A tale of two studies: is peripheral eosinophilia associated with Dientamoeba fragilis detection in adult stool samples?

The protozoan parasite Dientamoeba fragilis is a frequently isolated stool organism and postulated cause of gastrointestinal symptoms. Peripheral blood eosinophilia has been described. This is the first study amongst the Australasian adult population to assess the relationship between organism detection and eosinophilia. A case-control study took place over 7 years at a single Sydney laboratory site, evaluating patients with D. fragilis identified on stool using real-time PCR with a recent full blood count, to control groups with Giardia spp. and sequential negatives with neither organism. A nested study compared those with microscopic evidence of D. fragilis as a marker of disease burden, to molecular diagnosis alone. Sixty-four D. fragilis, 30 Giardia spp., and 94 sequential controls were enrolled. Only 60.1% of samples were preserved in sodium acetate-acetic acid formalin (SAF) fixative, indication mostly not documented. The major co-organism detected amongst all participants was Blastocystis sp., particularly in the D. fragilis cohort (37.2%). The most common pathogen amongst sequential controls was Campylobacter spp. (7.4%). Patients with D. fragilis were more likely (12.5%) to have a clinically significant eosinophilia (>0.5×109/L) compared to those with Giardia spp. (3.3%) or sequential controls (4.3%) (p=0.03). A significant difference was also noted in the overall median eosinophil count of those with D. fragilis versus all controls (0.2 vs 0.1×109/L, p=0.01); however, this was within the reference interval (where up to >0.5×109/L is accepted in healthy individuals within a typical population). No eosinophil difference was found between those with molecular versus additional microscopic detection of D. fragilis (0.1 vs 0.1×109/L). These results support an association between the identification of clinically significant peripheral blood eosinophilia and D. fragilis presence, which may impact the diagnostic approach to the patient with unexplained eosinophilia. Further prospective trials may help assess any significance further and the implication of co-carriage with other enteric organisms. The importance of clinical indication and need for appropriate fixative media in diagnostic parasitology are also highlighted.

Haemophilus influenzae blood-stream infection and third-generation cephalosporin susceptibility testing: a comparative case study using EUCAST and CLSI guidelines.

Introduction: In this comparative case study, we discuss clinically relevant discrepancies of antimicrobial susceptibility testing (AST) interpretation for ceftriaxone against a non-typable, beta-lactamase negative, ampicillin-resistant (BLNAR) Haemophilus influenzae isolated from a blood culture. Case report: A 74-year-old man presented with a 3 day illness characterized by shortness of breath and dry cough, and was noted to be febrile and hypoxic on admission. A blood culture bottle flagged positive with Gram-negative coccobacilli, later identified as Haemophilus influenzae with the patient commenced on ceftriaxone. The isolate was beta-lactamase negative and antibiotic susceptibility testing (AST) using disc diffusion revealed the isolate resistant to ceftriaxone and ampicillin by EUCAST methodology, with the patient subsequently changed to amoxicillin/clavulanate. Further AST using the CLSI methodology in parallel demonstrated discrepant results between the two susceptibility methods. The patient recovered without complications. Conclusion: This discrepancy could lead to inconsistent reporting of susceptibilities between laboratories, and consequently antibiotic prescribing, especially for invasive isolates. As more laboratories adopt EUCAST methodologies for AST interpretation in Australia and globally, it is important for clinicians to consider the clinical implications of these methodological discrepancies.

Longer-term Mortality and Kidney Outcomes of Participants in the Combination Antibiotics for Methicillin-Resistant Staphylococcus aureus (CAMERA2) Trial: A Post Hoc Analysis.

Background: The Combination Antibiotic Therapy for Methicillin-Resistant Staphylococcus aureus (CAMERA2) trial ceased recruitment in July 2018, noting that a higher proportion of patients in the intervention arm (combination therapy) developed acute kidney injury (AKI) compared to the standard therapy (monotherapy) arm. We analyzed the long-term outcomes of participants in CAMERA2 to understand the impact of combination antibiotic therapy and AKI. Methods: Trial sites obtained additional follow-up data. The primary outcome was all-cause mortality, censored at death or the date of last known follow-up. Secondary outcomes included kidney failure or a reduction in kidney function (a 40% reduction in estimated glomerular filtration rate to <60 mL/minute/1.73 m2). To determine independent predictors of mortality in this cohort, adjusted hazard ratios were calculated using a Cox proportional hazards regression model. Results: This post hoc analysis included extended follow-up data for 260 patients. Overall, 123 of 260 (47%) of participants died, with a median population survival estimate of 3.4 years (235 deaths per 1000 person-years). Fifty-five patients died within 90 days after CAMERA2 trial randomization; another 68 deaths occurred after day 90. Using univariable Cox proportional hazards regression, mortality was not associated with either the assigned treatment arm in CAMERA2 (hazard ratio [HR], 0.84 [95% confidence interval [CI], .59-1.19]; P = .33) or experiencing an AKI (HR at 1 year, 1.04 [95% CI, .64-1.68]; P = .88). Conclusions: In this cohort of patients hospitalized with methicillin-resistant S aureus bacteremia, we found no association between either treatment arm of the CAMERA2 trial or AKI (using CAMERA2 trial definition) and longer-term mortality.

