NATIONAL STANDARD METHOD

INVESTIGATION
OF ABSCESSES AND
DEEP-SEATED WOUND
INFECTIONS
BSOP 14

Issued by Standards Unit, Department for Evaluations, Standards and Training

Centre for Infections

INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS

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STATUS OF NATIONAL STANDARD METHODS
National Standard Methods, which include standard operating procedures (SOPs), algorithms and
guidance notes, promote high quality practices and help to assure the comparability of diagnostic
information obtained in different laboratories. This in turn facilitates standardisation of surveillance
underpinned by research, development and audit and promotes public health and patient confidence
in their healthcare services. The methods are well referenced and represent a good minimum
standard for clinical and public health microbiology. However, in using National Standard Methods,
laboratories should take account of local requirements and may need to undertake additional
investigations. The methods also provide a reference point for method development.

National Standard Methods are developed, reviewed and updated through an open and wide
consultation process where the views of all participants are considered and the resulting documents
reflect the majority agreement of contributors.

Representatives of several professional organisations, including those whose logos appear on the
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member. The current list of participating organisations can be obtained by emailing
[email protected].

The performance of standard methods depends on the quality of reagents, equipment, commercial
and in-house test procedures. Laboratories should ensure that these have been validated and shown
to be fit for purpose. Internal and external quality assurance procedures should also be in place.

Whereas every care has been taken in the preparation of this publication, the Health Protection
Agency or any supporting organisation cannot be responsible for the accuracy of any statement or
representation made or the consequences arising from the use of or alteration to any information
contained in it. These procedures are intended solely as a general resource for practising
professionals in the field, operating in the UK, and specialist advice should be obtained where
necessary. If you make any changes to this publication, it must be made clear where changes have
been made to the original document. The Health Protection Agency (HPA) should at all times be
acknowledged.

The HPA is an independent organisation dedicated to protecting people’s health. It brings together
the expertise formerly in a number of official organisations. More information about the HPA can be
found at www.hpa.org.uk.

The HPA aims to be a fully Caldicott compliant organisation. It seeks to take every possible
precaution to prevent unauthorised disclosure of patient details and to ensure that patient-related
records are kept under secure conditions1.

More details can be found on the website at www.evaluations-standards.org.uk. Contributions to the

development of the documents can be made by contacting [email protected].

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Suggested citation for this document:

Health Protection Agency (2009). Investigation of abscesses and deep-seated wound infections.
National Standard Method BSOP 14 Issue 5.
http://www.hpa.org.uk/srmd/div_esl_su/pdf_bacteriology.htm.

INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS

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INDEX

STATUS OF NATIONAL STANDARD METHODS ................................................................................. 2

INDEX...................................................................................................................................................... 3

AMENDMENT PROCEDURE ................................................................................................................. 4

SCOPE OF DOCUMENT ........................................................................................................................ 5

INTRODUCTION ..................................................................................................................................... 5

TECHNICAL INFORMATION/LIMITATIONS .......................................................................................... 5

1 SAFETY CONSIDERATIONS ......................................................................................................... 5

1.1 SPECIMEN COLLECTION ............................................................................................................... 5
1.2 SPECIMEN TRANSPORTATION AND STORAGE ................................................................................. 5
1.3 SPECIMEN PROCESSING............................................................................................................... 5
2 SPECIMEN COLLECTION .............................................................................................................. 5
2.1 OPTIMAL TIME FOR SPECIMEN COLLECTION ................................................................................... 5
2.2 CORRECT SPECIMEN TYPE AND METHOD OF COLLECTION ............................................................... 5
2.3 ADEQUATE QUANTITY AND APPROPRIATE NUMBER OF SPECIMENS .................................................. 5
3 SPECIMEN TRANSPORT AND STORAGE ................................................................................... 5
3.1 TIME BETWEEN SPECIMEN COLLECTION AND PROCESSING .............................................................. 5
3.2 SPECIAL CONSIDERATIONS TO MINIMISE DETERIORATION ............................................................... 5
4 SPECIMEN PROCESSING ............................................................................................................. 5
4.1 TEST SELECTION ......................................................................................................................... 5
4.2 APPEARANCE .............................................................................................................................. 5
4.3 MICROSCOPY .............................................................................................................................. 5
4.4 CULTURE AND INVESTIGATION ...................................................................................................... 5
4.5 IDENTIFICATION ........................................................................................................................... 5
4.6 ANTIMICROBIAL SUSCEPTIBILITY TESTING ..................................................................................... 5
5 REPORTING PROCEDURE............................................................................................................ 5
5.1 MICROSCOPY .............................................................................................................................. 5
5.2 CULTURE .................................................................................................................................... 5
5.3 ANTIMICROBIAL SUSCEPTIBILITY TESTING ..................................................................................... 5
6 REPORTING TO THE HPA (LOCAL AND REGIONAL SERVICES AND CENTRE FOR
INFECTIONS)................................................................................................................................... 5

7 ACKNOWLEDGEMENTS AND CONTACTS.................................................................................. 5

APPENDIX .............................................................................................................................................. 5

REFERENCES ............................................................................ERROR! BOOKMARK NOT DEFINED.

