Consultation outcome

S 11: closed consultation draft

Updated 13 June 2023

This draft document was consulted on between 27 May 2022 to 30 June 2022

Acknowledgments

UK Standards for Microbiology Investigations (UK SMIs) are developed under the auspices of the UK Health Security Agency (UKHSA) working in partnership with the partner organisations whose logos are displayed below and listed on the UK SMI page. UK SMIs are developed, reviewed and revised by various working groups which are overseen by a steering committee (see the Steering Committee).

The contributions of many individuals in clinical, specialist and reference laboratories who have provided information and comments during the development of this document are acknowledged. We are grateful to the medical editors for editing the medical content. UK SMIs are produced in association with:

  • The Association for Clinical Biochemistry Microbiology Group
  • British Infection Association (BIA)
  • The British Society for Antimicrobial Chemotherapy (BSAC)
  • British Society for Medical Mycology
  • British Society For Microbial Technology (BSMT)
  • British Society for Parasitology
  • Welsh Microbiological Association (WMA)
  • Healthcare Infection Society
  • Public Health Agency
  • Institute of Biomedical Science (IBMS)
  • Microbiology Society
  • National Services Scotland
  • Northern Ireland Microbiology Forum and Audit Group
  • Public Health Scotland
  • Public Health Wales
  • The Royal College of Pathologists
  • Society for Anaerobic Microbiology (SAM)
  • Society for Applied Microbiology (SfAM)
  • Scottish Microbiology and Virology Network
  • UK Clinical Mycology Network (UKCMN)
  • The UK Clinical Virology Network

Amendment table

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General information

View general information related to UK SMIs.

Scientific information

View scientific information related to UK SMIs.

Scope of document

This UK SMI describes the investigation of patients with red or painful eye. Such infection can occur in patients with a normal or impaired immune system often producing characteristic syndromes associated with specific underlying conditions and associated orbital tissue.

The document covers the investigation of red or painful eye covering common pathogens using molecular, culture and serology techniques. This UK SMI should be used in conjunction with other associated UK SMIs.

Please note in accordance with recent updates of fungal taxonomy Candida species are referred to as Candida and associated ascomycetous yeast.

Background

Red or painful eye can be caused by bacteria, viruses, fungi and parasites. They can be classified into infections affecting the external eye such as conjunctivitis, blepharitis, canaliculitis and orbital cellulitis or infections affecting the globe of the eye such as keratitis, endophthalmitis and uveitis.

These infections could be acute or chronic with varying severity. Exogenous organisms may be introduced to the eye via hands, fomites (for example contact lenses), traumatic injury, or following surgery. Haematogenous spread from a focus elsewhere in the body can also occur (1). Infections causing red or painful eye can rapidly progress and require urgent investigation and immediate treatment.

Red or painful eye

Red or painful eye can be caused by a variety of infectious and non-infectious conditions. It is a common ophthalmic presentation in primary care which can present with visual loss. Some patients may experience pain, which can be caused by severe conjunctivitis, corneal trauma, peripheral keratitis and orbital cellulitis.

Red eye with no pain can be a result of subconjunctival haemorrhage. Patients presenting with mild pain involving the cornea may be as a result of non-infectious keratitis. Mild pain in association with mucopurulent discharge may be caused by chlamydial or bacterial conjunctivitis.

Gonococcal conjunctivitis may give rise to hyperpurulent discharge whereas allergic or viral conjunctivitis may result in watery discharge or itching.

Early dacryocysititis results from mild pain that involves the eyelid (2). Dacryocystitis causes inflammation of the tear sac. It is common in infants and middle-aged women and can be classified into acute, chronic and congenital (3).

The most common organisms in acute infection include: Staphylococcus species and Streptococcus species, followed by Haemophilus influenza and Pseudomonas aeruginosa. Chronic dacryocystitis is a result of chronic obstruction due to systemic disease, repeated infection, dacryoliths, and chronic inflammatory debris of the nasolacrimal system (4).

An overarching algorithm of this syndrome is presented in the ‘presentation of red or painful eye’ algorithm.

Conjunctivitis

Conjunctivitis is a common eye infection caused by the inflammation of the conjunctiva (the thin layer of tissue that covers the front of the eye) due to viral and bacterial infections.

It occurs in people of all ages including neonates. Features such as the clinical appearance of the eye, the age of the patient, and the exposure history should be considered in assessing the likely cause of this condition. In most cases of conjunctivitis, the inflammation usually clears up in a few days and does not require treatment. Symptoms include itchiness, red eye, foreign body sensation, watery and sticky discharge.

In England the incidence of conjunctivitis is 13 to 14 in 1000 people per year. The rates are higher in children under 1 years of age (5).

The following types of conjunctivitis can occur:

Infective conjunctivitis

Infective conjunctivitis is caused by viruses and bacteria. In very rare cases (for example post use of corticosteroid eye drops) fungal infection may occur. Infective conjunctivitis is most commonly caused by viruses, especially adenovirus (6). Other viral causes include herpes simplex virus (HSV), varicella-zoster virus (VZV) and enterovirus (7). Acute contagious haemorrhagic conjunctivitis may be caused by enterovirus 70 and coxsackievirus A24 (6).

Bacterial conjunctivitis

Bacterial conjunctivitis is common in children. Symptoms include the eyelids being stuck together, particularly on waking, with crusting of the eyelids. The discharge can be purulent or mucopurulent. Most cases resolve in 5 to 10 days. Infection with Neisseria gonorrhoeae should be considered if mucopurulent (8). Studies have reported that 50 to 70% of eye infections are due to bacterial infections (9).

