A6-year-oldgirlisbroughtintotheofficeforevaluationofasorethroatandfever,whichshehashadforapproximately4days.HerparentshaveimmigratedtotheUnitedStatesfromRussia about 6 months ago. She has not had much medical care in her life, and herimmunization status is unknown.On examination the child is anxious, tachypneic, and illappearing.Hertemperatureis38.6°C(101.5°F),andhervoiceishoarse.Examinationofherpharynx reveals tonsillar and pharyngeal edema with the presence of a gray membranecoatingofthetonsil,whichextendsovertheuvulaandsoftpalate.Shehasprominentcervicaladenopathy.Herlungsareclear.

1. Whatisthesuspecteddiagnosisforthispatient?2. What Gram stain characteristics does Corynebacterium diphtheriae have on

microscopy?3. Whatfactorisrequiredfortheexpressionofdiphtheriatoxin?4. The mechanism of action of the exotoxin produced by C. diphtheriae can be

characterizedbywhichofthefollowing?a. Acting as a superantigen that binds toMHC class II protein and the T-cell

receptor.b. Blockingthereleaseofacetylcholinecausinganticholinergicsymptoms.c. Blockingthereleaseofglycine(inhibitoryneurotransmitter).d. InhibitsproteinsynthesisviaEF-2adenosinediphosphate(ADP)ribosylation.e. StimulationofadenylatecyclasebyADPribosylationofG-protein.

5. Which of the following most accurately describes the therapy available for thepreventionandtreatmentofC.diphtheriae?

a. Antimicrobialtherapyforprophylaxisonlyb. Antimicrobialtherapyandprophylaxis,antitoxin,andtoxoid(DPT)c. Antitoxinonlyd. Diphtheriatoxoid(DPT)boostervaccinationonly

A10-year-oldgirlwithanincompletevaccinationhistorypresentstoherpediatricianwithafeverof101.5°F,sorethroat,malaise,anddifficultybreathing.Physicalexaminationrevealscervical lymphadenopathyandagray, leatheryexudateintherearoftheoropharynx.Thearea bleeds profusely when disturbed with a tongue depressor. Which of the followingcorrectlydescribesthecausalagent?

a. Gram-negativerod;toxinthatinhibitsproteinsynthesisb. Gram-negativerod;toxinthatincreasescAMPc. Gram-positiveaerobicrod;toxinthatinhibitsproteinsynthesisd. Gram-positiveanaerobicrod;toxinthatinhibitsproteinsynthesise. Gram-positiveaerobicrod;toxinthatincreasescAMP

Ayoungboy,9yearsofage,isoutsideplayinginthesummerinTexasandstepsonaboardwitharustynailinit.Thenailgoesrightthroughhisgymshoeandentershisrightfoot.Hedoesnottellhisparentsaboutitbecauseheissurehismotherwillyellathimbecausesheisalwaystellinghimnottodowhathejustdid.Heisalsoafraidshewillcurtailhisplayingoutsideprivileges.Besides,itdidnotbleedmuchandthebleedingstoppedbeforehewent

inforsupper.Besides,theboy’sfamilydidnotbelieveingoingtothedoctorforeverylittlething.Infact,theboycannotrememberthelasttimehesawadoctor.Aboutaweeklater,theboydevelopedasorethroatandthen4dayslaterhisparentsdidtakehimtothehospital(reluctantly)withdifficultyinswallowing,talking,andbreathing.Also,theboybegantoexperiencemusclespasms.Thehospitaldoctorrecognizedthesignsoftetanusandimmediatelyadministeredtetanusimmuneglobulin.Thedoctoraskedtheparentswhenthelasttimetheboyhadreceivedatetanusshotandtheyrepliedthattheydidnotknow.Afterbeinginthehospitalforaweek,theboyunfortunatelydiedofrespiratoryfailure.Thisunfortunateincidentoccurredbecausewhichofthefollowingfactsrepresentsthebestanswer?

a.Thecausativeorganism,Clostridiumtetani,isastrictanaerobeb.Thecausativeorganism,Clostridiumtetani,isastrictaerobec.Thecausativeorganism,Clostridiumtetani,producesapotentheat-labileneurotoxind.Thecausativeorganism,Clostridiumtetani,isasporeproducer,astrictanaerobe,andproducesapotentheat-labileneurotoxin

e.Thecausativeorganism,Clostridiumtetani,isasporeformer,astrictaerobe,andproducesapotentheat-labileneurotoxin