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AMENDMENT PROCEDURE
Controlled document BSOP 14
reference
Controlled document title Investigation of abscesses and deep-seated wound infections

Each National Standard Method has an individual record of amendments. The current amendments are
listed on this page. The amendment history is available from [email protected].

On issue of revised or new pages each controlled document should be updated by the copyholder in the
laboratory.

Amendment Issue no. Insert Page Section(s) involved Amendment

Number/ Discarded Issue
Date no.
5/ 4 5 All All Department name
23.11.09 changed from ESL to
DEST

All All PDF links inserted to

cross-reference NSM
documents

All All The term “CE Marked

leak proof container”
replaces “sterile leak
proof container”;
endnotea added to
clarify the change; and
referenced to the IVD
Directive 98/79/EC.

1 Front Page NIMAG, Scottish Forum

and CMN logos added

2 Status Taxonomy sentence

inserted

12 Introduction Gangrene section

amended

17 4.4.3 Updated and amended

19 4.5.2 Links to reference

laboratory user manuals
inserted.

22 Appendix Flow chart inserted

23 References Reviewed and updated

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 INVESTIGATION OF
ABSCESSES AND DEEP-SEATED WOUND
INFECTIONS
Types of specimens: Abscess pus Post-operative wound swab
Abscess swab Deep-seated pus swab
Wound exudate

SCOPE OF DOCUMENT
This National Standard Method (NSM) describes the processing and bacteriological investigation of
specimens from abscesses and from post-operative wound and deep-seated infections.

INTRODUCTION
Abscesses are accumulations of pus in the tissues and any organism isolated from them may be of
significance. They occur in many parts of the body as superficial infections or as deep-seated infections
associated with any internal organ. Many abscesses are caused by Staphylococcus aureus alone, but
others are caused by mixed infections. Anaerobes are predominant isolates in intra-abdominal abscesses
and abscesses in the oral and anal areas. Members of the "Streptococcus anginosus" group and
Enterobacteriaceae are also frequently present in lesions at these sites.

Bartholin gland abscesses and tubo-ovarian abscesses are considered in BSOP 28 – Investigation of genital
tract and associated specimens. Processing of specimens for Mycobacterium species from, for example,
subcutaneous cold abscesses is described in BSOP 40 – Investigation of specimens for Mycobacterium
species.
Brain abscess2
Brain abscesses are serious and life-threatening.

Sources of abscess formation include:

• Direct contiguous spread from chronic otitic or paranasal sinus infection

• Metastatic haematogenous spread either from general sepsis or secondary to chronic
suppurative lung disease
• Penetrating wounds
• Surgery
• Cryptogenic (ie source unknown)

Treatment of brain abscesses involves the drainage of pus and appropriate antimicrobial therapy. Brain
stem abscesses have a poor prognosis due to their critical anatomical location3.

Bacteria isolated from brain abscesses are usually mixtures of aerobes and obligate anaerobes, and the
prevalent organism may vary depending upon geographical location, age and underlying medical conditions.
The most commonly isolated organisms include4-8:

• Anaerobic streptococci
• Anaerobic Gram-negative bacilli
• "Streptococcus anginosus" group
• Enterobacteriaceae
INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS
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 • Streptococcus pneumoniae
• β-haemolytic streptococci
• S. aureus
Organisms commonly isolated vary according to the part of the brain involved. Many other less common
organisms, for example Haemophilus species, may be isolated2,6-13. Nocardia species often exhibit
metastatic spread to the brain from the lung. Any organism isolated from a brain abscess must be regarded
as clinically significant.

Organisms causing brain abscesses following trauma may often be environmental in origin, such as
Clostridium species14 or skin derived, such as staphylococci and Propionibacterium species.

Brain abscesses due to fungi are rare. Aspergillus brain abscess can occur in patients who are neutropenic.
Zygomycosis is an uncommon opportunistic infection caused by Rhizopus and Absidia species and related
fungi. Scedosporium apiospermum (Pseudallescheria boydii) enters the lungs and spreads
haematogenously15.
Breast abscess
Breast abscesses occur in both lactating and non-lactating women. In the former infections are commonly
caused by S. aureus, but may alternatively be polymicrobial, involving anaerobes and streptococci16-18.
Signs include discharge from the nipple, swelling, oedema, firmness and erythema.
In non-lactating women a subareolar abscess forms often with an inverted or retracted nipple. Mixed
growths of anaerobes are usually isolated19. Some patients require surgery involving complete duct
excision19. Abscesses may also be caused by Pseudomonas aeruginosa and Proteus species20.
Carbuncles, furuncles, cutaneous, soft tissue and other abscesses
Carbuncles are deep and extensive subcutaneous abscesses involving several hair follicles and sebaceous
glands. Carbuncles are most often caused by S. aureus.

Furuncles are abscesses which begin in hair follicles as firm, tender, red nodules that become painful and
fluctuant. Furuncles are caused by the same pathogens as carbuncles. Recurrent staphylococcal
furunculosis is highly infectious and may be the first sign of an underlying disease such as diabetes mellitus.

