FEDERATION OF
infection societies
CONFERENCE 2018

13th – 15th November 2018 | Sage Gateshead – Newcastle

FEDERATION OF
infection societies
CONFERENCE 2018

13th – 15th November 2018 | Sage Gateshead – Newcastle

FEDERATION OF
infection societies
CONFERENCE 2018

13th – 15th November 2018 | Sage Gateshead – Newcastle

Posters

MYCOLOGY – Poster Nos. 078-085

078
Molecular characterization of Candida species associated with human urinary tract infection

Abstract - 078

Poster 078

Molecular characterization of Candida species associated with human urinary tract infection

Muhammad Mushtaq, Zil E Huma
Balochistan University of Information Technology, Information Technology, Engineering, Management Sciences (BUITEMS), Quetta, Pakistan

Introduction: Studies on fungal infection have shown the concomitant increase in the prevalence of candiduria as well as in the incidence of Candida UTIs along with the bactiuria. In the developing countries, clinicians have little confidence in the accuracy and quality of laboratory test results for their diagnosis. Physicians routinely prescribe costly antifungals without knowing the exact antifungal profile of the infectious agent; thereby increasing economic burden of the society as well as contributing to the emergence of resistant Candida spp. Resistance towards drugs is a major problem in treating yeasts and other infections. It is important to note that clinical isolates of Candida species differ in their virulence and antifungal susceptibility. Hence accurate species level identification has a direct impact for the choice of pragmatic antifungal treatment. This problem is leading to development of new drugs and delivery systems for which the quick and accurate identification of disease causing yeasts is the first step. Several techniques are used to identify Candida species from clinical specimens, however, molecular techniques are rapid, object oriented, yield results which are not influenced by growth conditions.

Methods: Candida sppp. were isolated from urine samples by cultural techniques using YM Agar medium and identified using CHROMagar technique, RapID Yeast Plus System, PCR-RFLP Assay using MspI restriction enzyme, DNA sequencing of ITS regions. The phylogeneitc tree was constructed using Mega 7 software to determine the erlationship among the identified Candida species.

Results: Results indicated that Fungal UTI is more prevalent in female patients (86%) as compare to male (14%). Candida albicans. C. tropicalis, C. parapsolosis and C. krusei were identified successfully on the basis of their colony color on CHROMagar while C. glabrata showed confusing results. Moreover some of the yeast cultures which did not produced colored colonies on CHROMagar were further identified using RapID Yeast Plus system, where all such unidentified yeast cultures were identified as C. glabtrata. The RapID yeast Plus System successfully identified all Candida spp., but not C. krusei and C. parapsilosis as they produced weak positive/ambiguous results. A total of 5 Candida species viz., C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis were finally identified on the basis of PCR RFLP and DNA sequencing of ITS regions. C. albicans appeared to be the most occurring pathogen with 53.16% as compared to C. glabrata (13.9%), C. tropicalis (16.45%), C. krusei (Pichia kudriavzevii) (5%) and C. parapsilosis (2.53%).

Discussion: The results showed that Candida albicans was the most prevalent Candida species, whereas, Candida glabrata also recorded as an emerging fungal pathogen in funguria. CHROMagar Candida medium was used as a primary medium for the presumptive identification of Candida species and found to be an appropriate and affordable diagnostics medium in a resource-limiting environment. RapID Yeast Plus system was found suitable for all Candida spp., including those which were difficult to identify by Chromagar technique, however it was not found suitable to distinguish C. krusei and C. parapsilosis. Candida glabrata also recorded as an emerging fungal pathogen in funguria. PCR-RFLP and DNA sequencing of PCR products appeared to be the most reliable techniques for the identification of Candida species. This finding is a conclusive reason to apply molecular methods as an easy, fast and reliable technique in comparison with other conventional techniques, which are insensitive, lacks of reproducibility and standardization and with the limited availability for determination and identification of medically important Candida spp. in clinical laboratory. In general, it seems that this genotyping system comparing the other molecular system and phenotyping system is a rapid, almost inexpensive and completely valid method for identification of Candida spp.

079
Laboratory evaluation of a new T2 Magnetic Resonance assay for rapid detection of Candida species in spiked human blood samples

Abstract - 079

Poster 079

Laboratory evaluation of a new T2 Magnetic Resonance assay for rapid detection of Candida species in spiked human blood samples

Alireza Abdolrasouli, Nita Fatania, Giovanni Satta
North West London Pathology, Imperial College Healthcare NHS Trust, London

Introduction: The mortality associated with invasive candidiasis remains high. The culture-independent T2 Magnetic Resonance (T2MR) system is a rapid diagnostic platform shown to detect target pathogens of interest from unprocessed blood samples in <5 hours. In clinical trials, T2MR has exhibited good clinical sensitivity and specificity. Potential benefits from the adoption of T2MR technology in the diagnostic and therapeutic algorithms for invasive candidiasis can arise from timely diagnosis of disease, increased case detection, tailored therapy and decrease in empiric antifungal treatment. Here we aimed to assess the performance of T2Candida in detection and identification of Candida species in spiked human blood samples.