A6-year-oldgirlinRussiadevelopedasorethroatandwastakentothedoctorbyherparents.Thedoctordiagnoseda“strepthroat”andgaveherashotofpenicillin.Thepenicillinshotdidnothelp,andthechild’shealthworsenedandshewasbroughtbacktothedoctor.Nowthechildcomplainedofmorethanasorethroat.Nowsherefusedtoeatandwasverylethargic.Shealsohadafeverof40°C.Whenthedoctorreexaminedthechild,heobservedanormalchestsound,aproductivepharyngitis,andinflamedcervicallymphnodes.AthroatculturedidnotrevealanyGroupAstreptococci,andthechildwasbecomingincreasinglylethargic.Thedoctorthennoticedastructureinthebackofthechild’sthroatthatlookedlikealeathermembrane.Theparentstoldthedoctorwhenheaskedthatthegirlhadreceivednovaccinations.Thedoctorthenknewwhatdiseasehewasobserving.Theorganismmostlikelytobethecausativeagentofthisinfectionwaswhichofthefollowing?

a.Bacillusanthracisb.Clostridiumbotulinumc.Clostridiumperfringensd.Clostridiumtetanie.Corynebacteriumdiphtheriae

206

Section II l Microbiology

GENUS: CLOSTRIDIUMGenus Features

l Gram-positive rods

l Spore forming

l Anaerobic

Species of Medical Importance

l Clostridium tetani

l Clostridium botulinum

l Clostridium perfringens

l Clostridium difficile

Clostridium tetaniDistinguishing Features

l Large gram-positive, spore-forming rods

l Anaerobes

l Produces tetanus toxin

Reservoirsoil

Transmission

l Puncture wounds/trauma (human bites)

l Requires low tissue oxygenation (Eh)

Pathogenesis

l Spores germinate in the tissues, producing tetanus toxin (an exotoxin also called tetanospasmin).

l Carried intra-axonally to CNS

l Binds to ganglioside receptors

l Blocks release of inhibitory mediators (glycine and GABA) at spinal synapses

l Excitatory neurons are unopposed → extreme muscle spasm

l One of the most toxic substances known

Diseasetetanus

l Risus sardonicus

l Opisthotonus

l Extreme muscle spasms

Key Vignette CluesClostridium tetani

l Dirty puncture wound

l Rigid paralysis

edically Important Bacteria

207

Diagnosisprimarily a clinical diagnosis; organism is rarely isolated.

Treatment of Actual Tetanus

l Hyperimmune human globulin (TIG) to neutralize toxin plus metronida-zole or penicillin

l Spasmolytic drugs (diazepam); debride; delay closure

Prevention

l Toxoid is formaldehyde-inactivated toxin.

l Important because disinfectants have poor sporicidal action

l Care of wounds: proper wound cleansing and care plus treatment

Table II-2-14. Wound Management

Patient Not Tetanus Prone Tetanus Prone

Linear, 1 cm deep cut, without devitalized tissue, without major contami-nants, less than 6 hours old

Blunt/missile, burn, frostbite, 1 cm deep; devitalized tissue present + contaminants (e.g., dirt, saliva); any wound 6 hours old

Not completed primary or vaccination history unknown

Vaccine Vaccine and TIG*

Completed primary series Vaccine if more than 10 years since last booster

Vaccine if more than 5 years since last booster

*TIG = tetanus immunoglobulin (human).

Clostridium botulinumDistinguishing Features

l Anaerobic

l Gram-positive spore-forming rods

Reservoirsoil/dust

Transmissionfoodborne/traumatic implantation

Pathogenesis

l Spores survive in soil and dust; germinate in moist, warm, nutritious but nonacidic and anaerobic conditions

l Botulinum toxin

− A-B polypeptide neurotoxin (actually a series of 7 antigenically different; type A and B most common)

− Coded for by a prophage (lysogenized Clostridium botulinum).

− Highly toxic

− Heat labile (unlike staph), 10 minutes 60.0°C

− Mechanism of action

º Absorbed by gut and carried by blood to peripheral nerve synapses

º Blocks release of acetylcholine at the myoneuronal junction resulting in a reversible flaccid paralysis

Key Vignette CluesClostridium botulinum

l Home-canned alkaline vegetables

l Floppy baby syndrome (infant with flaccid paralysis)

l Reversible flaccid paralysis

208

Section II l Microbiology

Disease(s)

Table II-2-15. Forms of Botulism

Disease Adult Infant

Acquisition Preformed toxin ingested (toxicosis)

Poorly canned alkaline vegetables (green beans), smoked fish

Spores ingested: house-hold dust, honey

Toxin produced in gut (toxi-infection)

Symptoms 1–2 day onset of weakness, dizziness, blurred vision, flaccid paralysis (reversible); ± diarrhea, nausea or vomiting

Constipation, limpness/flaccid paralysis (revers-ible): diplopia, dysphagia, weak feeding/crying; may lead to respiratory arrest