Cutaneous abscesses are usually painful, tender, fluctuant erythematous nodules often with a pustule on
top. In some cases they are associated with extensive cellulitis, lymphangitis, lymphadenitis and fever.
They are caused by a variety of organisms. The location of an abscess often determines the flora likely to
be isolated. Thus S. aureus is most often isolated from cutaneous abscesses of the axillae, the extremities
and the trunk, whereas cutaneous abscesses involving the vulva and buttocks may yield faecal or urogenital
mucosal flora.

Soft tissue abscesses involve one or more tissue planes underlying the epidermis, usually developing after
trauma to the skin. They may arise from animal bites, in which case common isolates include Pasteurella
and Actinobacillus species21 as well as other organisms of the HACEK group (Haemophilus, Actinobacillus,
Cardiobacterium, Eikenella and Kingella species).

Burkholderia pseudomallei causes melioidosis, but is rare in the UK. The disease may present in a variety
of forms with skin lesions and/or cellulitis. Diagnosis is made by blood culture, serology or culture of pus
(refer to BSOP 37 – Investigation of blood culture (for organisms other than mycobacterium species)).

Pyomyositis is a purulent infection of skeletal muscle in which solitary or multiple muscle abscesses form. It
most often occurs in tropical areas, and in HIV-infected or other patients who are immunocompromised.
The main causative organism is S. aureus22,23.

Abscesses in intravenous drug users

Cutaneous abscesses frequently occur as a complication of injecting drug use. They commonly result
INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS
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from the use of non-sterile solutions in which the drug is dissolved or from lubrication of the needle using
saliva.

Common bacterial isolates include24:

• Oral streptococci
• Streptococcus anginosus group
• Fusobacterium nucleatum
• Prevotella species
• Porphyromonas species
• Staphylococcus aureus
• Clostridium species

Dental abscess
Dental abscesses involve microorganisms colonising the teeth that may become responsible for oral and
dental infections, leading to dentoalveolar abscesses and associated diseases. They may also occur as a
direct result of trauma or surgery.

Periodontal disease involves the gingiva and underlying connective tissue25, and infection may result in
gingivitis or periodontitis.

Organisms most commonly isolated in acute dentoalveolar abscesses are facultative or strict anaerobes.
The most frequently isolated organisms are anaerobic Gram-negative rods, however other organisms have
also been isolated. Examples include23,25-28:

• α-haemolytic streptococci
• Anaerobic Gram-negative bacilli
• Anaerobic streptococci
• "S. anginosus" group
• Actinobacillus actinomycetemcomitans
• Spirochaetes
• Actinomyces species

Aspiration of dental abscesses is necessary to obtain samples containing the likely causative organisms.
Swabs are likely to be contaminated with superficial commensal flora.
Liver abscess
Liver abscesses can be amoebic or bacterial (so-called pyogenic) in origin or, more rarely, a combination of
the two.

Pyogenic liver abscesses usually present as multiple abscesses and are potentially life-threatening. They
require prompt diagnosis and therapy by draining and/or aspirating purulent material, although it is possible
to treat liver abscesses with antibiotics alone. They occur in older patients than those with amoebic liver
abscesses, and are often secondary to a source of sepsis in the portal venous distribution.
Examples of the sources of pyogenic liver abscess include27:

• Biliary tract disease

• Extrahepatic foci of metastatic infection
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 • Surgery
• Trauma
Many different bacteria may be isolated from pyogenic liver abscesses. The most common
include29-32:

• Enterobacteriaceae
• Bacteroides species
• Clostridium species
• Anaerobic streptococci
• "S. anginosus" group
• Enterococci
• P. aeruginosa
• B. pseudomallei (in endemic areas)

Other causes include Candida species.

Amoebic liver abscesses arise as a result of the spread of Entamoeba histolytica via the portal vein from the
large bowel which is the primary site of infection (investigation of amoebae is described in BSOP 31 –
Investigation of specimens other than blood for parasites).

Hydatid cysts may also occur as fluid-filled lesions in the liver. However, the clinical presentation is usually
different from that of liver abscesses (see BSOP 31 – Investigation of specimens other than blood for
parasites). Cysts may become super-infected with gut flora and progress to abscess formation.
Lung abscess33
Lung abscesses involve the destruction of lung parenchyma and present on chest radiographs as large
cavities often exhibiting air-fluid levels. This may be secondary to aspiration pneumonia, in which case the
right middle zone is most frequently affected. Other organisms may give rise to multifocal abscess
formation and the presence of widespread consolidation containing multiple small abscesses (<2 cm
diameter) is sometimes referred to as necrotising pneumonia. Pneumonia caused by S. aureus and
Klebsiella pneumoniae may show this picture. (See BSOP 57 – Investigation of brochoalveolar lavage,
sputum and associated specimens).

Lung abscesses most often follow aspiration of gastric or nasopharyngeal contents as a consequence of
loss of consciousness, resulting for example from alcohol excess, cerebrovascular accident, drug overdose,
general anaesthesia, seizure, diabetic coma, or shock. Other predisposing factors include oesophageal or
neurological disease, tonsillectomy and tooth extraction.

