James Branley

Australian human and parrot Chlamydia psittaci strains cluster within the highly virulent 6BC clade of this important zoonotic pathogen

Chlamydia psittaci is an avian pathogen and zoonotic agent of atypical pneumonia. The most pathogenic C. psittaci strains cluster into the 6BC clade, predicted to have recently emerged globally. Exposure to infected parrots is a risk factor with limited evidence also of an indirect exposure risk. Genome sequencing was performed on six Australian human and a single avian C. psittaci strain isolated over a 9 year period. Only one of the five human patients had explicit psittacine contact. Genomics analyses revealed that the Australian C. psittaci strains are remarkably similar, clustering tightly within the C. psittaci 6BC clade suggested to have been disseminated by South America parrot importation. Molecular clock analysis using the newly sequenced C. psittaci genomes predicted the emergence of the 6BC clade occurring approximately 2,000 years ago. These findings reveal the potential for an Australian natural reservoir of C. psittaci 6BC strains. These strains can also be isolated from seriously ill patients without explicit psittacine contact. The apparent recent and global spread of C. psittaci 6BC strains raises important questions over how this happened. Further studies may reveal whether the dissemination of this important zoonotic pathogen is linked to Australian parrot importation rather than parrots from elsewhere.

Prevalence of antiseptic resistance genes qacA/B and specific sequence types of methicillin-resistant Staphylococcus aureus in the era of hand hygiene

Sir, In an attempt to control the spread of methicillin-resistant Staphylococcus aureus (MRSA) and other multiresistant organisms in hospitalized patients, hand hygiene campaigns have promoted the widespread use of alcohol-based hand solutions containing antiseptic agents such as chlorhexidine and quaternary ammonium compounds. The use of these products, however, has led to increased resistance to these antiseptic agents in MRSA strains. – 5 qacA/B are plasmid-borne resistance genes coding for multidrug efflux pumps that confer high-level resistance to chlorhexidine, quaternary ammonium compounds and other antiseptic compounds commonly used in hand hygiene solutions. They are associated with higher MICs and tolerance of these antiseptic agents. qacA/B genes have been found in healthcare-associated MRSA (HA-MRSA) isolates from around the world. – 8 In addition, the prevalence of qacA/B genes has been associated with particular sequence types (STs) of HA-MRSA. To our knowledge, the prevalence of MRSA strains harbouring these genes has not been researched in Australia. In this study, we characterize the prevalence of qacA/B genes in HA-MRSA isolates in our institution spanning 10 years, from 2000 to 2009. We determined whether the national hand hygiene campaign implemented in 2006 had any impact on the prevalence of these genes and, in addition, if any relationship exists between the different STs of HA-MRSA and qacA/B gene prevalence. All (total 151) clinically significant unique HA-MRSA isolates from the period 2000–09 from Nepean Hospital, a 490 bed tertiary referral hospital in Sydney, were used in this study. The presence of qacA/B genes in these isolates was evaluated by real-time PCR using the following primers: qacA/B forward primer 5′-CTATGGCAATAGGAGATATGGTGT and reverse primer 5′-CCACTACAGATTCTTCAGCTACATG. The HA-MRSA isolates were categorized into STs using their antibiograms and correlations with pre-existing typing performed on HA-MRSA isolates in our institution by the Australian Group on Antimicrobial Resistance. Quantities of hand hygiene solutions used by the hospital were obtained via the pharmacy database. A sustained .6-fold increase in antiseptic-containing alcoholbased hand solution use occurred as a result of the hand hygiene campaign (‘Clean hands saves lives’) implemented in 2006 [amounts of 0.5% chlorhexidine gluconate and quaternary ammonium compounds used (average litres per year) were 968 and 150 L before the hand hygiene campaign (2004–06) and 3967 and 2958 L after the hand hygiene campaign (2007–09), respectively]. The yearly prevalence of qacA/B genes in HA-MRSA isolates from 2000 to 2009 ranged from 65.0% to 94.7% (mean 78.6%). This is higher than that previously found in the UK (8%–26%), Europe (63%) and Asia (33%–61%), but comparable to that found in a recent study in Geneva (79%). The HA-MRSA isolates in this study comprise two STs: ST239 MRSA-III Aus-2 EMRSA and ST22 MRSA-IV EMRSA-15. The ST239 type was the predominant strain throughout the study period (mean yearly prevalence of ST239 from 2000 to 2009 was 84.3%, range 75%–100%). Comparing the prevalence of the qacA/B genes before (2000– 06) and after (2007–09) the hand hygiene campaign, we did not find an increase in the prevalence of these genes despite the marked increase in antiseptic hand solutions after 2006 [mean yearly prevalence of qacA/B-positive MRSA isolates between 2000 and 2006 and between 2007 and 2009 was 78.6% and 73.5%, respectively (P1⁄40.53; x1⁄40.39, df1⁄41)]. Similarly, there was no change in the incidence of MRSA ST239 [mean yearly prevalence of MRSA ST239 between 2000 and 2006 and between 2007 and 2009 was 85.6% and 78.6%, respectively (P1⁄40.37; x1⁄40.81, df1⁄41)]. Interestingly, we found a significant association between qacA/B gene positivity and MRSA type ST239 [94.5% of qacA/Bpositive isolates were ST239 and 88.9% of ST239 isolates were qacA/B positive (OR1⁄429.09, CI1⁄48.93–94.82)]. This relationship between qacA/B and MRSA ST239 has been suggested in previous studies in the UK and Taiwan; however, the prevalence of this MRSA strain, as well as qacA/B genes, was much lower in these studies. MRSA ST239 is found in significantly high numbers in Eastern Australia, and is associated with a broad spectrum of antimicrobial resistance and epidemic potential. The qacA/B genes are thought to be located on multiresistance plasmids, such as pSK1, and co-transmitted with plasmid-mediated antimicrobial resistance genes; hence their association with this multiresistant strain of MRSA is logical. The high prevalence of this ST239 strain could be due to selective pressure from the use of antiseptic products and/or the widespread use of antimicrobial agents. Our findings suggest the latter, given the absence of a corresponding rise in qacA/B genes with increased antiseptic hand solution use; however, the baseline use of antiseptic agents within the hospital could also explain the high prevalence of qacA/B genes even prior to the hand hygiene campaign.

