbreak cases. Molecular and conventional epidemiology data were correlated on a just-in-time basis to improve understanding of coronavirus disease 2019 (COVID-19) transmission and reinforce or adapt control measures. Results: Between April 2020 and October 2021, 6 outbreaks including 59 nosocomial infections (per the epidemiological definition) were investigated. Genomic data supported 7 distinct transmission clusters involving 6 patients and 26 healthcare workers. We identified multiple distinct modes of transmission, which led to reinforcement and adaptation of infection control measures. Molecular epidemiology data also refuted (n = 14) suspected transmission events in favor of community acquired but institutionally clustered cases. Conclusion: SARS-CoV-2 genomic sequencing can refute or strengthen transmission hypotheses from conventional nosocomial epidemiological investigations, and guide implementation of setting-specific control strategies. Our study represents a template for prospective, on site, outbreak-focused SARS-CoV-2 sequencing. This approach may become increasingly relevant in a COVID-19 endemic state where systematic sequencing within centralized surveillance programs is not available. Trial registration: clinicaltrials.gov identifier: NCT05411562.

Ciprofloxacin-resistant Shigella sonnei among men who have sex with men, Canada, 2010.

In 2010, we observed isolates with matching pulsed-field gel electrophoresis patterns from 13 cases of ciprofloxacin-resistant Shigella sonnei in Montréal. We report on the emergence of this resistance type and a study of resistance mechanisms. The investigation suggested local transmission among men who have sex with men associated with sex venues.

vanA-containing Enterococcus faecium susceptible to vancomycin and teicoplanin because of major nucleotide deletions in Tn1546.

OBJECTIVES: During the course of routine screening for vancomycin-resistant enterococci (VRE), we found six Enterococcus faecium isolates positive for vanA by PCR, but susceptible to vancomycin and teicoplanin by phenotypic testing. The aim of this study was to characterize the genetic composition of the Tn1546 vanA gene cluster of these isolates. METHODS: The E. faecium isolates were characterized by antibiotic susceptibility, PFGE and structural analysis of the Tn1546 elements. Plasmids extracted from these isolates were used to determine the presence of the Tn1546 vanA gene cluster by PCR and the genomic organization of the deleted Tn1546 element by primer walking DNA sequencing. RESULTS: The vancomycin-susceptible vanA-positive E. faecium isolates showed three PFGE patterns, and were missing the vanR and vanS genes that are responsible for the activation of transcription of resistance genes. Primer walking sequencing revealed that these genes were completely deleted and that interruptions in the vanA cluster were in the vicinity of insertion sequence elements. CONCLUSIONS: The presence of vancomycin-susceptible vanA-positive E. faecium in clinical samples results from major deletions in the Tn1546 vanA operon. Our findings support the essential role of vanR and vanS for the expression of resistance to vancomycin in enterococci.

Subtype diversity associated with the development of HIV-1 resistance to integrase inhibitors.

We used genotypic and phylogenetic analysis to determine integrase diversity among subtypes, and studied natural polymorphisms and mutations implicated in resistance to integrase inhibitors (INI) in treatment-naïve persons (n = 220) and -experienced individuals (n = 24). Phylogenetics revealed 7 and 10% inter-subtype diversity in the integrase and reverse transcriptase (RT)/protease regions, respectively. Integrase sequencing identified a novel A/B recombinant in which all viruses in a male-sex-male (MSM) transmission cluster (n = 12) appeared to possess subtype B in integrase and subtype A in the remainder of the pol region. Natural variations and signature polymorphisms were observed at codon positions 140, 148, 151, 157, and 160 among HIV subtypes. These variations predicted higher genetic barriers to G140S and G140C in subtypes C, CRF02_AG, and A/CRF01_AE, as well as higher genetic barriers toward acquisition of V151I in subtypes CRF02_AG and A/CRF01_AE. The E157Q and E160Q mutational motif was observed in 35% of INI-naïve patients harboring subtype C infections, indicating intra-subtype variations. Thirteen patients failed raltegravir (RAL)-containing regimens within 8 ± 1 months, in association with the major Q148K/R/H and G140A/S (n = 8/24) or N155H (n = 5/24) mutational pathways. Of note, the remaining patients on RAL regimens for 14 ± 3 months harbored no or only minor integrase mutations/polymorphisms (T66I, T97A, H114P, S119P, A124S, G163R, I203M, R263K). These results demonstrate the importance of understanding subtype variability in the development of resistance to INIs.

Impact of cobas PCR Media freezing on SARS-CoV-2 viral RNA integrity and whole genome sequencing analyses.