Lung abscesses may arise from endogenous sources of infection. The bacteria involved in these cases are
generally from the upper respiratory tract and anaerobes are often implicated, secondarily infecting
consolidated lung after aspiration from the upper respiratory tract. Nosocomial infections involving S.
aureus, S. pneumoniae, Klebsiella species and other organisms may also occur.

B. pseudomallei may cause lung abscesses or necrotising pneumonia in those who have visited endemic
areas (mainly south east Asia and northern Australia)34 especially in diabetics.

Nocardia infection is most often seen in the lung where it may produce an acute, often necrotising,
pneumonia35. This is commonly associated with cavitation. It may also produce a slowly enlarging
pulmonary nodule with pneumonia, associated with empyema. Nocardiosis, almost always occurring in a
setting of immunosuppression, may present as pulmonary abscesses.

Abscesses as a result of blood borne spread of infection from a distant focus may occur in conditions
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such as infective endocarditis.

Lemierre's syndrome (or necrobacillosis) originates as an acute oropharyngeal infection usually in a young
adult. Infective thrombophlebitis of the internal jugular vein leads to septic embolisation and metastatic
infection. The lung is most frequently involved but multifocal abscesses may develop. Fusobacterium
necrophorum is the most common pathogen isolated from blood cultures in patients with this syndrome33.

Aspergilllus species have been isolated from lung abscesses in patients who are immunocompromised.
Pancreatic abscess
Pancreatic abscesses are potential complications of acute pancreatitis. Infections are often polymicrobial
and common isolates include Escherichia coli, other Enterobacteriaceae, enterococci and anaerobes:
longer-standing collections, especially after prolonged antibiotic therapy, are often infected with coagulase-
negative staphylococci and Candida species.
Perirectal abscess
Perirectal abscesses are encountered in patients with predisposing factors. These include36:

• Immunodeficiency
• Malignancy
• Rectal surgery
• Ulcerative colitis

They are often caused by37:

• Anaerobes
• Enterobacteriaceae
• Streptococci
• S. aureus

Pilonidal abscess
Pilonidal abscesses are common in children and result from infection of a pilonidal sinus. Anaerobes and
Enterobacteriaceae are usually isolated38, but they may be caused by S. aureus and β-haemolytic
streptococci.
Prostatic abscess
Prostatic abscesses may be caused by, or associated with39:

• Diabetes Mellitus
• Acute and chronic prostatitis
• Instrumentation of the urethra and bladder
• Lower urinary tract obstruction
• Haematogenous spread of infection

Organisms that may cause prostatic abscesses include40:

• E. coli and other Enterobacteriaceae

• Anaerobes

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 • Neisseria gonorrhoeae
• S. aureus

Prostatic abscesses can act as reservoirs for Cryptococcus neoformans resulting in relapses of infection
with this organism41.
Psoas abscess
Psoas abscesses may be seen as secondary infections to42:

• Appendicitis
• Diverticulitis
• Osteomyelitis of the spine
• Infection of a disc space
• Bacteraemia
• Perinephric abscess
Pus tracks under the sheath of the psoas muscle. Infection often occurs in drug abusers after injection into
the ipsilateral femoral vein.

Psoas abscesses are predominantly caused by43-45:

• Enterobacteriaceae
• Bacteroides species
• S. aureus
• Streptococci
• Mycobacterium tuberculosis

Renal abscess
Renal abscesses are typically caused by Gram-negative bacilli and result from ascending urinary tract
infection, pyelonephritis, renal calculi or septicaemia46. Diabetes mellitus can also occur in patients who are
immunocompromised. S. aureus has been replaced by E. coli (from urinary tract infections) as the most
prevalent organism found in these abscesses.

Perinephric abscesses are relatively uncommon, but serious, extensions of renal abscesses. Infection
spreads beyond the cortex and capsule into the perinephric fat. Causative organisms are the same as
those for renal abscesses.
Scalp abscess
Scalp abscesses are a recognised complication of electronic monitoring with fetal scalp electrodes during
labour. A localised collection of pus surrounded by inflamed tissue forms where the electrodes are inserted.
Anaerobes are most commonly isolated, probably as a result of contamination with vaginal organisms
during delivery.

Polymicrobial infections also occur, involving47:

• Anaerobes
• β-haemolytic streptococci
• S. aureus
• Enterobacteriaceae
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 • Enterococci
• Coagulase-negative staphylococci

Kerion is a pustular folliculitis of adjacent hair follicles, creating dense inflamed areas of the scalp, and is
caused by dermatophytes (see BSOP 39 – Investigation of dermatological specimens for superficial
mycoses). Secondary bacterial infection may occur.
Spinal epidural abscess
Spinal epidural abscesses may occur in patients with:

• Predisposing disease (such as diabetes)

• Prior infection elsewhere in the body which may serve as a source for haematogenous spread
• Abnormality of, or trauma to, the spinal column (often involving invasive medical procedures such
as epidural catheterisation)

The most common isolate is S. aureus48. Staphylococcus epidermidis may be isolated in patients following
invasive spinal manipulation. Streptococci (α-haemolytic, β-haemolytic and S. pneumoniae),
Enterobacteriaceae and pseudomonads may also be isolated48,49.
Subphrenic abscess
Subphrenic abscesses occur immediately below the diaphragm, often as a result of50:

• Gastric, duodenal or colonic perforation

• Acute cholecystitis
• Procedures on the liver and upper part of the gastrointestinal tract
• Ruptured appendix
• Trauma

Subphrenic abscesses are caused by mixed infections from the normal gastrointestinal flora50.
Unusual cases of abscess formation
Unusual cases of abscess formation can occur in patients with many underlying conditions and may be
caused by a vast range of organisms51-58. Any organism isolated from abscess pus is potentially significant.

