Where Are We? Pneumocystis Jirovecii (P

The 28th ECCMID 2018 in Madrid 23 Apr 2018, 09:00-11:00, Hall R

Molecular diagnosis of fungal infections: where are we? Pneumocystis jirovecii (P. jirovecii)

Norihiko Goto Nagoya Daini Red Cross Hospital, Transplant Nephrology, Nagoya, Japan

ESCMID eLibrary I have no financial relationships to disclose relevant to my presentation © by author In the 1980s, P. Jirovecii infection increased dramatically with the emergence of the HIV epidemic

P. Jirovecii infection in non-HIV has become an increasing problem

P. Jirovecii infection in HIV can be controlled by maintaining ESCMIDthe CD4 count and using eLibraryroutine prophylaxis © by authorAm J Med. 2014; 127(12): 1242.e11-7 Pneumocystis pneumonia (PCP) incidence rates in non-HIV-infected patients

ESCMID eLibrary Am J Med. 2014;127(12):1242.e11-7 © by author Proportion of all P. jirovecii–associated hospital admissions and change in population rates over time (England, UK, 2000–2010)

 The difference in rates between the 2 periods was most marked among ESCMIDpatients who had undergone transplantation.eLibraryEmerg Infect Dis. 2013; 19(3): 386-92  47% of whom had undergone kidney transplantation during 2000–2010. © by author What is the problem in non-HIV PCP?

ESCMID eLibrary © by author Non-HIV PCP presents with an abrupt onset of respiratory failure

ESCMID eLibrary Emerg Infect Dis. 2014; 20(9): 1490-7 © by author Clinical management of HIV PCP and non-HIV PCP

Emerg Infect Dis. 2014; 20(9): 1490-7

ESCMID eLibrary © by author Difference between HIV PCP and non-HIV PCP

Non-HIV HIV

Onset Sudden Slow

Number of pathogen Few Many (P. jirovecii)

Useful markers of CD4 cell counts No immunological status <200/mm3

Mortality High; 39% Not high; 6.6%

PCP rapidly progresses, is difficult to diagnose correctly, and causes severe ESCMIDClin Med eLibrary Insights Circ Respir Pulm Med. 2015; 9( Suppl 1): 81- 90 respiratory failure with a poor prognosisTranspl in Infect non -Dis.HIV 2011; 13(6): 551-8 Chest 2000; 118 (3): 704–711. © by author Host inflammatory response and number of organisms

HIV PCP

Host immune response Number of P. jirovecii ESCMID eLibrary © by author Host inflammatory response and number of organisms

Non-HIV PCP

Number of P. jirovecii Host immune response ESCMID eLibrary © by author Mortality and host immune response

Host Underlying disease Mortality immune response

HIV 6.6% Weak

Kidney transplantation 5-33%

Collagen disease 32-33% Strong

Transplantation 2009; 88 (3): 380–385 J Clin Microbiol 2008; 46 (3): 966–971 The outcome of PCP is inversely correlated withClin Infectintensity Dis 2007; of immunosuppression44 (9): 1143–1149 Transplant Proc 2009; 41 (1): 170–172 ESCMID eLibraryClin Infect Dis 2002; 34 (8): 1098 – 1107 J Rheumatol 1994; 21 (2): 246–251 © by author Adjunctive corticosteroids for severe host responses to P. jirovecii in non-HIV PCP are needed?

ESCMID eLibrary © by author Adjunctive corticosteroids for non-HIV PCP

Retrospective studies with small sample size

 Benefit  Decreased mortality Chest. 1998;113:1215–1224. BMC Infect Dis. 2011;11:76. Rheumatol Int. 2009;29:1327–1330.  No benefit Antimicrob Agents Chemother. 2011;55:4613–4618. Arthritis Rheum. 2009;61:305–312. Respir Res. 2013;14:87. Clin Infect Dis. 2002;35:929–934. ESCMID eLibrary © by author Impact of adjunctive corticosteroids on survival for non-HIV PCP (Systematic Review and Meta-Analysis)

