Public health microbiologyDisciplines and laboratory methods

Ones upon the time there was a microbiologist

Epidemiologist?????

Which one?????

And she found a way; PH microbiologist

Objectives of the lecture

Define public health microbiology (PHM)

Explain role of PHM

Give example of PHM disciplines

Understand basic methods of characterization of the microorganisms

What is Public Health Microbiology (PHM)?

“Microbiology is the study of microorganisms, including viruses, fungi, parasites and bacteria including immunity to these microorganisms.

Public health microbiology refers to a cross-cutting area that spans the fields of human, animal, food, water, and environmental microbiology, with a focus on human health and disease.

Public health microbiology laboratories play a central role in detection, monitoring, outbreak response, and providing scientific evidence to prevent and control infectious diseases.

Public health microbiology requires laboratory scientists with ability to work effectively across disciplines, particularly with epidemiologists and clinicians.” Consensus definition for PHM laid out by the group of microbiologists representing

the member states of the EU within the ECDC National Microbiology Focal Point Network

Why focus on this?

Public health is multidisciplinary

•Epidemiologists•Laboratory specialists•Clinicians•Veterinarians•Environmental specialists•Nurses•And more…

Activities must be coordinated to reach common goals!

The Lab – Epi challengeEpidemiologists and lab specialists are infectious disease experts with different:• Perspective and approach

• Skills and knowledge

• Working habits

“The two sides of the same medal”

Communication and understanding between Lab and Epi is crucial to the quality of public health

investigations!

Epi and lab – room for synergy?

Infecious disease epidemiology

– Hypothesis -> risk factors -> methods to make conlusions from incomplete data

Clinical microbiology

– Evidence of the presence of pathogen, but not everyone can be sampled and the problems don’t stop there...

Veterinary data

Environmental data

Different laboratories......with different roles

•Primary health care laboratories•Hospital laboratories •Independent diagnostic laboratories (state, regional or private)•Academic research laboratories•Veterinary Laboratories•Environmental Laboratories•Reference laboratories•Public health laboratories

Some important PH Laboratory tasks1. Confirm diagnosis for targeted interventions (detection,

monitoring, outbreak response, and providing scientific evidence)

2. Identify (new) types of pathogens

• Population-dynamics

• Virulence, persistence, resistance

• Implications for control measures

3. Microbiological safety of food and water

4. Quality assurance of diagnostic results

5. Information management, communication and coordination

6. Biosafety

7. Develop new tests/ Optimize existing tests

8. Basic/applied research for new insights and innovative solutions to health problems (vaccine and antibiotic development)

Where to find a public health microbiology laboratory regime

•Only integrated into the national PH institute, depending on size and development of country (eg. Netherlands)

•In a separate institution collaborating with the national PH institute (eg. France, Institute Pasteur)

•At the national PH institute and in regional laboratories, depending on infrastructure and size of country (eg. Germany, UK, Sweden)

Keep in mind

Essential functions of a PHL are not exclusive

Many public health laboratories conduct both public health and clinical diagnostic services

Many public health laboratories conduct both public health and research

Some public health laboratories produce and sell vaccines or biologicals (ex: Cantacuzino Institute, Roumania: diagnostic antisera; Pasteur Institute, Senegal: yellow fever vaccine)

Do you know your country's laboratory system?

Who is in charge of which disease?

Who do you contact in which case?• Local labs

• Regional labs

• Hospital labs

• Reference labs

• International lab networksFIND OUT!

http://ecdc.europa.eu/en/activities/microbiology/pages/microbiologicalcooperation_nationalmicrobiologicalfocalpoints.aspx

What disciplines do you need at a PH laboratory

Bacteriologists / Virologists / Parasitologist

Medical Microbiologists

Molecular Biologists

Immunologists

Post doctoral researchers / PhD students

Technicians / technical assistance / Analyst

Phylogenetic / molecular epidemiology specialists

Environmental specialists

Zoonosis specialists

Epidemiologists/ Statisticians

Public Health Microbiologists…..what is the difference and who is the best contact for what…

FIND OUT!

Conclusions part1:Conditions for successful collaboration between Lab and Epi ( Satu and Sabine share experience with you)Identify common goals

Understand that one is not only supporting the other, you work together for the same goalsEstablish and keep up lines of communication from the beginning to the endCommunicate expectationsAgree on authorship issues before the start of the projectShare data and information efficiently and openly; do not hide data and informationUnderstand that there are different perspectivesRecognize different skillsRespect different working cultures

Part 2: From story to reality Step by step

Species versus strains Discriminating features Discriminating features

ClassificationClassificationStrain: one single isolate or lineStrain: one single isolate or lineSpecies: related strainsSpecies: related strainsType: sub-set of speciesType: sub-set of speciesGenus: related speciesGenus: related speciesFamily: related generaFamily: related genera

Steps in isolation and Steps in isolation and identificationidentification• Step 1: Step 1: Streaking culture plates Streaking culture plates

– colonies on incubation (e.g 24 hr)colonies on incubation (e.g 24 hr)– size, texture, color, hemolysis size, texture, color, hemolysis – oxygen requirement oxygen requirement

21CDC/Dr. James Feeley

Sheep blood agar plate culture

Bacillus anthracisBacillus cereus.