Actinomycosis is a chronic suppurative infection characterised by chronic abscess formation with

surrounding fibrosis. It is rare and usually follows perforation of a viscous, trauma or surgery. It is caused
by Actinomyces israelii59, usually in mixed culture with other bacteria.

Abscess formation is most often associated with the gastrointestinal tract, the jaw and the pelvis. Other
areas of the body may be involved and the formation of abdominal abscesses may occur. Thoracic
involvement occurs in 15% of cases of actinomycosis. Pulmonary actinomycosis can be difficult to diagnose
prior to cutaneous involvement, which results in direct extension through the chest wall. The disease
progresses to form a chronic indurated mass with draining fistulae. Material should be drained from
abscesses and biopsies taken. Skin biopsies may reveal the presence of organisms (see BSOP 17 –
Investigation of tissues and biopsies).

"Sulphur granules" are sought in the pus specimen60. These are discharged from actinomycosis abscesses.
Sulphur granules are colonies of organisms forming a filamentous inner mass which is surrounded by host
reaction. They are formed only in vivo. They are hard, buff to yellow in colour, and have a clubbed surface.
Post-operative wound infections
Post-operative wound infections arise when microorganisms contaminate surgical wounds during an
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operation or immediately afterwards. Colonised body sites are frequent sources of pathogens, although
they may be transmitted via medical and nursing staff or via inanimate objects from other patients or
elsewhere in the hospital environment.

Organisms most commonly isolated include:

• S. aureus including MRSA

• Bacteroides species
• Clostridium species
• Enterobacteriaceae
• Pseudomonads
• β-haemolytic streptococci
• Enterococci
• Peptostreptococcus species

Coagulase-negative staphylococci and coryneforms isolated from post-operative sites overlying implants or
prostheses may indicate infection. This is particularly true in the presence of a sinus tract in direct
communication with the joint. However, with the exception of S. aureus, superficial flora do not necessarily
represent the flora deep inside a wound and cultures should be interpreted with care.

The following, though uncommon, are important clinical conditions and all require surgical debridgement as
a vital component of therapy:
Soft Tissue and other abscesses
Gangrene
There are four main types:

1) Meleney's progressive synergistic gangrene - presents as a burrowing lesion or chronic

gangrene of the skin following abdominal operations, and results from mixed infections by
organisms such as S. aureus, streptococci, Enterobacteriaceae, pseudomonads and anaerobic
Gram-negative bacilli61,62.

2) Gas Gangrene - is a necrotising process associated with systemic signs of toxaemia and gas is
present in the tissues. It often follows traumatic injuries such as penetrating wounds or crush
injuries. Gas gangrene is caused by clostridia, in particular Clostridium perfringens. However,
these organisms may colonise a wound without causing disease. Alternatively, they may cause a
spreading cellulitis, or extend into the muscle causing myonecrosis63. Classical gas gangrene is
associated with clinical shock, leakage of serosanguinous fluid, tissue necrosis and presence of gas
in the tissues.

3) Non-sporing anaerobes - are particularly important causes of infection in the pelvic and scrotal
areas (when the term Fournier's gangrene is applied) and are common causes of gangrene in
ischaemic and diabetic limbs. They often occur in infections mixed with Enterobacteriaceae,
streptococci and Clostridium species64.

4) Spontaneous gangrene occurs either with no apparent relation to trauma or following mild, non-
penetrating trauma. It is most commonly associated with patients with colonic carcinoma,
leukaemia or neutropenia. The main causative organisms are C. perfringens and septicum65.
Intra-abdominal sepsis
Intra-abdominal sepsis is infection occurring in the normally sterile peritoneal cavity66. The term covers
primary and secondary peritonitis, as well as intra-abdominal abscesses.
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Primary peritonitis is infection of the peritoneal fluid in which no perforation of a viscus has occurred.
Infection usually arises via haematogenous spread from an extra-abdominal source and is often caused by
a single pathogen66. It is common in patients with ascites following hepatic failure. In females it may also
result from organisms ascending the genital tract, for example N. gonorrhoeae and Chlamydia trachomatis
pneumococci, actinomycetes, enterobacteriacae and streptococci have been associated with peritonitis in
women with IUCDs but can cause primary peritonitis in any patient group at any age.