The most common organisms that cause this infection include: Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Enterobacterales, Pseudomonas aeruginosa, Neisseria gonorrhoeae and Chlamydia trachomatis (1),(7). A test of cure is recommended for all who have been treated for N. gonorrhoeae (10) (11).

Neonatal conjunctivitis is caused by pathogens commonly found in adult cases. C. trachomatis can be transferred to newborns during delivery from the infected mother. Chlamydial inclusion conjunctivitis and trachoma are caused by various serotypes of C. trachomatis. Trachoma is associated with serotypes A to C (A, B, Ba and C) (1). These serotypes are associated with sexual transmission and in adults, co-infection with other sexually transmitted pathogens may be present.

Meningococcal conjunctivitis may cause acute conjunctivitis especially in children, but this is not a common cause of conjunctivitis.

Gonococcal conjunctivitis is associated with sexually transmitted infections. Neonates can contract this disease through maternal transmission during birth and is referred to as ophthalmia neonatorum.

Allergic conjunctivitis occurs when the body’s immune system attacks allergens, such as pollen or make-up causing inflammation. The eyes are typically very itchy.

Dacryoadenitis

Dacryoadenitis is caused by inflammation of the lacrimal gland which results from bacterial or viral infections. In rare cases dacryoadenitis can result from fungal and parasitic infections.

The most common viral infection is caused by EBV. Other viruses such as adenovirus, VZV, HSV, rhinovirus, cytomegalovirus (CMV) or mumps are less common.

Bacterial causes include Staphylococcus aureus and Streptococcus species.

Symptoms of dacryoadenitis include pain in the superolateral orbit, pain with eye movements, droopy upper eyelid or difficulty opening the affected eye, redness of the eye, and occasionally double vision.

In some cases a conjunctival swab may be taken for culture or molecular testing.

Blepharitis

Blepharitis can be an acute or chronic inflammatory process affecting the margin of the eyelids. It can cause burning, itching, hyperaemia, foreign body sensation, burning and crusted eyelashes affecting both eyes.

It most commonly occurs in middle-aged patients but can occur at any age (12). It is estimated that blepharitis causes 5% of all eye problems reported to general practitioners in the UK (13) .

Organisms include:

  • Staphylococcus species
  • Streptococcus species including S. pneumoniae and S. pyogenes
  • Moraxella catarrhalis, Corynebacterium species including C. diphtheriae
  • C. bovis
  • Cutibacterium acnes

On rare occasions chronic blepharitis can be caused by fungi, such as Malassezia species, Trichophyton species, Cryptococcus and Blastomyces.

Canaliculitis

Canaliculitis is a rare condition causing chronic inflammation of the canaliculus, a short channel near the inner corner of the eyelid through which tears drain into the tear sac. Patients present with epiphora, mucopurulent discharge, eyelid erythema and recurrent conjunctivitis. Infection is most often caused by a bacterial pathogen but can also be caused by a viral or fungal infection.

Primary infection is due to coagulase negative staphylococci, streptococci, C. acnes, and Actinomyces species. Chronic infections may be due to anaerobic actinomycetes such as Actinomyces israelii or by Cutibacterium propionicum (14) (15).

Cellulitis

Preseptal cellulitis

Preseptal cellulitis or periorbital cellulitis is infection of the skin and soft tissue around the eye, anterior to the orbital septum. This condition most commonly occurs in children and may be as a result of trauma or contiguous sinusitis. Patients present with unilateral eyelid swelling and oedema. Causative organisms include H. influenzae, Staphylococcus aureus and Streptococcus species.

Preseptal cellulitis is commonly caused by bacterial organisms and in rare cases can be viral (16). Most cases resolve after 5 to 7 days of therapy but can progress to orbital cellulitis without appropriate treatment (17).

Orbital cellulitis

Orbital cellulitis is a severe condition. It affects the orbital tissue and is usually due to underlying bacterial sinusitis that requires urgent medical attention. Complications can result in sub-periosteal abscess formation, cavernous sinus thrombosis, intracranial abscess formation, loss of vision and death. Rhino-orbital cellulitis can present similarly to an orbital cellulitis although it has a different pathology.

The most common pathogens in adults are Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus species and anaerobes. In children H. influenzae still remains prevalent, but the capsulated (type b) strain is rarely seen following the introduction of the vaccine programme (18).

Treatment for periorbital and orbital cellulitis is different and depends on diagnosis.

Keratitis

Keratitisis an infection of the cornea. It is a serious condition requiring immediate attention as it may progress rapidly. Keratitis can cause blindness or visual impairment and may progress to endophthalmitis if not appropriately treated. Predisposing factors include contact lens use, pre-existing ocular disease including, ocular trauma, ocular surgery, laser refractive surgery, diabetes, immunosuppressive disease and the use of topical steroids (19).

Nature explorers, hikers and farmers can also be at risk. The condition may be caused by a range of bacteria, virus, fungi and parasites.

Bacterial keratitis

Bacterial keratitismay result in absence of anterior chamber activity and stromal infiltrates, which are usually multifocal. Symptoms include pain, photophobia, blurred vision, corneal opacity, hypopyon and red eyes in contact lens users (19). Organisms include: Staphylococcus species, Streptococcus species, P. aeruginosa, Enterobacterales, Corynebacterium diphtheriae, M. catarrhalis, H. influenzae, N. gonorrhoeae and C. acnes.

