Uk standards for Microbiology Investigations



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Salivary Gland Abscess

There are three pairs of major salivary glands; the parotid, submandibular and sublingual. Infection is generally a secondary infection with an orthodontic cause. Parotic abscesses are more commonly seen in the elderly. Common organisms include:



  • S. aureus (including MRSA)

  • Anaerobes

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. aureus52. 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 isolated52,53.

Subphrenic Abscess

Subphrenic abscesses occur immediately below the diaphragm, often as a result of54:


  • 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 flora54.

Throat Abscess

Throat abscess are relatively common. Add text from reference 55

Causative organisms include:


  • β haemolytic streptococci

  • Anaerobes

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 organisms56-63. 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 israelii, usually in mixed culture with other bacteria64.

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 (refer to B 17 – ).

"Sulphur granules" are sought in the pus specimen65. 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.

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.

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 polymicrobial and organisms that have been isolated include67:



  • 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 (refer to B 40 – Investigation of Specimens for Mycobacterium species).

Technical Information/Limitations



Limitations of UK SMIs

The recommendations made in UK SMIs are based on evidence (eg sensitivity and specificity) where available, expert opinion and pragmatism, with consideration also being given to available resources. Laboratories should take account of local requirements and undertake additional investigations where appropriate. Prior to use, laboratories should ensure that all commercial and in-house tests have been validated and are fit for purpose.



Selective Media in Screening Procedures

Selective media which does not support the growth of all circulating strains of organisms may be recommended based on the evidence available. A balance therefore must be sought between available evidence, and available resources required if more than one media plate is used.

Specimen Containers1,2

SMIs use the term “CE marked leak proof container” to describe containers bearing the CE marking used for the collection and transport of clinical specimens. The requirements for specimen containers are given in the EU in vitro Diagnostic Medical Devices Directive (98/79/EC Annex 1 B 2.1) which states: “The design must allow easy handling and, where necessary, 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”.



Rapid methods

To reduce turnaround times, rapid identification and sensitivity tests may be performed in conjunction with routine methods where appropriate. A variety of rapid identification and sensitivity methods have been evaluated; these include molecular techniques and the Matrix Assisted Laser Desorption Ionisation Time-of-Flight (MALDI-TOF)68,69. It is important to ensure that fresh cultures of pure single isolates are tested to avoid reporting misleading results.

Laboratories should follow manufacturers’ instructions and all rapid tests must be validated and be shown to be fit for purpose prior to use.

1 Safety Considerations1,2,70-84



1.1 Specimen Collection, Transport and Storage1,2,70-73

Use aseptic technique.

Collect specimens in appropriate CE marked leak proof containers and transport in sealed plastic bags.

Avoid accidental injury when pus is aspirated.

Collect swabs into appropriate transport medium and transport in sealed plastic bags.

Compliance with postal, transport and storage regulations is essential.



1.2 Specimen Processing1,2,70-84

Containment Level 2.

If infection with a Hazard Group 3 organism eg Mycobacterium species, Paracoccoides brasiliensis or Brucella species is suspected, all specimens must be processed in a microbiological safety cabinet under full Containment Level 3 conditions. 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 under full Containment Level 3 conditions.

It is recommended that all Gram-negative coccobacilli from should be processed in a Class I or Class II microbiological safety cabinet until Hazard Group 3 pathogens (ie Brucella) have been definitively excluded 85.

Laboratory procedures that give rise to infectious aerosols must be conducted in a microbiological safety cabinet76.

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 (65-75°C), under the hood, until dry. Then place in a rack or other suitable holder.

Note: Heat-fixing may not kill all Mycobacterium species86. Slides should be handled carefully.

Specimen containers must also be placed in a suitable holder.

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

The above guidance should be supplemented with local COSHH and risk assessments.

2 Specimen Collection

2.1 Type of Specimens

Abscess pus, abscess swab, deep-seated wound pus swab



2.2 Optimal Time and Method of Collection87

For safety considerations refer to Section 1.1.

Collect specimens before antimicrobial therapy where possible87.

Unless otherwise stated, swabs for bacterial and fungal culture should then be placed in appropriate transport medium88-92.

Collect specimens other than swabs into appropriate CE marked leak proof containers and place in sealed plastic bags.

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).

2.3 Adequate Quantity and Appropriate Number of Specimens87

Ideally, a minimum volume of 1mL of pus.

Swabs should be well soaked in pus.

Numbers and frequency of specimen collection are dependent on clinical condition of patient.

3 Specimen Transport and Storage1,2

3.1 Optimal Transport and Storage Conditions

For safety considerations refer to Section 1.1.

Specimens should be transported and processed as soon as possible87.

The volume of specimen influences the transport time that is acceptable. Large volumes of purulent material maintain the viability of anaerobes for longer93,94.

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

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

4 Specimen Processing/Procedure1,2

4.1 Test Selection

Divide specimen on receipt for appropriate procedures such as examination for parasites (B 31 – Investigation of Specimens other than Blood for Parasites) and culture for Mycobacterium species (B 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 Sample Preparation

4.3.1 Pre-treatment

Exudates


Centrifuge in a sterile, capped, conical-bottomed container at 1200 x g for 5-10 min.

Transfer the supernatant with a sterile pipette, leaving approximately 0.5mL, to another CE Marked leak proof container 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 container 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.

4.3.2 Specimen processing

Pus


Inoculate agar plates and enrichment broth with the pus or centrifuged deposit with a sterile pipette (refer to Q 5 – Inoculation of Culture Media for Bacteriology).

