Laboratory diagnosis of wounds, abscesses, plagues

Cutaneous infections

Primary cutaneous infections – occur on apparently lesion free skin:– impetigo, – foliculitis, – furuncle

Secondary infections – occur on damaged skin: – traumatic lesion, – chronic dermatosis, – viral infection.

Purulent skin infections (PSI)

Class I PSI: deeper, closed Class II PSI:

– deep and closed, the focus communicates or has communicated with an organ containing commensal flora

– deep and open, fistulas Class III PSI: superficial, open with heavy

contamination by commensal flora ; these sample have a high probability of containing both pathogenic and commensal flora.

Objectives

To identify the pathogenic bacteria in superficial primitive skin infections (anthrax, furuncle, erysipelas …)

To identify bacteria present in acute traumatic lesions (fractures, bites, burns, surgical wounds)

To identify bacteria causing secondary infections of surgical wounds

To identify the bacteria responsible for the signs of local chronic infections.

Physiopathology:

Puncture and pus aspiration from closed, undrained tissue doesn’t exclude the contamination during sampling.

Important is skin decontamination and drying of the skin before sampling. Any contaminant bacteria can be cause of false positive diagnosis.

Clinical significance of these isolates: cyto-bacterioscopic aspect of sample.

Physiopathology

Suppurations can be mono- or poly microbial.Any suppuration is a step of a inflammatory

process. Inflammation evolve only in a live tissue; never

in a necrotic tissue.The essential problem of the pus examination is

differentiation between bacteria with clinical significance and the specimen contamination.

Physiopathology:

The exudates and necrotic tissue from burns, bed sore, plagues are constantly contaminated with bacteria which usually are not involved in the inflammation’ s etiology.

Important is to remove the stagnant tissue!

Rather: specimens should be aspirated from the primary infection through non-infected tissue.

Sampling methods

sample from healthy skin or non-exsudative lesion: 2 swabs + transport medium

skin with supperficial cutaneous lesions: two swabs + transport medium

cutaneous inflammation, erysipelas, hypodermitis: skin disinfection, syringe aspiration / biopsy

bites: aspirate the liquid / swab for anaerobic bacteria supperficial wounds: aspiration using a syringe ulcerations, sores, necrotic cutaneous lesions: deep tissue

biopsy

Type of sampling:

Specimens for normal sterile cavities (ascitic fluid, peritoneal, pleural, synovial exudates);

Specimens from purulent collections after septic metastasis; evolve related to the anatomic site, from normal colonized regions (oral cavity, digestive system, genital areas);

Pus from the plagues (traumatic, surgical, bite) or abscesses contiguous with mucosal surfaces in open wounds.

These specimens can be contaminated with normal flora;

Infected burn lesion, bedsore are always contaminated.

Frequently, SAMPLING from the WOUNDS SPECIMEN IS UN-REPEATABLE

Procedure

Clean the wound Remove all exsudates Debride the necrotic tissue Apply cutaneous antiseptic and leave to dry Rinse with sterile saline Take a biopsy of the lesion or Curette the active edge of the lesion and Place the sample bottom of a sterile vial Aspirate the inflammatory liquid – fine needle Swabbing is not a adequate procedure and should be

avoided

Sampling technique:

skin decontamination (~ blood culture); remove of the stagnant exudates by washing,

from the burns; for biopsy: sterile cutaneous perforator - 4 mm sterile swabs; special transport culture media - from lab; slides for smears.

Puncture of the closed collections:

use fine needle to avoid false negative results, when the pus is viscous;

skin decontamination and drying;puncture and aspiring of the pus;decant the pus in a special recipient which

allow anaerobic bacteria to survive;“anaerobic syringe”: remove the air from the

syringe and put a rubber stopper in the needle. The time for analyzing is 30 minutes.

Skin lesion (blood culture if possible)

Sampling during surgery intervention:

for abscesses (pelvic, abdominal, chest) – sterile recipients for pus collection;

for purulent membranes: some fragments must be transported to laboratory in 30 minutes.

Abscesses sampling

Sampling from fistula:

Sampling with the swab it is not a good technique: the lack of a good identification of the microorganism with clinical significance (normal flora of the surface);

Preferable: washing, skin decontamination, deep curettage of the fistula and quick transport of the sample at the laboratory.

Big areas of eroded skin: more sampling from many places of the lesion;

Sampling of the primary infection through uninfected tissue– Remove the stagnant exudates or topics antibiotics, by

washing and using sterile swabs;– Spin the swab until a minimum bleeding.– Send the swab for examination.