A comparison of SARS-CoV-2 RNA extraction with the QuickGene-810 Nucleic Acid Isolation System compared to the EZ1 Advanced DSP Virus Kit.

The QuickGene-810 Nucleic Acid Isolation System is a semi-automated extraction platform which may be used for RNA extraction. New methods were required to support the rapid increase in respiratory virus testing during the SARS-CoV-2 pandemic. The aim of this study was to assess SARS-CoV-2 RNA extraction using the QuickGene-810 kit compared to the EZ1 Advanced Extraction Platform for use on the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well RT-PCR assay. Qualitative results from all clinical samples were concordant between the QuickGene-810 and the EZ1 extraction methods, demonstrating that the QuickGene-810 kit is suitable for use in pathogen diagnostics. However, there was an average difference of approximately two cycles between the cycle threshold (Ct) values for both SARS-CoV-2 targets, suggesting that the EZ1 kit yields a higher concentration of nucleic acid extract, possibly related to its use of carrier RNA and/or smaller elution volume, which infers the possibility of false negative results for samples with very low viral loads.

Clinical Performance of Real-Time Polymerase Chain Reaction for Strongyloides stercoralis Compared with Serology in a Nonendemic Setting.

Strongyloides stercoralis is a nematode endemic to subtropical and tropical regions that may cause asymptomatic carriage, peripheral eosinophilia, cutaneous, gastrointestinal, and pulmonary disease, or hyperinfection syndrome. Conventional diagnostic methods for strongyloidiasis include feces microscopy and culture, with low sensitivity in chronic infection due to the low helminth burden, and serology, which may be prone to false-negative results with immunocompromise and false-positive results with other infections and immunological disorders. We evaluated a laboratory-developed real-time polymerase chain reaction (RT-PCR), detecting the 18S SSU ribosomal RNA gene, compared with conventional diagnostic methods, using serology via ELISA as the gold-standard. The population studied included tertiary hospital inpatients and outpatients residing in a nonendemic area. Seven hundred fifty unfixed stool specimens submitted sequentially between 2014 and 2018 were tested for S. stercoralis via microscopy and RT-PCR. Agar plate culture (APC), Harada-Mori culture (HMC), and ELISA were performed in conjunction with 141, 135, and 177 of the specimens, respectively. RT-PCR yielded 13 positive and 730 negative results, with inhibition in seven specimens. ELISA yielded 53 positive, 18 equivocal, and 106 negative results. Results for direct diagnostic methods obtained after treatment with ivermectin were excluded from the performance analysis. Compared with ELISA, RT-PCR, microscopy, APC, and HMC exhibited sensitivities of 38%, 6%, 3%, and 0%, respectively, and specificities of 100%. Given the low sensitivities commensurate with testing a population with remote infection and thus low parasite burden, we recommend a combination of serological and molecular diagnostic testing to achieve the best balance of sensitivity and specificity.

Evaluation of the influenza and respiratory syncytial virus (RSV) targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay: sample pooling increases testing throughput.

During the COVID-19 pandemic, sample pooling has proven an effective strategy to overcome the limitations of reagent shortages and expand laboratory testing capacity. The inclusion of influenza and respiratory syncytial virus (RSV) in a multiplex tandem PCR platform with SARS-CoV-2 provides useful diagnostic and infection control information. This study aimed to evaluate the performance of the influenza and RSV targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay, including the effect of pooling samples on target detection. RSV target detection in clinical samples was compared to the Cepheid Xpert Xpress Flu/RSV assay as a reference standard. Samples were then tested in pools of four and detection rates were compared. Owing to the unavailability of clinical samples for influenza, only the effect of sample pooling on simulated samples was evaluated for these targets. RSV was detected in neat clinical samples with a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.5% compared to the reference standard, demonstrating 99.7% agreement. This study demonstrates that sample pooling by four increases the average Ct value by 2.24, 2.29, 2.20 and 1.91 cycles for the target's influenza A, influenza A typing, influenza B and RSV, respectively. The commercial AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay was able to detect influenza and RSV at an intermediate concentration within the limit of detection of the assay. Further studies to explore the applicability of sample pooling at the lower limit of detection of the assay is needed. Nevertheless, sample pooling has shown to be a viable strategy to increase testing throughput and reduce reagent usage. In addition, the multiplexed platform targeting various respiratory viruses assists with public health and infection control responses, clinical care, and patient management.

