Specimen collection – microbiology and virology

The purpose of this guideline is to provide guidance on the collection of microbiology and virology specimen at Great Ormond Street Hospital (GOSH).

Introduction

Rationale for specimen collection

Preparation (including transport)

Equipment

Blood samples

Biopsy material 

Bronchoalveolar lavage (BAL)

Buccal swab

Cerebrospinal fluid (CSF)

Ear swabs

Eye swabs

Fungal samples of hair, nail and skin

Gastric washings (lavage)

MRSA screening

Molecular bacteriology testing

Nose swabs 

Nasopharyngeal aspirate (NPA)

Oral fluid (saliva) test

Pernasal swabs 

Sputum

Stool samples

Throat swabs

Urine

Vaginal/vulval swabs 

Vesicular fluid for herpes polymerase chain reaction (PCR)

Wounds Swabs

Introduction

Microbiological and virological laboratory testing has a key role in the management of children with infection. Accurate and rapid identification of significant microorganisms is vital for guiding optimal antimicrobial therapy and improving outcome from infections and infectious disease. 

Laboratory diagnosis is also essential for effective infection prevention and control in both the hospital and community settings, as well as providing valuable epidemiological data. In addition, accurate diagnosis reduces the over-prescribing of antimicrobial agents and contributes significantly to reductions in the emergence and spread of antimicrobial resistance (Public Health England, 2015).

Clinicians (including healthcare assistants, nurses, doctors and professionals allied to medicine) have the responsibility of using the correct procedure during the collection and safe transportation of samples to the laboratory. The validity of test results largely depends on good practice in the ‘pre-test’ stage and it is essential that documentation is accurate and comprehensive (Higgins, 1994).

Microbiological tests are not as standardised as some other lab tests; the way in which a sample is processed and the results are interpreted depend significantly on the information provided with the specimen.  

Contamination of samples, especially those from normally sterile sites such as blood or cerebrospinal fluid, leads to misleading results, inappropriate antibiotic usage and unnecessary laboratory work. 

Prolonged periods of storage at ambient temperature and delay in transport of specimens to the laboratory may increase the number of contaminants present. 

It is therefore essential that every effort should be made to avoid these problems. 

Please note: While this guideline refers to the ‘child’ throughout, all activities are applicable to young people.

Rationale for specimen collection

Specimen collection is undertaken when laboratory investigation is required for the examination of material, eg blood, body fluid, tissue or faeces to aid diagnosis.  

Preparation

  • Laboratory request forms are printed from the GOSH Patient Information Management System (PIMS). The labels with the patient details at the bottom of the request form should be used to label the specimen accompanying the form. The forms are barcoded to aid the audit trail. 
  • All specimens must be clearly labelled to identify their source (eg nose swab, throat swab etc.). This is especially important when more than one specimen is sent with one form.
  • Do not pre-label specimen containers as this may increase the risk of errors. The specimen must be labelled next to the patient when the sample is taken. 
  • Before labelling the specimen, check the patient’s name band against the patient’s details on the laboratory request form to ensure they match.
  • A laboratory request form with the following information must accompany the specimen. This aids interpretation of results and reduces the risk of errors.  
    • Patient's name, DOB, ward/department and hospital number  
    • Type of specimen and the site from which it was obtained 
    • Date and time collected 
    • Diagnosis with history and reasons for request such as returning from abroad (specify country) with diarrhoea and vomiting, rash, pyrexia, catheters in situ or invasive devices used, or surgical details regarding postoperative wound infection 
    • Any antimicrobial drug(s) given 
    • Consultant's name 
    • Name/bleep number of the clinician who ordered the investigation, as it may be necessary to telephone preliminary results and discuss treatment before the final result is authorised 
  • In children with suspected infections of hazard group 3 and 4 pathogens (eg Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), viral haemorrhagic fevers such as Lassa fever, Marburg or Ebola virus) the Infection Prevention and Control Team or the Microbiologist on call (out of hours) MUST be consulted before any specimens are taken.
  • Always explain the procedure to the child and parent and the reasons for taking the specimen. Separate permission must be obtained from the child and parent if specimens are sought for research purposes. They have a right to refuse without any obligation (Ethics Advisory Committee, 2000).  
  • Hands should be cleaned before and after specimen collection (refer to GOSH clinical guideline ‘Hand hygiene’. Standard precautions should be applied and appropriate personal protective equipment (PPE) worn when collecting or handling specimen to protect the healthcare worker from exposure to blood and/or body fluids (Loveday et al, 2014). If an infection is suspected, eg when a patient has respiratory symptoms or loose stools, the appropriate isolation precautions should be applied even before the results of the specimen are available. The isolation precautions should be based on the symptoms the child is presenting with. Once the result of the specimen is available, the need and type of the isolation precautions can be reassessed according to the GOSH Standard and Isolation Precautions Policy (available on the GOSH intranet infection control website).
  • When collecting certain specimens (eg catheter urines, cerebrospinal fluid and blood) every effort should be made to minimise the risk of infecting the child. An appropriate aseptic or aseptic non-touch technique should be used. 
  • All pathological specimens must be treated as potentially infectious and local written laboratory protocols should be followed for the safe handling and transportation of specimens (Health and Safety Executive, 2003). Specimen should be collected in sterile containers with close fitting lids to avoid contamination and spillage. All specimen containers must be transported in a double sided, self-sealing polythene bag with one compartment containing the laboratory request form and the other the specimen. 
  • Ideally, microbiological specimen should be collected before beginning any treatment such as antibiotics or using antiseptics. However, treatment must not be delayed in serious sepsis. 
  • When collecting pus specimen obtain as much material as possible as this increases the chance of isolating microorganisms which may be difficult to grow or are minimal in number eg tuberculosis. Pus should be sent in a plain sterile specimen container, not on a swab. 