Toxin demon-strated in

Suspected food or serum Stool or serum

Treatment Respiratory support

Trivalent (A-B-E) antitoxin

Respiratory support in monitored intensive care; hyperimmune human serum

Antibiotics generally not used as may worsen or prolong

Prevention Proper canning; heat all canned foods

No honey first year

Clostridium perfringensDistinguishing Features

l Large gram-positive, spore-forming rods (spores rare in tissue), nonmotile

l Anaerobic: “stormy fermentation” in milk media

l Double zone of hemolysis

Reservoirsoil and human colon

Transmissionfoodborne and traumatic implantation

Key Vignette CluesClostridium perfringens

l Contaminated wound

l Pain, edema, gas, fever, tachycardia

l Food poisoning: reheated meats, non-inflammatory diarrhea

Chapter 2 l Medically Important Bacteria

209

Pathogenesisl Spores germinate under anaerobic conditions in tissue.

l Vegetative cells produce:

– Alpha toxin (phospholipase C) is a lecithinase. It disrupts membranes, damaging RBCs, platelets, WBCs, endothelial cells → massive hemolysis, tissue destruction, hepatic toxicity.

l Identified by Nagler reaction: egg yolk agar plate—one side with anti- α-toxin; lecithinase activity is detected on side with no antitoxin.

l Twelve other toxins damage tissues.

l Enterotoxin produced in intestines in food poisoning: disrupts ion trans-port → watery diarrhea, cramps (similar to E. coli); resolution <24 hours.

Disease(s)

l Gas gangrene (myonecrosis)

– Contamination of wound with soil or feces

– Acute and increasing pain at wound site

– Tense tissue (edema, gas) and exudate

– Systemic symptoms include fever and tachycardia (disproportionate to fever), diaphoresis, pallor, etc.

– Rapid, high mortality

l Food poisoning

– Reheated meat dishes, organism grows to high numbers; 8–24 hour incubation

– Enterotoxin production in gut; self-limiting noninflammatory, watery diarrhea

Diagnosis—clinical

Treatment

l Gangrene

– Debridement, delayed closure, clindamycin and penicillin, hyperbaric chamber

l Food poisoning

– Self-limiting

Prevention—extensive debridement of the wound plus administration of penicillin.

210

Section II l Microbiology

Clostridium difficileReservoir—human colon/gastrointestinal tract

Transmission—endogenous

Pathogenesis

l Toxin A: enterotoxin damaging mucosa leading to fluid increase; granulocyte attractant

l Toxin B: cytotoxin: cytopathic

Disease(s)—antibiotic-associated (clindamycin, cephalosporins, amoxicillin, ampicillin) diarrhea, colitis, or pseudomembranous colitis (yellow plaques on colon)

Diagnosis

l Culture is not diagnostic because organism is part of normal flora

l Stool exam for toxin production

Treatment

l Severe diseasemetronidazole: use vancomycin only if no other drug avail-able; to avoid selecting for vancomycin-resistant normal flora

l Mild diseasediscontinue other antibiotic therapy

Prevention

l Caution in overprescribing broad-spectrum antibiotics (limited-spectrum drugs should be considered first)

l In the nursing home setting, patients who are symptomatic should be iso-lated.

l Autoclave bed pans (treatment kills spores)

Key Vignette CluesClostridium difficile

l Hospitalized patient on antibiotics

l Develops colitis, diarrhea

Chapter 2 l Medically Important Bacteria

213

GENUS: CORYNEBACTERIUMGenus Features

l Gram-positive rods

l Non−spore forming

l Aerobic

Species of Medical Importance

l Corynebacterium diphtheriae

l Diphtheroids (normal flora)

Corynebacterium diphtheriaeDistinguishing Features

l Gray-to-black colonies of club-shaped gram-positive rods arranged in V or L shapes on Gram stain

l Granules (volutin) produced on Loeffler coagulated serum medium stain metachromatically

l Aerobic, non−spore forming

l Toxin-producing strains have β-prophage carrying genes for the toxin (lysogeny, β-corynephage). The phage from one patient with diphtheria can infect the normal nontoxigenic diphtheroid of another person and cause diphtheria.

Reservoirthroat and nasopharynx

Transmissionbacterium or phage via respiratory droplets

Pathogenesis

l Organism not invasive; colonizes epithelium of oropharynx or skin in cuta-neous diphtheria

l Diphtheria toxin (A-B component)—inhibits protein synthesis by adding ADP-ribose to eEF-2

l Effect on oropharynx: Dirty gray pseudomembrane (made up of dead cells and fibrin exudate, bacterial pigment)

l Extension into larynx/trachea → obstruction

l Effect of systemic circulation → heart and nerve damage

Disease—diphtheria (sore throat with pseudomembrane, bull neck, potential respira-tory obstruction, myocarditis, cardiac dysfunction, recurrent laryngeal nerve palsy, and lower limb polyneuritis)

Diagnosis

l Elek test to document toxin production (ELISA for toxin is now the frontline)

l Toxin produced by toxin-producing strains diffuses away from growth.

l Antitoxin diffuses away from the strip of filter paper.

l Precipitin lines form at zone of equivalence.