Herpes keratitis

Herpes keratitisis an infection with HSV or HZV. The predominant strain responsible for herpes simplex ocular disease is herpes simplex virus type 1 (HSV-1) (20). Most cases of ocular HSV are recurrent and presents classically with unilateral corneal defects, although the lid and conjunctiva may be involved. These recurrent attacks may lead to corneal scarring, decreased corneal sensation, and visual loss. The global incidence of HSV keratitis was calculated at approximately 1.5 million with 40 000 new cases of severe monocular visual impairment or blindness per year (19).

Acanthamoeba keratitis

Acanthamoeba keratitis (21) to (23) is a rare and serious condition and usually occurs in contact lens users or after ocular trauma. Acanthamoeba keratitis has been linked to prolonged exposure to contaminated water, especially in contact lens users and in individuals who use hot tubs or swimming pools. However it should be noted that there have been reports to suggest that Acanthamoeba keratitis has been seen in non-contact lens users in recent years (24).

Fungal keratitis

Fungal keratitis (25) is also known as ‘mycotic keratitis’ or ‘keratomycosis’. It is an infection of the cornea caused by any of the multiple fungi capable of invading the ocular surface. Non-specific symptoms include redness, pain, photophobia, impaired vision. Clinical features such as non-yellow infiltrates with feathery edges or raised surface, intact epithelium with deep stromal involvement, satellite lesions, endothelial plaques, lack of improvement with antibiotics and worsening with steroids are suggestive of fungal keratitis. There are 2 types: Keratitis caused by yeast like infections and filamentous keratitis.

Keratitis caused by yeast like infections can be caused by insufficient tear secretion, defective eyelid closure, diabetes, immunosuppression or contaminated contact lenses (26).

Filamentous fungal keratitis usually occurs in agricultural or outdoor workers where there is a risk of accidental eye injury. The risk factors in suspected fungal keratitis are ocular trauma, previous ocular surgery, contact lens use, topical or systemic corticosteroid use, evidence of ocular surface disease and contact lens use. Organisms include: Fusarium, Aspergillus species, ascomycetous yeasts, Dermatophytes, Cladosporium, Curvularia, and Mucorales (26).

Retinitis

Retinitis is caused by inflammation of the retina. Viral retinitis is rare but vision threatening and is usually seen in individuals with weakened immune system. The most common viruses associated with retinitis are HSV, VZV, Herpes Zoster Virus (HZV), CMV and possibly Epstein-Barr virus (EBV) (27). Bacterial retinitis can also occur.

Other organisms associated with retinitis include: Mycobacterium species, Treponema pallidum, Toxocara species, Toxoplasma pallidum, Bartonella, Aspergillus species, Ascomycetous yeasts and Blastomyces species.

Endophthalmitis

Endophthalmitis is a relatively uncommon but potentially sight-threatening infection of intraocular fluids and tissue. Infectious endophthalmitis can develop as a result of haematogenous spread of organisms from an endogenous source or from an exogenous source following trauma, surgery or intraocular injection.

Causative organisms include Staphylococcus species, Streptococcus species, C. acnes, Aspergillus species, Fusarium species, Candida and associated ascomycetous yeasts, dematiaceous fungi and Scedosporium apiospermum (28) (29).

Post-traumatic endophthalmitis

Post-traumatic endophthalmitis occurs after penetrating or perforating ocular injuries. Risks include metal or blunt trauma injuries, retained intra-ocular foreign bodies, disruption of the lens, and delay in primary repair of greater than 24 hours (32). Poorly treated fungal keratitis can also lead to fungal endophthalmitis.

Organisms include Staphylococcus species, Bacillus cereus, Streptococcus species, Clostridium perfringens, Microsporidium species, Fusarium species, Candida and associated ascomycetous yeasts, Aspergillus species, Acremonium and Paecilomyces species.

Acute post-operative endophthalmitis

Acute post-operative endophthalmitis occurs within 1 to 7 days of intraocular surgery (33)The source of infection is usually the patient’s own ocular or eyelid surface flora making diagnosis by culture difficult, although virtually any organism may be introduced and can cause infection. To address these issues molecular testing can now be used to diagnose infection and can help to clarify the causative organisms (34).

Bacterial organisms are more common and include Enterobacterales, Staphylococcus species, Streptococcus species, P. aeruginosa and C. acnes. Fungal postoperative endophthalmitis is less common. Organisms include Aspergillus species and Ascomycetous yeasts. Fungal endophthalmitis usually has a late onset of 1 to 2 months after surgery but in tropical countries it may present acutely within 4 weeks of surgery (35).

Glaucoma filtering-bleb-associated endophthalmitis

Glaucoma filtering-bleb-associated endophthalmitis is a surgically created scleral defect, covered only with conjunctiva, that allows excess aqueous humour to be absorbed into the systemic circulation. Because only conjunctiva separates the ocular surface flora from the aqueous humour at the bleb, endophthalmitis may occur at any time (36).

It can occur within weeks or years after the original surgery including pars plana vitrectomy, penetrating keratoplasty and glaucoma drainage implant. Organisms include Enterobacterales, Staphylococcus species, Streptococcus species, H. influenzae, P. aeruginosa C. acnes and M. catarrhalis (32), (36).