There was no association between corticosteroids and survival in non-HIV PCP

(OR, 0.66; 95% CI, 0.38-1.15;Transplant P = 0.14 Direct.) 2017; 3(3): e137 ESCMIDBut data are limited and findings eLibrary should not be considered conclusive © by author Dose and duration of corticosteroids

mg HIV PCP Cochrane Database Syst Rev 2006; 3: CD006150 100 80

50 40 20

150 80-120 ; 5-7days Non-HIV PCP Am J Transplant. 2013; 13(Suppl 4): 272–279 100 7-14 days 50

0 ESCMID40–60 mg of prednisone eLibrary given twice daily for 5–7 days before being tapered over a period of at least 7–14 days © by author  Corticosteroids are best administered within 72 hours in the setting of hypoxia (PaO2 < 70 mmHg)

 Commonly used but not well studied in transplantation

Am J Transplant. 2013; 13 Suppl 4: 272-9 mg 200 160 150 Our kidney Center

100 80

50 40 5 ESCMID0 eLibrary © by author Anti-pneumocystis agents for treatment

Agents Dosing

15–20 mg/kg/day of the TMP component given IV in divided doses every Trimethoprim-sulfamethoxazole 6–8 hours often in combination with corticosteroids (see below); for (TMP-SMX) milder disease, two double-strength tablets can be given po bid-tid 4 mg/kg/day IV initially over 1–2 hours; dose reduction to 2–3 mg/kg/day Pentamidine isesthionate if needed Atovaquone 750 mg po bid (optimal dose uncertain; 1500 bid used anecdotally) Primaquine 15–30 mg po qd in combination with clindamycin 600–900 Primaquine and clindamycin mg IV or po q6–8 hours Dapsone 100 mg po qd used in combination with trimethoprim 15 Dapsone and trimethoprim mg/kg/day po divided tid P. Jirovecii isTrimetrexate now classified45 mg/m2/day as a fungus, IV (or not 1.5 mg/kg/daya protozoan IV in patients <50 kg) Trimetrexate with folinic acid with folinic acid 20 mg/m2 po or IV every 6 hours (80 mg/m2 total daily); But most ofFolinic themacid for therapytreatment extends are ≥antiprotozoal 3 days beyond agentstrimetrexate therapy Pyrimethamine and Pyrimethamine load of 100–200 mg po, followed by 50–100 mg po qd in sulfadiazine combination with sulfadiazine 4 g po qd in divided doses Macrolides such as clarithromycin or azithromycin in combination with Macrolide and SMX sulfamethoxazole may be synergistic in vivo (49) 70 mg IV loading dose of caspofungin on day one, followed by 50 mg IV Caspofungin ESCMIDand TMP-SMX daily after in combinationeLibrary with TMP-SMX (dose reduced in the setting of moderate to severe hepatic dysfunction) © by authorAm J Transplant. 2013; 13 Suppl 4: 272-9 Life cycle of P. jirovecii remains poorly defined

 Pneumocystis organisms in different mammals are quite different, and strains from one host animal do not infect other animal species

 P. jirovecii can only survive in the respiratory organ of human beings ESCMID eLibraryInfect Immun. 1993;61(7):2886 -90 © by authorMem Inst Oswaldo Cruz. 2009; 104(3): 419-26 Trophic forms or cyst forms?

 In the lungs of PCP, trophic forms are the most abundant (90%–95%)

Mem Inst Oswaldo Cruz. 2009; 104(3): 419-26

 Cysts are detected in the bronchial lumen

Med Mycol. 2000; 38 Suppl 1: 23-32

TMP-SMX is effective for PCP (trophic forms) treatment ESCMID eLibrary © by author PCP prophylaxis is required for the poor prognosis of non-HIV PCP?