Key Vignette CluesCorynebacterium diphtheriae

l Gram (+), aerobic, non−spore forming rods

l Bull neck, myocarditis, nerve palsies

l Gray pseudomembrane → airway obstruction

l Toxin produced by lysogeny

l Toxin ribosylates eEF-2; heart, nerve damage

214

Section II l Microbiology

Filter paper strip with C. diphtheriae antitoxin

Precipitin line

Known toxigenic C. diphtheriae

Known nontoxigenic C. diphtheriae

Unknown (pt's isolate)

Figure II-2-6. Elek Test

Treatment

l Erythromycin and antitoxin

l For endocarditis, intravenous penicillin and aminoglycosides for 4–6 weeks

Preventiontoxoid vaccine (formaldehyde-modified toxin is still immunogenic but with reduced toxicity), part of DTaP, DTP, or Td, boosters 10-year intervals

• Introduction

• Organisms and epidemiology

• Specific issues associated with the chronic leg ulcer swab

• Primary care

• The patient

• Collecting the specimen

• The request form

• The laboratory report

• Treatment

• Summary and key points

5

The Swab of theChronic Leg Ulcer

85

Medical Microbiology Testing in Primary Care 86

key to management; staff must be trained to knowwhen it is appropriate to take a specimen.Inappropriate collection of samples should not beaccepted; this is a quality issue because it is wastefulof resources, and can be potentially harmful topatients.

The most common bacteria causing infectionare Staphylococcus aureus and β-haemolyticstreptococci. Pseudomonas aeruginosa is frequentlyfound in chronic ulcers, but is rarely the cause ofinfection. However, it is able to impair host defencesby inducing inappropriate or premature apoptosisin macrophages and neutrophils.2, 3 It interferes withhost defences, which delays healing. On occasion,less common but important pathogens are isolatedfrom an ulcer swab. Pasteurella multocida is oneexample; this gram-negative bacillus which is acommensal in the mouth of kittens and cats, isoccasionally identified. The likely source is a kittenor cat of the elderly owner licking/desloughing theulcer, at some point inoculating it with thepathogen! In addition to cellulitis, life-threateningsystemic infection can occur.

Wound swabs account for about 10% of thebacteriology specimens received from primarycare, and unlike urine and genital swab specimens,there is no significant bias in terms of sex.

INTRODUCTIONThis chapter is primarily concerned with the‘wound swab’ collected from the chronic leg ulcer.The key points here are:1. Knowing when to swab an ulcer.2. How to swab an ulcer.3. Interpreting the laboratory report.Figure 86 highlight these points, and with the QuickAction Guides can be used as a training tool tohighlight the critical steps in the collection of aswab from an ulcer.1 The principles that arediscussed here apply to taking a swab from post-surgical wounds.

ORGANISMS AND EPIDEMIOLOGYA list of the common bacteria that are associatedwith ulcers is shown in 87 (page 90). A wide rangeof organisms can be grown from ulcers, many ofwhich are unlikely to cause infection. However,even the isolation of a well-recognized pathogensuch as Staphylococcus aureus from a wound doesnot mean it is causing infection. An organism caneither colonize a wound surface, being residentthere, or causing infection, to which the bodymounts an inflammatory response. Distinctionbetween the two requires a clinical assessment ofthe wound/ulcer to determine if there is evidence ofinfection. Hence critical step 1 in the Introduction is

86 The chronic leg ulcer swab. An example where a swab is taken where there is a clear evidence of infection. The steps in theprocess can be divided into seven critical steps (see Chapter 1, 1). In this scenario, the chronic leg ulcer has surrounding painful cellulitis, and a swab should be taken. The primary care nurse carefullywashes the ulcer, and removes some slough to obtain a sample adjacent to viable tissue. The specimen grows group A streptococcus, andthe result is telephoned by the laboratory. The primary care physician telephones the patient, who relates that after 24 hours of treatment,the pain and redness is not settling. On discussion with the microbiologist, the decision is made to change to doxycycline, as the organismmay be resistant to macrolides; this is confirmed the next day with the susceptibility result. In this example, there was correctmanagement from the outset through to critical step 7, where assessment of the patient after review of the laboratory report promptedthe appropriate change of treatment.

Storage and transport of the routine swab from an ulcer or woundBuffered semi-solid charcoal ‘transport swabs’ are usually used. The environment in these ‘transport swabs’ is such that it canmaintain the viability of both aerobes and anaerobes for several days, without allowing their multiplication. The swab should be wellloaded with as much of the appropriate sample as possible. If there is a delay in transport to the laboratory, the specimen should berefrigerated; viable organisms can be recovered for up to 72 hours at least. Note that if there is pus present/likely to be present, thisshould be collected as pus, rather than a swab of the fluid. Any pus should be carefully aspirated, and placed in a sterile white-toppedcontainer. If there is a delay in transport, the sample should be refrigerated.

The Swab of the Chronic Leg Ulcer 87

CRITICAL STEP 1:Is collection of a specimen indicated?