During surgery intervention: specimens from diagnostic tissue biopsies, from the plague;

Oblique puncture and aspiration to normal skin: for chronic skin ulcers.

Sampling from burns, plagues and chronic skin ulcers:

Burned wards:

• Superficial skin samples: any topical agents are removed with a sterile compress soaked in normal sterile water;

• the area of interest can be swabbed using two sterile swabs that are placed in transport culture medium to preserve fragile bacteria;

• We can use two techniques:– Z stroke technique: a specified surface area of skin, generally

2-4 cm2, is swabbed using several zigzag movements;– Levin technique: the tip of the swab is rotated several times

over a localised area.

Burned wards:

• Skin biopsy: should be used a disposable biopsy punch (3 – 5 mm) or by cutting area measuring 1-2 cm by 1,5 cm

• sterile scalpel and forceps, • detaching the tissue along the subcutaneous fat

layer, and also • sampling healthy, unburned tissue; fragments

usually weigh 20-50 mg.

Wrong techniques:

sampling on swab for anaerobic bacteria;

sampling on swab, instead of syringe, biopsy or curettage;

sampling of necrotic tissue, expose to contamination

Results and interpretations

Clinical information is essential to the interpretation of the microbiological examination:– nature and site of the lesion– sampling method– global context: age, immune status, diabetes, ongoing

infections, corticotherapy,– recent antibiotic treatment.

Only comparison of the microscopic examination and results of culture with the symptoms can lead to reliable interpretation

Mixed culture (>3 bacterial species): contamination

Results’ s interpretation:

Puncture / aspiration of the closed collections: – has clinical significance the isolated with the same

microscopic appearance like bacteria from directly smear;

– when the patient has systemic infection, isolating of the same bacteria from the blood culture can be a major criteria of clinical significance of the bacteria from the pus;

Specimen during surgery intervention:– has clinical significance the bacteria observed

through direct examination;

Results’ s interpretation: Specimens obtained by curettage:

– has clinical significance bacteria associated with PMNs, in direct microscopy.

Specimens from plagues and burns: – has clinical significance the conditioned pathogens ≥ 105

CFU/g tissue; – β-hemolitic streptococci always has clinical significance;– the presence of epithelial cell means inefficient removing

of the stagnant exudates;– for the correct sampling: quantitative criteria: ≥ 106 CFU/

swab.

1st Case

34 years old patients, with no significant history, present a furuncle at the right arm. After surgical incision, S. aureus from pus was isolated.

Disk diffusion susceptibility test for S. aureus was:

Penicilline - R Kanamicyn - SOxacilline – S Tobramicyn - SEritromycin– S Gentamcyn - SClindamicyne – S Vancomicyn - STetracicline - RCotrimoxazol - SCiprofloxacin - S

1st Case: comments

• Strain from community, resistance to penicilline (penicillase production);

• No Vancomycin test diffusion method (the method is no appropriate for this test);

• Susceptibility test - it is NOT necessary

(treatment = surgery + local antibiotics or antiseptics).

2nd Case8 years old child with osteomyelitis (traumatism in

recent past). S. aureus was isolated.

Results of disk diffusion susceptibility test for S. aureus:

Penicilline – R Kanamycin - SOxacilline – R Tobramycin - RCefoxitin – R Gentamycin - SCeftriaxone – S Vancomycin - SEritromycine– R Linezolid - SClindamicine - S (inducible resistance)Tetracycline - RCotrimoxazol - SCiprofloxacine - S

2nd Case: comments

• Resistance to Oxacilline – confirmation by Resistance to Cefoxitine;

• False sensitivity to Ceftriaxone (treatment failure when we use beta – lactamase antibiotics for MRSA);

• Low level resistance strains to Vancomycin can’t be identified by disk diffusion test;

• Clindamycin – the best for ostheomyelitis’ s treatment – high level in the inflammatory focus;

• Inducible resistance can induce treatment failure.

Acute diarrhea

Causative agents:

Choleric syndrome: – Vibrio cholerae– NAG Vibrio– ETEC, EPEC, ECAD, ECEAg

Dysenteric syndrome:– Shigella spp.– EIEC– Campylobacter– Non - typhoid Salmonella – Yersinia enterocolitica

Hemorrhagic diarrhea:– EHEC

Enteric fever diarrhea:– Salmonella enterica serovar

Typhi, Paratyphi A, B, C Diarrhea post antibiotics

treatment:– Clostridium difficile– Staphylococcus aureus

Viruses:– rotavirus,– enteroviruses, – adenoviruses,– coronaviruses, etc.

Parasites:– Giardia duodenalis, – Cryptosporidium

parvum, – Entamoeba.

