THE NEW KIDS ON THE FUNGAL BLOCK

Professor Deborah Marriott, Clinical Director, Antimicrobial Therapeutic Drug Monitoring Unit, Senior Specialist in Clinical Microbiology and Infectious Diseases, St Vincent’s Hospital Sydney

PNEUMOCYSTIS JIROVECII

The emergence of a new

nosocomial pathogen

PNEUMOCYSTIS: HISTORY

• described by Carlo Chagas (1909) while working on a guinea pig model of trypanosome infection and believed to be new form of trypanosome

• 1910 – Antonio Carini identified similar organisms in rat lungs

• 1912 – pneumocystis in the rat recognised as a unique species and christened ‘P. carinii’ in honour of Antonio Carini

• first human cases described by Dutch investigators Van de Meer and Brug, 1942

PNEUMOCYSTIS: HISTORY

• 1952 – Vanex and Jirovec reported that Pneumocystis ‘carinii’ caused interstitial pneumonia in neonates

• first cases of PCP recognised in premature and malnourished infants in Central Europe after the Second World War – ‘plasma cellular interstitial pneumonitis of the newborn’ (Baar, 1955)

• 1953 – Giese proposed pneumocystis may be a yeast and that the foamy material seen in the airways was spores

• 1960-70’s – rare cases described in children and adults with haematologic malignancy

PNEUMOCYSTIS: HISTORY

• 1980’s – HIV-related opportunistic infection causing severe pneumonia

• prophylaxis (1980’s) and highly active antiretroviral therapy (late 1996) resulted in a decline in HIV-related PCP where access to treatment is available or is accessed

• 2000’s - ongoing rise in PCP in other immuno-suppressed patients including bone marrow and solid organ transplantation, malignancy and autoimmune diseases

PNEUMOCYSTIS: TAXONOMY

• considered to be a protozoan until 1988 – two life forms morphologically parallel to other protozoans and response to antiprotozoal drugs - pentamidine and cotrimoxazole

• 1988 – Edman et al sequenced the small ribosomal RNA subunit and pneumocystis was reclassified as ascomycetous fungi

P. JIROVECII: “YEW ROW VET ZEE”

Kingdom: Fungi

• Phylum: Ascomycota (Schizosaccharomyces)

• Class: Archia-ascomycetes

• Order: Pneumocystidales

• Family: Pneumocystidaceae

• Genus: Pneumocystis

Named after Otto Jirovec (Czech parasitologist): human lungs

PneumoCystis Pneumonia = PCP

PNEUMOCYSTIS: EPIDEMIOLOGY

• ubiquitous organisms that can colonise and infect almost every species of mammalian host

• RNA of some species (not P. jirovecii) found in ambient air and pond water (Wakefield, 1998) but true environmental reservoir of viable organisms not yet established

• unique specificity for a mammalian host –

- P. jirovecii only capable of infecting human

- P. wakefieldiae infects rats

- P. murina found in mice

(NOTE – unusual for a fungus to be this host specific!!)

• very low numbers or absent in the normal host

PNEUMOCYSTIS : LIFE CYCLE

Excystment

• challenging – no environmental niche and no long-term in

vitro culture system.

• animal model or molecular techniques only

Cyst form

• 8-10µm

• contains up to

8 intracystic

bodies

• thick cyst wall

Trophic form

• 2µm

•single nucleus

•plasma

membrane

two separate life forms

PNEUMOCYSTIS CELL WALL

• provides rigidity and viability of organism against environment

• composed of:-

- β1, 3-glucan

- mannoproteins

- chitins

PNEUMOCYSTIS CELL WALL

• β-glucans induce multiple inflammatory pathways

- exaggerated inflammatory response elicited by alveolar macrophages and respiratory epithelial cells resulting in respiratory failure (Carmoan, 2006; Lebron, 2003)

- activate dendritic cells and modulate T-cell response crucial for pneumocystis clearance (Carmona 2006)

- prime dendritic cells to polarise T-cells towards Th17 differentiation (inflammatory cytokines, key mediators of host defence , auto-immune and inflammatory disorders), increasing antifungal immune resistance (Zelante, 2007)

