Emerging pathogens 2007Emerging pathogens 2007

• Peter H. Gilligan PhD• Clinical Microbiology-Immunology Labs• UNC Hospitals

How I became a clinical microbiologistHow I became a clinical microbiologist• Obtained doctoral degree in microbiology at the University

of Kansas• Did post-doctoral training (2 years) in medical and public

health microbiology at UNC Hospitals• Director of Microbiology Labs at St Christopher’s Hospital

for Children (Philadelphia) for 4 years• Past 20+ years, Associate Director then Director of the

Clinical Microbiology-Immunology Labs at UNC Hospitals• Have served on medical school admission committee for

approximately 15 years and the MD/PhD advisory (admissions) committee for the past 10 years

What do clinical microbiologists do?What do clinical microbiologists do?

• We serve:» our patients

» our health care-providing colleagues, physicians, nurses, physician assistants, pharmacy colleagues

» hospital administrators• We make money for the institution

» general public by insuring the public health• Involved in studying outbreaks of several emerging

infectious diseases

How do we serve?How do we serve?• central role in the diagnosis and management of

infectious diseases• central role in infection control and antimicrobial use• recognize emerging disease threats and outbreaks

including bioterrorism events• we educate & train health care providers• we create new knowledge (research) to deal with

practical problems

Best things about my jobBest things about my job• Direct impact on patient care and public health of the

community• Intellectually challenging job requiring a broad fund of

knowledge-need to know a little about a lot of things –I am never bored!!!!!!!

• Work with highly motivated and intelligent individuals• Get to be at the cutting edge of infectious disease

diagnosis

Worst things about my jobWorst things about my job• Incredible amounts of governmental oversight• Increasing emphasis on financial aspects of the job• Declining talent pool of technologists• Need to be responsible for an organization that run

24/7/365-we never close. Personally have worked through ice storms, blizzards, and hurricanes.

How you can become a clinical How you can become a clinical microbiologistmicrobiologist

• CLS programs available here, ECU, WCU, WSSU, Wake Forest, UNC-CH» Education is also available on line

• 2 more years of school to get a BS in CLS • Take ASCP certification exam to become certified as a

MT.» Starting salary is 38,000 and up

» Career options are amazingly diverse; many former UNC students work in leadership positions in the pharmaceutical and biotech industries

Emerging/Re-emerging Infectious Diseases in the Emerging/Re-emerging Infectious Diseases in the past 25 yearspast 25 years

• HIV*

• Avian influenza

• SARS*

• Cryptosporidium*

• E. coli O157:H7*• Nipah virus

• nv Creutzfeldt-Jakob disease

• Sin Nombre Virus

• West Nile Virus

• Clostridium difficle*

• Bacillus anthracis (BT agent) Cyclospora

• CA-MRSA*

• Rapidly growing mycobacterium*

• Rotavirus*

• BK virus*

• Chlamydia pneumoniae

• Pencillinum marneffei

• Legionella*

• MDR- TB and pneumococcus*

• Burkholderia cepacia complex*

• VRE/VRSA*

• Helicobacter pylori*

• Invasive Group A streptococcal disease*

• HHV-6*

• HPV*

• HCV*

How do new pathogens emergeHow do new pathogens emerge• Organisms that jump species barriers• Changing ecosystems• Changes in food production techniques• Evolution of medical devices and care

» Long term survival of immunosuppressed

• Pathogens that are detected because of new technology

• Misuse of micro-organisms» Biocrime/bioterrorism

• Organism evolution as a result of human intervention» Antibiotic pressure

How do microbes change?How do microbes change?• Bacteria, because they evolve very quickly, can readily

adapt to hostile environments» Assume a generation time for a bacteria of 50 minutes

» 30 generations/day; or 220,000 bacterial generations for each human generation (assume generation is 20 years)

» Bacteria have a huge evolutionary advantage over humans

How emerging pathogens develop?How emerging pathogens develop?