Secondary peritonitis is acute, suppurative inflammation of the peritoneal cavity usually resulting from bowel
perforation or postoperative gastrointestinal leakage. Secondary peritonitis is most often treated with a
combination of surgery and antibiotics.

The most frequent isolates encountered in intra-abdominal sepsis with secondary peritonitis are derived
from the normal gastrointestinal flora. Anaerobic bacteria are isolated from the majority of cases with
Bacteroides species being isolated. However, infections are usually polymicrobial67 and organisms that
have been isolated include:

• Enterococcus species
• Bacteroides species
• Pseudomonads
• Peptostreptococcus species
• Yeasts
• β-haemolytic streptococci
• Clostridium species
• Enterobacteriaceae

Tuberculous peritonitis is a rare disease in the UK. It is more common on the Indian sub-continent, so it is
important to consider this in immigrants from that area. In most cases a primary pulmonary focus is present
with secondary spread of Mycobacterium tuberculosis (see BSOP 40 – Investigation of specimens for
Mycobacterium species).

TECHNICAL INFORMATION/LIMITATIONS
In National Standard Methods, the term “CE marked leak proof container” is used to describe containers
bearing the CE marking and which are used for the collection and transport of clinical specimens. The
requirements of the EU in vitro Diagnostic Medical Devices Directive (98/79/EC Annex 1 B 2.1)68 state that
such devices must “reduce as far as possible contamination of, and leakage from, the device during use
and, in the case of specimen receptacles, the risk of contamination of the specimen. The manufacturing
processes must be appropriate for these purposes”.

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Reference no: BSOP 14i5
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1 SAFETY CONSIDERATIONS69-74
1.1 SPECIMEN COLLECTION
Avoid accidental injury when pus is aspirated
1.2 SPECIMEN TRANSPORTATION AND STORAGE
Pus in a CE Marked leak proof containera in a sealed plastic bag
1.3 SPECIMEN PROCESSING
Containment Level 2 unless infection with a Hazard Group 3 organism is suspected on clinical
grounds, in which case Containment Level 3 is required

All specimens likely to contain Hazard Group 3 organisms must be processed in a

microbiological safety cabinet in a Containment Level 3 (CL3) laboratory.

Thus initial examination and all follow up work on specimens from patients with suspected
Mycobacterium species, or suggesting a diagnosis of blastomycosis, coccidioidomycosis,
histoplasmosis, paracoccidioidomycosis or penicilliosis must be performed inside a microbiological
safety cabinet in a CL3 laboratory.

Any grinding of sulphur granules should be performed in a microbiological safety cabinet

Prior to staining, fix smeared material by placing the slide on an electric hotplate (65 to 75°C), under
the hood, until dry. Then place in a rack or other suitable holder

Note: Heat-fixing may not kill all Mycobacterium species75. Slides should be handled carefully

Centrifugation must be carried out in sealed buckets, which are subsequently opened in a
microbiological safety cabinet

Specimen containers must also be placed in a suitable holder

Laboratory procedures that give rise to infectious aerosols must be conducted in a microbiological
safety cabinet

Refer to current guidance on the safe handling of all organisms documented in this NSM

The above guidance should be supplemented with local COSSH and risk assessments

Compliance with postal and transport regulations is essential

2 SPECIMEN COLLECTION
2.1 OPTIMAL TIME FOR SPECIMEN COLLECTION
Before antimicrobial therapy where possible
2.2 CORRECT SPECIMEN TYPE AND METHOD OF COLLECTION
The specimen will usually be collected by a medical practitioner

Samples of pus are preferred to swabs. However, pus swabs are often received (when using
swabs, the deepest part of the wound should be sampled, avoiding the superficial microflora)

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2.3 ADEQUATE QUANTITY AND APPROPRIATE NUMBER OF SPECIMENS
Ideally, a minimum volume of 1mL of pus

Swabs should be well soaked in pus

3 SPECIMEN TRANSPORT AND STORAGE

3.1 TIME BETWEEN SPECIMEN COLLECTION AND PROCESSING
Specimens should be transported and processed as soon as possible
The volume of specimen influences the transport time that is acceptable. Large volumes of purulent
material maintain the viability of anaerobes for longer76,77

The recovery of anaerobes is compromised if the transport time exceeds 3 h

3.2 SPECIAL CONSIDERATIONS TO MINIMISE DETERIORATION
Swabs should be transported in Amies transport medium with charcoal78

If processing is delayed, refrigeration is preferable to storage at ambient temperature. Delays of

over 48 h are undesirable

4 SPECIMEN PROCESSING
4.1 TEST SELECTION
Divide specimen on receipt for appropriate procedures such as examination for parasites (BSOP 31
– Investigation of specimens other than blood for parasites) and culture for Mycobacterium species
(BSOP 40 – Investigation of specimens for Mycobacterium species), depending on clinical details
4.2 APPEARANCE
Describe presence or absence of sulphur granules (if sought)
4.3 MICROSCOPY
4.3.1 STANDARD
Swab
Prepare a thin smear on a clean microscope slide for Gram staining after performing culture (see
QSOP 52 – Inoculation of culture media)
Pus
Using a sterile pipette place one drop of neat specimen or centrifuged deposit (see 4.4.1), as
applicable, on to a clean microscope slide

Spread this using a sterile loop to make a thin smear for Gram staining
(see BSOPTP 39 – Staining procedures)
4.3.2 SUPPLEMENTARY
Gram stain of sulphur granules
With care, either squash the sulphur granules that have been washed in saline (see Section 4.4.1)
between two slides using gentle pressure, or use the homogenised granules (see Section 4.4.1)
and make a thin smear for Gram staining.