An outbreak of psittacosis at a veterinary school demonstrating a novel source of infection

In November 2014, New South Wales Health was notified of a cluster of respiratory illness in a veterinary school. Active case finding identified another case at a local equine stud. All cases had exposure to the equine fetal membranes of Mare A. This tissue subsequently tested positive for Chlamydia psittaci using quantitative real-time polymerase chain reaction. We conducted a cohort study of the university and stud farm staff to determine risk factors for disease. Nine people were exposed to the fetal membranes of Mare A. Of these, five cases of psittacosis were identified. Two required hospital admission. Contact with birds was not associated with illness (RR = 0.5, 95% CI = 0.09–2.73). People who had direct contact with the abnormal fetal membranes were more likely to develop disease (RR = 11.77, 95% CI = 1.02–∞). The emergence of an association between horse exposure and C. psittaci infection has important implications for the prevention and control of psittacosis. Article summary line: Investigation of an outbreak of psittacosis in a rural veterinary school demonstrates novel source of infection for psittacosis through exposure to abnormal equine fetal membranes.

Multilocus sequence typing identifies an avian-like Chlamydia psittaci strain involved in equine placentitis and associated with subsequent human psittacosis

Emerging Microbes & Infections (2017) 6, e7; doi:10.1038/emi.2016.135; published online 15 February 2017

Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens: Chlamydia psittaci and Chlamydia pecorum