Success of ceftazidime-avibactam and aztreonam in combination for a refractory biliary infection with recurrent bacteraemia due to blaIMP-4 carbapenemase-producing Enterobacter hormaechei subsp. oharae.

BACKGROUND: Infections due to metallo-beta-lactamase (MBL)-producing organisms are becoming a significant problem, and antibiotic treatment options are limited. Aztreonam inhibits MBLs, and its use in combination with ceftazidime-avibactam (CAZ-AVI-AZT) to inhibit other beta-lactamases shows promise. METHODS: A 45-year-old woman suffered from recurrent and sustained MBL (blaIMP-4)+ Enterobacter cloacae complex bacteraemia from an undrainable biliary source, and had failed nine alternative antibiotic regimens over a 5-month period. The 10th episode was successfully treated with CAZ-AVI-AZT, and she has had no further relapses. Three of the isolates underwent whole-genome sequencing (WGS) on the MiSeq platform and were analysed with the Nullarbor pipeline. RESULTS: A layered Etest method for synergy between CAZ-AVI and aztreonam demonstrated an MIC of 2 mg l-1 for the combination. Isolates were identified by WGS as Enterobacter hormaechei subsp. oharae . All three of the isolates had blaTEM-4 ESBL, blaOXA-1 and blaACT-25. Two of the carbapenem-resistant isolates contained blaIMP-4. CONCLUSION: While aztreonam inhibits MBLs, MBL-positive isolates often express other beta-lactamase enzymes. Avibactam inhibits ESBLs and other beta-lactamases, and its use in this case possibly contributed to therapeutic success due to inhibition of the concomitant blaTEM-4 in the isolates. This case demonstrates that phenotypic antimicrobial susceptibility testing (layered Etests for synergy), backed up by WGS, can produce results that allow tailored antimicrobial therapy in difficult infections. This case adds to the evidence for using CAZ-AVI-AZT in serious MBL infections.

Efficacy of aerosolized hydrogen peroxide (Deprox) cleaning compared to physical cleaning in a Burns Unit.

BACKGROUND: The performance of Deprox aerosolized hydrogen peroxide (aHP) has not been extensively studied in real-world clinical settings. A comparative study of aHP terminal disinfection was conducted in a Burns Unit and its performance compared to physical cleaning alone. METHODS: Environmental surfaces were sampled pre-cleaning, post-cleaning and post-aHP disinfection. Samples were cultured for MRSA, VRE, Gram-negative multi-resistant organisms and other Gram-negative bacilli. RESULTS: 310 sites were sampled. There was a reduction in the rates of contaminated surfaces post-aHP, though pathogens were still recoverable in most cases, except for VRE. There was a marked reduction in MRSA contamination of soft surfaces (12% post-clean vs 6% post-aHP), and patient room surfaces (8.3% post-clean vs 2.8% post-aHP). It does not work as well for MRSA in bathrooms: 7% of surfaces were positive post-clean, and 9% post-aHP. There was a reduction in multiresistant Gram-negative bacteria (7%-3%), mostly due to drains (33%-13%). CONCLUSION: aHP is a useful method of environmental disinfection, especially for Gram-negative pathogens in drains and MRSA on hard and soft surfaces. Where ongoing acquisition of MRSA is a problem, an adjunctive method of terminal disinfection in bathrooms could be considered.

Reactivation of Q fever: case report of osteoarticular infection developing at the site of a soft tissue injury.

Coxiella burnetii , the causative agent of Q fever, is known to cause acute and persistent infection, but reactivation of infection is rarely reported. This case demonstrates reactivation of a distant, untreated Q fever infection after a relatively innocuous soft tissue injury in an adjacent joint without pre-existing pathology. A 52-year-old male abbatoir worker sustained an adductor muscle tear in a workplace injury. He was unable to walk thereafter, and developed a chronic, progressive, destructive septic arthritis of the adjacent hip with surrounding osteomyelitis of the femur and acetabulum. He had evidence of prior Q fever infection, with a positive skin test and serology 15 years beforehand. He was diagnosed with chronic osteoarticular Q fever on the basis of markedly elevated phase I antibodies, and symptomatic and serological response to prolonged antibiotic treatment with doxycycline and hydroxychloroquine. He required a two-stage hip arthroplasty. This case illustrates reactivation of latent C. burnetii infection at the site of a soft tissue injury. Clinicians need to be aware of this possibility in patients with previous Q fever infection, and in the setting of undiagnosed osteoarticular pathology following soft tissue injury.