The Practice team collects a swab from an ulcer of a 75-year-old patient. The skin bordering the ulcer is red and painful. As thepatient is penicillin-allergic, a prescription for erythromycin 500 mg q6h and analgesia, is written up.

CRITICAL STEP 2:Obtain a good quality specimen.

The nurse carefully washes the ulcer with saline to remove surface contaminants and slough with a swab to reveal viable tissue. Afresh swab is taken from this area.

CRITICAL STEP 3:

Record the full patient andclinical details and type ofspecimen on the requestform. Where necessary,record the specific test

request.

CRITICAL STEP 4:

How is the specimenstored and what is the

frequency of transport tothe laboratory?

CRITICAL STEP 5:

What is the quality of thelaboratory process,

including turnaroundtimes?

CRITICAL STEP 6:

Is immediate or routinereporting of this result

required by thelaboratory?

86

Report on AB, 75-year-old:Leg ulcer MC&SProfuse growth of group A streptococcus:Penicillin SErythromycin RTetracycline S

Phoned report from microbiology: Profuse growth of group A streptococcus;Sensitivities to follow

Primary care physician telephones the patient. The patientreports that the ulcer is still painful, and there is noreduction in the redness. On discussion with themicrobiologist, doxycycline is prescribed. This change isconfirmed with the susceptibility profile available the next day

CRITICAL STEP 7: How is the laboratory report interpreted?

The result should becommunicated to thepractice/clinic now.

Medical Microbiology Testing in Primary Care 88

The chronic leg ulcer; reviewing the patient at the first visit.

NO YES

(Adapted in part from Venous Leg Ulcers: Infection, Diagnosis and Microbiology Investigation; Quick Reference Guide for Primary Care. www.hpa.org.uk.1

With permission of the UK Health Protection Agency.)

Quick Action Guide - 1

Do not sample ulcer (unless as part ofMRSA screening).

Collect sample from the infected ulcer.

• Clean with sterile water or saline to remove surface bacteria.• Where possible, remove slough (and necrotic material) with a swab (discard).• Sample an area of exposed viable tissue displaying evidence of infection,

rotating the swab here to load it well.

Review previous culture results on the patient before determining antibioticprescription. For example, has the patient had MRSA in the (recent) past? If not

consider empirical treatment with either flucloxacillin or a macrolide.

Increased painEnlarging ulcerIncreased exudateA new sinus

CellulitisFeverLoss of diabetic controlIncreased or new confusion

IS THE PATIENT’S LEG ULCER INFECTED?

The Swab of the Chronic Leg Ulcer 89

The chronic leg ulcer; reviewing the laboratory report, with the reason for the swab being taken.

YES

Laboratory culture report received.

You are asked to review the report on a patientof a staff partner:

This is your patient; the ulcer was infectedwhen the swab was collected.

(This Quick Action Guide is adapted, in part, from Venous Leg Ulcers: Infection, Diagnosis and Microbiology Investigation; Quick Reference Guide for Primary Care.

www.hpa.org.uk.1 With permission of the UK Health Protection Agency.)

Quick Action Guide - 2

• Check the relevance of organism(s), e.g. MSSA, group A streptococcus versuspseudomonas, skin flora, faecal flora.

• Check antibiotic susceptibility profile wherereported, e.g. erythromycin was prescribed. Forexample, if a group A streptococcus isreported is it erythromycin sensitive?

• Review the patient to determine the responseto treatment; consider suitable alternativeantibiotic(s).

• MRSA has been isolated, but flucloxacillin wasprescribed at the first visit.

Review the patient:• Give patient advice about MRSA, and

necessary actions.• Good response to flucloxacillin: complete

course.• No improvement/deteriorating: stop

flucloxacillin; consider doxycycline plusrifampicin and discuss with themicrobiology/infectious diseases physician.

NO

Was there evidence that the ulcer was infectedwhen the swab was taken?

• If there is no evidence of infection, do notprescribe antibiotics, especially on the basis of thereport alone. The exception is ‘A equals always’for group A streptococcus.

• Review criteria for collecting swabs.

Relatively more swabs are received from individualsover 50 years of age (88). Clinical details include‘Leg ulcer-? infected’, (this in itself indicates theswab was taken inappropriately), ‘Leg ulcer+++slough’, ‘Infected post operation wound’, and‘Paronychia’. Swabs taken from chronic leg ulcerscan account for over 20% of wound swabssubmitted to the laboratory, based on the clinicaldetails recorded on the request form.