PNEUMOCYSTIS: PATHOGENESIS

• route of infection not well understood ?aerosol from host to host

Initial infection in

childhood (>80%

antibody positive)

later reactivation

during immune

suppression

new acquisition of

the organism in T-

cell impaired host

TRANSMISSION OF DISEASE

• latency: supported by early exposure and infection • high prevalence anti-Pneumocystis antibodies (83% age 4)

• high rate PCP in immuno-compromised infants

• de novo infection (exogenous) • animal model studies (rats, mice, macaques)

• Pneumocystis DNA on cage filters of rats

• Pneumocystis cleared after infection

• presence of genotype switching in repeat episodes

ACQUISITION DE NOVO

?? environmental exposure (air-borne)

?? patient-to-patient

?? asymptomatic carriers (colonised persons)

PNEUMOCYSTIS: IMMUNITY

• CD4 T-cells essential for clearance of the organism

• CD8 T-cells mediate acute inflammatory response in immune reconstitution syndrome

• role of antibody in immunity uncertain

• HIV infected versus other immunocompromised patients – substantial difference:-

• demography

• clinical presentation

• therapy

• outcome

• mortality

CLINICAL PRESENTATION

HIV • subacute – onset

• progressive dyspnoea

• non or minimally productive cough

• fever

• headache

• diarrhoea

• plentiful organisms

on BAL

• less inflammatory response

Non-HIV • acute onset

• marked dyspnoea

• fever

• chills

• rapid progression to respiratory failure

• few organisms on BAL

• more marked inflammatory response

Get ready for voting…

Go to webpage: pollev.com/mmtn1

Wifi network: MMTN2016 Password: 2016mmtn

Voting question on the next slide

QUESTION 1

Have you seen an increase in incidence of PCP in:-

1. Renal transplant recipients

2. Other solid organ transplant recipients

3. HIV

4. Other immuno-compromised patients

5. Some or all of the above

PCP IN AUSTRALIAN RENAL TRANSPLANT UNITS

• nosocomial clusters first recognised in 2010

• since then it has affected half of the renal units on the eastern seaboard and subsequently Adelaide

• overall 83 cases between 2010 and 2012

• considerable heterogeneity in clinical presentation

• 13 deaths

• 9 additional graft losses

AUSTRALIAN OUTBREAK: 2010-2012 Hospital Organ No. cases Deaths Graft losses

1 [1st, 2nd) K-P 14+ 8 2 5

2 K-P 17 1 0

3 K 8 0 1

4 K 2 1 1

5 K 14 5 1

6 K 3 1 0

7 K 1 0 0

8 K 2 0 0

9 K,K-P 5 1 0

10 K,K-P 1 0 1

11 K 1 0 0

12 Liver 4 1 0

13 Heart 1 0 1

14 Lung 2 1 0

PHIPPS et al. Transplantation 2011 PCP Controls p values

Numbers 14 324

Age

Sex (n, % males)

Deceased donors (n, %)

Years after transplant

HLA Mismatch (out of 6)

Prior rejections (numbers)

Anti-lymphocyte preparations (n, %)

Recent pulse steroids

PJP prophylaxis

46.6 +/- 13.3

10 (36%)

10 (71%)

6.3 +/- 5.3

3.8 +/- 1.9

0.5 +/- 0.5

3 (21.4%)

0 (0%)

13 (93%)

46.0 +/- 13.4

193 (59.6%)

204 (62.9%)

5.5 +/- 6.1

3.5 +/- 1.9

0.6 +/- 1.0

50 (15.4%)

36 (11%)

302 (93%)

NS

NS (0.08)

NS

NS

NS

NS

NS

NS

NS

Tacrolimus Dose (mg/day)

Tacrolimus Levels (ng/day)

MMF Dose (g/day)

Prednisolone Dose (mg/day)

4.0 +/- 2.3

6.7 +/- 1.8

1.6 +/- 0.5

8.9 +/- 2.5

5.4 +/- 3.5

7.5 +/- 3.8

1.7 +/- 0.6

11.3 +/- 5.9

NS

NS

NS

NS

Serum creatinine (umol/L)

eGFR (mls/min/1.73m2)

Prior CMV disease

Pulmonary Disease

Former Smoker

267 +/- 193

31+/- 23

4 (28.6%)

4 (28.6%)

4 (28.6%)