Stool sampling:

Necessary:– sterile recipient –Cary Blaire transport

culture media–1g of feces (blood, pus,

mucus).

Sampling by swab:

Useful for patient with dysenteric syndrome;

After sampling the swab is introduced in transport culture media (Cary Blaire).

Transport

Maximum 1 hour.

If the transport need to be delayed, the sample must be preserved at 4°C for maximum 24 hours.

Requesting paper must specify the clinical syndrome.

Stool culture = 4 days

3rd Case

7 years old child, went a party in a fast food restaurant. After 12 hours, the child present numerous stools (8 / 24 hours), yellow, with mucus. The same symptoms were noted fort other 8 children who were at the same party.

Pathologic product: feces.

Suspected food?

Hamburger

Coprocitogram: ~ 10 PMN s / field (x400)

Stool culture: present Salmonella group B

Antibiogram:

Ampicilin - R Cotrimoxazol - R

Ciprofloxacine - S Ceftazidime – S

Nalidixic acid– R

Treatment?

Causative agents of food borne diseases

Superior GI syndrome – Bacillus cereus– Staphylococcus aureus

Inferior GI syndrome– Clostridium perfringens– Escherichia coli ( ECET,

ECEI, ECEH)– Non typhoid Salmonella – Shigella– Vibrio cholerae– V. parahaemolyticus– Yersinia enterocolitica– Campylobacter

Neurologic syndrome– Clostridium botulinum

Pharynx syndrome– Streptococi group A, C, G– S. zooepidemicus

4th Case

23 years old, in last 24 hours presented 6 stools semi-consistent.

Pathologic product: feces.

Coprocitogram: ~ 20 PMN s/ field (x400)

Stool culture: present in high quantity:

- Proteus vulgaris,

- Yersinia enterocolitica.

Ethiology?

5th Case

January, 2007

2 years old patient, 12 watery stool / 24 hours.

Pathologic product: stool.

Coprocitogram: inflammatory reaction – absent (x400).

Stool culture: Proteus mirabilis in high quantity.

Latex agglutination – positive for rotaviruses.

The final conclusions !!!

Why did you made Clinical Microbiology ?

You have refreshed your memory regarding:

The main rules for sampling pathologic products;

The diagnostic methods for the most important infectious;

The interpretation of the results;The types of antibiograms and Interpretative antibiogram.

Why performin vitro susceptibility testing?

DUAL PURPOSE OF SUSCEPTIBILITY TESTING:– Individual (to guide the selection and

modification of antimicrobial therapy)– Epidemiological.

When should a susceptibilitytest be performed?

Susceptibility testing should not be routinely performed on organisms that are part of the normal bacterial flora and usually not considered pathogenic.

Establishing the need for susceptibility testing requires a close working relationship between microbiologists and clinicians.

Can susceptibility and/orresistance of bacteria to an

antibiotic be predicted?NATURAL RESISTANCE:

– permanent characteristic of the species, which is known and predictable.

ACQUIRED RESISTANCE:– characteristic of some bacterial strains, which is

evolutionary, unpredictable and justifies the need for susceptibility testing.

What criteria are used to select

the antibiotics to be tested?

The choice of antibiotics to be tested is made in relation to their therapeutic value and their usefulness to detect resistance mechanisms.

Some current resistance issues

healthcare-associated MRSA (HA-MRSA)community-associated MRSA (CA-MRSA)extended spectrum -lactamase (ESBL)

Conclusion

– Antibiotic susceptibility testing, at the interface between the diagnosis and the therapeutic decision, is a key element essential for guiding both microbiologically-documented and empiric antibiotic therapy.

– Not only is the susceptibility test result of immediate interest for the clinician to guide selection of antimicrobial therapy, but it also plays a role as an epidemiological surveillance tool for local bacterial.

Conclusion

The evolution of resistance, as well as the development of new antibiotics and laboratory techniques make a close working relationship between the microbiologist and the clinician more necessary now than ever before.

Antimicrobial Stewardship:Measuring, Auditing and

ImprovingHow to measure antimicrobial consumptionHow to audit antimicrobial prescriptions in

a hospitalLinking antimicrobial resistance to

antimicrobial exposure with patient or aggregate data

Educational interventions to improve hospital prescribing

Antimicrobial Stewardship:Measuring, Auditing and

ImprovingAudit of antibiotic surgical prophylaxisWhat are the competencies required to be a

good antibiotic prescriber Increase of antimicrobial consumption in a

medical intensive care unitSocial and behavioural approach to

antibiotic prescribing in hospitals

Thank you !