SARS-CoV-2 whole genome sequencing is a molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swabs and shipment to reference laboratories is usually required for sequencing. Cobas PCR Media transport medium facilitates high throughput SARS-CoV-2 RT-PCR analyses on cobas platforms. The manufacturer doesn't recommend freezing this transport medium because of risks of degrading molecular templates and impairing test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Sequencing performance, quality and results did not significantly differ between fresh and frozen samples (n = 10). Freezing of cobas PCR Media does not negatively affect SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories.

Clinical Evaluation of In-House-Produced 3D-Printed Nasopharyngeal Swabs for COVID-19 Testing.

3D-printed alternatives to standard flocked swabs were rapidly developed to provide a response to the unprecedented and sudden need for an exponentially growing amount of diagnostic tools to fight the COVID-19 pandemic. In light of the anticipated shortage, a hospital-based 3D-printing platform was implemented in our institution for the production of swabs for nasopharyngeal and oropharyngeal sampling based on the freely available, open-source design provided to the community by University of South Florida's Health Radiology and Northwell Health System teams as a replacement for locally used commercial swabs. Validation of our 3D-printed swabs was performed with a head-to-head diagnostic accuracy study of the 3D-printed "Northwell model" with the cobas PCR Media® swab sample kit. We observed an excellent concordance (total agreement 96.8%, Kappa 0.936) in results obtained with the 3D-printed and flocked swabs, indicating that the in-house 3D-printed swab could be used reliably in the context of a shortage of flocked swabs. To our knowledge, this is the first study to report on autonomous hospital-based production and clinical validation of 3D-printed swabs.

Performance of an agar dilution method and a Vitek 2 card for detection of inducible clindamycin resistance in Staphylococcus spp.

The D-zone test detects inducible clindamycin resistance in Staphylococcus spp. Two other methods not described by the Clinical and Laboratory Standards Institute (CLSI) are available to test for this resistance mechanism: an agar dilution method and new Vitek 2 cards. This study evaluated the performance of both methods in detecting inducible clindamycin resistance. Nonduplicate clinical strains of Staphylococcus spp. (111 Staphylococcus aureus and 52 coagulase-negative staphylococcus strains), intermediate or resistant to erythromycin but susceptible to clindamycin, were obtained from three hospitals in Montreal, Quebec, Canada. Molecular analysis to detect resistance genes was conducted on all strains. A Mueller-Hinton agar containing 1 mg of erythromycin and 0.5 mg of clindamycin/liter was used for the dilution method, and two inocula were tested: 10(4) and 10(5) CFU per spot. Plates were read at 24 and 48 h. The Vitek 2 AST-P580 card was used according to the manufacturer's recommendations. The results were compared to those of the D-zone test. The D-zone test was positive in 134 of 163 (82%) strains. With the 10(4) CFU inoculum, the sensitivities were 84 and 99% at 24 and 48 h, respectively. The 10(5) CFU inoculum increased the sensitivities at 24 and 48 h to 91 and 100%, respectively. The specificity was 100% for the 10(4) CFU inoculum at 24 h and 97% for the other combinations. The sensitivity and specificity for the Vitek 2 card were 93 and 100%, respectively. The performance of both the agar dilution method and the Vitek 2 card was good, but these methods were not as sensitive as the D-zone test at 24 h.

Polymorphism of the capsid L1 gene of human papillomavirus types 31, 33, and 35.

The L1 gene encodes for the major capsid protein of human papillomaviruses (HPV). There is limited information on the polymorphism of L1 for types related to HPV-16. This report explores the polymorphism of L1 in phylogenetically related types 31, 33, and 35 compared to HPV-16. Genital specimens collected from 732 HIV-seropositive and 323 HIV-seronegative women were screened for HPV DNA with consensus L1 PCR. Cervical samples positive for HPV-16 (n = 74), HPV-31 (n = 78), HPV-33 (n = 37), and HPV-35 (n = 58) were further characterized by PCR-sequencing of the complete L1 gene. The number of nucleotide substitutions within L1 ranged from 19 for HPV-33 to 52 for HPV-31. The ratio of the number of variants/number of isolates tested was higher for HPV-31 (56.4%, P = 0.05) and HPV-35 (60.3%, P = 0.04) compared to HPV-16 (40.5%), while this ratio was lower for HPV-33 (24.3%), although not significantly (P = 0.14). The maximal distance between HPV variants was greater in the five putative surface-exposed loops of L1 than in sequences outside the loops (P < 0.01). Synonymous variations were encountered in 1.7% (95% CI 1.1-2.3) of nucleotides inside the L1 loops and 2.4% (95% CI1.2-3.7) of nucleotides outside the L1 loops. Non-synonymous variations were encountered in 1.8% (95% CI 1.1-2.5) of nucleotides within the L1 loops and 0.2% (95% CI 0-0.4) of nucleotides outside the loops. dN/dS ratios were below 1.0 in extra-loop and intra-loop regions, but they were lower in extra-loop regions. These results suggest that sequences within and outside the hypervariable loops of L1 were under selective constraint.