Chronic endophthalmitis

Chronic endophthalmitis occurs months to years after intraocular surgery. Patients typically present with a persistent low grade uveitis or they may present the same way as acute endophthalmitis. Organisms include C. acnes, Staphylococcus species, Corynebacterium species, Mycobacterium species, Pseudomonas aeruginosa, Aspergillus species, Ascomycetous yeasts and Fusarium species.

Endogenous endophthalmitis

Endogenous endophthalmitis is rare and occurs in patients with bacteraemia or fungaemia, often associated with treatment of immunosuppressive therapy, recreational drug use or invasive surgical procedures. The use of broad-spectrum antibiotics can predispose the patient to increased risk of fungaemia and fungal endophthalmitis (30).

Patient presentation ranges from asymptomatic to symptoms typical of severe uveitis, including a red, painful eye with photophobia, floaters, or reduced vision (31). Other important risk factors include sepsis, malignancy, hepatitis B, hepatitis C, pneumonia, and urinary tract infection (30). Organisms include Bacillus cereus, Enterobacterales, Staphylococcus species, Streptococcus species, Candida and associated ascomycetous yeasts, and Aspergillus, Cryptococcus and Paecilomyces species.

Uveitis

Uveitis is the inflammation of the uveal tract, iris, ciliary body and choroid. It is an uncommon eye condition which can result in visual impairment. Uveitis can be caused by inflammation, autoimmunity, infections, tumours, trauma or toxins that may penetrate the eye. Infectious organisms include: HSV, HZV, CMV, HIV, Lyme disease, toxoplasmosis, tuberculosis and syphilis (37).

In the developing world uveitis accounts for 25% of legal blindness cases and 5 to 10% of visual impairment cases worldwide. Uveitis causes pain, red eye, blurred vision, watering photophobia, flashes and floaters (38).

Uveitis can be classified as follows (39):

Anterior uveitis is the most common infection occurs at the front of the eye, and is caused by HSV, VZV, CMV and syphilis.

Intermediate uveitis and panuveitis infection is caused by syphilis and Lyme disease.

Posterior uveitis is the least common infection occurs at the back of the eye and can be caused by toxoplasmic retinitis, syphilis, TB, CMV retinitis, HSV, VZV retinitis, Bartonella and Lyme disease.

Travellers are also at risk from ocular infections from exposure to external or environmental factors such as high temperatures, humidity, air pollution, sleeping with contact lenses, exposure to air conditioning, swimming or bathing in contaminated water. This can lead to risk of conjunctivitis, keratitis and uveitis. In some countries (39) where there are less resources available, healthcare may not be easily accessible (40).

Toxoplasmosis results from infection with the parasite Toxoplasma gondii. Toxoplasma eye infections cause significant inflammation and subsequent scarring which may cause temporary or permanent impairment of vision. Ocular toxoplasmosis can either be congenital (transmitted from mother to child during pregnancy) or acquired (introduced into the body by ingestion of undercooked or raw food). Infection is life-long and asymptomatic.

Clinical presentations of red and painful eye

Multiplex molecular testing may give results for organisms not requested. Under these circumstances, laboratories should follow local procedures.

Presentation of red or painful eye

Red or painful eye is a common ophthalmic presentation in primary care. Below is an overarching algorithm of this syndrome (2) with further testing shown in the algorithms below. An accessible text description of this flowchart is provided with this document.

Notes

Patients with infectious keratitis, anterior uveitis, acute angle closure glaucoma orbital cellulitis and gonococcal conjunctivitis usually require urgent referral to ophthalmology.

Patients with severe conjunctivitis, corneal trauma may require referral.

Patients with non-infectious keratitis, early dacryocystitis, chlamydia, bacterial, allergic or viral conjunctivitis and subconjunctival haemorrhage can be managed in primary care settings (2).

Patients with endophthalmitis can present with red eye, blurred or visual loss or eye pain after surgery (41).

Conjunctivitis

In cases of uncomplicated conjunctivitis, diagnostic samples may not be required prior to empirical treatment. Conjunctival swabs should normally be collected in neonatal sticky eye, especially if ophthalmia neonatorum is suspected, in patients who fail to respond to first-line antibiotics, in severe disease, particularly if referral to ophthalmology is being considered and in immunocompromised patients. An accessible text description of this flowchart is provided with this document.

Notes

Culture or molecular swab maybe required depending on circumstances.

Haemorrhagic conjunctivitis-enterovirus 70 and coxsackievirus A24 (6).

Adenovirus 8, 19 associated with keratoconjunctivitis. Notify laboratory if an outbreak is suspected.

Suspected neonatal HSV should trigger urgent paediatric referral. If C. trachomatis or N. gonorrhoeae detected, mother and her sexual contacts should be offered testing and the baby will require paediatric referral due to risk of severe local or systemic infection.

Consider BASHH guidelines on NAAT for non-genital samples.

Please refer to the relevant UK SMI for additional information.

Blepharitis

Patients are not routinely swabbed for blepharitis unless associated with other ocular infections. An accessible text description of this flowchart is provided with this document.

Malassezia species and Trichophyton species are associated with chronic disease.

Please refer to the relevant UK SMI for additional information.

Canaliculitis

Canalicular pus is preferable to eye swabs for canaliculitis. An accessible text description of this flowchart is provided with this document.

Please refer to the relevant UK SMI for additional information.