Background Chlamydia psittaci and Chlamydia pecorum are important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robust C. psittaci-specific and C. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens. Methods and Materials The LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of the C. psittaci-specific Cps_0607 gene or a 209 bp region of a C. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity for C. psittaci and C. pecorum LAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples, C. psittaci LAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while for C. pecorum LAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36 C. pecorum samples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays. Results Both LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min for C. psittaci and C. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% for C. psittaci (91.7% sensitivity and 92.9% specificity); and 84.1% for C. pecorum (90.6% sensitivity and 77.4% specificity). For a subset of 36 C. pecorum samples tested in a thermal cycler using newly developed LAMP, we observed 34/36 (94.4%) samples result being congruent between LAMP performed in fluorometer and in thermal cycler. Rapid swab processing method evaluated in this study also allows for chlamydial DNA detection using LAMP. Discussion In this study, we describe the development of novel, rapid and robust C. psittaci-specific and C. pecorum-specific LAMP assays that are able to detect these bacteria in clinical samples in either the laboratory or POC settings. With further development and a focus on the preparation of these assays at the POC, it is anticipated that both tests may fill an important niche in the repertoire of ancillary diagnostic tools available to clinicians.

Chlamydia trachomatis in fallopian tubes of women undergoing laparoscopy for ectopic pregnancy

To study whether Chlamydia trachomatis is absent or persists in a latent state in the fallopian tube at the time of laparoscopic salpingectomy for tubal ectopic pregnancy (EP).

Biomarkers for high-risk influenza patients: what are the next steps?

Treatment of infectious disease often focuses on a single cure, based on the assumption that a singular, dominant pathophysiological process (pathogen virulence or load) drives disease progression and that reversing of this process may alleviate most, if not all, of the mortality caused by the disease. However, in infectious diseases with high fatality rates, both the pathogen and the host response contribute to disease progression/death. Identifying the biomarkers of the host’s response is an emerging field that is rapidly growing in both scope and depth, reflecting the increasing awareness that host response is an important determinant of patient outcomes, especially in lethal viral infections (e.g. influenza and SARS). In this Editorial, influenza infection is used as an illustrative example to demonstrate that host response biomarkers could play an important role in guiding the management of lethal viral infections.

Analysis of positive bacteriology cultures at a regional microbiology in view of understanding local epidemiology and improving the quality of services

Aim Analysis of positive bacteriology specimens performed over one year to determine local epidemiology data and to improve methods and reporting procedure. Methods The study was performed at a regional microbiology laboratory servicing a large number of rural health facilities. All positive bacteriology cultures for 7467 organisms were extracted from the laboratory information system and analysed using Microsoft Excel. This included 354 positive blood cultures, 1696 wound swabs, 2293 urine cultures and 512 sputum samples. Susceptibility data were analysed for Staphylococcus aureus, Enterococci, ESCPPM and non-ESCPPM groups of Enterobacter-iaceae and Pseudmonas aeruginosa. Observations were made regarding current methodology and opportunities for improvements with ongoing support of supervising laboratory were identified. Results Direct susceptibility data for urine specimens for ampi-cillin, amoxycillin-clavulanate, cephalothin, gentamicin and tri-methoprim for non-ESCPPM organisms were 43%, 76%, 45%, 98% and 80%, respectively; the ESCPPM group of organisms showed susceptibilities of 93% for gentamicin and 78% for trimethoprim and 88% for norfloxacin. The single anti-Pseudomonal drug tested, gentamicin, showed 99% susceptibility. Eighty percent of Enterococci remained susceptible to ampicillin. Forty percent of urinary S. aureus were methicillin-resistant Staphylococcus aureus (MRSA) with the majority community (CMRSA). Susceptibilities for bacteraemic S. aureus isolates included 88% of methicillin-sensitive Staphylococcus aureus (MSSA), 10% of CMRSA and 2% of nosocomial MRSA. Non-ESCPPM organisms showed susceptibilities of 48%, 67%, 92%, 92% 98% and 100% for ampicillin, amoxycillin-clavulanate, gentamicin, cefotaxime, ciprofloxacin and meropenem, respectively. Susceptibilities of bacteraemic ESCAPPM isolates for gentamicin, ciprofloxacin and meropenem were 100%, 88% and 100%. Sixty-two percent of Enterococci remained susceptible to ampicillin. Conclusions A number of analytical and post-analytical factors were identified with potential improvements to be done in view of upgrading the quality of services.