SPECIFIC ISSUES ASSOCIATED WITH THECHRONIC LEG ULCER SWABColonization versus infectionAs highlighted in Chapter 1, differentiatingcolonization from infection is fundamental inproviding correct patient care. This is particularlyimportant in primary care, where apart from theoccasional specimen collected from a ‘true sterilesite’ such as a bursa, all others, with the exception

Medical Microbiology Testing in Primary Care 90

Commonly considered as a pathogen in an infected ulcer, when there is evidence of infection: Staphylococcus aureus (MSSA or MRSA)Group A streptococcusGroup C streptococcusGroup G streptococcus

Commonly considered as secondary colonizers, and can be important in the development of critical colonization. They canoccasionally be the causative agent:Pseudomonas aeruginosaAnaerobes/mixed anaerobes

Colonizers of ulcers with unlikely pathogenic potential:Coagulase-negative staphylococciDiphtheroidsSkin flora‘Coliforms’Escherichia coli (including ESBL-producers)

87

87 A list of the bacteria that are associated with the infected chronic leg ulcer. See Appendix, Chapter 5 for more detailed information.

88

88 The typical age and sex distribution of patients submitting wound swab specimens to the laboratory over a period of 1 week.

25

20

15

10

5

00–9 10–19 20–29 30–39 40–49 50–59 60–69 70–79 80–89 90–99

Age (years)

Num

ber o

f pat

ient

s Females

Males

of urines, are collected from sites that have, or passover, a resident bacterial flora. Careful assessmentof the chronic leg ulcer, and making a clinicaldiagnosis of whether it is infected or not is soimportant; the result of a microbiology swab cannotdetermine if there is infection.4, 5 If a swab is takenunnecessarily or incorrectly, it can initiate a chain ofevents that waste resources, and by givingantibiotics unnecessarily, can be detrimental to thepatient.

Chronic leg ulcers and pressure ulcers willalways be colonized with bacteria, as there is a richsource of nutrients supporting biofilm growth,consisting of a mixed population of aerobic,facultative, and anaerobic bacteria. At some stage,the bacterial population in the ulcer progresses to a‘critical colonization’ stage.6 The ulcer looksunhealthy, and is not healing, but there are noobvious signs of infection, either locally orsystemically. Pseudomonas aeruginosa is consideredto play a key role in critical colonization byproducing subclinical damage to tissue andconsequent delay in healing. As a result biofilm

formation, and chronic inflammation stimulated by the organism, may give rise to a Trojan horseeffect for other pathogens.7 In malodorous ulcers, anaerobes such as Bacteroides spp. andPeptostreptococcus spp. are likely to be present,and can be found in up to 30% of venous ulcers.8The critically colonized ulcer can provide theenvironment for a pathogen such as Staphylococcusaureus or group A streptococcus to initiate infectionin the ulcer base and edge. The changes fromcolonization to critical colonization then toinfection, and the modalities that can be used to reverse this, are shown in 89. The key to reducing the chance of critical colonization andthus infection in ulcers is regular washing to removedebris and bacteria. In assessment of the infectedulcer, one needs to consider the depth of infectionand whether any deep-seated structures areinvolved, such as bone which would necessitatespecialist referral. (The smell of an ulcer can also bevery useful in assessment, as a ‘rotten flesh’ smellindicates that there is underlying necrosis, anddebridement is needed).

The Swab of the Chronic Leg Ulcer 91

89

89 The chronic ulcer and the progression from colonization to infection. (a) Colonization of the ulcer. All ulcers will be colonized bybacteria. Initially various organisms such as CNS, MSSA, and pseudomonas take up residence. (b) Critical colonization of the ulcer. Herethe bacterial flora has increased to a degree that tissue viability becomes compromised. At this stage, the situation is salvageable.However, if not remedied, it can progress to infection. (c) Infection of the ulcer. Local invasion by MSSA occurs, with increased pain,purulent exudate, and surrounding erythema/cellulitis. Correct swabbing samples the pathogen. The progression from the colonized ulcerto critical colonization can be prevented by regular washing with water. Antibiotics have no role here. Appropriate antibiotics givenpromptly for infection against the most likely pathogen should resolve the situation.

Washing Antibiotics

Slough

Inflammation

(b) (c)(a)

PRIMARY CAREThe key issue in primary care is to have definedguidelines for the care of the patient with chronicleg ulcers, developed in conjunction with thecommunity tissue viability nurse. These guidelinesshould detail all the aspects of care that can preventinfection in the ulcer, including ulcer washing atregular intervals, and close monitoring of bloodglucose in the diabetic patient. There should beclear protocols for when and how to collect swabs,and for interpreting the laboratory report. Ofoverriding importance is that staff receive ongoingtraining otherwise such protocols become yetanother policy that gathers dust on the shelf.

THE PATIENTThe patient with chronic leg ulcers is usually older,and will often have other co-morbidities such asdiabetes and/or peripheral vascular disease. Withdiabetes, the reaction to infection may be modified,with neuropathy, ischaemia, and a reduced painresponse making assessment more difficult.Monitoring full blood count (FBC), C-reactiveprotein (CRP), and blood glucose can help indecision making. There should be close cooperationwith the community tissue viability nurse, thediabetic physician and dermatologist, with aprompt referral system to manage the patient whois not responding to standard treatment. Thevascular surgeons would be involved as needed. Inorder to reduce the ‘critical colonization’ stage andthus infection, regular washing of the ulcer shouldbe done. A litre of water can be boiled, and when ithas cooled, one heaped tablespoon of aqueouscream is mixed into this. The use of aqueous creamis important as it moistens the surrounding skin; dryscaly skin is more likely to be colonized withStaphylococcus aureus.