Cross-Cultural Adaptation and Validation of the Voice Handicap Index in the Quebec French Population (VHI-QF).

BACKGROUND AND OBJECTIVES: The Voice Handicap Index (VHI) is a widely recognized, self-administered questionnaire, designed to evaluate patients' perception of voice-related disability. It takes into consideration the physical, functional and emotional impacts of dysphonia. The VHI has been translated and validated in many languages, including European French. The purpose of our study is to translate, adapt and validate a new version of the VHI in Quebec French. METHODS: The original VHI was translated into Quebec French (QF) by forward and backward translations by four professional translators, including a speech-language pathologist. The content validity of the resulting VHI-QF was examined in focus groups with six patients and seven speech-language pathologists. Another sample of 154 patients with voice disorders and 150 healthy controls allowed evaluation of the new questionnaire's convergent and discriminant validity, and internal consistency. Satisfaction toward the questionnaire was also evaluated for all patients, as well Test-retest reliability and responsiveness for a sub-sample. RESULTS: The VHI-QF showed a moderate correlation with dysphonia severity level, indicating adequate convergent validity. Both total and subscale scores also exhibited adequate ability to discriminate between patients and controls (discriminant validity), high internal consistency, and good test-retest reliability. The analysis of pre- and post-treatment VHI-QF scores revealed adequate responsiveness to voice treatment. Patients were overall satisfied with the questionnaire. CONCLUSION: The VHI-QF is a valid, reliable and clinically useful self-reported tool to evaluate the severity and change of voice disorders in Quebec French population. Therefore this questionnaire can be used in clinical and research contexts.

Comparison of SARS-CoV-2 detection from combined nasopharyngeal/oropharyngeal swab samples by a laboratory-developed real-time RT-PCR test and the Roche SARS-CoV-2 assay on a cobas 8800 instrument.

OBJECTIVE: Although several assays have been developed to detect SARS-CoV-2 RNA in clinical specimens, their relative performance is unknown. METHODS: The concordance between the cobas 8800 SARS-CoV-2 and a laboratory developed (LD) reverse transcriptase-polymerase chain reaction (RT-PCR) assay was assessed on 377 combined nasopharyngeal/oropharyngeal swabs in Hanks medium. RESULTS: The positive and negative agreement between these assays were 99.3 % (95 % CI, 97.3-99.9) and 77.1 % (95 % CI, 67.7-84.4), respectively, for an overall agreement of 93.6 % (95 % CI, 90.7-95.7) beyond random chance (kappa of 0.82, 95 % CI, 0.75-0.85). Of the 22 samples positive by cobas SARS-CoV-2 only, 9 were positive only for ORF-1 gene and had Cycle thresholds (Ct) > 35.1, 8 were positive only for the E gene with Ct > 35.5 and 5 were positive for both targets with Ct > 33.9. Samples positive only with the cobas assay were more often positive with only one gene target (77.3 %) than samples positive in both assays (16.9 %, p < 0.0001). Ct values in the cobas SARS-CoV-2 assay were significantly higher in the 279 samples testing positive in both assays (32.9 %, 95 % CI 32.3-33.6) compared to the 22 samples with discordant results (36.6 %, 95 % CI 36.2-37.1; p = 0.0009). An excellent correlation (r2 = 0.98) was obtained between Ct values of the ORF-1 and E targets in the cobas assays and a good correlation was obtained between LD RT-PCR test and cobas SARS CoV-2 ORF-1 target (r2 = 0.82). CONCLUSION: Our study demonstrated an excellent concordance between a LD RT-PCR and the cobas SARS-CoV-2 tests on the 8800 platform.

Viral load of episomal and integrated forms of human papillomavirus type 33 in high-grade squamous intraepithelial lesions of the uterine cervix.