Note: Any grinding of sulphur granules should be performed in a microbiological safety

cabinet

For microscopy, Mycobacterium species (BSOP 40 – Investigation of specimens for Mycobacterium

INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS
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 species) and parasites (BSOP 31 – Investigation of specimens other than blood for parasites). For
fungi and other staining procedures see BSOPTP 39 – Staining procedures.
4.4 CULTURE AND INVESTIGATION
4.4.1 PRE-TREATMENT
Standard
Exudates
Centrifuge in a sterile, capped, conical-bottomed container at 1200 x g for 5-10 mins
Transfer the supernatant with a sterile pipette, leaving approximately 0.5 mL, to another CE Marked
leak proof containera in a sealed plastic bag for additional testing if required
Resuspend the deposit in the remaining fluid
Supplementary
Wash any sulphur granules that are present in saline
Suspend an aliquot of pus containing sulphur granules in sterile water or saline in a CE Marked
leak proof containera in a sealed plastic bag. Agitate gently to wash pus from the granules
Grind the washed granules in a small amount of sterile water or saline, with a sterile tissue grinder
(Griffiths tube or unbreakable alternative) or a pestle and mortar
Use this homogenised sample to make a smear for Gram staining and to inoculate agar plates
Note 1: All grinding of sulphur granules should be performed in a microbiological safety cabinet
Note 2: If a fungal infection is suspected then grinding of the whole specimen should be avoided.
This is to prevent damaging hyphae that would result in a reduced yield, particularly with
zygomycetes.
4.4.2 SPECIMEN PROCESSING
Pus
Inoculate agar plates and enrichment broth with the pus or centrifuged deposit with a sterile pipette
(see QSOP 52 – Inoculation of culture media)
If sulphur granules are present, these should be ground (see Section 4.4.1) and included in the
culture
For the isolation of individual colonies, spread inoculum with a sterile loop
All additional pus/fluid from the specimen should be stored for up to 7 days at 4°C
Swabs
Inoculate each agar plate with swab (see QSOP 52 – Inoculation of culture media)
For the isolation of individual colonies, spread inoculum with a sterile loop

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Reference no: BSOP 14i5
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4.4.3 CULTURE MEDIA, CONDITIONS AND ORGANISMS FOR ALL SPECIMENS
Clinical details/ Standard media Incubation Cultures Target organism(s)
conditions read
Temp Atmos Time
°C

Blood agar 35-37 5 – 10% 40-48 h daily S. aureus

β-haemolytic
CO2
streptococci
Enterococci
CLED/
35-37 air 16-24 h ≥16 h Enterobacteriaceae
MacConkey agar
Pseudomonads
All specimens
Neomycin 35-37 anaerobic 5d ≥40 h and Anaerobes
fastidious at 5 d
anaerobe agar
with
metronidazole 5
µg disc

Fastidious 35-37 air 5d N/A Any organism

anaerobe broth
then subcultured
All pus and exudates
where appropriate
(not swabs)
on to above media
(excluding CLED/ 35-37 as above 40-48 h ≥40 h
MacConkey agar)

For these situations, add the following:

Clinical details/ Supplementary Incubation Cultures Target organism(s)

conditions media read
Temp Atmos Time
°C

Submandibular abscess Fastidious 35-37 anaerobic 5d ≥40 h and at Anaerobes

Empyema anaerobe agar 5d

Normally sterile sites

such as:

Brain abscess Chocolate agar 35 – 37 5 – 10% 40 – 48 h ≥40 h Fastidious organisms

Liver abscess CO2
Lung abscess
Psoas abscess
Spinal abscess

Actinomycosis Blood agar 35-37 anaerobic 10 d ≥40 h, at 7 d Actinomyces species

(or where microscopy supplemented and 10 d
suggestive of with
actinomycetes) metronidazole and
nalidixic acid

Nocardiosis Blood agar 35-37 air up to 7 d at 3 d and 7 Nocardia species

d

Immunocompromised Sabouraud agar 35-37 air 5d ≥40 h Fungi

Prostatic abscess GC selective/ 35-37 5-10% CO2 40-48 h ≥40 h N. gonorrhoeae

Primary peritonitis in Chocolate agar
females

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 Optional media Incubation Cultures Target organism(s)
read
Temp Atmos Time
°C

Staph/strep 35-37 air 40-48 h daily S. aureus

When clinical details or selective agar Streptococci
microscopy suggestive
of mixed infection GN medium (NAV) 35-37 anaerobic Up to 5 d 48 h and Gram-negative
5d anaerobes