Orbital cellulitis

Preseptal cellulitis is commonly caused by bacterial organisms and in rare cases can be viral. Eye swabs and aspirates from the affected orbital tissues should be sampled. An accessible text description of this flowchart is provided with this document.

Please refer to the relevant UK SMI for additional information.

Keratitis

Corneal scrapings are sampled for keratitis (42). Occasionally corneal biopsy or anterior chamber aspirate may also be needed. Keratitis may progress to endophthalmitis if inappropriately treated. Acanthamoeba investigation is carried out by a specialist or referral laboratory.

Microscopy is an essential investigation for fungal keratitis. Species identification is critical to treatment, as antifungal susceptibility profiles will vary. An accessible text description of this flowchart is provided with this document.

Please refer to the relevant UK SMI for additional information.

Retinitis

NAAT testing of aqueous/vitreous fluid samples are primarily used for the investigation of retinitis.An accessible text description of this flowchart is provided with this document.

Notes

If there is no specimen available from the eye, send a blood specimen for serology testing of Toxocara species.

Negative serology for anti Toxocara antibodies in peripheral blood samples cannot exclude ocular Toxocariasis. Testing of vitreous fluid for anti Toxocara antibodies may be considered if clinical suspicion is high.

Toxoplasma and Treponema pallidum serology may be a useful adjunct to molecular testing.

In ocular toxoplasmosis negative results are uncommon and therefore useful in helping to exclude the disease.

Endophthalmitis

Specimens include intraocular fluids aqueous humour from the anterior chamber and vitreous humour from the vitreous cavity/body. Eye swabs may also be taken for visible pus. For Endogenous Endophthalmitis blood cultures are required. An accessible text description of this flowchart is provided with this document.

Please refer to the relevant UK SMI for additional information.

Uveitis

Aqueous and vitreous humour fluids are sampled for molecular and serology testing. Uveitis is the inflammation of the uveal tract, iris, ciliary body and choroid. It is an uncommon eye condition which can be a cause of visual impairment. An accessible text description of this flowchart is provided with this document.

Please refer to the relevant UK SMI for additional information.

Pre-laboratory processes (pre-analytical stage)

Specimen type

The types of specimens include:

  • aspirates and tissue for:

  • swabs and scrapings for

    • bacterial and fungal culture
    • microscopy for parasites
    • molecular testing for viruses (PCR), bacteria (16S sequencing) and fungi (18S sequencing) as appropriate
  • blood for

    • blood culture when suspecting disseminated infection and in cases of endophthalmitis
    • molecular testing for viruses (PCR), bacteria (16S sequencing) and fungi (18S sequencing) as appropriate
  • serum for

    • fungal biomarker tests (beta-D-glucan and Aspergillus galactomannan) when suspecting disseminated infection – please note that to avoid false positive results, the BDG test should be taken into a separate tube and only processed at the laboratory performing the test in BDG free environment and equipment; also, please note that Aspergillus galactomannan ELISA testing has a poor sensitivity in non-neutropenic patient setting
    • infectious diseases serology
  • contact lens cleansing and storage fluid

Note:

On occasions, laboratories may wish to test environmental specimens, but this is outside the scope of this document. These environmental specimens may include contact lens cases and cleaning fluids such as contact lens solution, irrigation solution and dye used during eye surgery or corticosteroids injected directly into the eye. Generally, these are tested by culture methods for detection of bacteria, fungi and parasites. Smears are not recommended.

Gram negative organisms can be isolated from contact lens disinfecting solutions for example P. aeruginosa and Serratia marcescens which have been reported to be commonly resistant to contact lens disinfecting solutions (43), (44).

Superficial swabs, although not ideal, may be all that is available. Deep-seated samples such as tissue or aspirates, if available, should be sought as they are superior to swabs depending on clinical syndrome.

Specimen collection and handling

Collect specimens as soon as possible after onset of symptoms. Specimens should be transported and processed as soon as possible.

Refer to current guidance on the safe handling of all organisms documented in the UK SMI general safety document.

Collect specimens before antimicrobial therapy where possible. If the patient is already on antimicrobial treatment, please provide the details of this to the laboratory. Swabs for bacterial and fungal culture should be placed in appropriate transport medium (45) to (49).

Corneal scraping and intraocular fluids for keratitis are collected by an ophthalmic surgeon. Because of the small amounts of material involved, inoculation of transport media for molecular testing, culture plates and preparation of slides may need to be done at the patients’ bedside or in clinic.

Laboratories should agree a protocol for the collection of specimens, inoculation of media, and transport to the laboratory with their local ophthalmologists, and supply kits for this purpose when required.

The direct detection of the causative agent in a corneal scrape specimen is the only reliable diagnostic method for Acanthamoeba keratitis. Several molecular based techniques are well established and usually increase sensitivity significantly and are becoming the tool of choice for diagnosis (50).

Sample collection requires anaesthetic eye drops, needle, cotton or dacron swab, surgical blade, spatula, glass slide and cover slips. Liquid or solid culture media is required.

Two conjunctival swabs are required. First swab for direct microscopy and the second swab for molecular testing.

Separate samples must be collected into appropriate transport media for detection of viruses or chlamydia (refer to UK SMI V 37 – Chlamydia trachomatis infection – testing by nucleic acid amplification tests (NAATs)).

Specimen transport and storage

This section covers specimen transport and storage consideration related to this UK SMI, and should be read in conjunction with the scientific information on GOV.UK.