144 +/- 94

52 +/- 20

2 (0.6%)

10 (3.1%)

128 (49.5%)

< 0.01

< 0.001

< 0.001

< 0.001

NS

*Multivariate: sig risk factors for PCP - underlying lung disease (OR10.1), previous CMV infection (OR 65.9), impaired eGFR (OR1.61 /10mls/min/1.732)

Transmission map: cases and contacts

B.Nankivell, 2011

Hospital 1

MLST (stored extracts, 11 unrelated “control DNA” same period)

four genetic loci: β-tubulin

ITS1/2 region**

DHPS

mtLSU

Carolina Firacative, Wieland Meyer

ST1

ST2

Contemporaneous

patients, Sydney

Outbreak

patients

THE WESTMEAD EXPERIENCE

• universal prophylaxis for all patients (approximately 500) in the renal transplant unit for ≥ 12 months and ≥ 6 months after last case diagnosed

• subsequent prophylaxis for patients determined to be at highest risk after initial outbreak:-

• within 1 year of transplantation

• CMV disease

• chronic respiratory disease

• late treated graft rejection

THE WESTMEAD EXPERIENCE

Despite the initiatives.......

......second outbreak 6 months after cessation of universal prophylaxis

• further 8 patients with PCP

• 2 patients critically ill but no deaths or graft loss

THE OUTCOME OF THE TSANZ/ANZMIG/ASID MEETING

• ACT QUICKLY

• introduce universal prophylaxis

• reduce patient-to-patient transmission

• examine genotypes

Duration of prophylaxis • kidney – 6-12 months

• liver – minimum 3 months

• heart – minimum of 12 months

• lung - indefinite

• retrospective 3 year review of PCP in Northern Ireland

• 51 cases over 3 years (2008-2011) v. 10 cases in the preceding 7 years

• 23 (43%) required intensive care

• 30% mortality rate

J Med Micro 2012

Discussion

• BAL had highest DNA load but nose and throat swab a simple test at presentation

• asymptomatic carriage uncommon – 0.4%

• risk factors changing

- less HIV

- more iatrogenic risk groups – transplantation, autoimmune disease, chemotherapy

• in the transplant cohort, increased risk only seen in renal transplant recipients

Background

• blinded RFLP analysis used to type Pneumocystis isolates from outbreaks in Zurich, Munich and Nagoya, Japan in addition to sporadic isolates

Results

• Zurich

– 7/7 renal transplant patients identical RFLP

- 3/3 sporadic cases different

• Munich

- 14/14 identical and also identical to Swiss isolate

• Nagoya

– 10 samples

- 4 able to be amplified from renal transplant patients

- 4/4 identical but 0/4 identical to European strain

CID 2012

Conclusion

• outbreak strain in renal transplant recipients identical and different from predominant local circulating strain

• European strain appears to differ from Australian strains based on MLST analysis

Background • 18 renal transplant recipients developed PJP over a

2 year period at Brest University Hospital, France

• no cases identified in the unit from 2002-2008

• ITS sequencing typing

Methods • 18 renal transplant recipients

- 12 clinical disease

- 6 colonized

• 22 unlinked control patients

- 18 clinical disease

- 4 colonized

• 69 patients from Brest geographic area

- 34 clinical disease

- 35 colonized

CID 2012

Discussion

• frequency of type Eg significantly higher in renal transplant group

• 14 encounters between renal transplant recipients who had identical fungus

• 10 possible index patients of whom 3 were colonised by the fungus and 7 presented with PCP

• highlights potential role of colonised patients in transmission of PCP

Background

• single centre study at the University of Frankfurt, Germany

• 2002-2007 – outbreak in renal transplant recipients with 29 cases reported (plus one with anti-GBM disease)

• case control study undertaken

Euro J Clin Microbiol Infect Dis 2012

Results

• 25/30 patients attended the same outpatient clinic within 3 months prior to PCP with 22±12.3 visits each (range 1-48)

• 29/30 had prior contact

• genotype – 4/4 identical and different from 2 control strains

• 27 patients not on dialysis including 3 who died

• 12/30 patients had CMV viraemia prior to PCP diagnosis

- 4/12 developed CMV disease

- 2/4 co-infected patients died

Discussion

• high incidence of concurrent CMV and PCP in case control study

• higher incidence of CMV in PCP patients compared with controls (40% v. 20%)