Antimicrobial stewardship audit and feedback rounds: the impact of electronic systems and moving beyond the restricted antibiotic list.

BACKGROUND: Current methods of antimicrobial usage surveillance have limited efficacy in changing practice due to delayed reporting to clinicians and the inability to stratify by medical specialty. This study was undertaken in a tertiary teaching hospital with a well established antimicrobial stewardship (AMS) programme and electronic medicines management (eMM) system in Sydney, Australia. AIMS: To describe and analyse the implementation of a novel AMS audit and feedback method, in the context of an eMM system. METHODS: The AMS team conducted the audit weekly, and the study design was a prospective, observational study. All acute, adult inpatients were included in this intervention. All active systemic antimicrobial prescriptions on the day of the rounds were included. RESULTS: The prevalence of patients on antimicrobial therapy was 37%. The median time taken per round was 44 min for eMM compared to 58 min for paper. All key performance indicators improved over the study period. Appropriateness compared to guidelines increased from 55% to 71%, and documentation of an indication increased from 75% to 98%. There were 1413 recommendations made, with the most common being to cease an antimicrobial agent. The recommendation uptake rate was 47% at 24 h post-round. CONCLUSIONS: AMS rounds are an effective tool for auditing and providing feedback on antimicrobial use and should include all antimicrobials rather than solely 'restricted' agents. These rounds had a high uptake rate, improvements in the appropriateness of antimicrobial use, and a planned duration or review date. A benefit of eMM was improvement in the documentation of indication for antimicrobial agents, and reduced time taken to audit.

Hospital MRSA outbreaks: Multiplex PCR-reverse line blot binary typing as a screening method for WGS, and the role of the environment in transmission.

BACKGROUND: Whole-genome sequencing (WGS) can provide useful information on methicillin-resistant Staphylococcus aureus (MRSA) transmission in hospitals. However, it is expensive and laborious, especially for environmental screening programs which generate large numbers of isolates. Multiplex PCR-reverse line blot binary typing (mPCR-RLB BT) is a rapid, high throughput, inexpensive typing method which could be useful to screen isolates for WGS. This study assessed the strategy of screening isolates with mPCR-RLB BT to reduce the need for WGS; and to assess the role of the environment in transmission. METHODS: MRSA transmission in a Burns Unit and its related Intensive Care Unit was studied. mPCR-RLB BT was performed on 238 isolates; this, combined with epidemiological data, was used to choose 97 isolates for WGS. RESULTS: Relationships between isolates by WGS demonstrated several outbreaks. There was a significant contribution of environmental isolates to transmission, and several problem areas were identified. There was a substantial cost saving from screening isolates with mPCR-RLB BT. CONCLUSIONS: The use of an inexpensive, rapid screening method for MRSA typing is useful to reduce expenditure and time spent on hospital infection control programs, and reductions are likely to be even more considerable in a non-outbreak setting. Environmental screening and WGS are useful to determine exact sources of transmission to focus mitigation strategies.

Secondary Syphilis with Eosinophilia Complicated by Severe Jarisch-Herxheimer Reaction.

It has long been acknowledged that syphilis is a disease with a diverse range of presentations. We herein describe a case of a young man who presented with fever, rash, and eosinophilia following the commencement of allopurinol, only to be diagnosed with secondary syphilis on histopathology. His treatment was complicated by a severe exacerbation of his cutaneous eruption following the commencement of penicillin, likely secondary to a Jarisch-Herxheimer reaction, an entity often overlooked by clinicians managing syphilis.

Effect of Vancomycin or Daptomycin With vs Without an Antistaphylococcal ß-Lactam on Mortality, Bacteremia, Relapse, or Treatment Failure in Patients With MRSA Bacteremia: A Randomized Clinical Trial.

