Agatha N. Jassem

In Vitro Susceptibility of Burkholderia vietnamiensis to Aminoglycosides

ABSTRACT Burkholderia cepacia complex (BCC) bacteria are opportunistic pathogens that can cause severe disease in cystic fibrosis (CF) patients and other immunocompromised individuals and are typically multidrug resistant. Here we observed that unlike other BCC species, most environmental and clinical Burkholderia vietnamiensis isolates were intrinsically susceptible to aminoglycosides but not to cationic antimicrobial peptides or polymyxin B. Furthermore, strains acquired aminoglycoside resistance during chronic CF infection, a phenomenon that could be induced under tobramycin or azithromycin pressure in vitro. In comparing susceptible and resistant B. vietnamiensis isolates, no gross differences in lipopolysaccharide structure were observed, all had lipid A-associated 4-amino-4-deoxy-l-arabinose residues, and all were resistant to the permeabilizing effects of aminoglycosides, a measure of drug entry via self-promoted uptake. However, susceptible isolates accumulated 5 to 6 times more gentamicin than a resistant isolate, and aminoglycoside susceptibility increased in the presence of an efflux pump inhibitor. B. vietnamiensis is therefore unusual among BCC bacteria in its susceptibility to aminoglycosides and capacity to acquire resistance. Aminoglycoside resistance appears to be due to decreased cellular accumulation as a result of active efflux.

Interim estimates of 2016/17 vaccine effectiveness against influenza A(H3N2), Canada, January 2017

Using a test-negative design, the Canadian Sentinel Practitioner Surveillance Network (SPSN) assessed interim 2016/17 influenza vaccine effectiveness (VE) against dominant influenza A(H3N2) viruses considered antigenically matched to the clade 3C.2a vaccine strain. Sequence analysis revealed substantial heterogeneity in emerging 3C.2a1 variants by province and over time. Adjusted VE was 42% (95% confidence interval: 18–59%) overall, with variation by province. Interim virological and VE findings reported here warrant further investigation to inform potential vaccine reformulation.

Early season co-circulation of influenza A(H3N2) and B(Yamagata): interim estimates of 2017/18 vaccine effectiveness, Canada, January 2018

Using a test-negative design, we assessed interim vaccine effectiveness (VE) for the 2017/18 epidemic of co-circulating influenza A(H3N2) and B(Yamagata) viruses. Adjusted VE for influenza A(H3N2), driven by a predominant subgroup of clade 3C.2a viruses with T131K + R142K + R261Q substitutions, was low at 17% (95% confidence interval (CI): −14 to 40). Adjusted VE for influenza B was higher at 55% (95% CI: 38 to 68) despite prominent use of trivalent vaccine containing lineage-mismatched influenza B(Victoria) antigen, suggesting cross-lineage protection.

Investigation of aminoglycoside resistance inducing conditions and a putative AmrAB-OprM efflux system in Burkholderia vietnamiensis

BackgroundBurkholderia cepacia complex (BCC) bacteria are highly virulent, typically multidrug-resistant, opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. B. vietnamiensis is more often susceptible to aminoglycosides than other BCC species, and strains acquire aminoglycoside resistance during chronic CF infection and under tobramycin and azithromycin exposure in vitro, apparently from gain of antimicrobial efflux as determined through pump inhibition. The aims of the present study were to determine if oxidative stress could also induce aminoglycoside resistance and provide further observations in support of a role for antimicrobial efflux in aminoglycoside resistance in B. vietnamiensis.FindingsHere we identified hydrogen peroxide as an additional aminoglycoside resistance inducing agent in B. vietnamiensis. After antibiotic and hydrogen peroxide exposure, isolates accumulated significantly less [3H] gentamicin than the susceptible isolate from which they were derived. Strains that acquired aminoglycoside resistance during infection and after exposure to tobramycin or azithromycin overexpressed a putative resistance-nodulation-division (RND) transporter gene, amrB. Missense mutations in the repressor of amrB, amrR, were identified in isolates that acquired resistance during infection, and not in those generated in vitro.ConclusionsThese data identify oxidative stress as an inducer of aminoglycoside resistance in B. vietnamiensis and further suggest that active efflux via a RND efflux system impairs aminoglycoside accumulation in clinical B. vietnamiensis strains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but not hydrogen peroxide, in vitro. Furthermore, the repressor AmrR is likely just one regulator of the putative AmrAB-OprM efflux system in B. vietnamiensis.

Identification of synergists that potentiate the action of polymyxin B against Burkholderia cenocepacia

Burkholderia cenocepacia and other members of the Burkholderia cepacia complex (BCC) are highly multidrug-resistant bacteria that cause severe pulmonary infections in patients with cystic fibrosis. A screen of 2686 compounds derived from marine organisms identified molecules that could synergise with polymyxin B (PMB) to inhibit the growth of B. cenocepacia. At 1 μg/mL, five compounds synergised with PMB and inhibited the growth of B. cenocepacia by ≥70% compared with growth in PMB alone. Follow-up testing revealed that one compound from the screen, the aminocoumarin antibiotic novobiocin, synergised with PMB and colistin against tobramycin-resistant clinical isolates of B. cenocepacia and Burkholderia multivorans. In parallel, we show that novobiocin sensitivity is common among BCC species and that these bacteria are even more susceptible to an alternative aminocoumarin, clorobiocin, which also had an additive effect with PMB against B. cenocepacia. These studies support using aminocoumarin antibiotics to treat BCC infections and show that synergisers can be found to increase the efficacy of antimicrobial peptides and polymyxins against BCC bacteria.

Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

ABSTRACT Shiga toxin-producing Escherichia coli (STEC)-associated enteric illness is attributed to O157 and non-O157 serotypes; however, traditional culture-based methods underdetect non-O157 STEC. Labor and cost of consumables are major barriers to implementation of the CDC recommendation to test all stools for both O157 and non-O157 serotypes. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10 samples each. The sensitivity was 83% for the preenrichment pooling strategy and 100% for the postenrichment pooling strategy compared with those for individual NAAT results. The difference in cycle threshold (CT ) between individual and pooled NAAT results for specimens was significantly lower and more consistent for postenrichment pooling (stx 1 mean = 3.90, stx 2 mean = 4.28) than those for preenrichment pooling (excluding undetected specimens; stx 1 mean = 9.34, stx 2 mean = 8.96) (P ≤ 0.0013). Cost of consumables and labor time savings of 48 to 81% and 6 to 66%, respectively, were estimated for the testing of 90 specimens by the postenrichment pooled NAAT strategy on the basis of an expected 1 to 2% positivity rate. A 30-day prospective head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the postenrichment pooled NAAT strategy. The postenrichment pooled NAAT strategy described here is suitable for efficient large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimation of STEC burden and, consequently, appropriate public health interventions.

Measles Lymphadenopathy in a Child With PFAPA Syndrome

Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is a common cause of periodic fever in children. The pathogenesis of PFAPA is unknown but likely involves immune system dysregulation and may be initiated by an environmental trigger. Tonsillectomy resolves or improves symptoms in some patients, but the reason for this is unknown; moreover, specific abnormalities in tonsillectomy specimens from PFAPA patients have not been described. Here, we report measles virus in tonsil from a child with PFAPA. Measles-type viral cytopathic effect was discovered on histological examination of tonsillar tissue after therapeutic tonsillectomy for PFAPA. Molecular testing showed the left tonsil was positive for measles RNA by reverse transcription polymerase chain reaction (RT-PCR) while the right tonsil was inconclusive (weakly positive). Real-time RT-PCR specific for measles vaccine strain RNA (genotype A) was weakly reactive in the left tonsil tissue when tested in 3 independent replicates, but this result could not be confirmed with conventional genotyping by sequencing. The relationship and clinical significance between measles virus and PFAPA in this case is unclear but may be related to PFAPA-associated immune dysregulation. Additional investigation of measles virus in PFAPA patients would be helpful in further exploring this potential association.

Diagnostic Yield of Laboratory Methods and Value of Viral Genotyping during an Outbreak of Mumps in a Partially Vaccinated Population in British Columbia, Canada

ABSTRACT Mumps remains endemic in North America despite routine use of the measles, mumps, and rubella (MMR) vaccine. In 2016, an outbreak of mumps in British Columbia, Canada, provided an opportunity to determine the diagnostic utility of laboratory testing methods. Specimens from patients with clinical mumps were tested for infection using a commercial enzyme-linked immunosorbent assay (ELISA) for antibody detection and an in-house reverse transcriptase PCR (RT-PCR) targeting viral fusion and small hydrophobic (SH) genes. Viral genotyping was performed by SH gene sequencing. Laboratory data was linked with epidemiologic case data. Of the 139 confirmed cases, 94 (68%) had reported or documented history of MMR vaccination. Specimens were typically collected 1 day (for buccal and IgM tests) or 2 days (for urine tests) after symptom onset. Most confirmed cases (69%) were confirmed by buccal swab RT-PCR. Among cases tested by multiple methods, the percent positivity for buccal swab RT-PCR was 90% (96/107) compared to 43% (30/69) for both IgM ELISA and urine RT-PCR. Mumps IgM detection was higher in confirmed cases with no history of vaccination than in those with history (64% versus 34%, P = 0.02). The outbreak strain was identified as genotype G related to MuVi/Sheffield.GBR/1.05 but with conserved variations in five nucleotides within the SH gene that allowed linkage of geographically distinct cases. In conclusion, RT-PCR of buccal specimens had the highest diagnostic yield during a mumps outbreak in a partially vaccinated population. To optimize mumps diagnostic potential, clinicians should collect specimens depending on when the patient presents for care and their immunization history.

Vaccine effectiveness against lineage matched and mismatched influenza B viruses across 8 seasons in Canada, 2010–11 to 2017–18

Abstract Vaccine effectiveness (VE) against influenza B was derived separately for Victoria and Yamagata lineages across 8 seasons (2010–2011 to 2017–2018) in Canada when trivalent influenza vaccine was predominantly used. VE was ≥50% regardless of lineage match to circulating viruses, except when the vaccine strain was unchanged from the prior season.

Development and validation of a real-time, reverse transcription PCR assay for rapid and low-cost genotyping of hepatitis C virus genotypes 1a, 1b, 2, and 3a

Hepatitis C virus (HCV) infection affects millions of people and leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Treatment regimen selection requires HCV genotype (Gt) and Gt 1 subtype determination. Use of a laboratory developed, reverse transcription (RT)-PCR assay was explored as a low-cost, high-throughput screening approach for the major HCV genotypes and subtypes in North America. A commercial line probe assay (LiPA) was used for comparison. Sequencing and/or an alternative PCR assay were used for discordant analyses. Testing of 155 clinical samples revealed that a paired, duplex real-time RT-PCR assay that targets Gts 1a and 3a in one reaction and Gts 1b and 2 in another had 95% overall sensitivity and individual Gt sensitivity and specificity of 98-100% and 85-98%, respectively. The RT-PCR assay detected mixed HCV Gts in clinical and spiked samples and no false-positive reactions occurred with rare Gts 3b, 4, 5, or 6. Implementation of the RT-PCR assay, with some reflex LiPA testing, would cost only a small portion of the cost of using LiPA alone, and can also save 1.5h of hands-on time. The use of a laboratory developed RT-PCR assay for HCV genotyping has the potential to reduce cost and labour burdens in high-volume testing settings.