Emerging pathogens 2008Emerging pathogens 2008

• 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» There is no unemployment in this group

• 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

Antibiotic associated adverse effectsAntibiotic associated adverse effects• Antimicrobial toxicity and allergic reactions

» Anti-parasitic>anti-fungal>anti-viral>antibacterials

» 20% of ER visits for drug adverse events are due to antimicrobials

• Alteration in the microbial flora» Candida vaginitis and thursh

» C. difficile infection

» Salmonellosis

• Emergence of resistance» Few organisms where resistance is not clinically important

CaseCase• Patient is an 80 yr female with primary pulmonary

hypertension who present with SOB, DOE and chest radiograph consistent with lobar pneumonia

• Treated for 14 days with levofloxacin and discharged to a skilled nursing facility

• 3 weeks later she has has the onset of loose watery stools up to 10 episodes a day

CaseCase• She develops generalized weakness, abdominal pain, and

is unable to walk• She develops vomiting and nausea and can’t eat• She is re-admitted to the hospital after three weeks of

diarrhea; she presents with fever, chill, malaise, myalgias, and dizziness

• PE is significant for a pulse rate of 120, RR of 24, and BP of 82/45

CaseCase• Her abdominal examination is significant for decreased

bowel sounds, tenderness and mild distension.• She had a white blood cell count of 29,000/ul• Imaging studies were consistent with pseudomembranous

colitis• Over a four day period her white blood cell count rose from

29,000 to 127,000 ul; she had expanding abdominal girth and decreasing bowel sounds.

• She died on the 4th hospital day

Clostridium difficileClostridium difficile• General characteristics

» Gram positive rod

» Spore former» Anaerobic» Can be part of human

microflora» Pathogenicity due to the

production of two protein exotoxins A and B

Chance favors Chance favors only only the prepared mindthe prepared mind

Louis Pasteur

Key events in the discovery of Key events in the discovery of C. difficileC. difficile

• Larson and colleagues describe a toxin in the feces of a child with pseudomembranous colitis (1977)

• Bartlett and colleagues show that C difficile can induce colitis in hamsters given clindamycin and then a variety of antibiotics and then proves that the organism can cause the same disease in humans (1978)» Serendipity is important- showed that C. sordelli antitoxin could

neutralize toxins produced by C. difficile in a tissue culture cyotoxicity assay.

Key events in the discovery of Key events in the discovery of C. difficileC. difficile

• Among others, Gilligan and colleagues show that C. difficile is the most common bacterial agent in a general population (1980)

• Lylerly and colleagues purify two toxins, A and B, from C. difficile and also produce an important anti-toxin against these organisms (1982)

What makes What makes C difficile C difficile an important an important pathogen in the industrialized world?pathogen in the industrialized world?

• Important ideas» Organism can survive in the environment for months as spores;

spores are refractory to disinfectants especially alcohol and all antimicrobials

» Alternation in the gut flora is important in predisposing patient’s to disease with this organism- antibiotics mediate this change

» Most common diarrheal disease etiology in the industrialized world requiring specific antimicrobial interventions

Changing Changing C. difficile C. difficile epidemiologyepidemiology

Changing Changing C. difficile C. difficile epidemiologyepidemiology

• Beginning in 2002, a highly virulent strain of C. difficile (NAP1/027) emerged in the Quebec Province in Canada, several US states, Britain, and just recently in Netherlands» The disease is seen primarily in those over 65 years of age» The incidence in one Canadian region in patients over 65 went

from:• 102/100,000 in 1991-2

• 210/100,000 in 2002

• 866/100,000 in 2003

What factors has resulted in the re-emergence of What factors has resulted in the re-emergence of

Clostridium difficileClostridium difficile????• Better case ascertainment

» Improvement in lab diagnosis• Aging population

» Decline in Bifidobacterium with age, an organism important in colonization resistance, in gut flora may create more permissive environment for C. difficile

• Increased use of antimicrobials especially fluoroquinolones with anti-anaerobic activity to which C. difficile is resistant» This is being debated in the infectious disease community» 90% of C. difficile isolates are fluorquinolone resistant

What factors has resulted in the re-emergence of What factors has resulted in the re-emergence of Clostridium difficileClostridium difficile??