Methods: 4mL sterile human blood samples in K2EDTA BD vacutainer tubes were spiked with five Candida ATCC type strains (C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis) and each strain was tested in triplicate. Yeast isolates were cultured on Sabouraud dextrose agar and incubated at 37°C for 24 hours. Yeast cells were serially diluted in PBS and each blood tube was spiked to a final concentration of 5-20 CFU/mL. In total, five negative control samples were tested simultaneously with spiked blood samples. Samples were tested following T2 Biosystems instructions.

To compare the performance of T2Candida with BD FX automated blood culture system, 20 mL of human blood was spiked with each yeast suspension at the same concentration used to spike the T2Candida tubes. Aerobic and anaerobic blood culture bottles were inoculated with 10 mL of blood per bottle and incubated for up to 5 days. If flagged positive, each bottle was sub-culture to confirm to identity and purity of isolated yeasts.

To test for potential cross-reactivity, five wild-type yeast isolates including Candida metapsilosis, Candida dubliniensis, Candida auris, Candida niveriensis and Trichosporon asahii were included for the analysis. These isolates were tested as described above, however, the final concentration of yeast cells for four Candida species were increased to 50-200 CFU/mL when blood samples were spiked.

Results: Overall, 20 blood samples were tested from which 15 contained Candida species and 5 did not have any Candida (negative controls). All 15 blood samples contained target Candida species, produced positive results on T2Candida system. All five tested species were correctly identified to corresponding groups. No false-negative or miss-identification was detected. Concentration of yeast cells varies from 5 to 17 CFU/mL of blood per tube. All negative controls consistently generated negative result with no false-positive reaction observed. All spiked blood culture sets which were tested in parallel with T2Candida system flagged positive at 1-3 days on automated blood culture system. Results were in agreement with T2Candida system.

Analysis of three closely related species to the T2Candida targets revealed that C. metapsilosis was detected and identified as C. parapsilosis, however C. dubliniensis and C. niveriensis were not detected. C. auris and T. asahii did not cause a false-positive result.

Discussion: Our pre-clinical laboratory evaluation demonstrated that T2Candida Panel provides consistent and accurate performance in the detection and identification of five most common Candida species directly from blood samples.

080
Candida auris molecular detection in epidemiological surveillance samples by OLM Diagnostics AurisID kit

Abstract - 080

Poster 080

Candida auris molecular detection in epidemiological surveillance samples by OLM Diagnostics AurisID kit

Carme Salvador Garcia1, Nuria Tormo Palop1, Roberto Olmos Arenas1, Juan Vicente Mulet Bayona1, Gemma Johnson2, Concepción Gimeno Cardona1,3
1Consorcio Hospital General Universitario de Valencia, Spain. 2OLM Diagnostics, Newcastle. 3Medicine School. University of Valencia, Spain

Introduction: Candida auris has emerged as a resistant fungal pathogen responsible for hospital outbreaks, especially in risk care units. C. auris has the capacity to survive in the environment and to colonize biomedical devices as well as skin and mucosa of patients that could be implicated in maintenance of reservoirs and patient cross-infection, and therefore persistence of an outbreak.

The control of C. auris outbreak is important because comorbidity and clinical severity of infected patients in addition to fungal resistance lead to high mortality rates and presence of frequent complications. A major point is the epidemiological surveillance of C. auris in colonized patients to avoid infection and cross-infections. In this setting, molecular methods are a useful tool in order to detect C. auris in colonized patients in a quick and easy way.

The aim of this study was to test the OLM Diagnostics AurisID kit in epidemiological surveillance samples.

Methods: We performed the OLM Diagnostics AurisID kit in epidemiological surveillance samples (axillary-rectal and oropharynx swabs) of 5 patients. The samples were collected in a tube with gel Amies transport media (Deltalab®). We tested three different pre-treatment DNA extraction protocols: i) dissolve in sterile water, concentration, lysozyme (Sigma) and proteinase K (Roche) treatment ii) dissolve in sterile water and concentration iii) dissolve in sterile water. After that, DNA extraction was carried out by MagNA Pure Compact (Roche®) according to manufacturer´s instructions using DNA Blood protocol with 400ul of sample and 100ul of elution.