ESCMID eLibrary © by author Anti-pneumocystis agents for prophylaxis

Agents Dosing

Trimethoprim-sulfamethoxazole Can be given at 80 mg TMP/400 mg SMX or 160 mg TMP/800 mg SMX (TMP-SMX) po (single or double strength) daily or three times weekly

Dapsone 50–100 mg po qd (4,4- diaminodiphenylsulfone)

Atovaquone 1500 mg po qd (as single dose)

Pentamidine 300 mg administered through aerosolized nebulizer q 3–4 weeks

Up to 300 mg of clindamycin po qd with 15 mg of pyrimethamine po qd Clindamycin and pyrimethamine (some clinicians have administered this regimen 3 times weekly instead of daily) 70 mg IV loading dose of caspofungin on day one, followed by 50 mg IV Caspofungin ESCMIDand TMP-SMX daily after in eLibrarycombination with TMP-SMX (dose reduced in the setting of moderate to severe hepatic dysfunction) © by authorAm J Transplant. 2013; 13 Suppl 4: 272-9 TMP/SMX prophylaxis is highly effective for prevention of PCP In patients with or without HIV

For PCP prevention, prophylactic TMP/SMX should be considered ESCMIDwhen the risk of eLibraryPCP is moreCochrane than Database 6.2%. Syst Rev. 2014; 10: CD005590 © by author What are clinical conditions in which the rates of PCP for patients not receiving prophylaxis might be above 6.2%?

 Allogeneic bone marrow transplantation, during the six months post- transplantation and afterwards with continued immunosuppression

 Solid organ transplantation, in the first six months after transplantation

 Acute lymphoblastic leukemia

During outbreaks of PCP among immunocompromised patients the incidence of PCP might be high enough to justify prophylaxis

Clin Microbiol Rev. 2004; 17(4): 770-82 ESCMID eLibraryCochrane Database Syst Rev. 2014; 10: CD005590 © by author Changing timeline of infection after organ transplantation

Nosocomial, technical Activation of latent infection Donor-Derived Community-acquired Infection (donor or recipient) (relapsed, residual, opportunistic)

Dynamic assessment of risk of infection Transplantation Common Infections in Solid-Organ Transplant Recipients

Recipient-Derived <1Month 1–6Months >6Months Infection Infection with antimicrobial resistant With PCP and antiviral (CMV,HBV) Community-acquired pneumonia, species: prophylaxis: urinary tract infection MRSA Polyomavirus BK infection, Infection with aspergillus, atypical VRE nephropathy molds, mucor species Candida species (non-albicans) C. difficile colitis Infection with nocardia, rhodococcus Aspiration HCV infection species Catheter infection Adenovirus infection, influenza Late viral infections: Wound infection Cryptococcus neoformans infection CMV infection (colitis and retinitis) Anastomotic leaks and ischemia Mycobacterium tuberculosis infection Hepatitis (HBV, HCV) Clostridium difficile colitis Anastomotic complications HSV encephalitis Donor-derived infection (uncommon): Without prophylaxis: Community-acquired (SARS, HSV, LCMV, rhabdovirus Pneumocystis West Nile virus infection) (rabies), West Nile virus, Infection with herpesviruses (HSV, JC polyomavirus infection (PML) HIV,Trypanosoma cruzi VZV, CMV, EBV) Skin cancer, lymphoma (PTLD) Recipient-derived infection HBV infection (colonization): Infection with listeria, nocardia, Aspergillus, pseudomonas toxoplasma, strongyloides, leishmania, ESCMID eLibraryT. cruzi © by authorN Engl J Med. 2007; 357(25): 2601-14 Primary prevention of PCP after solid transplantation

PCP prophylaxis for

 At least 4 months in the EBPG guidelines

Nephrol Dial Transplant. 2002;17(Suppl 4):36–39

 3 to 6 months in the KDIGO guideline

Kidney Int. 2010;77:299–311.

 6 to 12 months in the AJT guideline

Am J Transplant. 2013;13(Suppl 4):272–279

These guidelines are only directed at prevention of PCP in individuals ESCMIDsoon after transplantation, not eLibrary for prevention of a PCP outbreak © by author PCP caused by P. jirovecii

cyst forms trophic forms

N Engl J Med. 2004; 350(24): 2487-98.

 Reactivation  By four years of age, two-thirds of normal children who have been exposed to the respiratory-aerosol route are found to have antibodies to P. jirovecii.