Not infrequently a patient is diagnosed ashaving bilateral cellulitis. True bilateral cellulitis isuncommon, and conditions such as venoushypertension, or varicose eczema, with associatedswelling and skin discolouration, should beconsidered.9 This can be improved by leg elevation,compression bandages, and ankle exercises.Increased nonpurulent exudate from ulcers can alsobe mistaken as infection, and in addition to theinterventions above, increasing the frequency ofwashing the affected limb(s), and of dressingchanges is useful. Cellulitis can also be confusedwith an allergic reaction when there is erythemalocalized to, and taking the shape of the dressing;this points to an allergic reaction.9 The likely causeshould be removed, and ‘patch testing’ done asrequired. Beware of the patient with suchconditions who has true cellulitis of one leg.

COLLECTING THE SPECIMEN When infection is identified, a specimen should becollected using care to choose the area likely tocontain the probable pathogen. A swab collectedfrom the superficial layer of the ulcer, where sloughor exudate is present is of little use, as the bacteriaidentified may not represent the organismresponsible for the infection. The ulcer area shouldbe washed first with tap water or saline, in order todetach any surface material, such as slough andnecrotic material, thus reducing the likelihood ofsampling surface contaminants.10, 11 Where possible,a swab should be used to remove this material, inorder to expose viable tissue. A fresh swab shouldthen collect the sample from the infected area,rotating it to maximize the loading (90).

Medical Microbiology Testing in Primary Care 92

The Swab of the Chronic Leg Ulcer 93

90

90 (a) Sampling the surface slough of an ulcer will produce a report showing a profuse growth of pseudomonas. A decision to treat withoral ciprofloxacin is incorrect, and puts the patient at risk of CDAD. (b) Washing with sterile saline, followed by careful removal of theslough obtains a deeper sample, which grows group A streptococcus, the pathogen.

Skin flora

Group A streptococcus

Pseudomonas aeruginosa

+++ Pseudomonas aeruginosa

+++ Group A streptococcus

(b)

(a)

THE REQUEST FORMThe relevant clinical details should be included on the request form. If methicillin-resistantStaphylococcus aureus (MRSA) screening only isrequired, this information should be clearly visibleto the laboratory staff. In order to readily identifyMRSA, a selective agar medium is used thatprevents the growth of most other bacteria,including other pathogens; the standardmicroscopy, culture, and sensitivity (MC&S) cannotbe done. Any relevant history of travel, exposure towater (fresh or sea) should be given. While Vibriovulnificus is often a rapidly progressing infection,other marine vibrios, such as Vibrio alginolyticuscan also cause skin and soft tissue infection. Thisinformation is a useful guide for the laboratorystaff.

THE LABORATORY REPORTThe key point when reviewing the report is firstly toconfirm if the swab was taken for the correctreason, i.e. clinical evidence of infection. Only if this

is the case should one proceed to correlate this withthe bacteria reported and the antibiotics prescribed.Quick Action Guide 2 should be referred to.

Interpreting a report can be difficult if theprimary care physician has not seen the patient,unless clear clinical reasons have been recorded inthe patient’s record. As a minimum, assessingwhether there were signs of infection, and theappropriateness of the antibiotics should beconfirmed; 91 gives guidance. If the result showedthat methicillin-sensitive Staphylococcus aureus(MSSA), group A, group C, or group Gstreptococcus was isolated, and flucloxacillin wasgiven, all four bacteria would be susceptible to thisagent. If MRSA was isolated, prompt review of thepatient would be needed. The infection may in facthave improved, with the flucloxacillin treating asusceptible pathogen, indicating that MRSA was acolonizer! If there is no improvement, doxycyclinecan be given to treat the MRSA; rifampicin can beadded as a second agent. (Also see Chapter 2, 33,34).

Medical Microbiology Testing in Primary Care 94

91 The laboratory will report a limited number of antibiotics for organisms such as MSSA, MRSA, and streptococci. The appropriateness ofthe antibiotic prescribed should be reviewed with the information in this Figure, which shows the susceptibility of the organism to otheragents as well (See Chapter 2, 33 and 34). In the diagram, left panels are the laboratory report showing the susceptibility of the organism,and right (arrowed) panels other agents to which the organism will either be sensitive or resistant.