In addition, all the swabs were cultured in CHROMagar Candida medium (Becton Dickinson). The plates were incubated at 36ºC, 24-48 hours. Candida isolates were identified by proteomic profiling (MALDI-TOF, Bruker) according to clinical laboratory practice.

Results: A total of 5 samples from 5 patients (4 skin-rectal and 1 oropharynx swabs) were analyzed by OLM Diagnostics AurisID. OLM Diagnostics AurisID kit showed the same results with the three different pre-treatment DNA extraction protocols in all samples. Moreover, the AurisID qPCR Kit results were positive in 4/5 patient samples and were in concordance with Candida culture results (4 patients were colonized by C. auris and the other one was negative). 

Discussion: C. auris is difficult to treat and to eradicate. Despite all efforts, new colonized and/or infected patients appear, that´s why this emergent resistant fungal is considered a worrisome healthcare problem.

Rapid epidemiological surveillance results are necessary to improve the clinical dealing with these patients. OLM Diagnostics AurisID kit detects C. auris in surveillance samples within 45 minutes of nucleic acid extraction (without pre-treatment protocol), thus it seems to be an interesting tool to improve quick C. auris colonized patients detection.

081
Comparison of three commercial susceptibility testing panels for the determination of minimum inhibitory concentrations of antifungals against Candida species

Abstract - 081

Poster 081

Comparison of three commercial susceptibility testing panels for the determination of minimum inhibitory concentrations of antifungals against Candida species

Jane Johnson, Susie Batley, James Cargill
Alder Hey Children’s Hospital, Liverpool

Introduction: Alder Hey is a specialist paediatric hospital in Liverpool, providing clinical services across north-west England and north Wales. Basic microbiology and virology services are provided by the on-site laboratory.

The YeastOne Sensititre broth dilution plate (Thermo Scientific) was originally introduced into the Alder Hey laboratory in 2012, when they replaced the bioMérieux antifungal etest MIC strips. Following the laboratory’s transition to EUCAST susceptibility testing repeat verification exercises were performed for all our antimicrobial tests, including the Sensititre. It had also been noted that the in-house echinocandin MIC results were higher compared to results from the Mycology Reference Centre at the Manchester Medical Microbiology Partnership.

At the same time as reassessing the Sensititre plate, the Vitek YST07 antifungal card (bioMérieux) and SensiQuattro Candida EU broth dilution plate (Liofilchem) were also evaluated.

Methods: Twenty replicates were performed using two EUCAST control organisms; Candida albicans CNM-CL F8555 and Candida krusei ATCC 6258. Results were compared to the EUCAST control values; accuracy was interpreted by agreement with the EUCAST MIC range and reproducibility by the number of tests within 1 doubling dilution of the mode MIC.

The results for Fluconazole, Micafungin and Amphotericin B were considered to be the most relevant when selecting a test when the hospital antimicrobial formulary was taken into account.

Results: Results are reported below as:

  • Test: Mode MIC C. albicans / EUCAST control range / mode MIC C. krusei / EUCAST control range

Micafungin

  • Sensititre: 0.03mg/L / no EUCAST range / 0.12mg/L / EUCAST 0.03-0.125mg/L
  • YST07: <=0.06mg/L / no EUCAST range / 0.12mg/L / EUCAST 0.03-0.125mg/L
  • SensiQuattro: >=0.25mg/L / no control / >=0.25mg/L / EUCAST 0.03-0.125mg/L

Knowing that the Sensititre gave results typically within 1 doubling dilution of the reference laboratory and the agreement between the Sensititre and YST07 results, the SensiQuattro evaluation was halted after ten replicates.

All tests showed good reproducibility, with all MIC values within a doubling dilution of the mode.

Amphotericin B

  • Sensititre: 0.5mg/L / EUCAST 0.06-0.5mg/L / 1mg/L / EUCAST 0.125-1mg/L
  • YST07: 1mg/L / EUCAST 0.06-0.5mg/L / 0.5mg/L / EUCAST 0.125-1mg/L

The results were less reproducible than for micafungin; Sensititre against C. albicans 100% within one doubling dilution of the mode, against C. krusei 95%, YST07 against C. albicans 85%, and against C. krusei 100%.

Fluconazole

  • Sensititre: 32mg/L / EUCAST 32-128mg/L / 32mg/L / EUCAST 16-64mg/L
  • YST07: >=64mg/L / EUCAST 32-128mg/L / 16mg/L / EUCAST 16-64mg/L

The results were less reproducible than for micafungin; Sensititre against C. albicans 95% within one doubling dilution of the mode, against C. krusei 100%, YST07 against C. albicans 100%, and against C. krusei 90%.

