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LIFE Newsletter - Leading International Fungal Education

December 2016

Molecular Identification of clinically important invasive fungal pathogens directly from formalin fixed paraffin-embedded tissue

The frequency and diversity of lethal invasive fungal pathogens has moved beyond Aspergillus fumigatus to include a plethora of pathogens including the non-fumigatus species of Aspergillus, the Mucormycetes, Fusarium and Scedosporium species and a variety of melanised fungi. Identification by histopathology to genus or species level is limited and fungal cultures from tissue biopsy specimens are often negative.

mucoralesA recent article by Salehi et al (2016) in a multicentre retrospective study in Iran, has evaluated a real-time qPCR assay which targets the internal spacer (ITS) region of ribosomal DNA (rDNA) to detect and identify genus and species of Aspergillus, the Mucormycetes, Fusarium and Scedosporium from FFPE tissue specimens in patients with histologically proven invasive fungal infections.
The qPCR assay evaluated showed an overall sensitivity of 64% for the identification of fungi from FFPE and highlighted that histopathological features of moulds may be easily confused in tissue sections:

Mucorales

  • Fusarium oxysporum and Fusarium solani DNA was detected in five specimens but diagnosed as Aspergillus by histopathology
  • Aspergillus flavus, Scedosporium apiospermum and Syncephalastrum were detected from samples classified as mucormycosis by histopathology
  • Molecular results  (R. oryzae and A flavus) correlated with only one of four tissue samples histologically suspected of having concomitant aspergillosis and mucormycosis; two with A. flavus only and one Mucor isolate

A key finding was the high sensitivity (94%) of the DNA extraction method utilised (automated EZ1 extraction instrument (Qiagen) in combination with a DNA tissue kit). This method gave an excellent yield compared to the 60-80% amplification success rates of other current methods. Direct identification by qPCR assays such as these demonstrate promise for a rapid, reliable adjunctive tool for the accurate identification of clinically relevant fungal pathogens. Sequence based analysis should also be possible, using a similar protocol.
Salehi et al 2016:  J Clin Microbiol 54:2798–2803. doi:10.1128/JCM.01185-16
Guarner et al (2011) Histopathological Diagnosis of Fungal Infections in the 21st century.
More information

E-learning course in direct microscopy for identifying fungal infections
Experts in Manchester have recently launched the first e-learning course in direct microscopy for identifying fungal infection, also teaching histological staining methods and interpretation of fungal elements. The course is accredited by the University of Manchester & will teach not only how to rapidly and accurately diagnose life-threatening fungal infections, but also how to set up direct microscopy in a diagnostic laboratory. It is available at www.microfungi.net. The first 50 students to complete this course will be offered free ESCMID membership for one year.
ESCMID


NEWS

Over a million AIDS deaths preventable by 2020

Fungal infection causes around half of AIDS-related deaths, of which there were 1,100,000 in 2015 (UNAIDS fact sheet). An novel analysis: Modelling Reduction in AIDS deaths in conjunction with GAFFI (global action fund for fungal infections) suggests that the opportunity to save lives is being missed.

With improved access to antiretroviral therapy and a focus on diagnosing TB co-infection, deaths from AIDS have been falling.  However, progress is slower than anticipated across the world. The UNAIDS aspirational target of zero AIDS deaths by 2015 was not met, however from UNAIDS numbers, there was a 41% reduction in lives lost (from 2010 at 1.76 m to 1.1 m in 2015). aids day

Continued failure to focus efforts on advanced HIV infection and the 47% who have fungal infections means the current UNAIDS target of fewer than 500,000 annual deaths by 2020 will almost certainly be missed, as was the aspirational target of zero AIDS deaths by 2015.  Retention in care is a major factor, but it is late presentation with overwhelming infection that is GAFFI’s primary concern. 

GAFFI's projections reveal that by improving access to just 60% of those who need it, over 300,000 lives could be saved per year. By 2020 a total of over a million lives could have been saved, helping to meet the UNAIDS mortality reduction target reducing AIDS deaths to 500,000 per year.

South Africa & Rwanda are leading the world in screening for cryptococcal meningitis using a simple blood dipstick test. In South Africa, an estimated 250 000 patients living with HIV and with a CD4 count below 100 will be screened for the cryptococcal antigen annually. link

More information; Article

 

Micafungin is effective for candiduria

A recent shift in the epidemiology of candidiasis has been noted with an increase in the frequency of urinary tract infections (UTIs) caused by non-albicans Candida spp. commonly resistant to fluconazole. Echinocandins have fungicidal activity against Candida and penetrate well into the renal parenchyma. However, these agents achieve poor concentrations in the urine and are generally considered ineffective in the treatment of Candida UTIs.

A study of 33 hospitalized patients with candiduria or Candida UTI who received micafungin and had follow-up urine cultures were evaluated. The most commonly recovered species were C. albicans (n=13) and C. glabrata (n=10). Twenty-five patients (75%) had an indwelling urinary catheter. UTI was diagnosed in 16 (48%). Only 3 (10%) had concomitant Candida bloodstream infection. Micafungin was used for a mean duration of 6 days.
Gabardi and colleagues show that intravenous micafungin eradicates candiduria in 75% of patients, there is little else published on the use of echinocandins for candiduria.
More information, Article

 

Fungal Mycobiome is linked to Crohn's disease

Crohn’s disease (CD) is a relapsing inflammatory bowel disease that is driven by an abnormal immune response to gut microbial antigens, suggesting a complex interplay between host genetic factors and endogenous microbial communities. It is only recently that sequencing-based investigations of the gut microbial community have included the  fungal community (mycobiome) confirming the importance of  fungal component of the microbiome and confirmed its involvement in Candida-host interplay in CD.