Pediatrics. 1978;61(1):35–41  Reinfection with different genotypes ESCMIDDe novo infection is the maineLibrary reason rather than reactivation. J Infect Dis. 2003;187(6):901–8. © by author Transmission route

 Person-to-person transmission

 From patients with active PCP

Emerg Infect Dis. 2004;10:1766–1773.

 From asymptomatic carriers

Microbes Infect. 2002;4:95–103 J Clin Microbiol. 1996;34:1754–1759 Clin Infect Dis. 2010;51:259–265

 Environmental exposure

J Infect Dis. 2008;197:10–17

ESCMIDUnlike other fungi, P. jirovecii eLibrarycan be transmitted from humans. © by author A large outbreak of PCP in kidney transplant recipients (n1-27)

The number of kidney transplantation at that time was more than 1800. ESCMIDPCP prophylaxis had not been prescribedeLibrary to recipients before this outbreak. © byTime ( 2004 -author2005 ) Transplantation 2009; 88: 380-385 ESCMID eLibrary © byTime ( 2004 -author2005 ) Transplantation 2009; 88: 380-385 Diagnosis of PCP

 Clinical features  fever, nonproductive cough, and/or dyspnea

 Imaging findings  X-ray and high-resolution CT

 Blood test  high serum levels of [1–3]-β-D-glucan

 Diagnosis  P. jirovecii with specific staining and/or P. jirovecii DNA by PCR in BALF ESCMID eLibrary  All BALF samples were stored for genotyping © by author Genotyping of P. jirovecii

Method Gene Number of genotypes

PCR + Sequence mt LSU rRNA 4

PCR + Sequence or RFLP DHPS 4

PCR + Sequence ITS1, ITS2 More than 130

PCR + SSCP ITS1, 26s rRNA, mt 26s rRNA, β-tublin More than 35

MLST（PCR + Sequence） ITS1, 26s rRNA, mt 26s rRNA, β-tublin More than 35 ESCMIDITS; internal transcribedeLibrary spacer J Clin Microbiol 1998; 36(3): 734-41 © by author Treatment

 TMP-SMX  Treatment dose is adjusted for kidney graft function

 Adjunctive corticosteroids

mg 200 160 Our kidney Center 150

100 80

50 40 5 ESCMID0 eLibrary © by author Outpatient examination rooms and waiting space

Waiting space

② ③

ESCMID④ ① eLibrary Kidney transplant department© by author Patient demographics (1st outbreak)

ESCMID eLibrary © by author Patient demographics (1st outbreak)

Long incubation period suggest transmission from asymptomatic carriers

Unknown incubation period suggests transmission from environment

ESCMID eLibrary © by authorTransplantation 2009; 88: 380-385 Number of P. jirovecii of air sample (inside and outside the room of patients with PCP)

ESCMID eLibraryClin Infect Dis. 2010 1; 51(3): 259 - 65 © by author Incubation period of PCP

Patients (n)

10 Median: 53 days (range; 7- 188) 9

2 The average incubation period of Pneumocystis ranged from 124 to 172 days 1 Transplantation 2011; 92(12): 1327-34

0 ESCMID～2W 2W～1M 1M～ 2MeLibrary2M～3M 3M～4M 4M～5M 5M～ 6M 6M ～ 7M Incubation time

Transpl Infect Dis 2011; 13: 551–558 © by author The first outbreak involved 33 cases and lasted 42 months

The first outbreak of PCP was controlled by

 TMP-SMX prophylaxis in the first 12 months after transplantation  No prophylaxis before July 2004

 A universal 3-month TMP-SMX prophylaxis for all recipients

Transplantation 2009; 88: 380-385 Transplant Direct. 2017; 3(5): e151

ESCMID eLibrary © by author Infection control when an outbreak has occurred

1. Treat PCP patients as soon as possible

 abrupt onset of respiratory distress  a delay in diagnosis and treatment may lead to an increase in the number of other PCP patients transmitted by direct or from carrier.