Discussion: The selection of an appropriate antifungal test panel depends on the antimicrobial formulary (i.e. what antifungals need testing) and the equipment available in the laboratory.

The high micafungin results seen with the SensiQuattro in our hands could not be explained. The performance of the amphotericin and fluconazole tests was in keeping with the expected results (not shown), and all tests (eight antifungals in total) had all results within one dilution of the mode MIC.

The Vitek YST07 card is the easiest test to set up and report as the incubation and reading are automated. The reproducibility of the results was however the lowest on evaluation (85% of amphotericin results against C. albicans were within one doubling dilution of the mode).

We chose to continue with the Sensititre plate; it showed good reproducibility although for micafungin and amphotericin the mode MICs were at the top of the EUCAST expected range. Borderline results are therefore referred to a reference centre for confirmation.

082
Case study of Malassezia pachydermatis in an adolescent undergoing chemotherapy

Abstract - 082

Poster 082

Case study of Malassezia pachydermatis in an adolescent undergoing chemotherapy

Amy Aice Carson
University Hospitals Bristol NHS Foundation Trust

Introduction: Malassezia are basidiomycetous yeasts that are mostly lipid dependent, inhabiting the mucosa of humans and other mammals, as well as forming a major part of the normal human skin microbiome. Malassezia pachydermatis falls under the genus Malassezia, and is considered a zoophilic, being the most common veterinary Malassezia species found on the skin and in the ear canal of dogs and cats, frequently causing seborrhoic dermatitis and otitis externa. M. pachydermatis is only occasionally found on human skin and, if it is, it is normally associated with pet owners. M. pachydermatis is a rare cause of systemic infection, and if it does occur it is most commonly associated with neonates receiving lipid supplementation. As far as we know M. pachydermatis is very rarely implicated in systemic infection in adults.

Here we describe a case of M. pachydermatis fungaemia in an adolescent currently undergoing chemotherapy.

Methods: A 16-year-old white British male presented with a one week history of a rapidly enlarging neck swelling, progressing to the development of cervical and inguinal lymphadenopathy and an enlarged right testis. A computed tomography (CT) scan of his neck, chest, abdomen and pelvis demonstrated multifocal large lymph nodes in the neck, inguinal region and mesentery. A left cervical biopsy was performed that demonstrated a lymphoid malignancy, with CD20 positive cells in keeping with a high grade B (non-Hodgkin) cell lymphoma: Burkitts lymphoma. He was then started on steroids and a combination of cyclophosphamide and vincristine (COP) chemotherapy via a peripherally inserted central catheter (PICC). The chemotherapy was subsequently converted to Rituximab, Cyclophosphamide, Vincristine, Prednisolone, Doxorubicin and Methotrexate (R-COPADM) with curative intent. As he failed to progress on this regimen it was converted to cyclophosphamide, vincristine, doxorubicin and methotrexate (CYVE).

Eighteen days after initiating the first cycle of CYVE the patient was admitted with febrile neutropenia (white cell count 0.36 x 109/L (4.0-11.0), neutrophil count 0.01 x 109/L (1.5-8.0), lymphocytes 0.32 x 109/L (1.0-4.0), CRP 17mg/L. (<6). There were no localising symptoms or signs. Blood cultures (BacT/ALERT®, bioMérieux) were taken from his PICC line and he was initiated on empirical treatment of piperacillin/tazobactam (4.5g every 8 hours). After 3 days of incubation a yeast was isolated from the aerobic bottle. His PICC line was removed and he was commended on micafungin empirically pending identification of the yeast. The isolate was referred to the Mycology Reference Laboratory, Bristol. The isolate was purple on CHROMagar™, and confirmed to be Malassezia pachydermatis by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry (ID score 2.063). 

Discussion: Information about M. pachydermatis fungaemia remains limited. Due to the lipid-dependent nature of the species, diagnosis of Malassezia remains challenging. Special media are required such as Dixon or modified Leeming and Notham agar. The species may then be confirmed and distinguished using MALDI-TOF mass spectrometry as it was in this case. M. pachydermatis is easier to grow than the more commonly found M. furfur. Unlike other Malassezia species, M. pachydermatis has the ability to grow without fat, and hence it is able to grow on Sabouraud glucose agar (SGA) without lipid supplementation. However, precisely due to the ability of M. pachydermatis to grow without lipids, it can be misidentified as a Candida species which can then lead to inappropriate therapy, which becomes problematic as Malessezia spp. are intrinsically resistant to echinocandins.

Data remains limited surrounding M. pachydermatis infections in humans with regards to its recognition, diagnosis and management. In particular, little systematic data is available beyond the neonatal age. 