P16.18: Chlamydia trachomatis in fallopian tubes of women undergoing laparoscopy for ultrasound diagnosed ectopic pregnancy

Objectives: Chlamydia trachomatis is common among young and sexually active people. Most often infections are asymptomatic but have potential long-term consequences for the reproductive health. The pathogenesis of tubal ectopic pregnancy (EP) in the context of chlamydia trachomatis infection is poorly understood. We aimed to study whether chlamydia trachomatis is absent or persists in a latent state in the fallopian tube at the time of laparoscopy for tubal EP. Methods: We examined tissue of the fallopian tubes for the presence of chlamydia trachomatis from women who underwent laparoscopic salpingectomy for EP. Results: Fresh tubal tissue from 16 women with histological confirmation of EP were examined in a hospital setting for the presence of chlamydia trachomatis. The presence of chlamydia trachomatis DNA was confirmed by polymerase chain reaction (PCR) using a commercial test (BD ProbeTecTM ET System) and a real-time enhanced PCR able to detect few copies of the organism. Chlamydial DNA was detected in 0 of the 15 tubal specimens. In 1 case the PCR analysis was not possible for presence of inhibitors. Conclusions: We did not find any evidence of latent infection of chlamydia trachomatis in the fallopian tube at the time of laparoscopy for EP in our study. This suggests that EP can be considered a late complication of the tubal damage resulted from a previous acute chlamydia infection, and that EP may not be related to a latent persistence of chlamydia in the fallopian tube.

Phenotypic and molecular characterisation of metallo-beta lactamases (MBLs) producing bacteria at a tertiary care hospital

Introduction In 2005–2006 at the department of Microbiology, Nepean Hospital, multiresistant Gram negative rods were increasingly encountered at the sensitivity bench. They carried phenotypic markers of resistance to cefotaxime and ceftazidime and gentamicin and were not extended spectrum beta lactamases (ESBLs). Isolates originated mainly from the neonatal intensive care unit (NICU) and were identified as carrying IMP-4 gene. These organisms present a challenge to the routine microbiology laboratory because of variable phenotypic expression, the range of organisms involved and the need for rapid results. As this genetic element was new, a greater understanding of the problem in the local environment was required and this prompted us to look at improved screening for these organisms using a mixture of phenotypic and molecular methods. Aims (1) To establish rapid and low cost PCR to detect IMP-4 gene and low cost phenotypic test/s to screen for metallo-beta lactamases (MBLs). (2) To evaluate the performances of phenotypic tests. Methods A total of 276 Gram negative clinical isolates recovered from the sensitivity bench were studied. The 276 isolates comprised of 100 isolates sensitive to third generation cephalosporins and aminoglycosides, 83 stored historical isolates resistant to either/both drugs and 93 isolates from the surface swabs and urine of NICU babies where it was thought an ongoing outbreak of MBL positive bacteria was occurring. The bacterial isolates were identified and tested using VITEK and CLSI disk diffusion. Phenotypic testing for MBL detection was performed using EDTA and 2-Mercaptopropionic acid (2-MPA) as described in the literature. Real time PCR was performed for IMP-4 gene. For the isolates with discrepant phenotypic and molecular tests, a multiplex PCR was performed to detect the majority of described MBLs. Results IMP-4 gene was detected in 20.2% of the study population. EDTA has sensitivity of 95% and specificity of 89% and 2-MPA has sensitivity of 95% and specificity of 70% for MBL detection. Sixty- seven false positives were detected in 2-MPA test and 26 in EDTA test. No MBLs other than IMP-4 were detected by multiplex PCR. Conclusions Phenotypic tests are useful as they are cheap, quick and easy to perform in a routine microbiology laboratory. They are highly sensitive but the higher number of false positives among non-fermenters is a cause for concern. MBL gene detection by real time PCR is highly sensitive and specific, results can be obtained within a few hours, and they are therefore extremely useful in outbreak situations to initiate infection control measures. However, geographically prevalent MBL genes should be targeted in a multiplex PCR.