• mortality rate 10%

• low CD4 cell count of time of admission

• strong evidence for inter-patient transmission of PJP

CLUSTERS IN KIDNEY TRANSPLANTS

Now reported worldwide; 1 systematic review

• 16 outbreaks, 15 articles (since then >10 pubs)

• median 12 cases (up to 28 cases)

• median time after transplant 12 mo (IQR 7-21 mo) vs. 6.3 yrs for Westmead outbreak

THREE common features

• preceding prevalence low (<2%)

• 12/15 studies no chemoprophylaxis

• no patient cohorting !

De Boer MJ, et al, Med Mycol 2011;49: 673

THE FIRST OUTBREAK DESCRIBED IN NORTH AMERICA

2016

• 4 cases over 5 months in liver

transplant recipients

• 25 unrelated cases of PCP

• 4 liver transplant cases identical

on multi-locus sequence typing;

all others different

• hospitalisation mapping

demonstrated several possible

encounters including an

out-patient clinic when all

were present on one day

69 patients over 10 years.

Mortality rate 32%

Women receiving intense dosing adriamycin,

cyclophosphamideand corticosteroids for

nausea at greatest risk

Outbreak with 9 patients colonised and 5 cases

over 2 years with no prior cases

29 cases (16 renal, 13 liver)

caused by 3 strains not seen

In sporadic cases. Strong

association with CMV.

UNANSWERED QUESTIONS

Why renal transplant recipients

Why a single strain

? altered pathogenicity

?virulence factors

Why now

SUMMARY

• Pneumocystis infections are increasing in frequency and occurring in a new range of hosts

• outbreaks have occurred in renal transplant units – mode of transmission uncertain but may be person to person

• early intervention is essential as morbidity and mortality are high

• strong association with CMV in many studies

• prophylaxis should be continued for at least 6 months post transplant – possibly longer??

AND ANOTHER NEW KID ON THE FUNGAL BLOCK

.......or when the human petri dish serves up an unexpected guest!

THE CASE

• 1999 – bilateral single lung transplant for emphysema

• progressive renal failure secondary to hypertension and calcineurin toxicity

• progressed to intermittent haemodialysis

• August 2011-episode of nausea, abdominal pain and diarrhoea

• weight loss 3kg; now 64kg

• investigations non-diagnostic

• symptoms gradually resolved

• February 2012 - nausea/dry retching

• lower abdominal pain

• sweats

• weight now 57kg

Renal transplant May 2012

THE CASE

• seen in transplant clinic 8 weeks later complaining of:-

- worsening peripheral oedema

- decreased muscle strength

- myalgias, particularly small muscles of the hand

- difficulty swallowing

- low grade fevers

- further weight loss

THE CASE

•admitted to hospital

•progressive weakness

•only able to swallow thickened feeds

A DIAGNOSTIC TEST WAS PERFORMED BY THE RENAL TRANSPLANT PHYSICIAN

DELTOID MUSCLE BIOPSY

Warthin-Starry H and E

THE NEW KID ON THE FUNGAL BLOCK

A newly recognised

Australian

pathogen –

Anncaliia algerii

AUSTRALIAN CASES OF ANNCALIIA ALGERAE

Patient Risk factor Myositis, especially small muscles of hands

Bulbar weakness

Preceding GIT symptoms, weight loss

1

2

3

4

Lung transplant

Lung and kidney transplant

Rheumatoid arthritis

Rheumatoid arthritis

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Patient 3 – courtesy of Elaine Cheong; patient 4 courtesy of Matthew Watts

MICROSPORIDIA: HIGHLY REDUCED AND DERIVED RELATIVES OF FUNGI

• unusual intracellular organisms that infect a wide range of animal species, both vertebrate and invertebrate

• very small genomes encoding few proteins

• minimal functional pathways making them a model system for reductive evolution of eukaryotic cellular function

• now accepted as fungi or a ‘closely related sister lineage’

MICROSPORIDIA: HIGHLY REDUCED AND DERIVED RELATIVES OF FUNGI

•unique infection strategy involving the rapid expulsion of a polar tube from a spore to pierce a host cell, allowing the direct transfer of the spore contents into the host cell cytoplasm