Bacterial cultures should be submitted in a suitable bacteriological transport media such as Amies Agar gel transport swab/medium with charcoal. The presence of charcoal in medium neutralises the bacterial toxins and inhibitory substances. Amies transport medium with charcoal has been shown to increase the recovery of N. gonorrhoeae.

Specimens should be transported and processed as soon as possible (51). If processing is delayed, the specimens should be refrigerated (51) to (53).

Speed of delivery of specimens to the laboratory is paramount in certain specimens such as post-operative specimens.

If specimens for fungal molecular testing cannot be performed on the same day, they should be frozen and defrosted only on the day of testing.

If specimens for investigation for amoebae cannot be processed immediately, it is preferable to store them at ambient temperature for up to 48 hours. Refer to the UK SMI B31: Investigation of specimens other than blood for parasites for more information.

Relevant clinical history details needed on patient request forms when referring samples to the laboratory

Full clinical details and information on patient history should be provided with clinical requests.

These details should include:

  • specimen date and time of collection
  • where the sample has taken from such as the left or right eye
  • type of infection suspected
  • type of swab/sample sent to the laboratory
  • immune status
  • other relevant information (contact lens wearer, water exposure)

Safety considerations

The section covers specific safety considerations (54) to (76) related to this UK SMI, and should be read in conjunction with the general safety considerations on GOV.UK.

All Hazard group 2 organisms must be confirmed at Containment Level 2.

All work on suspected N. meningitidis which is likely to generate aerosols must be performed in a microbiological safety cabinet (77).

If infection with a Hazard Group 3 organism, for example Mycobacterium tuberculosis, or an agent of endemic mycosis, is suspected, all work must be undertaken in a microbiological safety cabinet under full Containment Level 3 conditions (78).

Laboratory processes (analytical stage)

Microscopy

Many laboratories utilise molecular methods for the detection of organisms. However, microscopy is still useful in primary identification. Refer to TP 39 – Staining procedures.

For safety considerations refer to Section 2.

Sample preparation for eye swabs

Perform a Gram stain on all eye swabs including those from neonates with sticky eyes and canalicular pus. Prepare a thin smear from the swab or pus on a clean microscope slide for Gram staining.

Sample preparation for corneal scraping for parasites and fungi

Corneal scrapings and eye fluid or tissue require lactophenol cotton blue stain or potassium hydroxide. Calcofluor wet mount preparation is optimal for visualization of fungal elements.

Prepare the specimen on a clean slide and then add stain to slide. Make a thin mount and then examine the prepared slide under low power (×100).

Note:

  1. In KOH wet mount, Acanthamoeba cysts appear typically as star or hexagonal shaped and double celled wall structures.

  2. Fungal species stained with lactophenol stain appear as fungal hyphae or yeast cells (26). Both microscopy and culture should be done whenever possible, as microscopy cannot differentiate between genera and species of fungi (79).

  3. Other alternative special stains that could be used include Periodic Acid Schiff (PAS) reaction, Giemsa, Ziehl-Neelsen or Gomori methenamine silver stains, when indicated (26).

Other stains include Acid fast (detection of mycobacteria and Nocardia) and Acridine orange (Fluorescent stain that interacts with microbial DNA and RNA).

Supplementary

For microscopy of Mycobacterium species, refer to UK SMI B 40 – Investigation of specimens for Mycobacterium species.

Molecular testing

When performing such syndromic molecular testing, knowledge of the detection range, sensitivity and specificity individual to a specific assay is required to identify any potential pathogens that have been missed and subsequently require additional testing.

Molecular assays for the detection of pathogens directly from ocular samples are now widely available.

Please refer to UK SMI Q4 Good practice when performing molecular amplification assays.

Culture

For safety considerations refer to Section 2.

Culture can be used by laboratories for the diagnosis of an eye infection. Refer to appendix A for sample preparation of contact lens solution.

Culture of Acanthamoeba requires a lawn of E.coli or of certain other coliforms on nutrient agar for growth. Refer to appendix B for sample preparation of Acanthamoeba species.

Supplementary

For Mycobacterium species, refer to UK SMI B 40 – Investigation of specimens for Mycobacterium species.

Culture media, conditions and organisms

Table 1: Culture media, conditions and organisms

Clinical details / conditions Specimen type Standard media Incubation Cultures read Target organism (s)
      Temperature °C Atmosphere Time    
Conjunctivitis Eye swab Culture Chocolate agar



Blood agar
35 to 37



35 to 37
5 to 10% CO2


5 to 10% CO2
40 to 48 hour

40 to 48 hour
Daily



Daily
Staphylococcus aureus Streptococcus pneumoniae Haemophilus influenzae Moraxella catarrhalis Enterobacteriales Pseudomonas aeruginosa
Conjunctivitis Neonates Eye swab Culture or Molecular GC selective agar 35 to 37 5 to 10% CO2 40 to 48 hour Greater than or equal to 40 hour Neisseria gonorrhoeae
  Eye swab Molecular           Adenovirus Herpes Simplex Virus Enterovirus Varicella Zoster Virus Chlamydia trachomatis
Blepharitis Eye swab Culture or molecular Chocolate agar