• Mutation drives evolution» constantly occurring» usually silent or lethal» environmental pressure such as antibiotics may select

“resistance” mutation• Key feature of success of antibiotic resistant strains is their

genetic fitness I.e. their ability to compete in a complex microbial environment

» Recognition that certain bacteria may be hypermutators because of mutation in DNA repair genes

• These strains may not be as “fit” as wild-types but may predominant in certain chronic infections such as P. aeruginosa causing chronic pulmonary infections in CF patients

How do emerging pathogens develop?How do emerging pathogens develop?

• Recombination

» Resistance genes from antibiotic producing organisms

» genetic exchange of resistant genes can occur among organisms which are genetically diverse

• Think Cholera toxin genes to E. coli

» transfer of resistance/virulence genes can be mediated by plasmids/phage/transposons/ integrons

Organisms that jump species barriersOrganisms that jump species barriers• HIV, SARS, Avian flu

» HIV likely jumped from primates to humans

» SARS from pigs(?)

» Avian flu-hasn’t yet made the jump from birds to humans because human to human spread is rare, if it occurs at all. However mutation may result in that occurring.

» Technology allows us to quickly develop diagnostics for new pathogens

• Took years to develop HIV diagnostics• Took weeks to develop SARS diagnostics

Changing ecosystemsChanging ecosystems• Lyme disease

» A perfect storm• Farmland in New England returned to forest• Natural predators for deer were eliminated• Deer populations and the ticks they carried increased

because of ecosystem changes• People built homes and spent increasing amounts of time

in the woods• This resulted in increased exposure to deer ticks that

carried Borrelia» Ticks were pencil point in size and often difficult to see

Changes in food production techniquesChanges in food production techniques• Increased use of factory farming• Feedlots bring together large numbers of animals who

produce large amounts of waste» Waste can lead to run-off of EHEC that can contaminant

adjacent fields as was seen in recent spinach outbreaks

• Large meat packing operations can result in 50 ton lots of ground meat containing 100s of animals» Meat can be distributed throughout the US

» Contaminated lots can then lead to large scale outbreaks

Changes in medical careChanges in medical care• Immunosuppression either as a result of HIV or medically

therapy (ex. transplants) results in emerging infections» Pneumocystis, MAC, toxoplasma and CMV in HIV patients

» CMV, adenovirus and HHV-6 in transplant patients

• The use of indwelling artificial materials such as catheters, shunts, artificial joints present new ecosystems and new organisms» Examples-coagulase negative staphylococci growing as a

biofilm on artificial joints/catheters/shunts

» Rapidly growing mycobacteria causing keratitis following LASIK surgery

Pathogens detected with new technologyPathogens detected with new technology• Prime example is HCV

» Viral genome elucidated using molecular cloning techniques

• Broad range 16S RNA primers are used to detect non-cultivable bacteria

• Next big thing- application of molecular tools to understand how mixed microbial populations cause disease» Likely diseases caused by mixed microbial populations are

bacterial vaginosis, peridontal disease, inflammatory bowel disease, CF lung disease

How does bacterial resistance How does bacterial resistance develop?develop?

• Bacterial resistance develops in response to antimicrobial pressure» It is estimated that 3 million lbs of antimicrobials are used

each year in the US• Much of it is used in children to treat viral respiratory

illness• Estimated that 3/4 of children in US younger than two

receive antimicrobials• Children then may serve as a key role for the emergence

of antimicrobial resistance

» 10x that amount are used in animals

» End result- tremendous selective pressure that results in the emergence of bacterial resistance

UNC-EDUNC-ED• 6% of wounds from ED in 1st quarter of 2005 grew MRSA

• 45% of wounds from ED in 2nd quarter of 2005 grew MRSA

• ? Due to proliferation of CA-MRSA?

