changing u.s. distribution of nocardia clinical isolates: importance of new molecular methods

04/19/23 1

Michael M. McNeil

May, 2007

Changing U.S. Distribution of Changing U.S. Distribution of NocardiaNocardia Clinical Isolates: Clinical Isolates:

Importance of New Molecular Importance of New Molecular MethodsMethods

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Epidemiology of NocardiosisEpidemiology of Nocardiosis

Estimated 500-1,000 infections per year

Possible increasing incidence due to rising number of immunocompromised patients

No national surveillance system

Rare nosocomial outbreaks

Therapy is prolonged trimethoprim-sulfamethoxazole (TMP-SMX) may result in treatment failure - alternative treatment

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Epidemiology of NocardiosisEpidemiology of Nocardiosis Nocardiosis

Respiratory infections

CNS - brain abscess

Cutaneous and lymphocutaneous disease

Most human cases of nocardiosis caused by:

Nocardia asteroides complex (N. abscessus, N. cyriacigeorgica, N. farcinica, and N. nova complex), N. brasiliensis and N. otitidiscaviarum

63 Nocardia species validated – 49 in the last 7 years

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MicrobiologyMicrobiology

Ubiquitous in soil, dust, organic matter

Weakly acid-fast, gram positive, branched filamentous rods

Slow growing - requires special media

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Colony of Colony of NocardiaNocardia

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Microbiology - IdentificationMicrobiology - Identification Before 2000, Nocardia species were identified using

morphology, biochemical methods and antimicrobial susceptibility testing (AST) profiles

Since 2000, coincident with the introduction of 16S rRNA gene sequencing methods, there have been many changes in the taxonomy of Nocardia

AST and 16S are not routinely performed in clinical or state laboratories

In 2005-2006, 16S rRNA gene sequencing replaced biochemicals and was used in combination with morphology and AST to identify all Nocardia species

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CDC CDC N. novaN. nova Study StudyM. Murph et al.M. Murph et al.

N. nova misidentified as NAC. Also, U.S. studies find recent increase in proportion of N. nova among NAC clinical isolates

1991 (Wallace et al) 20% N. nova 1986—1992 (CDC) 18% N. nova 1993—2000 (CDC) 42% N. nova

Several reports find N. farcinica is most frequent pathogen outside U.S. and N. nova in U.S. Hypothesized differences in geographic distribution and/or laboratory diagnostic procedures

1991—1993 UK, 21% NAC were N. farcinica 1979—1991 Germany, 79% NAC were N. farcinica 1987—1990 France, 67% N. asteroides and 24% were N. farcinica 1982—1992 Italy, N. asteroides most frequent, then N. farcinica

N. farcinica and N. nova cause pulmonary infections. Lung is the

commonest site of N. farcinica (Germany, France & Italy), then CNS.This CDC study found 15% CND isolates were N. farcinica and only 4% N. nova, suggesting N. farcinica is the more pathogenic species

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CDC CDC N. novaN. nova Study - Results Study - Results U.S. N. nova and N. farcinica infections vary (e.g., N. nova in AL, MA; N.

farcinica in GA, MT). May be selective referral and/or awareness by the state laboratories.

Age: N. nova peak ~70-79 years (ageing ICH population?)In contrast, N. farcinica peak ~40-59 years (transplants?)

Gender: N. nova, M1:F1; N. farcinica, M2.2:F1 (Germany, 1979-1988 M3.1:F1 and 1988-1991 M1.5:F1)

Drugs of choice: sulfonamides or TMP-SMX. However, reports of drug resistance/intolerance.