If sulphur granules are present, these should be ground 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 (refer to Q 5 – Inoculation of Culture Media for Bacteriology).

For the isolation of individual colonies, spread inoculum with a sterile loop.

4.4 Microscopy

4.4.1 Standard

Swab


Prepare a thin smear on a clean microscope slide for Gram staining after performing culture (refer to Q 5 – Inoculation of Culture Media for Bacteriology).

Pus


Using a sterile pipette place one drop of neat specimen or centrifuged deposit (see 4.5.1), as applicable, on to a clean microscope slide.

Spread this using a sterile loop to make a thin smear for Gram staining (refer to TP 39 – Staining Procedures).



4.4.2 Supplementary

Gram stain of sulphur granules


With care, either squash the sulphur granules that have been washed in saline between two slides using gentle pressure, or use the homogenised granules (see section 4.5.1) 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 (B 40 – Investigation of Specimens for Mycobacterium species) and parasites (B 31 – Investigation of Specimens other than Blood for Parasites). For fungi and other staining procedures refer to TP 39 – Staining Procedures.

4.5 Culture and Investigation

Inoculate each agar plate using a sterile pipette (Q 5 - Inoculation of Culture Media for Bacteriology).



For the isolation of individual colonies, spread inoculum with a sterile loop.

4.5.1 Culture media, conditions and organisms

Clinical details/

conditions

Specimen

Standard media

Incubation

Cultures read

Target organism(s)

Temp °C

Atmos

Time

All clinical conditions


Pus

Swab


Blood agar

35-37

5–10% CO2

40-48hr

daily

Any organism


CLED/

MacConkey agar



35-37

Air

18-24hr

18hr

Selective anaerobe agar with a metronidazole 5 µg disc

35-37

Anaerobic

5 d

40hr and at 5 d

Anaerobes

All pus and exudates (not swabs)

Fastidious anaerobic, cooked meat broth or equivalent.

Subculture if evidence of growth (≥40hr), or at day 5

to above media (excluding MacConkey agar)


35-37

35-37


Air

As above


5 d

40-48hr


N/A

40hr


Any organism




For these situations, add the following:

Clinical details/

conditions

Specimen

Supplementary media

Incubation

Cultures read

Target organism(s)

Temp °C

Atmos

Time

Submandibular abscess

Empyema


Normally sterile sites such as:

Brain abscess

Liver abscess

Lung abscess

Psoas abscess

Spinal abscess



Pus

Swab


Selective anaerobe agar

35-37

Anaerobic

5 d

40hr and at 5 d

Anaerobes

Chocolate agar

35–37

5–10% CO2

40 – 48hr

40hr

Fastidious organisms

Actinomycosis

(or where microscopy suggestive of action-mycetes)



Pus

Swab


Blood agar supplemented with metronidazole and nalidixic acid

35-37

Anaerobic

10 d

40hr, at 7 d and 10 d

Actinomyces species

Nocardiosis

Pus

Swab


Blood agar

35-37

Air

up to 7 d

at 3 d and 7 d

Nocardia species

Immunocompromised

Pus

Swab


Sabouraud agar

28-30

Air

14 d

daily

Yeast

Mould


Prostatic abscess

Primary peritonitis in females



Pus

Swab


GC selective/ Chocolate agar

35-37

5-10% CO2

40-48hr

40hr

N. gonorrhoeae

Clinical details/

conditions

Specimen

Optional media

Incubation

Cultures read

Target organism(s)

Temp °C

Atmos

Time

When clinical details or microscopy suggestive of mixed infection

Pus

Swab


Staph/strep selective agar

35-37

air

40-48hr

daily

S. aureus

Streptococci



Gram negative medium

(NAV)


35-37

anaerobic

Up to 5 d

40hr

and 5 d


Gram negative anaerobes

Other organisms for consideration - Fungi (B 39 – Investigation of Dermatological Specimens for Superficial Mycoses) and Mycobacterium species (B 40 – Investigation of Specimens for Mycobacterium species)

4.6 Identification

Refer to individual SMIs for organism identification.



4.6.1 Minimum level of identification in the laboratory

Actinomycetes

species level

ID 10 – Identification of Aerobic Actinomycetes

ID 15 – Identification of Anaerobic Actinomyces species


Anaerobes

"anaerobes" level

β-haemolytic streptococci

species 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

(consider Panton-Valentine leukocidin (PVL) and toxin testing if appropriate clinical details)



"S. anginosus" group

"S. anginosus" group level

Mycobacterium

B 40 - Investigation of Specimens for Mycobacterium species

Parasites

B 31 - Investigation of Specimens other than Blood for Parasites

Organisms may be further identified if this is clinically or epidemiologically indicated.

4.7 Antimicrobial Susceptibility Testing

Refer to British Society for Antimicrobial Chemotherapy (BSAC) and/or EUCAST guidelines. Prudent use of antimicrobials according to local and national protocols is recommended.



4.8 Referral for Outbreak Investigations

N/A


4.9 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.

Contact appropriate devolved national reference laboratory for information on the tests available, turn around times, transport procedure and any other requirements for sample submission:

England and Wales

https://www.gov.uk/specialist-and-reference-microbiology-laboratory-tests-and-services

Scotland

http://www.hps.scot.nhs.uk/reflab/index.aspx

Northern Ireland

http://www.publichealth.hscni.net/directorate-public-health/health-protection

5 Reporting Procedure

5.1 Microscopy





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