In a recently published article on CD patients and their healthy relatives (NCDR) vs a control group without CD or history of CD, significant microbial interactions were identified and validated using single- and mixed-species biofilms. The abundance of the fungus Candida tropicalis was significantly higher in CD than in NCDR (p0.003) samples and positively correlated with levels of anti-Saccharomyces cerevisiae antibodies (ASCA). The abundance of C. tropicalis was positively correlated with S. marcescens and E.coli, suggesting that these organisms interact in the gut. CD and NCDR groups clustered together in the mycobiome but not in the bacteriome.

Biofilms comprising three species ie. C. tropicalis with S. marcescens and E. coli  had a greater mass and thickness than those of single-and double-species biofilms. Notably C. tropicalis biofilms comprised blastospores, while double and triple species biofilms were enriched in hyphae.
The results of this study indicate that the interplay between fungi and bacteria in the gut of Crohn’s disease patients may be very significant. 
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LIFE would also appreciate your help to tell us if you have antifungal eyedrops in your country. GAFFI is seeking to map the availability of eyedrops globally.
Do you have these eyedrops for fungal keratitis in your country: - natamycin, voriconazole, econazole or chlorhexidine? Mail your reply here. Thank you we really need your help!

 

Featured LIFE website section: Skin testing for allergy to fungi

 Fungal sensitisation is much less common than other sensitisations in the general population in Europe, but more common in those with severe asthma. Approximately 25% of adult asthma patients referred to specialists and up to 75% of patients with severe asthma requiring multiple hospital admissions are sensitized to one or more fungi.  
Skin test responses may vary over time in the same individual even if performed in an identical manner with the same reagent. Asthma may also ‘go through good and bad periods’, possibly consistent with this. While the correlation between positive skin tests and IgE RAST test on blood is reasonable, there is enough discordance to require both to be done to identify all patients who are sensitized to fungi. Skin tests tend to be more sensitive. Some clinics test for a wide range of fungal sensitivity, others test for a limited number or only for Aspergillus fumigatus sensitivity. A reasonable minimum testing set is A. fumigatusCandida albicans, Cladosporium herbarumAlternaria alternataPenicillium chrysogenum (notatum) and Trichophyton mentagrophytes
Commercial skin tests include kits from BencardThermo Scientific (previously Phadia), Stallergenes and  Allergopharma. View LIFE section.

The following video demonstrates the technique of skin testing for all types of allergen including fungal. (Flash)




Top Diagnostic Tip: AST to ALT ratio is elevated in disseminated histoplasmosis

Histoplasma antigen tests may take up to 7 days for results. In searching for assistance with diagnosis it was found that an elevated level of aspartate aminotransferase (AST) to alanine aminotransferase (ALT) at a mean level of 2.69 (95% CI:1.22, 4.16) for disseminated histoplasmosis, while for other fungi the mean ratio ranged from 0.38 to 1.14 with disseminated coccidioidomycosis and blastomycosis respectively (P < 0.0001). The ratio in patients with bacterial sepsis was 0.84. AST/ALT ratios may be a useful indicator to suggest disseminated histoplasmosis compared to other endemic fungal infections.

Article Spec et al 2016 doi: 10.1093/mmy/myw106

 

Really Important Review: Pulmonary cryptococcosis in a 35 year old non-immunocompetent man raised many questions.

1. Does symptomatic pulmonary cryptococcosis resolve in most individuals without antifungal therapy?
2. Does asymptomatic pulmonary cryptococcosis resolve in most individuals without antifungal therapy?
3. Does antifungal therapy hasten clinical and radiographic resolution of pulmonary disease in both symptomatic and asymptomatic individuals?
4. How often can negative fungal cultures be expected when pulmonary tissues or secretions reveal encapsulated yeast and what is their significance?
5. Can antifungal therapy be discontinued despite the presence of significant radiographic abnormalities?
6. How long do pulmonary lesions persist with and without therapy?
7. What is the significance of persistently positive serum cryptococcal antigen testing with pulmonary cryptococcosis? 
The authors addressed these questions in the review: Fisher et al.

i Book

 

Illustrative cases in Medical Mycology: by the Univerity of Manchester

This book is available for download on Mac or iphone devices from the Apple store. An interactive learning platform for Masters in Mycology students but also a a practical resource for anyone to develop skills in Medical Mycology. Interactive images, videos and many resources.

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Courses

 

Master Class on Primary Immunodeficiencies will take place between the 7th and 8th of April 2017 in Prague, The Czech Republic. More information

A web-based course 'Invasive Mycoses: Emerging Paradigms & Practical Applications' is running between 25th September 2016 and 24th September 2017. More information

Masters in medical Mycology at Manchester University.
An Msc programme designed for graduates who wish to develop skills as mycologists. The course can be full or part time taught programme combining much time in the  laboratory, with work experience in the NHS Mycology Reference Laboratory and clinics and ward rounds. More information
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Global burden@LIFE copyright

We are looking for volunteers to assist with estimating the burden of fungal infection in the following countries:
Bhutan, Bolivia, Burkina Faso, Chad, Chad, DRC, Gabon, Guinea and Guinea Bissau, Papua New Guinea, Rwanda and Sierra Leone.
Can you help? Contact us

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