Long, 1994,TMP-SMX in AIDS patients - only 50% response and 90% fatality rate

Hill et al. 2007, in UK 45% NAC isolates were resistant to TMP-SMX

If standard therapy is not tolerated/ineffective Optimal therapy for N. farcinica infection is parenteral imipenem or

imipenem in combination with amikacin or amoxicillin-clavulanate In contrast, N. nova responds to more cost effective macrolides

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Distribution of Distribution of Nocardia asteroidesNocardia asteroides complex isolates, 1986-1992, by speciescomplex isolates, 1986-1992, by species

N. asteroides

Type 628%

N. nova25%

N. farcinica

36%

N. asteroides

Type 111%

N=91

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N. asteroides Type 611%

N. farcinica40%


N. nova42%

N=271

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N. nova44%

N. farcinica25%



N. farcinica27%


N. asteroides Type 1

9%

N. nova38%

2002-2004 2005-2006

N=174/341 N=109/211

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NocardiaNocardia isolates received by CDC, isolates received by CDC, 2002-2006, by species2002-2006, by species

0

5

10

15

20

25

Species

Per

cen

t

2002-2004 (n=341)

2005-2006 (n=211)

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Nocardia farcinicaNocardia farcinica isolates received isolates received by CDC, 1993-2000, by siteby CDC, 1993-2000, by site

CNS15%

Blood6%

Pulmonary36%

Wound18%

Other25%

N=108

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Nocardia novaNocardia nova isolates received by isolates received by CDC, 1993-2000, by siteCDC, 1993-2000, by site

Blood 4%

Wound 17%

Other12%

Pulmonary63%

CNS4%

N=114

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Distribution of Distribution of Nocardia asteroidesNocardia asteroides complex isolates, 2002-2006, by sitecomplex isolates, 2002-2006, by site

2002-2004 (n=341) 2005-2006 (n=211)

Wound39%

Unk6%

Blood3%CNS

4%

Pulmon 48%

Pulmonary51%

CNS5%

Blood4%

Unknown13%

Wound27%

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Distribution ofDistribution of Nocardia novaNocardia nova andand Nocardia farcinicaNocardia farcinica,, 1993-20001993-2000

0

5

10

15

20

25

30

Ala. Fla. Ga. Mass. Mont. Penn. S.C. Canada Foreign

Origin

Nu

mb

er

Nocardia farcinica

Nocardia nova

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Nocardia Nocardia isolates received by isolates received by CDC, 2002-2006, by originCDC, 2002-2006, by origin

0

10

20

30

40

50

60

Species

Nu

mb

er

2002-2004 (n=341)

2005-2006 (n=211)

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Distribution of Distribution of Nocardia novaNocardia nova and and Nocardia farcinicaNocardia farcinica, 1993-2000, by age, 1993-2000, by age

Nocardia farcinica

02468

101214161820

<19 20-29 30-39 40-49 50-59 60-69 70-79 80-89

Age group (years)

Nu

mb

er

Nocardia nova

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Distribution of Distribution of N. novaN. nova and and N. N. farcinica, farcinica, 1993-2000, by sex1993-2000, by sex

N. farcinica (n=45)

N. nova(n=79)

Females49%

Males51%

Males69%

Females31%

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Nocardia spp. Nocardia spp. andand N. farcinica and N. farcinica and N. nova, N. nova, 2005-2006, by sex2005-2006, by sex

Males66%

Females34%

Males59%

Females41%

Males62%

Females38%

Nocardia farcinica (n=29)

Nocardia nova (n=36)

Nocardia spp. (n=211)

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Alabama Isolates StudyAlabama Isolates StudyJ. BrownJ. Brown et al. et al.

Objective To evaluate and compare phenotypic

and genotypic identification of Nocardia isolates (n=69) submitted to CDC during 2000-2004 from Alabama

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Phenotypic IdentificationPhenotypic Identification Biochemicals

Decomposition tests adenine, casein, esculin, hypoxanthine, tyrosine, and xanthine

Oxidative acid production from 23 carbohydrates Utilization of acetamide and citrate Arylsulfatase production Growth in lysozyme Growth at 45oC

Antibiogram MICs to 11 antimicrobial agents – two fold dilutions Interpretive criteria for resistance according to CSLI (formerly

NCCLS) guidelines recommended for Nocardia species

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Genotypic IdentificationGenotypic Identification 16S rRNA gene sequencing of 69 isolates

16S rRNA gene sequencing methods were those used routinely in the SBRL

Phylogenetic analysis of ~1440 base pairs were blasted against a database dominated by Roth et al., J Clin Microbiol 2003;41:851-856

Isolates designated as “Nocardia species” if % similarity to type strain of the closest species was <99.5% as suggested by Roth for genus Nocardia

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0 10 20 30 40 50

Other

N. veterana

N. otitidiscaviarum

N. nova

N. cyriacigeorgica

N. brevicatena

N. brasiliensis

N. beijingensis

N. arthritidis

N. africana

N. abscessus

N. asteroides complex

Nocardia spp.

