Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Multidrug-resistant OXA-48/CTX-M-15 Klebsiella pneumoniae cluster in a COVID-19 intensive care unit: salient lessons for infection prevention and control during the COVID-19 pandemic.

BACKGROUND: Wards caring for COVID-19 patients, including intensive care units (ICUs), have an important focus on preventing transmission of SARS-CoV-2 to other patients and healthcare workers. AIM: To describe an outbreak of carbapenemase-producing Enterobacterales (CPE) in a COVID-19 ICU and to discuss key infection control measures enabling prompt termination of the cluster. METHODS: CPE were isolated from clinical specimens and screening swabs from intensive care patients with COVID-19 disease and from environmental screening. Whole-genome sequencing analysis was instrumental in informing phylogenetic relationships. FINDINGS: Seven clinical isolates and one environmental carbapenemase-producing Klebsiella pneumoniae isolate - all carrying OXA-48, CTX-M-15 and outer membrane porin mutations in ompK35/ompK36 - were identified with ≤1 single nucleotide polymorphism difference, indicative of clonality. A bundle of infection control interventions including careful adherence with contact precautions and hand hygiene, twice weekly screening for multidrug-resistant organisms, strict antimicrobial stewardship, and enhanced cleaning protocols promptly terminated the outbreak. CONCLUSION: Prolonged use of personal protective equipment is common with donning and doffing stations at the ward entrance, leaving healthcare workers prone to reduced hand hygiene practices between patients. Minimizing transmission of pathogens other than SARS-CoV-2 by careful adherence to normal contact precautions including hand hygiene, even during high patient contact manoeuvres, is critical to prevent outbreaks of multidrug-resistant organisms. Appropriate antimicrobial stewardship and screening for multidrug-resistant organisms must also be maintained throughout surge periods to prevent medium-term escalation in antimicrobial resistance rates. Whole-genome sequencing is highly informative for multidrug-resistant Enterobacterales surveillance strategies.

Evidence for decontamination of single-use filtering facepiece respirators.

Single-use filtering face respirators (FFRs) are critical pieces of personal protective equipment for healthcare workers treating patients with suspected upper respiratory tract pathogens. Experiences during pandemics in the 2000s, as well as the ongoing COVID-19 pandemic caused by the SARS-2-CoV-2, have highlighted concerns over the pressures that sustained respiratory virus pandemics may have on supplies of FFRs globally. Decontamination of FFRs has been posited as one solution to support the re-use of FFRs with a growing body of literature over the last 10+ years beginning to examine both the efficacy of disinfection of contaminated FFRs but also the impact of the decontamination process on the FFR's performance. Physical and chemical methods of decontamination have been tested for treatment of FFRs with ultraviolet germicidal irradiation, sterilization by steam, ethylene oxide and vaporous hydrogen peroxide, demonstrating the most promising results thus far. Many of these methods utilize existing equipment that may already be available in hospitals and could be re-purposed for FFR decontamination. Importantly, some methods may also be replicated on household equipment, broadening the utility of FFR decontamination across a range of healthcare settings. Utilizing techniques to experimentally contaminate FFRs with a range of microorganisms, most decontamination methods appear to reduce the risk of the mask as a source of infection to the wearer and others to negligible levels. The performance of the filter, especially the efficiency of particle penetration following treatment, varied greatly depending on the processing method as well as the model of the filter itself, however. Urgent regulatory body-supported research is required to endorse the routine decontamination of FFRs. In emergency settings, these methods should nevertheless be carefully considered as one strategy to address potential shortfalls in supplies of FFRs for healthcare workers.

Equine chlamydiosis-An emerging infectious disease requiring a one health surveillance approach.

Psittacosis is a rare but potentially fatal zoonosis caused by Chlamydia psittaci, an organism that is typically associated with bird contact. However C. psittaci is capable of infecting other non-avian hosts, such as horses, sheep, cattle and goats. Stud staff and veterinarians have significant exposure to parturient animals and reproductive materials in their routine work. To investigate the zoonotic potential associated with the emergence of C. psittaci as an abortifacient agent in horses, we established a programme of joint human and animal surveillance in a sentinel horse-breeding region in Australia. This programme comprised cross-notification of equine cases to public health agencies, and active follow-up of known human contacts, including stud workers, foaling staff, veterinarians and laboratory staff. We identified no confirmed cases of acute psittacosis despite intensive surveillance and testing of heavily exposed contacts; however, further work in the area is needed.

Clinical features of endemic community-acquired psittacosis.

