Simon Gagnon

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. J. Med. Virol. 83:751–759, 2011.

Prosthetic hip joint infection with a Streptococcus agalactiae isolate not susceptible to penicillin G and ceftriaxone

Microbiologie medicale et infectiologie, Centre hospitalier de l’Universite de Montreal (CHUM)-Hopital Saint-Luc, 1058 rue Saint Denis, Montreal, Quebec, Canada, H2X 3J4; Departement de microbiologie et immunologie, Universite de Montreal, CP 6128 succ. Centre-Ville, Montreal, Quebec, Canada, H3C 3J7; Medecine interne, CHUM-Hopital Saint-Luc, 1058 rue Saint Denis, Montreal, Quebec, Canada, H2X 3J4; Laboratoire de sante publique du Quebec/Institut national de sante publique du Quebec (LSPQ/ INSPQ), 20045 chemin Sainte-Marie, Sainte-Anne-de-Bellevue, Quebec, Canada, H9X 3R5; Microbiologie medicale et infectiologie, CHUM-Hopital Notre-Dame, 1560 rue Sherbrooke Est, Montreal, Quebec, Canada, H2L 4M1

Human Papillomavirus Type 33 Polymorphisms and High-Grade Squamous Intraepithelial Lesions of the Uterine Cervix

BACKGROUND We investigated the association between polymorphisms of human papillomavirus (HPV)-33 and squamous intraepithelial lesions (SILs). METHODS Endocervical specimens from 89 women infected with HPV-33, out of a total of 5347 recruited for 2 case-control and 2 cohort studies, were further analyzed by polymerase chain reaction sequencing of the long control region (LCR), E6, and E7. RESULTS Of the 89 samples, 64 were normal, 7 had low-grade SILs (including 3 determined by histopathologic analysis), 15 had high-grade SILs (HSILs, including 14 determined by histopathologic analysis), and 3 had an unknown diagnosis. Non-prototype-like LCR variants were significantly associated with HSILs (age- and study site-adjusted odds ratio [OR], 9.2 [95% confidence interval {CI}, 1.8-45.9]). The C7732G variation, which results in the loss of a putative binding site for the cellular upstream stimulatory factor, was associated with HSILs (age- and site-adjusted OR, 8.0 [95% CI, 1.5-42.8]). E6 and E7 polymorphisms were not associated with HSILs. Samples collected at 6-month intervals from 14 participants contained the same variant. The HPV-33 MT 1-0-0 variant carrying the G7584A variation was detected more frequently in women from Brazil (7/20 [35%]) than in women from Canada (1/65 [1.5%]; P=.001). CONCLUSION Intratypic LCR variants of HPV-33 seem to vary geographically and to differ with respect to their oncogenic potential.

Viral Polymorphism in Human Papillomavirus Types 33 and 35 and Persistent and Transient Infection in the Genital Tract of Women

BACKGROUND Genetic polymorphism in human papillomavirus (HPV)-33 and -35 was investigated in 1055 sexually active women (732 human immunodeficiency virus [HIV] seropositive and 323 HIV seronegative). METHODS Consecutive genital specimens obtained at 6-month intervals were screened for HPV-33 and -35 by use of MY09-MY11. HPV-33 and -35 isolates from 95 women were analyzed by polymerase chain reaction sequencing of the long control region (LCR), E6, and E7. RESULTS For HPV-33, 101 (20%) of 506 nucleotides in the LCR were variable, compared with 10 (2.1%) of 483 nucleotides in E6 (P<.001) and 6 (1.9%) of 324 nucleotides in E7 (P<.001). For HPV-35, the proportion of variable nucleotide sites was similar between the LCR and both E6 (P=.54) and E7 (P=.33). The presence of a 78-base pair deletion in HPV-33 (relative risk [RR], 1.8 [95% confidence interval [CI], 1.2-2.7]) and the presence of nonsynonymous E7 variations in HPV-35 (RR, 2.6 [95% CI, 1.4-4.6]) were associated with persistence. When the data for HPV-33 and -35 were combined, infection by HPV isolates with nonsynonymous E7 variations (RR, 2.3 [95% CI, 1.6-3.4]; P=.001) and ethnicity (P=.04) were associated with persistence, whereas age (P = .14) and HIV infection/CD4 cell count status (P=.12) were not significantly associated with persistence, by logistic regression analysis. CONCLUSION HPV-33 and -35 polymorphism was different between types and was associated with persistence of HPV infection.

Performance of an Agar Dilution Method and a Vitek 2 Card for Detection of Inducible Clindamycin Resistance in Staphylococcus spp.

ABSTRACT 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: 104 and 105 CFU per spot. Plates were read at 24 and 48 h. The Vitek 2 AST-P580 card was used according to the manufacturers 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 104 CFU inoculum, the sensitivities were 84 and 99% at 24 and 48 h, respectively. The 105 CFU inoculum increased the sensitivities at 24 and 48 h to 91 and 100%, respectively. The specificity was 100% for the 104 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.

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.

Clinical Prediction and Diagnosis of Neurosyphilis in HIV-Infected Patients with Early Syphilis

ABSTRACT 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.

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.

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 β‐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 (≥≥106.69 copies/μg 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 (≥≥106.64 copies/μg 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.

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. J. Med. Virol. 82: 1168–1178, 2010.