083
Impact of invasive Candidiasis in a Paediatric Transplant Unit

Abstract - 083

Poster 083

Impact of invasive Candidiasis in a Paediatric Transplant Unit

Richard Capstick, Isha Rizal, Caroline Williams, Hloniphani Mpofu, Joanna Lumb, Yamura Thiru, Julie Samuel
Newcastle upon Tyne Hospitals

Introduction: We report a case series of five patients with invasive Candida infections between February 2017 and June 2018. All five isolates were Candida parapsilosis; three isolated from blood culture and two from mediastinal tissues. All 5 of these patients were admitted to the Paediatric Cardiothoracic Intensive care unit at the time of their Candida infection. Three of these patients died during their admission, two of which were heart transplants. The other three patients all had underlying cardiac problems and underwent a number of procedures during their admissions.

Methods: A retrospective review of the isolates showed that all samples were fully sensitive. Two of the isolates from February this year were determined to be indistinguishable from each other when sent for typing to Bristol Mycology Reference Laboratory. The 2 isolates from 2017 were not available for typing. Subsequent environmental screening of the Intensive Care Unit and a fingertip screen from a member of staff also isolated Candida parapsilosis. The fifth patient had a distinct strain to the ones previously isolated as was the isolate from the HCW. This was based on multilocus sequencing of 4 different microsatellite regions (CP1, CP4, CP6 and B5) within C. parapsilosis.

Discussion: Invasive Candida infections represent a significant problem in paediatric patients especially those with long inpatient ICU stays and those that are immunocompromised. This case series highlights the importance of basic infection control measures to prevent cross contamination and environmental pathogens causing significant infection in susceptible paediatric patients.

084
An unusual cause of intracranial mass lesions in an immuno-competent adult

Abstract - 084

Poster 084

An unusual cause of intracranial mass lesions in an immuno-competent adult

Phoebe Cross, Hugh McGann, Penelope Lewthwaite
Leeds Teaching Hospitals

Introduction: We present an unusual cause of intracranial mass lesions in an immuno-competent adult.

Case Description: A 55 year old female presented with right arm sensory loss, weakness, speech disturbance and diplopia progressing over weeks.

She had a background of hidradenitis suppurativa, smokes, drinks alcohol and works in IT. She is British and has travelled to the Canary Islands and Aruba.

On examination she had an intranuclear ophthalmoplegia, dysarthria, word finding difficulties, facial droop and right arm weakness with sensory loss. Other examination and bloods were unremarkable.

MRI brain showed lesions in right frontal area, left internal capsule and posterior fossa with leptomeningeal enhancement.

CT body normal.

Lymphoma was high on the differential so dexamethasone was commenced pending brain biopsy.

Biopsy revealed active granulomatous inflammatory reaction secondary to protozoa, probably toxoplasma. No malignancy.

Toxoplasma IgG and PCR on the specimen was negative. HIV was negative and normal immune system markers.

Discussion: The biopsy specimen was referred to London for a second opinion as toxoplasma was felt to be unlikely.

Histology showed histiocytic-multi nucleated giant cells and macrophages containing large circular structures with a clear outer wall and red circular and granular bodies identified. Outer walls stain positively with Grocott silver and PAS stains.

085
Common commensal or uncommon calamity?

Abstract - 085

Poster 085

Common commensal or uncommon calamity?

Aarti Velani, Liz Hart
Nottingham University Hospitals

Introduction: A 21 year old lady presented to the Acute Medical Unit with symptoms consistent with a lower respiratory tract infection. She had recently returned from a 2 week holiday to Florida. Over the next 18 months she developed recurrent disfiguring skin lesions associated with systemic features of fever, headache and fatigue. These occurred on the face and scalp and healed without scarring – pictures are included. She was referred to a number of specialities and had two skin biopsies and a variety of treatments before being referred to Infectious Diseases.

Methods: A repeat skin biopsy and pan fungal PCR revealed the diagnosis. A prolonged course of treatment with itraconazole was commenced with resolution of the skin lesions and improvement in the patient’s quality of life. A treatment break at 6 months was associated with recurrence of the lesions. The treatment was complicated by interactions with over the counter slimming tablets and concerns with regards to adequate contraception.

Discussion: A query had been raised as to the possibility of blastomycosis. The second skin biopsy had seen one spore of Malassezia furfur (previously Pityrospoum furfur) which is often found on skin biopsies and was dismissed as a skin commensal. A further biopsy was performed and samples were sent to microbiology for culture and sensitivity and pan fungal PCR. This biopsy suggested the diagnosis and eventual treatment plan.

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