(a) With MSSA, flucloxacillin, erythromycin, and tetracycline should be reported by the laboratory as a minimum. *Co-amoxiclav is broader spectrum than flucloxacillin, and for an infected leg ulcer only, it would be reasonable determine why this wasprescribed, and to consider changing the treatment to flucloxacillin. **A cephalosporin, such as cefalexin, can be given in the penicillin-allergic patient, when the nature of the allergy allows the use of acephalosporin. There is a CDAD risk to consider, but this is a useful option, when for example the organism is resistant to botherythromycin and tetracycline, and when the benefit is considered to outweigh the risk. ***This includes the other macrolides. ****This includes the other tetracyclines. (‼: Note that some laboratories test, and then report clindamycin separately for Staphylococcus aureus; a proportion of organisms thatare resistant to erythromycin are clindamycin sensitive.)

(b) With MRSA, flucloxacillin, erythromycin and tetracycline should be reported as a minimum. If the organism is resistant to erythromycinand/or tetracycline, the laboratory should ‘release’ another antibiotic, such as trimethoprim or co-trimoxazole (TMP - SMX). *This includes the other macrolides. **This includes the other tetracyclines. (‼: Note that some laboratories test, and then report clindamycin separately for Staphylococcus aureus; a proportion of organisms thatare resistant to erythromycin are clindamycin sensitive.)

(c) For group A, C, and G streptococci, penicillin, erythromycin, and tetracycline should be reported by the laboratory. *Co-amoxiclav is broader spectrum than amoxicillin (and flucloxacillin), and for an infected leg ulcer only, it would be reasonable todetermine why this was prescribed, and to consider changing the treatment to amoxicillin. **A cephalosporin, such as cefalexin, can be given in the penicillin-allergic patient, when the nature of the allergy allows the use of acephalosporin. There is a CDAD risk to consider, but this is a useful option, when for example the organism is resistant to botherythromycin and tetracycline, and when the benefit is considered to outweigh the risk. ***This includes the other macrolides. ****This includes the other tetracyclines.

The Swab of the Chronic Leg Ulcer 95

91

Flucloxacillin

Erythromycin

Erythromycin

Tetracycline

Tetracycline

Clarithromycin***

Clarithromycin***

Doxycycline****

Doxycycline****

Co-amoxiclav* Cefalexin**

Clindamycin

!! Clindamycin

Flucloxacillin

Penicillin

Erythromycin

Erythromycin

Tetracycline

Tetracycline

Erythromycin

Erythromycin

Tetracycline

Tetracycline

Clarithromycin*

Clarithromycin*

Doxycycline**

Doxycycline**

Amoxicillin Flucloxacillin Co-amoxiclav* Cefalexin**

Clindamycin

Clindamycin

Clarithromycin***

Clarithromycin***

Doxycycline****

Doxycycline****

Co-amoxiclav Cefalexin

Clindamycin

!! Clindamycin

Sensitive to the antibiotic(a) MSSA

(b) MRSA

(c) Streptococci A,C,G

Resistant to the antibiotic

If the report states growth of a ‘coliforms’,pseudomonas, or faecal flora, and the infection hasimproved, for example with flucloxacillin, there isno need to take further action. It is likely that theantibiotic has treated an underlying MSSA or β-haemolytic streptococcus, which was not sampledand subsequently cultured because of poorcollection. If there is no improvement, appropriateresampling of the ulcer would be indicated as wellas discussing any empirical treatment pending theresult with a medical microbiologist/infectiousdiseases physician.

TREATMENTThe Antibiotic Guidelines in Chapter 11 gives moredetails of common antibiotic regimes used in thecommunity. It is useful to refer to 91 again, as thisdiscusses further options to consider in thepenicillin-allergic patient, based on the antibioticsusceptibilities reported.

SUMMARY AND KEY POINTSThis chapter is mainly concerned with the woundswab in the setting of chronic leg ulcers. The sameprinciples of colonization and infection apply topost-surgical wounds, with specimen collection andtreatment only undertaken when there is evidenceof infection. Following cardiothoracic, orthopaedic,or neurosurgical procedures, it is important tocontact the specialist team promptly by telephone ifthe patient has evidence of anything more than aminor infection at the operation site.

In summary, the following points must be noted:• The practice/clinic should establish a set of

guidelines for clinical and nursing staff to useso that there are set criteria, centred on QuickAction Guides and the critical steps of 86.

• These should be used regularly as a trainingtool to give guidance on when and how tocollect specimens from the various patient agegroups, storage and transport of specimens,and how the laboratory report should beinterpreted.

• It is essential to be familiar with the principlesof colonization and infection. Sampling anysuperficial wound should only be done wherethere is clear evidence of infection. Highlightingthis will save valuable resources in thepractice/clinic and laboratory. (Essentially theonly other requirement for swabbing is MRSAscreening pre-hospital admission.)

• The laboratory report must be interpreted inlight of the organisms identified, and thusdistinguish likely pathogens from bacteriacolonizing the ulcer. This will preventunnecessary and incorrect antibioticprescribing.

• Patients who are MRSA-positive should be toldto inform staff at any other health-care centrethey may attend of their MRSA status.

Medical Microbiology Testing in Primary Care 96