• Increased contamination of health care setting with C. difficile spores making infections more likely» Cleanliness of British Public Health Service hospitals has

become a major political issue there

• Shared rooms and bathroom facilities» Particular issue in Canada and Britain

Pathogenesis of Pathogenesis of C. difficileC. difficile• Key steps in pathogenesis

» Anaerobic gut flora confers “colonization resistance” to the host from infection with C. difficile

» Alteration of this gut flora by antimicrobial therapy creates a permissive environment for the vegetation of C. difficile spores

» Spores are either present in gut at time of alteration of gut flora or are obtained from the hospital environment during the period of gut flora alteration

• May take as long as six weeks for gut flora to return to normal• Antimicrobials most impacting gut flora include clindamycin,

cephalosporins, and perhaps newer fluoroquinolones

Pathogenesis of Pathogenesis of C. difficileC. difficile• Organism grows and begins to produce both toxin A & B

» Toxins have high degree of sequence similarity

• Toxins bind to specific receptors on surface of the cell and enter cell via receptor mediated endocytosis

• Toxins acts as a glucosyltransferase inactivating small GTPase

Pathogenesis of Pathogenesis of C. difficileC. difficile

• GTPase control a variety of cell functions resulting in: actin condensation which leads to cell rounding, membrane blebbing, apoptosis, and cell death

» increased permeability of the colonic epithelium» Chemokine expression which increases inflammatory

response » Increases neutrophil infiltration » Loss of tight junctions resulting in neutrophil migration into

the intestine

C. difficile: C. difficile: Spectrum of diseaseSpectrum of disease

Asymptomatic carriage

Mild diarrhea

Profuse diarrhea with non-specific colitis

Pseudomembranous colitis

Toxic megacolon

frequency

Clostridium difficile Clostridium difficile PMC and toxic PMC and toxic megacolonmegacolon

Is there an emerging pandemic of a Is there an emerging pandemic of a highly virulent highly virulent C. difficile C. difficile strain?strain?

• Recent outbreak in Canada, the regions of the US, Britain, and now the Netherlands have recognized the emergence of a highly virulent strain of C. difficile

• Characteristics of this new strain include:» Mutation in tcdC gene which down-regulates toxin

production during logarithmic phase » Mutation results in approximately 20-fold increase in

toxin production

Is there an emerging pandemic of a Is there an emerging pandemic of a highly virulent highly virulent C. difficile C. difficile strain?strain?

» Organism also produce binary toxin, a 2nd virulence factor typically produced by a small number of isolates

» Poor response to metronidazole has also been reported

» Higher morbidity and mortality particular among those over >75.

Treatment of Treatment of C. difficile C. difficile diseasedisease• Initial studies showed that metronidazole and vancomycin

had similar initial response to therapy (90%) and similar disease recurrence rates (5 to 12%)

• Metronidazole became the drug of choice because it was much cheaper and because of concerns of vancomycin use resulting in increased rates of VRE and concerns about the emergence of VRSA

Treatment of Treatment of C. difficile C. difficile diseasedisease• Two recent studies (published 6/05) have shown much higher rates of

treatment failures/recurrences than previously reported with metronidazole» One study (CID 40:1586, 2005) only 50% of patients were cured,

22% had symptoms continuous for > 10 days and 28% had recurrences

» In a Canadian survey (Pepin et al. CID 40:1591-7), recurrence rates increased from 21% in 1991-2002 to 48% in 2003-2004; in those over 65 y.o., that rate was close to 60% in 2003-4

Treatment of Treatment of C. difficile C. difficile diseasedisease» Whether resistance to metronidazole is the reason is not

reported in these studies; most recent report puts metronidazole resistance at 6% ( Pelaez et al. AAC 46: 1647, 2002)

• Isolates were recovered from 1993-2000 before emergence of the NAP 1/027 strain in 2002

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