• GOAL» To characterize and determine the prevalence of CA-MRSA

isolates at UNC hospitals

Molecular analysis: CA- vs. HA-MRSAMolecular analysis: CA- vs. HA-MRSA

Adapted from Weber, CID, 2005:41S

Virulence of CA-MRSAVirulence of CA-MRSA• Panton-Valentine leukocidin (PVL)

» Hemolysin first reported in 1932 by Panton and Valentine» Located on mobile phage» 2 co-transcribed genes, lukS-PV and lukF-PV» The two subunits form a hexameric pore-forming cytolytic toxin with

a high affinity for PMNs and macrophages

•PVL producing strains associated with skin and soft tissue infections and necrotizing pneumonia

•Rarely associated with osteomyelitis, septicemia, or endocarditis

•Rare HA-MRSA strains with PVL have similar clinical syndrome

•Usually only 2% of all S. aureus isolates produce PVL but found in the majority of epidemic CA-MRSA strains

Adapted from Diederen and Kluytmans, JID, 2005

SCCSCCmecmec types types

( (SStaphylococcal taphylococcal cchromosome hromosome ccassette)assette)

21-24

Susceptibility PatternsSusceptibility Patterns

HA-MRSA CA-MRSA

pen

doxy

cefox

tmp-smz

clinda

erygentvanc

vancery

clinda

gent

pen

tmp-smz

doxy

cefox

93% are erythromycin resistant16% clindamycin resistant

CA-MRSA TimelineCA-MRSA Timeline

Necrotizing pneumonia,United States and EuropeNecrotizing pneumonia,

United States and Europe

1980

Outbreak in Detroit2/3 of patients were IVDU

Outbreak in Detroit2/3 of patients were IVDU

Mid 1990s

Children without identifiable risk factors

Children without identifiable risk factors

Late 1990s

1998 - Athletes/sports teams 1999 - Native Americans

1998 - Athletes/sports teams 1999 - Native Americans

2000

Prison and jail populations

Prison and jail populations

2003

IVDU=intravenous drug users

Groom AV et al. JAMA. 2001;286:1201-1205. Herold BC et al. JAMA. 1998;279:593-598. CDC. Morb Mortal Wkly Rep. 2001;50:919-922.

Naimi TS et al. JAMA. 2003;290:2976-2984.Zetola N et al. Lancet Infect Dis. 2005;5:275-286.Levine DP et al. Ann Intern Med. 1982;97:330-338. CDC. Morb Mortal Wkly Rep. 2003;52:793-795.

Gillet Y et al. Lancet. 2002;359:753-759. CDC. Morb Mortal Wkly Rep. 1999;48:707-710.

Clinical presentation CA-MRSAClinical presentation CA-MRSA• CA-MRSA

» SSTIs (abscesses, cellulitis, folliculitis, impetigo, furunculosis*)

• Typically treated with excision and drainage; +/- oral antibiotics• Occasionally require IV antibiotics, hospitalization and surgical

intervention

» Necrotizing pneumonia especially in young people secondary to influenza was reported this flu season

• Mortality was over 50%- median time to death 3.5 days• Median age was 17.5 years• 5 isolates from Louisiana were CA-MRSA genotype of the

same PFGE type• Both levofloxacin and inducible clindamycin resistance seen in

these isolates

Case 5Case 5• The patient is a 16 yo who presents with shoulder and left

chest wall pain• An MRI is ordered because of concerns about a abscess• The patient becomes hypotensive, SOB, is intubated and

admitted to the MICU.• Prior to admission, he denied fever, chills, cough and night

sweats• He lives on a farm in rural central NC with exposures to

dogs cats and horses» In the past year a horse had been put done due to

“strangles.” Strangles is a respiratory infection caused by Streptococcus equi

Case 5Case 5• No contributory travel or sexual history. Does not use

drugs or alcohol • Two months previously he had a right-sided preauricular

abscess incised and drained» Treated with Augmentin and infection resolved

• On PE, afebrile, pulse was 103 bpm, RR 30 and BP 99/62• Skin examination was significant for a small violaceous

lesion at the site of the prior abscess• Had several pustules on his leg and a hyperpigmented

macules on his left great toe• LDH was highly elevated, he was anemic and had a sed

rate of 60

Gram stain of sputumGram stain of sputum

Culture from blood bottleCulture from blood bottle

Study DesignStudy DesignI. Outpatient wound cultures (SSTIs) with MRSA (6/05 to 3/06), n=233