Percent

Distribution of 69 Nocardia Species Isolates by Phenotypic Identification

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0 10 20 30 40 50

Other

N. veterana

N. otitidiscaviarum

N. nova

N. cyriacigeorgica

N. brevicatena

N. brasiliensis

N. beijingensis

N. arthritidis

N. africana

N. abscessus

Nocardia sp.

Percent

Distribution of 69 Nocardia Species Isolates byGenotypic Identification

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Unidentified Nocardia species (<99.5% similarity with the closest type strain)

Unidentified Nocardia sp.

20% Identified Nocardia

sp.

80%

Strain no. % Similarity Closest type strain

045-00 98.75 N. otitidiscaviarum

149-00 99.60 Nocardia sp. (DSM 43253)

150-01 99.38 N. africana

156-01 98.05 N. africana

007-02 99.31 N. brasiliensis

055-02 98.75 N. pseudobrasiliensis

175-02 98.89 N. asteroides

176-02 98.87 N. farcinica

216-02 99.38 N. africana

013-03 97.48 N. beijingensis

034-03 99.21 N. transvalensis

108-03 98.54 N. brevicatena

188-03 99.38 N. africana

129-04 98.82 N. farcinica

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N. nova (148-02, n=3) N. nova (133-03) N. nova (052-00, n=11)

N. nova (043-00, n=6) N. nova JCM 6044T (AF430028)

N. nova (103-01, n=2) N. nova (040-02)

N. nova (226-00, n=3) N. nova (119-00)

N. otitidiscaviarum (095-00) N. otitidiscaviarum DSM 43242T (AF430067)

N. otitidiscaviarum (185-00, n=4) Nocardia sp. (045-00)

N. pseudobrasiliensis DSM 44290T (AF430042) N. pseudobrasiliensis (022-00)

Nocardia sp. (055-02) N. transvalensis DSM 43405T (AF430047)

Nocardia sp. (034-03) Nocardia sp. (156-01)

N. africana DSM 44491T (AF430054) N. africana (197-01)

N. veterana DSM 44445T (AF430055) N. veterana (212-03)

N. veterana (076-03) N. veterana (119-01) N. veterana (134-02)

Nocardia sp. (188-03, n=3) N. paucivorans DSM 44386T (AF430041)

N. abscessus (054-00, n=2) N. abscessus DSM 44432T (AF430018)

Nocardia sp. (175-02) N. asteroides DSM 43757T (AF430019)

N. cyriacigeorgica (006-00, n=5) N. cyriacigeorgica DSM 44484T (AF430027)

Nocardia sp. (149-00) Nocardia sp. DSM 43253 (AF430021)

Nocardia sp. (108-03) N. takedensis MS1-3T AB158277

N. takedensis (183-01) N. brasiliensis (234-00)

N. brasiliensis (072-00) N. brasiliensis DSM 43758T (AF430038)

Nocardia sp. (007-02) N. tenerifenis GW39-1573T (AJ556157)

Nocardia sp. (176-02) Nocardia sp. (129-04)

N. farcinica (062-00) N. farcinica DSM 43665T (AF430033)

N. arthritidis IFM 10035T N. arthritidis (191-00, n=4)

N. beijingensis (072-04) N. beijingensis AS4.1521T (AF154129)

Nocardia sp. (013-03)

0.002

Phylogenetic tree of Nocardia species

N. nova complex

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Study of Sulfa Resistance Among Study of Sulfa Resistance Among U.S. Isolates, 1995-2004U.S. Isolates, 1995-2004

K. UdheK. Udhe et al. et al.

Objective To evaluate prevalence of sulfonamide

resistance among isolates submitted to CDC during 1995-2004 and identified by phenotypic methods as Nocardia (n=964)