Blood agar
35 to 37



35 to 37
5 to 10% CO2


5 to 10% CO2
40 to 48 hour

40 to 48 hour
Daily



Daily
Staphylococcus species Streptococcus species Moraxella catarrhalis Corynebacterium species Cutibacterium acnes
    Sabouraud agar (supplemented with olive oil) 28 to 30 Air 5 day Greater than or equal to 40 hour Malassezia species Trichophyton species
Canaliculitis Canalicular pus, eye swab Culture Fastidious anaerobe agar 35 to 37 Anaerobic 40 to 48h 7 day and 10 day Greater than or equal to 40 hour Actinomyces israelli Cutibacterium propionicum
Orbital cellulitis Orbital tissue fluids, blood cultures, eye swabs Blood agar



Chocolate agar



Sabouraud agar


Fastidious anaerobe agar
35 to 37



35 to 37



28 to 30



35 to 37
5 to 10% CO2


5 to 10% CO2


Air



Anaerobic
40 to 48 hour

40 to 48 hour

14 day

40 to 48 hour*
Daily



Daily



Greater than or equal to 40 hour
Staphylococcus aureus Streptococcus species Haemophilus influenzae Pseudomonas aeruginosa Peptostreptococcus species
  Molecular           Adenovirus
Keratitis Corneal scrapings Culture Blood agar



Chocolate agar
35 to 37



35 to 37
5 to 10% CO2


5 to 10% CO2
40 to 48 hour

40 to 48 hour
Daily



Daily
Staphylococcus species Streptococcus species Pseudomonas aeruginosa Enterobacterales Corynebacterium diphtheriae Haemophilus influenzae Moraxella catarrhalis
    GC selective agar 35 to 37 5 to 10% CO2 40 to 48hr Greater than or equal to 40 hour Neisseria gonorrhoeae
  Corneal scrapings Culture or Molecular Fastidious anaerobe agar

Sabouraud agar
35 to 37


28 to 30
Anaerobic


Air
40 to 48hr*

14 day
Greater than or equal to 40 hour Cutibacterium acnes Fusarium species Ascomycetous yeasts Aspergillus species
  Corneal scrapings, pus Culture or Molecular Non—nutrient agar with E.coli** 28 to 30 Air 10 day 24 hour and daily up to 7 day Acanthamoeba
  Eye swab Molecular           Herpes Simplex Virus Varicella Zoster Virus Adenovirus
Retinitis Vitreous and aqueous humour fluid Microscopy and Culture Blood agar



Sabouraud agar
35 to 37



28 to 30
5 to 10% CO2


Air
40 to 48 hour

14 day
Daily Greater than or equal to 40hour Mycobacterium species Ascomycetous yeasts Aspergillus species
  Vitreous and aqueous humour fluid Molecular           Cytomegalovirus Herpes Simplex Virus Varicella Zoster Virus Mycobacterium species Toxocara species Treponema pallidum Toxoplasma Bartonella Ascomycetous yeasts Blastomyces species Aspergillus species
Endophthalmitis Vitreous and aqueous humour fluids or taps or biopsy Culture or Molecular Blood agar



Chocolate agar



Sabouraud agar

Fastidious anaerobe agar
35 to 37



35 to 37



28 to 30


35 to 37
5 to 10% CO2


5 to 10% CO2


Air


Anaerobic
40 to 48 hour

40 to 48 hour

14days


40 to 48hr*
Daily



Daily



Greater than or equal to 40hr
Staphylococcus species Bacillus cereus Streptococcus species Enterobacteriales Pseudomonas aeruginosa Corynebacterium species Mycobacterium species Ascomycetous yeasts Aspergillus species Dematiaceaous fungi Scedosporium apiospermum Clostridium perfringens Cutibacterium acnes Fusarium species Microsporidium species
Uveitis Vitreous and aqueous fluids Molecular and serology           Toxoplasma Cytomegalovirus Herpes Simplex Virus Varicella Zoster Virus
If Gram negative rods seen in Gram film All samples CLED agar 35 to 37 Air 16 to 24 hour Greater than or equal to 16 hour Enterobacterales

Identification

Refer to individual UK SMI for organism identification.

All clinically significant isolates should be identified to species level.

Note:

Any organism considered to be a contaminant may not require identification to species level. Organisms may be identified further if clinically or epidemiologically indicated. Any organism isolated from a normally sterile site should be identified to species level.

Any organism considered to be a contaminant (or part of normal flora) may not require identification to species level.

All work on suspected N. meningitidis and Acanthamoeba species and isolates which is likely to generate aerosols must be performed in a microbiological safety cabinet.

Post laboratory processes (post analytical stage)

Reporting microscopy

Report organism as detected or fungal elements seen.

For fungal infection, please refer to the British Society for Medical Mycology Best practice guidelines.

Refer to: Microscopy for Mycobacterium species (UK SMI B 40 – Investigation of specimens for Mycobacterium species) and parasites (UK SMI B 31 – Investigation of specimens other than blood for parasites).

Reporting molecular results

Interpretation of the tests requires understanding of the normal flora of the conjunctiva and the eye lids.

Report bacterial, fungal or viral DNA/RNA as ‘detected’ (state the organism).

Report bacterial, fungal or viral DNA/RNA as ‘not detected’.

Reporting culture results

Positive results should be released immediately.

Report clinically significant organisms isolated as growth detected. State the species level identified.

Growth not detected report as ‘Absence of growth’.

Reporting time

Interim or preliminary results should be issued on detection of clinically significant isolates as soon as growth is detected, unless specific alternative arrangements have been made with the requestors. Positive results for microscopy should be released immediately, following local policy. Many preliminary results require specialist interpretation before they are released.