Definition of CA-MRSA

Panton-Valentine leukocidin positive

SCCmec type IV

III. Wound cultures with MRSA regardless of location (6/06-7/06), n=100

IV. Respiratory cultures with MRSA from Cystic Fibrosis (CF) patients (10/05 to 4/07), n=339

V. Child care centers

II. Nosocomial MRSA isolates (blood) (6/05 to 4/06), n=76

VI. All isolates recovered at Lilongwe Medical Center (6-06-2-07) n>100

I. PVL and SCCI. PVL and SCCmec mec CharacterizationCharacterizationof outpatient wound isolatesof outpatient wound isolates

22 26

168

15

1 10

20

40

60

80

100

120

140

160

180

PVL positive PVL negative

SCCmec II

SCCmec IV

SCCmec undetermined

IV II500

SCCmec typing**

(n= 191) (n= 42)

72%

9% 11% 6%

0.5%0.5%

n=233

** Oliveira and Lencastre (2002) Antimicrob Agents Chemother 46, 2155-61.

Study DesignStudy DesignI. Outpatient wound cultures (SSTIs) with MRSA (6/05 to 3/06), n=233

Definition of CA-MRSA

Panton-Valentine leukocidin positive

SCCmec type IV

III. Wound cultures with MRSA regardless of location (6/06-7/06), n=100

IV. Respiratory cultures with MRSA from Cystic Fibrosis (CF) patients (10/05 to 4/07), n=339

V. Child care centers

II. Nosocomial MRSA isolates (blood) (6/05 to 4/06), n=76

VI. All isolates recovered at Lilongwe Medical center (in June 07)

II. PVL and SCCII. PVL and SCCmec mec CharacterizationCharacterizationnosocomial blood isolatesnosocomial blood isolates

(n= 16) (n= 60)

0

42

16

11

0

7

0

5

10

15

20

25

30

35

40

45

PVL positive PVL negative

SCCmec II

SCCmec IV

SCCmec undetermined

SCCmec typing

IV II500

# o

f is

ola

tes

55%

15%9%

21%

n=76

II. Clinical and Molecular AnalysisII. Clinical and Molecular Analysisnosocomial blood isolatesnosocomial blood isolates

Clinical Characterization

Mo

lecu

lar

Ch

ar a

c te r

i za t

i on

CACA

HAHA

II

42

16

18

14

41

17

72

1

0

1

2

HAHACACA II

1

1

0

2

N=76

Study DesignStudy DesignI. Outpatient wound cultures (SSTIs) with MRSA (6/05 to 3/06), n=233

Definition of CA-MRSA

Panton-Valentine leukocidin positive

SCCmec type IV

III. Wound cultures with MRSA regardless of location (6/06-7/06), n=100

IV. Respiratory cultures with MRSA from Cystic Fibrosis (CF) patients (10/05 to 4/07), n=339

V. Child care centers

II. Nosocomial MRSA isolates (blood) (6/05 to 4/06), n=76

VI. All isolates recovered at Lilongwe Medical center (in June 07)

9 10

72

80 1

0

10

20

30

40

50

60

70

80

PVL positive PVL negative

SCCmec II

SCCmec IV

indeterminate

III. PVL and SCCIII. PVL and SCCmec mec CharacterizationCharacterizationof 2006 wound isolatesof 2006 wound isolates

n=100

# o

f is

ola

tes

SCCmec typing

IV II500

(n= 81) (n= 19)

10%9% 8%

72%

• Thanks to:» Melissa Miller» Jennifer Goodrich» Joel Wedd» Mwai Makoka and the UNC project Lilongwe» Tameaka Sutton-Shields» Kyle Rodino» All the CMIL technologists who identify, save and

freeze isolates so we can do this research

As Brian the scientist would say, “As Brian the scientist would say, “Any Questions?”Any Questions?”