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Patient Demographics and Site of IsolationPatient Demographics and Site of Isolation

Patient Demographics Age (n=700)

Median: 63 yrs (Range: <1 – 95 yrs) Gender (n=738)

Male = 44/738 (61%)

Site of specimen (n= 964) Pulmonary (50%) Wound (22%) CNS (7%) Other (16%) Unknown (5%)

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NocardiaNocardia Species (n=964) Species (n=964)

N.nova

N.farc inica

N.asteroides

N.brasiliensis

N.brevicatena

N.cyriac igeorgica

N.otitidiscaviarum

N.transvalensis

N.abscessus

N.pseudobrasiliensis

Other

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ProportionProportion of Selected U.S.of Selected U.S. Nocardia Nocardia species species

Found to be Sulfa-Resistant, 1995-2004Found to be Sulfa-Resistant, 1995-2004

020406080

100

Species

Per

cent

N=748 isolates

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Geographic Distribution of U.S. Geographic Distribution of U.S. NocardiaNocardia species speciesFound to be Sulfa-Resistant, 1995-2004 (n=748)Found to be Sulfa-Resistant, 1995-2004 (n=748)

47% (24/51)

56% (22/39)

61% (17/28) 79%

(33/42)

63% (136/216)

73% (35/48)

68% (132/195)

73% (70/96)

51% (17/33)

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Additional findingsAdditional findings

71/130 (55%) of N. brasiliensis from wound 50-70% of Nocardia were from pulmonary site 82% of N. brevicatena were from males 63% of N. farcinica were from males N. nova isolates male:female ratio = 1:1 All species more prevalent in persons >40 years 16S identification on a subset 69 AL isolates 99% (870/878) isolates susceptible to: amikacin,

amoxicillin/clavulanate and imipenem (only 1% were resistant to all three drugs)

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Proportion of Proportion of NocardiaNocardia spp. isolates spp. isolates Found to be Sulfa-Resistant, 2002-2006Found to be Sulfa-Resistant, 2002-2006

020406080

100

Species

Per

cent

2002-2004

2005-2006

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LimitationsLimitations

Role of selective referral of isolates to CDC

CDC may receive isolates from more severely ill patients

No data is available on clinical outcomes, and use of antibiotic prophylaxis or specific antimicrobial drug treatment in patients

No standard and accurate (molecular) laboratory method of identification was used for most of these Nocardia species isolates

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ConclusionsConclusions New molecular identification methods have contributed

to a change in the distribution of clinical Nocardia sp. isolates

Our data confirms that sulfa-resistance among clinical Nocardia isolates is widespread in the U.S.

Sulfa-resistance is most common among the most pathogenic species, N. farcinica and N. nova

Our results confirm the importance of antimicrobial susceptibility testing and speciation of clinical Nocardia isolates

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Recommendations for Future StudyRecommendations for Future Study Whether in vitro sulfa resistance is associated with

more serious patient outcomes? Whether all clinically significant Nocardia isolates

need speciation and susceptibility testing to guide treatment?

Whether antimicrobial susceptibility testing is useful for effective drug treatment?

Additional antimicrobial susceptibility studies may allow broader therapy options?

What is the possible mechanism of Nocardia resistance to sulfa?

What is the potential role of TMP-SMX prophylaxis in the immunocompromised host and does it select for infection with sulfa resistant Nocardia species?

Has increased antibiotic use in agriculture selected for sulfa-resistant infections in humans?

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Future PlansFuture Plans

Update and maintain in-house 16S rRNA gene database

Improve rapid identification methods

Identify novel strains and clusters of strains

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Public Health ImpactPublic Health Impact

Centralized capacity to reliably identify aerobic actinomycetes

Recognition of drug resistance to first line antimicrobial therapy in clinically significant nocardiae

Rapid identification of sources of infection allowing for implementation of appropriate therapy

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AcknowledgementsAcknowledgements

Actinomycete and Special Bacteriology Reference Laboratory, CDC

June Brown Kristin Udhe Mandi Murph Gerald Pellegrini Arnie Steigerwalt Roger Morey

changing u.s. distribution of nocardia clinical isolates: importance of new molecular methods

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