Other organisms for consideration - Fungi (BSOP 39 – Investigation of dermatological specimens for superficial mycoses) and
Mycobacterium species (BSOP 40 – Investigation of specimens for Mycobacterium species)

4.5 IDENTIFICATION
4.5.1 MINIMUM LEVEL IN THE LABORATORY
Actinomycetes species level
BSOPID 10 – Identification of aerobic Actinomycetes
species
BSOPID 15 – Identification of anaerobic Actinomycetes

Anaerobes "anaerobes" level

β-haemolytic streptococci Lancefield group level

Coagulase-negative staphylococci "coagulase-negative" level

Enterobacteriaceae "coliforms" level

Fungi species level (except yeast to yeast level)

Neisseria species level

Pseudomonads species level

S. aureus species level

"S. anginosus" group "S.anginosus" group level

Mycobacterium BSOP 40 - Investigation of specimens for Mycobacterium

species

Parasites BSOP 31 - Investigation of specimens other than blood for

parasites

Organisms may be further identified if clinically or epidemiologically indicated or if isolated from

normally sterile site.

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4.5.2 REFERRAL TO REFERENCE LABORATORIES
For information on the tests offered, turn around times, transport procedure and the other
requirements of the reference laboratory click here for user manuals and request forms.
Organisms with unusual or unexpected resistance, and whenever there is a laboratory or clinical
problem, or anomaly that requires elucidation should be sent to the appropriate reference
laboratory.

4.6 ANTIMICROBIAL SUSCEPTIBILITY TESTING

Refer to NSM on Antimicrobial Susceptibility Testing (BSOP 45 - Susceptibility Testing).

5 REPORTING PROCEDURE
5.1 MICROSCOPY
Report on WBCs and organisms detected

For the reporting of microscopy for fungi, Mycobacterium species and parasites (BSOP 40 –
Investigation of specimens for Mycobacterium species) and parasites (BSOP 31 – Investigation of
specimens other than blood for parasites).
5.1.1 MICROSCOPY REPORTING TIME
Urgent microscopy results to be telephoned or sent electronically

Written report, 16 – 72 h
5.2 CULTURE
Report clinically significant organisms isolated or

Report other growth or

Report absence of growth

Report on the presence of sulphur granules

Also, report results of supplementary investigations

5.2.1 CULTURE REPORTING TIME
Clinically urgent culture results to be telephoned or sent electronically

Written report, 16 – 72 h stating, if appropriate, that a further report will be issued

Supplementary investigations: Fungi, Mycobacterium species and parasites

(BSOP 31 – Investigation of specimens other than blood for parasites)
5.3 ANTIMICROBIAL SUSCEPTIBILITY TESTING
Report susceptibilities as clinically indicated

INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS

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6 REPORTING TO THE HPA (LOCAL AND REGIONAL
SERVICES AND CENTRE FOR INFECTIONS)79
Refer to the following:

Individual NSMs on organism identification

Health Protection Agency publications:

"Laboratory reporting to the HPA: A guide for diagnostic laboratories"

"Hospital infection control: Guidance on the control of infection in hospitals"

Local guidelines

Report all clinically significant isolates from deep-seated abscesses and metastatic infections to
CDSC

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7 ACKNOWLEDGEMENTS AND CONTACTS
This National Standard Method has been developed, reviewed and revised by the National
Standard Methods Working Group for Clinical Bacteriology
(http://www.hpa-standardmethods.org.uk/wg_bacteriology.asp). The contributions of many
individuals in clinical bacteriology laboratories and specialist organisations who have provided
information and comment during the development of this document, and final editing by the
Medical Editor are acknowledged.

The National Standard Methods are issued by Standards Unit, Department for Evaluations,
Standards and Training, Centre for Infections, Health Protection Agency, London.

For further information please contact us at:

Standards Unit
Department for Evaluations, Standards and Training
Centre for Infections
Health Protection Agency
Colindale
London
NW9 5EQ

E-mail: [email protected]

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Reference no: BSOP 14i5
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www.evaluations-standards.org.uk
Email: [email protected]
APPENDIX80

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Issue no: 5 Issue date 23.11.09 Issued by: Standards Unit, Department for Evaluations, Standards and Training. Page 22 of 27
Reference no: BSOP 14i5
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: [email protected]
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Reference no: BSOP 14i5
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: [email protected]
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Email: [email protected]
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Reference no: BSOP 14i5
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www.evaluations-standards.org.uk
Email: [email protected]
 Anaerobe 1999;5:1-3.

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INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS

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Reference no: BSOP 14i5
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: [email protected]
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a
The requirements of the EU in vitro Diagnostic Medical Devices Directive68 (98/79/EC Annex 1
B 2.1) state that such devices must “reduce as far as possible contamination of, and leakage from, the
device during use and, in the case of specimen receptacles, the risk of contamination of the specimen.
The manufacturing processes must be appropriate for these purposes”.

INVESTIGATION OF ABSCESSES AND DEEP-SEATED WOUND INFECTIONS

Issue no: 5 Issue date 23.11.09 Issued by: Standards Unit, Department for Evaluations, Standards and Training. Page 27 of 27
Reference no: BSOP 14i5
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: [email protected]