2. Protect other transplant recipients  For all transplant recipients that share a waiting room with a PCP patient, starting transient universal prophylaxis with TMP-SMX  Hospitalized patients with PCP should be managed with standard precautions

3. Education of medical staff in transplant centers  Medical staff such as doctors, nurses, and medical clerks who are infected by PCP patients or from environmental exposure can act as a transient ESCMIDreservoir. eLibrary © ClinbyMed Insights authorCirc Respir Pulm Med. 2015; 9(Suppl 1): 81-90 Additional 2 PCP outbreaks despite a universal short-term prophylaxis

ESCMID eLibrary © by author Patients demographics at onset of PCP (n = 48)

ESCMID eLibrary © by authorTransplant Direct. 2017; 3(5): e151 Genotype of P. jirovecii (n1-33)

E; environmental samples KRT; mouthwash samples obtained from asymptomatic kidney transplant recipients

E1; lp E2; Bi 7 KRT1; ln KRT2; Bi 6 3-month universal Bi prophylaxis 5

4 Prophylaxis in the first 12 months for a limited (36%) number of recipients 3

2 No. of RTRs diagnosed PCP diagnosed RTRs of No. 1

Jul-04 Jul-05 Jul-06 Jul-07

Jan-05 Jan-06 Jan-07 Jan-08

Jun-04 Jun-05 Jun-06 Jun-07

Oct-04 Oct-05 Oct-06 Oct-07

Apr-06 Apr-04 Apr-05 Apr-07

Sep-05 Sep-04 Feb-05 Feb-06 Sep-06 Feb-07 Sep-07 Feb-08

Dec-04 Dec-05 Dec-06 Dec-07

Aug-04 Aug-05 Aug-06 Aug-07

Nov-04 Nov-05 Nov-06 Nov-07

Mar-05 Mar-06 Mar-07

May-05 May-06 May-07 ESCMIDMay-04 eLibrary

Transplant Direct. 2017; 3(5): e151 © by author Genotype of P. jirovecii (n33-37)

6 3-month universal 3-month universal prophylaxis prophylaxis

PCP 5

4 diagnosed 3 Prophylaxis in the first 12 months for all recipients 2

Eb No. of RTRs RTRs of No. 1

Jul-09 Jul-08 Jul-10 Jul-11

Jan-08 Jan-09 Jan-10 Jan-11

Jun-10 Jun-08 Jun-09 Jun-11

Oct-11 Oct-08 Oct-09 Oct-10

Apr-08 Apr-09 Apr-10 Apr-11

Feb-08 Sep-08 Feb-09 Sep-09 Feb-10 Sep-10 Feb-11 Sep-11

Dec-07 Dec-08 Dec-09 Dec-10 Dec-11

Aug-08 Aug-09 Aug-10 Aug-11

Nov-08 Nov-09 Nov-10 Nov-11

Mar-08 Mar-09 Mar-10 Mar-11

May-09 May-10 May-11 ESCMIDMay-08 eLibrary

M; mouthwash survey obtained from a medical staff member M3m; mouthwash samples obtained from the same medical staff member 3 months later 7 M; Ne M3m; negative 6 3-month universal 3-month universal 6-month universal Life long prophylaxis prophylaxis prophylaxis prophylaxis 5

3 Prophylaxis in the first 12 months for all recipients 2 No. of RTRs diagnosed PCP diagnosed RTRs of No. Ne 1

Jul-12 Jul-13 Jul-14 Jul-15

Jan-12 Jan-13 Jan-14 Jan-15

Jun-12 Jun-13 Jun-14 Jun-15

Oct-13 Oct-11 Oct-12 Oct-14

Apr-12 Apr-13 Apr-14 Apr-15

Feb-12 Sep-12 Feb-13 Sep-13 Feb-14 Sep-14 Feb-15 Sep-15

Dec-11 Dec-12 Dec-13 Dec-14

Aug-12 Aug-13 Aug-14 Aug-15

Nov-11 Nov-12 Nov-13 Nov-14

Mar-12 Mar-13 Mar-14 Mar-15

May-14 May-13 May-15 ESCMIDMay-12 eLibrary

Transplant Direct. 2017; 3(5): e151 © by author Time to onset of PCP after kidney transplantation (All PCP cases were not on prophylaxis at the onset of PCP) Number of PCP