Mixed colonies

Isolation and identificationIsolation and identification

Step 2: Colonies Gram Step 2: Colonies Gram stained stained • cells observed microscopicallycells observed microscopically

Gram negativeGram negative Gram positiveGram positiveHeat/DryHeat/Dry

Crystal violet stainCrystal violet stain

IodineIodine FixFix

Safranin stainSafranin stain

AlcoholAlcohol dede-stainstain

25

26

Gram stain morphologyGram stain morphology

Gram positive or negativeGram positive or negative

ShapeShape• cocci (round)cocci (round)• bacilli (rods)bacilli (rods)• spiral or curved (e.g. spirochetes)spiral or curved (e.g. spirochetes)

Single or multiple cellsSingle or multiple cells• clusters (e.g. staphylococci)clusters (e.g. staphylococci)• chains (e.g. streptococci) chains (e.g. streptococci)

Step 3:Step 3: Isolated bacteria are speciatedIsolated bacteria are speciated Generally using biophysiological testsGenerally using biophysiological tests

Example Salmonella and E-coli

Step 4:Step 4: Antibiotic susceptibility Antibiotic susceptibility

testing testing

No No growthgrowth

SusceptibleSusceptibleNot susceptibleNot susceptible

BacterialBacterial lawnlawn

GrowthGrowth

Antibiotic diskAntibiotic disk

http://en.wikipedia.org/wiki/File:Rosalind_Franklin.jpg

DNA structure

DNA is usually a double-helix and has two strands running in opposite directions. (There are some examples of viral DNA which are single-stranded). Each chain is a polymer of subunits called nucleotides (hence the name polynucleotide).

Molecular differentiationMolecular differentiation

GenomicsGenomics

• Gene characterization Gene characterization – SequencingSequencing– PCR (PCR (Polymerase chain reaction )Polymerase chain reaction )• Specific part of a gene Specific part of a gene • 16SrRNA16SrRNA

– Restriction digestsRestriction digests

• HybridizationHybridization

Genotypic typing methods

Fingerprint-based methods– Plasmid profile, RFLP(restriction

fragment length polymorphism), PFGE, AFLP

Character-based methods – MLVA (Multiple Loci VNTR Analysis),

ribotyping (restriction fragments that contain all or part of the genes coding for the 16S and 23S rRNA ), microarray’s

Sequence-based methods– MLST– SNP=single nucleotide

polymorphism typing

Minimum spanning tree of 240 strains Salmonella Enteritidis by MLVA

MRSA typed with PFGE & MLST

McDougal LK et al, 2003, J Clin Microbiol 41:5113-20

PFGE

Noroviruses

Norwalk virus

Hawaii virus

Snow Mountain virus

Mexico virus

Desert Shield virus

Southampton virus

Lordsdale virus

GI GI.1, GI.2

GII

GIII

Protein profiling: defining a Protein profiling: defining a species by characteristic proteinsspecies by characteristic proteins

Proteomics: defining all proteins Proteomics: defining all proteins expressed by a species under expressed by a species under specific growth conditionsspecific growth conditions

Rapid diagnosis without cultureRapid diagnosis without culture

• WHEN AND WHY?• grow poorly• can not be cultured• Need speedy results

Bacterial DNA sequences amplified directly Bacterial DNA sequences amplified directly from human body fluidsfrom human body fluids

• Polymerase chain reaction (PCR) Polymerase chain reaction (PCR)

• Great success in rapid diagnosis Great success in rapid diagnosis of tuberculosis.of tuberculosis.

Serologic Serologic identificationidentification

• antibody response to the infecting agent

• several weeks after an infection has occurred

Virus detection time of diagnosis sensitivity specificity

- virus isolation 1 – 7 days high* high**

- hybridisation 3 – 4 hours high1 good

- PCR 3 – 4 hours high2 good

- Electronmicroscopy 30 min low3 high

- capture ELISA 3 – 5 hours good4 high

Serology

- ELISA 3 – 4 hours high low

- Immunofluorescence (IFA)

2 – 4 hours good good

- Immunoblot 2 – 4 hours good good

- Neutralisation/ compliment fixation

4 – 7 days good high

- HIA 2 – 4 hours low good 1 ca. 104 particle/ml,

2 ca. 200 genome equivalent/ml, 3 106 particle/ml, 4 ca. 0.01 µg antigen/ml

* depending on cultivation system

** depending on detection System

Diagnostic methods time line

Prof. Matthias Niedrig, RKI

Conclusion part2:Choice of typing methodPathogenReproducibility

Discriminatory power

Exchangeability of data!

Study question• Local/global and short/long term epidemiology

Availability and resources

?

Acknowledgment