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Changing U.S. Distribution of Nocardia Clinical Isolates: Importance of New Molecular Methods

Changing U.S. Distribution of Nocardia Clinical Isolates: Importance of New Molecular Methods. Michael M. McNeil May, 2007. Epidemiology of Nocardiosis. Estimated 500-1,000 infections per year Possible increasing incidence due to rising number of immunocompromised patients

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Changing U.S. Distribution of Nocardia Clinical Isolates: Importance of New Molecular Methods

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  1. Changing U.S. Distribution of Nocardia Clinical Isolates: Importance of New Molecular Methods Michael M. McNeil May, 2007

  2. Epidemiology 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

  3. Epidemiology of Nocardiosis • Nocardiosis • Respiratory infections • CNS - brain abscess • Cutaneous and lymphocutaneous disease • Most human cases of nocardiosis caused by: • Nocardiaasteroides 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

  4. Microbiology • Ubiquitous in soil, dust, organic matter • Weakly acid-fast, gram positive, branched filamentous rods • Slow growing - requires special media

  5. Colony of Nocardia

  6. Microbiology - 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

  7. CDC N. nova StudyM. 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

  8. CDC N. nova 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

  9. Distribution of Nocardia asteroides complex isolates, 1986-1992, by species N=91

  10. Distribution of Nocardia asteroides complex isolates, 1993-2000, by species N=271

  11. Distribution of Nocardia asteroides complex isolates, 2002-2006, by species N=174/341 N=109/211 2002-2004 2005-2006

  12. Nocardia isolates received by CDC, 2002-2006, by species 2002-2004 (n=341) 2005-2006 (n=211)

  13. Nocardia farcinicaisolates received by CDC, 1993-2000, by site N=108

  14. Nocardia novaisolates received by CDC, 1993-2000, by site N=114

  15. Distribution of Nocardia asteroides complex isolates, 2002-2006, by site 2002-2004 (n=341) 2005-2006 (n=211)

  16. Distribution ofNocardia novaandNocardia farcinica,1993-2000 Nocardia nova Nocardia farcinica

  17. Nocardia isolates received by CDC, 2002-2006, by origin 2002-2004 (n=341) 2005-2006 (n=211)

  18. Distribution of Nocardia nova and Nocardia farcinica, 1993-2000, by age Nocardia nova Nocardia farcinica

  19. Distribution of N. nova and N. farcinica, 1993-2000, by sex N. farcinica (n=45) N. nova (n=79)

  20. Nocardia spp. and N. farcinica and N. nova, 2005-2006, by sex Nocardia farcinica (n=29) Nocardia spp. (n=211) Nocardia nova (n=36)

  21. Alabama Isolates StudyJ. Brown et al. Objective • To evaluate and compare phenotypic and genotypic identification of Nocardia isolates (n=69) submitted to CDC during 2000-2004 from Alabama

  22. Phenotypic 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

  23. Genotypic 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

  24. Distribution of 69 Nocardia Species Isolates by Phenotypic Identification

  25. Distribution of 69 Nocardia Species Isolates byGenotypic Identification

  26. Unidentified Nocardia sp. 20% IdentifiedNocardia sp. 80% Unidentified Nocardia species(<99.5% similarity with the closest type strain)

  27. 0.002 Phylogenetic tree of Nocardia species N. nova (148-02, n=3) N. nova (133-03) N. nova (052-00, n=11) N. novacomplex 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)

  28. Study of Sulfa Resistance Among U.S. Isolates, 1995-2004K. Udhe 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)

  29. Patient 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%)

  30. Nocardia Species (n=964)

  31. Proportionof Selected U.S. Nocardia species Found to be Sulfa-Resistant, 1995-2004 N=748 isolates

  32. Geographic Distribution of U.S. Nocardia speciesFound to be Sulfa-Resistant, 1995-2004 (n=748) 51% (17/33) 73% (35/48) 73% (70/96) 61% (17/28) 79% (33/42) 56% (22/39) 68% (132/195) 63% (136/216) 47% (24/51)

  33. Additional 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)

  34. Proportion of Nocardia spp. isolates Found to be Sulfa-Resistant, 2002-2006 2002-2004 2005-2006

  35. Limitations • 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

  36. Conclusions • 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

  37. Recommendations 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?

  38. Future Plans • Update and maintain in-house 16S rRNA gene database • Improve rapid identification methods • Identify novel strains and clusters of strains

  39. Public 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

  40. Acknowledgements Actinomycete and Special Bacteriology Reference Laboratory, CDC • June Brown • Kristin Udhe • Mandi Murph • Gerald Pellegrini • Arnie Steigerwalt • Roger Morey

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