Importance: Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is associated with mortality of more than 20%. Combining standard therapy with a ß-lactam antibiotic has been associated with reduced mortality, although adequately powered randomized clinical trials of this intervention have not been conducted. Objective: To determine whether combining an antistaphylococcal ß-lactam with standard therapy is more effective than standard therapy alone in patients with MRSA bacteremia. Design, Setting, and Participants: Open-label, randomized clinical trial conducted at 27 hospital sites in 4 countries from August 2015 to July 2018 among 352 hospitalized adults with MRSA bacteremia. Follow-up was complete on October 23, 2018. Interventions: Participants were randomized to standard therapy (intravenous vancomycin or daptomycin) plus an antistaphylococcal ß-lactam (intravenous flucloxacillin, cloxacillin, or cefazolin) (n = 174) or standard therapy alone (n = 178). Total duration of therapy was determined by treating clinicians and the ß-lactam was administered for 7 days. Main Outcomes and Measures: The primary end point was a 90-day composite of mortality, persistent bacteremia at day 5, microbiological relapse, and microbiological treatment failure. Secondary outcomes included mortality at days 14, 42, and 90; persistent bacteremia at days 2 and 5; acute kidney injury (AKI); microbiological relapse; microbiological treatment failure; and duration of intravenous antibiotics. Results: The data and safety monitoring board recommended early termination of the study prior to enrollment of 440 patients because of safety. Among 352 patients randomized (mean age, 62.2 [SD, 17.7] years; 121 women [34.4%]), 345 (98%) completed the trial. The primary end point was met by 59 (35%) with combination therapy and 68 (39%) with standard therapy (absolute difference, -4.2%; 95% CI, -14.3% to 6.0%). Seven of 9 prespecified secondary end points showed no significant difference. For the combination therapy vs standard therapy groups, all-cause 90-day mortality occurred in 35 (21%) vs 28 (16%) (difference, 4.5%; 95% CI, -3.7% to 12.7%); persistent bacteremia at day 5 was observed in 19 of 166 (11%) vs 35 of 172 (20%) (difference, -8.9%; 95% CI, -16.6% to -1.2%); and, excluding patients receiving dialysis at baseline, AKI occurred in 34 of 145 (23%) vs 9 of 145 (6%) (difference, 17.2%; 95% CI, 9.3%-25.2%). Conclusions and Relevance: Among patients with MRSA bacteremia, addition of an antistaphylococcal ß-lactam to standard antibiotic therapy with vancomycin or daptomycin did not result in significant improvement in the primary composite end point of mortality, persistent bacteremia, relapse, or treatment failure. Early trial termination for safety concerns and the possibility that the study was underpowered to detect clinically important differences in favor of the intervention should be considered when interpreting the findings. Trial Registration: ClinicalTrials.gov Identifier: NCT02365493.

Australian consensus statements for the regulation, production and use of faecal microbiota transplantation in clinical practice.

OBJECTIVE: Faecal microbiota transplantation (FMT) has proved to be an extremely effective treatment for recurrent Clostridioides difficile infection, and there is interest in its potential application in other gastrointestinal and systemic diseases. However, the recent death and episode of septicaemia following FMT highlights the need for further appraisal and guidelines on donor evaluation, production standards, treatment facilities and acceptable clinical indications. DESIGN: For these consensus statements, a 24-member multidisciplinary working group voted online and then convened in-person, using a modified Delphi approach to formulate and refine a series of recommendations based on best evidence and expert opinion. Invitations to participate were directed to Australian experts, with an international delegate assisting the development. The following issues regarding the use of FMT in clinical practice were addressed: donor selection and screening, clinical indications, requirements of FMT centres and future directions. Evidence was rated using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system. RESULTS: Consensus was reached on 27 statements to provide guidance on best practice in FMT. These include: (1) minimum standards for donor screening with recommended clinical selection criteria, blood and stool testing; (2) accepted routes of administration; (3) clinical indications; (4) minimum standards for FMT production and requirements for treatment facilities acknowledging distinction between single-site centres (eg, hospital-based) and stool banks; and (5) recommendations on future research and product development. CONCLUSIONS: These FMT consensus statements provide comprehensive recommendations around the production and use of FMT in clinical practice with relevance to clinicians, researchers and policy makers.

Actinomyces cavernous sinus infection: a case and systematic literature review.

A 63-year-old man presented with a 2-month history of progressive right-sided exophthalmos, painful ophthalmoplegia and fevers. As more features developed, he was diagnosed with giant cell arteritis, then Tolosa-Hunt syndrome, and transiently responded to corticosteroids. A bland cerebrospinal fluid and highly metabolically active brain (18F)-fluoro-D-glucose-positron emission tomography suggested lymphoma. Biopsy of the mass showed sulphur granules with Gram-positive filamentous bacteria with Actinomyces-like colonies. Actinomyces cavernous sinus infections are rare and indolent. They often mimic non-infective causes including other inflammatory and infiltrative conditions, vascular and neoplastic causes, particularly lymphoma. Clinicians should consider infective cavernous sinus syndromes in people with a fluctuating painful ophthalmoplegia that responds poorly to corticosteroids. The term Tolosa-Hunt syndrome is problematic and should be retired or used only with reservation.