MICROSPORIDIUM:

ULTRASTRUCTURE

Trends in Parasitology 2016 32, 336-348DOI: (10.1016/j.pt.2015.12.004)

MICROSPORIDIUM: EXTRUDED POLAR TUBULE

MICROSPORIDIA: HIGHLY REDUCED AND DERIVED RELATIVES OF FUNGI

•8 genera and 14 species associated with human infection

•Enterocytozoon bieneusi, Encephalitozoon cuniculi/intestinalis and Nosema spp. most common human pathogens

MICROSPORIDIA: GEOGRAPHIC AND CLINICAL DIVERSITY

• emerging microsporidial infection in Russian HIV-infected patients (Sokolova, 2011)

• E. cuniculi as a causative agent of brain abscess in an immuno-competent patient, Czech Republic (Ditrich, 2011)

• identification and genotyping of E. bieneusi in China (Zhang, 2011)

• first cases of microsporidiosis in transplant recipients in Spain (Galvan, 2011)

MICROSPORIDIA: GEOGRAPHIC AND CLINICAL DIVERSITY

• inapparent microsporidial disease in immunocompetent people, Czech Republic (Sal, 2011)

• four cases of microsporidial myositis in Australia (Watts 2014)

1/16 immunocompetent patients with diarrhoea

3% of 100 diabetic patients

Asymptomatic microsporidiosis

in 65/255 children

16/272 vs 0/170 P<0.001

Encephalitozoon spp from diarrhoea

(5%) and farm soil (8.8%)

MICROSPORIDIOSIS: CLINICAL PRESENTATION

Enteric disease

Enterocytozoon bieneusi

Encephalitozoon hellum

Encephalitozoon intestinals

Disseminated disease

• encephalitis

• keratoconjunctivitis

• sinusitis

• pneumonia

• myositis

• peritonitis

• nephritis

• hepatitis

TRANSMISSION OF MICROSPORIDIA – FAECAL-ORAL?

• Enterocytozoon bieneusi, Encephalitozoon hellum, Encephalitozoon intestinals

• report of enteric microsporidia in municipal waste water/effluent and biosolids in warmer months in Ireland. Positive correlation with levels of enterococci (Cheng et al 2011)

• 50 sewerage outlet samples from Shanghai – E.bieneusi detected in 35/50 by PCR (Ma et al 2016)

• Swedish food-borne outbreak of E. bieneusi attributed to contaminated ready-to-eat, pre-washed cucumber slices (Decraene et al 2011)

Microsporidia – Emergent Pathogens in the Global Food Chain

G.D. Stentiford, J.J. Becnel, L.M. Weiss, P.J. Keeling, E.S. Didier, B.A.P. Williams, S. Bjornson, M.L. Kent, M.A. Freeman, M.J.F. Brown,

E.R. Troemel, K. Roesel, Y. Sokolova, K.F. Snowden, L. Solter

Trends in Parasitology

Volume 32, Issue 4, Pages 336-348 (April 2016) DOI: 10.1016/j.pt.2015.12.004

MICROSPORIDIA – EMERGENT PATHOGENS IN THE GLOBAL FOOD CHAIN

THE HISTORY OF MICROSPORIDIA AT SVH

• late 1980’s – recognised as a cause of diarrhoea and modified trichrome stain introduced for diagnosis

• during the heyday of HIV – up to 10% of faecal specimens positive for E. bieneusi

• review in 2006 – only 4 positive specimens in the preceding 3 years and microscopy skills being lost so test discontinued

• recent cases of disseminated microsporidiosis in transplant recipients and new cases of E. bieneusi in HIV patients…..

THE HISTORY OF MICROSPORIDIA AT SVH

Should we start looking again?????

We are – and we have developed a PCR to make looking easier!

AND FINALLY…….

• the world of fungi is forever changing

• expect the unexpected and look hard for it

• obtain tissue whenever possible – you never know what it may reveal

Interactive discussion

Your questions are welcome

Please raise your hand for a microphone or submit a question card

Break

Refreshments are available outside the room

5th MMTN Conference 5-6 November 2016, Bangkok, Thailand

Brought to you by the Asia Fungal Working Group, an ISHAM working group www.AFWGonline.com