0 ～3m 3～6m 6m～1y 1～2y 2～5y 5～10y 10y～ ESCMIDTime to onset ofeLibrary PCP after kidney transplantation © by author Outcome

Characteristic PCP patients (n=48)

6 (13%) Death 4 in the first outbreak 2 in the third outbreak

Graft loss 3 (6%)

ICU admission 10 (21%)

Mechanical ventilation

NPPV 3 ESCMIDEndotracheal incubation eLibrary7 ICU; intensive care unit , NPPV; noninvasive positive pressure ventilation © by author  We experienced 3 PCP outbreaks in a single kidney transplant center over a period of 10 years

 Each outbreak was controlled by a universal short-term prophylaxis

 Molecular analysis demonstrated that each intraoutbreak was caused by identical P. jirovecii; whereas interoutbreaks were by different genotypes, indicating that each outbreak was a completely separate event.

cyst forms

N Engl J Med. 2004; 350(24): 2487-98

Only the cystic forms (not the trophic forms) can be transmitted by aerial route from host to host. PLoS One. 2010;5(1):e8524 PLoS One. 2013;8(11):e79958 ESCMID eLibrary © by author Cysts are sensitive to TMP-SMX?

 Colonization of P. jirovecii is detected in 18.6% of kidney transplant recipients

Nephrology (Carlton). 2013;18(5):382–7

 Cysts may be the major form in colonization

 Cysts are not sensitive to TMP-SMX

 TMP-SMX is an antiprotozoal agent and not an antifungal agent

 Colonization can be cleared only by suppressed macrophages ESCMID eLibrary © by author PCP outbreak reported from Europa

2001 Sweden; Parasite 2008; 15: 359-365 2004 France; Emerg Infect Dis 2004; 10: 1766-1773 2007 Netherland; Clin Infect Dis 2007; 44: 1143-1149 2008 Germany; J Clin Microbiol 2008; 46: 966-971 2009 Switzerland; Transpl Infect Dis 2010; 12: 1-10 2011 Netherland; Nephrol Dial Transpl 2011; 26: 3391-3398 ESCMID2012 France; Clin Infect Dis eLibrary2012; 54: e62-e71 2012 Germany; Eur J Clin Microbiol Infect Dis 2012; 31(9): 2429-37 2013 Denmark ©; Transplantation. by 2013; author 96(9): 834-42 PCP outbreak reported from Japan

2009 Japan; Transplantation 2009; 88: 380-385 2009 Japan; Transpl Proc 2009; 41: 170-172 ESCMID eLibrary © by author PCP outbreak reported from East Asia

2014 Australia; Transplantation 2011; 92: 1327-1334

2016 Canada; Am J Infect Control. 2016; 44(4): 425-31

 Japanese kidney recipients under current immunosuppressive therapy have a greater chance of sharing time and space in outpatient clinics. This situation is similar to European outpatient clinics.

Waiting space

② ③

 Japanese kidney recipients under current immunosuppressive therapy have a greater chance of sharing time and space in outpatient clinics. This situation is similar to European outpatient clinics.

 TMP-SMX remains the first agent of choice for PCP prophylaxis as well as treatment. It is antiprotozoal agent, not to target fungus, but is highly effective for PCP (trophic forms) treatment.

 Unlike trophic forms, as cysts are not sensitive to TMP-SMX, colonization of P. jirovecii in the bronchial lumen cannot be eliminated.

 Macrophages which are phagocytic immune cells have a function for clearance of activated P. jirovecii, but those in kidney recipients are basically suppressed.

 Susceptible kidney recipients are ready to import any genotypes of P. jirovecii ESCMIDat outpatient clinic. eLibrary © by author Conclusion

 Although short-term administration of TMP-SMX effectively block the onset of PCP which changes from cysts to trophic forms, it is impossible to eradicate the nest of P. jirovecii.

 Therefore, lifelong administration could be approved to protect recipients from PCP outbreaks by different genotypes in a transplant facility where many of immunosuppressed patients visit.

No PCP outbreaks have been observed over a 40-month period after a lifelong prophylaxis strategy was adopted in our center.