Following a large outbreak of community-acquired psittacosis in 2002 in residents of the Blue Mountains, New South Wales, Australia, we reviewed new cases in this area over a 7-year period from 2003 to 2009. Using the 2010 criteria from the Centers for Disease Control National Notifiable Diseases Surveillance System, 85 patients with possible psittacosis were identified, of which 48 were identified as definite or probable infection. Clinical features of these cases are summarized. In addition to Chlamydia-specific serology, specimens, where available, underwent nucleic acid testing for chlamydial DNA using real-time PCR. Chlamydophila psittaci DNA was detected in samples from 23 patients. Four of 18 specimens were culture positive. This is the first description of endemic psittacosis, and is characterized in this location by community-acquired psittacosis resulting from inadvertent exposure to birds. The disease is likely to be under-diagnosed, and may often be mistaken for gastroenteritis or meningitis given the frequency of non-respiratory symptoms, particularly without a history of contact with birds. Clinical characteristics of endemic and outbreak-associated cases were similar. The nature of exposure, risk factors and reasons for the occurrence of outbreaks of psittacosis require further investigation.

Real-time PCR detection and quantitation of Chlamydophila psittaci in human and avian specimens from a veterinary clinic cluster.

We report three cases of psittacosis in staff working in a veterinary surgery, which was related to exposure to a sick, wild psittacine bird. Chlamydial genus- and chlamydial species-specific DNA was detected in clinical specimens, including throat swabs, whole blood and urine. The organism load was quantified by real-time PCR (RT-PCR), which revealed 10(5)-fold more organisms in conjunctival swabs from the source bird than in the human samples. One clinic attendant was infected despite using personal protective equipment when handling the bird. This is the first report of PCR analyses of blood and urine samples being used to diagnose human psittacosis, and the first time that the organism load in humans has been compared to that of the infecting bird.

Cat scratch disease. A cause of regional lymphadenitis.

BACKGROUND: In the last decade, the microbiological cause of cat scratch disease (CSD) has been determined using a combination of traditional culture and modern molecular techniques. A bacterium known as Bartonelia henselae is responsible for the vast majority of cases. The natural history of the disease is being reinterpreted in the light of more sophisticated diagnostic tools. OBJECTIVE: To enable practitioners to have a sound basis for the diagnosis and treatment of cat scratch disease. DISCUSSION: Bartonelia henselae is ubiquitous in the domestic feline and causes zoonotic infection in humans. Although this infection is usually self limiting and benign, it may cause more extensive disease in the immunosuppressed. Antibiotic therapy may hasten recovery.

The Etest for antimicrobial susceptibility testing of Bartonella henselae.

The in-vitro susceptibility of 10 isolates of Bartonella henselae was assessed using the Etest. The organisms, one reference human strain and nine feline isolates, were grown on chocolate agar and the Etests read at days 5, 8 and 11. Six antibiotics, erythromycin, azithromycin, doxycycline, ciprofloxacin, rifampicin and vancomycin were evaluated. The results correlated well with published results using agar dilution. The results confirmed the high in-vitro susceptibility of B. henselae to erythromycin, azithromycin, doxycycline and rifampicin and to a lesser extent ciprofloxacin. The majority of isolates were resistant to vancomycin. Although in-vitro results of B. henselae susceptibility testing may not necessarily correlate with clinical response, the Etest may be a simpler way for laboratories to monitor for the development of resistance particularly in the setting of relapsing infection.

Prevalence of Bartonella henselae bacteremia, the causative agent of cat scratch disease, in an Australian cat population.

In order to determine the prevalence of Bartonella henselae becteremia in an Australian cat population we examined blood cultures on a group of Sydney cats. Cats referred to the Concord Animal Hospital for euthanasia were selected randomly for blood culture and serum sampling. Blood samples were lysed and centrifuged and then cultured for up to five weeks. Suspicious colonies were identified biochemically as probable B. henselae. Selected isolates were confirmed as B. henselae using the polymerase chain reaction. Of the cats accrued throughout Sydney, 27/77 (35%) were culture positive for B. henselae, of these 24/59 (40%) were feral cats and 3/18 (16%) were domestic. Most cats in the study were younger than one year (mean 9.9 months). Our study demonstrates that bacteremia with B. henselae is common in the metropolitan cat population and suggests that it is particularly prevalent among feral animals. By contrast Cat Scratch Disease (CSD) is a relatively uncommon clinical diagnosis in the Australian population. Explanations for this discrepancy may include poor transmission, low bacterial virulence and underdiagnosis. It is possible that feral animals are a greater potential risk source for this infection than domestic cats.