Final reports should follow as soon as possible.

Results are communicated in accordance with local policy.

Antimicrobial susceptibility testing

For interpretation of susceptibility testing results, laboratories should test and interpret using the criteria in the criteria in the EUCAST topical agents tab, refer to EUCAST guidelines for breakpoint information.

Alternatively, isolates can be sent to an appropriate specialist or reference laboratory.

Clinical review and authorisation of the report is required before susceptibility results are released to check that all reported drugs have efficacy against infections of this type.

Reporting of antimicrobial susceptibility testing

Report susceptibilities as clinically indicated. Prudent use of antimicrobials according to local and national protocols is recommended.

Referral to reference laboratories

For information on the tests offered, turnaround times, transport procedure and the other requirements of the reference laboratory see 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.

Adenoviruses can cause outbreaks and so presumptive isolates should be sent to laboratories for further identification and confirmation.

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

Note:

In case of sending away to laboratories for processing, ensure that specimen is placed in appropriate package and transported accordingly.

Appendix A: Sample preparation of contact lens solution

  1. Transfer fluid from contact lens storage case to a sterile universal container. Rub the inside of the storage case with a sterile cotton-tipped swab moistened with sterile distilled water. Express the liquid from the swab into the fluid in the sterile universal container.
  2. Centrifuge at 800 × g for 5 mins.
  3. Using a sterile pipette, discard the supernatant into disinfectant, leaving approximately 0.5mL of centrifuged deposit.
  4. Resuspend the centrifuged deposit in the remaining fluid and place 2 drops in the centre of a bacteria-coated purified agar plate.
  5. After the fluid has been absorbed, incubate and examine the plate as described for corneal scrapings (above).

Appendix B: Investigation of Acanthamoeba species

Cultures for Acanthamoeba species must be processed in a microbiological safety cabinet.

  1. Autoclave a 1.5% concentration (15g/L) of purified non-nutrient agar in quarter-strength Ringer’s solution (or preferably Page’s saline if available). Allow to cool and pour into small Petri dishes. Dry plates before use.

  2. Flood the blood agar plate with Escherichia coli* (NCTC 10418) and incubate at 37°C overnight.

  3. Recover all the growth with a sterile cotton-tipped swab and suspend in 2mL of the preferred saline (as used in 1.) or sterile distilled water.

  4. Add 2 drops of the suspension to the surface of the purified agar plates and spread evenly over the surface with a swab. The plates are now ready for inoculation with the specimen. If not used for testing at time of preparation, these plates may be stored at 2 to 8ºC and should be used for testing within a week of inoculation.

  5. On grounds of patient safety, it is preferred to spread the coliform suspension after receipt of plates by the laboratory after sampling has been performed, in which case, the plates should be flooded on their return to the laboratory and excess fluid removed.

Note:

An even distribution of the organisms is required on the plate.

Klebsiella species (K. pneumoniae) and Enterobacter aerogenes are acceptable alternatives to E. coli. Laboratories may wish to use other alternative organisms apart from those mentioned in this document. They should however ensure that they have validated these prior to use in their work.

Sample processing of Acanthamoeba species

  1. Inoculate the specimen to a clean microscope slide (examine with a low-power objective) and to the surface of a bacteria-coated purified agar plate. Ring the inoculated area on the base of the plate with a permanent felt-tipped pen for easy reference. Include a plate with a non-pathogenic Acanthamoeba control.
  2. Place the plate in a sealable bag or moist box.
  3. Incubate plates at 30°C. Incubation at 37°C is not recommended as some strains grow poorly at this temperature.
  4. Examine the surface of the plate after 24 hours and then daily for up to 7 days with an inverted microscope with a low-power objective. The plate need not be opened.
  5. Trophozoite stage amoebae may be seen to have made tracks in the bacterial layer. Characteristic polygonal cysts may also be seen.

Supplementary

Culture for free-living amoebae

  1. Cultures for Acanthamoeba species must be processed in a microbiological safety cabinet.
  2. A non-pathogenic control strain of Acanthamoeba species, A. castellani (Neff strain), can be obtained from the Culture Collection of Algae and Protozoa (CCAP) on request (CCAP Ref. 1501/1A).

Sample processing of corneal scrapings

Corneal scrapings should be inoculated to media for culture of Acanthamoeba species if indicated by clinical details (for example the use of contact lenses or corneal ulceration).

Appendix C: Sample processing of swab or pus

Inoculate each agar plate with swab or pus (UK SMI Q 5 – Inoculation of culture media for bacteriology).

Agar plates for bacterial culture, inoculated directly at the patient’s bedside, should be streaked out with a sterile loop for the isolation of individual colonies, and incubated immediately on receipt in the laboratory.

For inoculation methods performed at the patient’s bedside, refer to local protocols.

E. coli seeded plates should not be sent to the patient’s bedside.

Appendix D: Sample processing of aqueous and vitreous humour

If fluids are received, one or 2 drops of fluid should be inoculated to each of the agar plates and streaked out with a sterile loop for the isolation of individual colonies. Enrichment media should also be inoculated if sufficient specimen is available.

Agar plates maybe inoculated directly at the patient’s side and should be streaked out with a sterile loop for the isolation of individual colonies, and immediately incubated on receipt in the laboratory.

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An explanation of the reference assessment used is available in the scientific information section on the UK SMI website.

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