The association between total and integrated HPV-33 DNA loads and high-grade squamous intraepithelial lesions (HSIL) of the uterine cervix was investigated. Of 5,347 women recruited in 4 studies, 89 (64 without SIL, 7 low-grade SIL (LSIL), 15 HSIL, 3 unknown grade) were infected by HPV-33. HPV-33 E6, HPV-33 E2 and beta-globin DNA were measured with real-time PCR that allowed to assess total (E6), episomal (E2) and integrated (E6-E2) HPV-33 viral loads. HPV-33 E6/E2 ratios >/=>/=2.0 suggesting the presence of integrated HPV-33 were obtained for 28.6% (n = 18) of women without SIL and 21.4% (n = 3) of women with HSIL (p = 0.74). Although median viral loads were similar, there was a trend toward having a greater proportion of women with HSIL in the fourth quartile (>/=>/=10(6.69) copies/mug DNA) of total HPV-33 viral loads compared to normal women. Controlling for age, site, ethnicity and LCR polymorphism by logistic regression, HPV-33 total loads in the fourth quartile {odds ratio (OR) 4.5 [95% confidence interval (CI) 1.2-17.3]; p = 0.03} and episomal loads in the fourth quartile (>/=>/=10(6.64) copies/mug DNA) [OR 3.9 (95% CI 1.1-13.2); p = 0.05] but not integrated HPV-33 load in the fourth quartile [OR 1.0 (95% CI 0.3-3.3); p = 0.50] were associated with HSIL. Controlling for age, study site and SIL grade, HPV-33 episomal load [OR 0.2 (95% CI 0.1-0.5), p = 0.0004] was associated with the presence of HPV-33 integration. High episomal loads in HSIL and the presence of integration in women without SIL are likely to weaken the usefulness of HPV load of integrated forms in clinical practice.

Diversity of cutaneous human papillomavirus types in individuals with and without skin lesion.

BACKGROUND: Human papillomavirus (HPV) is ubiquitous on the skin of normal and immunosuppressed hosts. OBJECTIVE: We describe the diversity of HPV types in skin specimens using PCR-sequencing directly and after cloning with FAP59/64 or HVP2/B5 primers. STUDY DESIGN: Cross-sectional analysis of skin swabs. RESULTS: Seventy-five (92.6%) of 81 subjects provided samples that could be analysed with PCR (34 healthy controls <50 years old, 13 healthy controls > or =50 years old, 12 with actinic keratosis (AK), 8 with squamous cell carcinoma (SCC) and 8 renal transplant recipients). HPV DNA was detected more frequently with FAP59/64 (68/75, 91%) than with HVP2/B5 (9/75, 12%) (p<0.001). Agreement of typing results using FAP59/64 primers with both sequencing strategies was fair (mean kappa 0.56+/-0.19, 95% CI: 0.46-0.65). HPV species 1 and 2 of the beta-papillomavirus genus were associated with the presence of AK (OR=24.8, 95% CI: 2.3-262.6). A greater number of HPV types per sample was found in individuals with AK or SCC (p=0.046) or AK alone (p=0.02), than in healthy participants. CONCLUSION: HPV infection on the skin is best evaluated with a combination of primers and sequencing strategies. Novel putative types were frequently detected in SCC. Skin lesions have a greater number of HPV types than normal skin.

High levels of HPV-16 DNA are associated with high-grade cervical lesions in women at risk or infected with HIV.

OBJECTIVE: To examine associations between levels of episomal and integrated human papillomavirus (HPV) 16 DNA and the grade of cervical disease. DESIGN: Cross-sectional data were obtained from a cohort of women with and without HIV infection and with high-risk sexual behaviour. METHODS: Episomal and integrated HPV-16 DNA loads were measured in cervicovaginal lavages collected from 75 women (58 HIV seropositive, 17 HIV seronegative) using real-time polymerase chain reaction assays, controlling for cell content and the presence of inhibitors. RESULTS: HPV-16 viral loads were significantly higher in women with high-grade squamous intraepithelial lesions (n = 6) than in women with normal cytology (n = 44), whether total (10(8.28) versus 10(5.10) HPV-16 DNA copies/microg DNA), episomal (10(7.99) versus 10(4.61)) or integrated (10(7.95) versus 10(4.77)) HPV-16 viral loads were measured (P < 0.02 for each comparison). Thirty-nine women had colposcopy [11 normal cervix, 16 cervical intraepithelial neoplasia (CIN) 1, six CIN 2, six CIN 3] and 24 additional women had three consecutive normal cytology smears. Controlling for age, race, CD4 cell count and HIV status, total (OR 3.5, 95% CI 1.2-10.4; P = 0.02), episomal (OR 2.9, 95% CI 1.2-7.4; P = 0.02,) and integrated (OR 1.6, 95% CI 1-2.6; P = 0.05) HPV-16 DNA loads were significantly associated with CIN 2,3, but the differences between CIN 1 and CIN 2,3 were not significant (P > 0.06). A greater amount of cellular DNA was collected from women with CIN 2,3 (P = 0.007). CONCLUSION: Higher HPV-16 DNA loads are associated with cervical lesions detected by either histology or cytology. No additional information is gained by measuring integrated or episomal over total HPV-16 DNA loads.