Transport medium may be used to preserve microorganisms during transportation:

  • Charcoal medium improves the isolation of bacteria by neutralising toxic substances such as naturally occurring fatty acids found on the skin. 
  • For virology specimen, a viral transport medium should be used as many viruses do not survive well outside the body. This can be obtained from the Virology Laboratory, Level 4 Camelia Botnar Laboratories. It may be stored at room temperature on theward, but should only be used for viral investigation. The viral transport medium must not be used after the expiry date. 
  • Samples submitted for bacterial polymerase chain reaction (PCR) should NOT be in transport medium.

Transport to the laboratory:

  • At GOSH routine specimen are delivered to the laboratories by porters, and more urgent specimens via the pneumatic tube delivery system (‘chute’).
  • NPA specimen must not be sent to the laboratory via the pneumatic tube delivery system (‘chute’) due to the risk of leakage. They must be transported to the laboratory by a porter.
  • Specimen from children suspected of suffering from hazard group 3 and 4 pathogens MUST be discussed with the laboratory before delivery and transported by hand in a safe transport container. GOSH laboratories have no Level 4 containment facilities, therefore special precautions will need to be taken and specimen transported off-site for testing (Advisory Committee on Dangerous Pathogens, 2013).
  • Specimens sent by post outside GOSH must be packed and sent by microbiology as they need to be sent according to UN3373 regulations (United Nations Economic Commission for Europe, 2017). The specimen must be wrapped in a plastic bag, encased in an absorbent material within another plastic bag, placed in an approved plastic container within an approved cardboard box with the appropriate addressed infectious diseases stickers. A warning ‘Pathological sample’ along with the sender’s name and address must be visible on the outside. Specimens should not be sent to a laboratory outside the Trust direct from a ward, but sent through the microbiology laboratory to ensure correct packing and audit trail. 
  • Spillage kits (eg Clinell® spill wipes) must be available for decontamination of any spillages of blood or body fluids. The manufacturer’s instructions for use must be followed (Health and Safety Executive, 2001). Instructions how to use a spillage kit are available on the GOSH intranet infection control web page.  

Equipment

This will vary according to the specimen required but must include:  

  • disposable gloves
  • additional personal protective equipment (PPE) where applicable (eg apron/gown, respirator, visor) 
  • a plastic tray 
  • a sterile container for the specimen 
  • appropriate transport medium, if required 
  • laboratory specimen form 
  • a polythene transportation bag 
  • biohazard label, if required.

Specimen collection

Blood samples

Blood sampling should be performed by a healthcare worker trained and competent in the procedure. As there are many different microbiological and virological blood tests the person should seek information as to the appropriate laboratory containers required for specific tests and the amount of blood required. 

This information is available on PIMS (when printing the appropriate lab form) and the individual departmental user manuals. Protective clothing such as gloves and aprons (and facial protection when appropriate) must be used along with the aseptic non-touch technique. 

The ’Broken Needle Technique’ (breaking the hub of the needle to obtain blood from small infants) poses an additional risk of injury to the child and user and must not be used (Safety Action Notice, 2001)

Analysis of antibiotic levels 

The relationship between drug dose, drug concentration in biological fluid and the individual child’s metabolic process must be understood for interpreting results. The results may be affected by the route of administration, age of the child and disease process such as liver and renal disease. The analysis involves testing levels in blood serum or plasma in direct relationship to drug administration.

Routine in-house drug level assay is available for amikacin, gentamicin, tobramycin and vancomycin. Other levels are referred and should be discussed.

  • Antibiotic policies (including specialty-specific policies) are available on the GOSH intranet pharmacy web page.
  • Record on the laboratory form/sample the drug, dose and mode of administration, the time the drug is given, the time the level is taken and whether the sample is a ‘peak’, ‘trough’ or random level. 
  • Trough levels are taken immediately prior to the time a drug is due; peak levels are taken one hour after bolus or infusion finished. 
  • Blood for antibiotic assay should preferably be taken from venepuncture, heel/finger prick or arterial line; they must not be taken through the same catheter which has been used to give the antibiotic at any time. Antibiotics bind to plastic and the drug may release intermittently giving false results. 
  • Spurious low or high-level results may occur if blood is drawn from any central venous catheter and ideally levels should not be drawn from a central venous catheter. However, if an individual risk assessment necessitates sampling from a central venous catheter the GOSH clinical guideline ‘Blood sampling from central venous access devices’ should be followed to minimise false results.
Blood culture  

Detection of microorganisms by culture of blood is essential in the diagnosis of bloodstream infections, including infective endocarditis, infections presenting as pyrexia of unknown origin, prosthetic material infections and intravenous catheter infections. Blood culture may also detect bacteraemia associated with primary infections such as pneumonia and septic arthritis. Accurate positive results provide valuable information to guide optimal antibiotic therapy early on which can improve outcome from these conditions. Contaminated blood cultures can, however, cause considerable diagnostic confusion and lead to unnecessary or sub-optimal antimicrobial therapy. This can be prevented by careful collection of the blood using the aseptic non-touch-technique (Rowley and Clare, 2011). The specimen should preferably be taken during pyrexial episodes as more bacteria may be present at that time. 

Blood cultures should be taken when there is a clinical need to do so in response to any of the following clinical signs suggestive of sepsis and a deteriorating clinical picture including: 

  • Abnormalities in heart rate, core temperature, leucocyte count 
  • Presence of rigors or chills 
  • Other focal signs of infection, such as pneumonia, septic arthritis, meningism, urinary tract infection including pyelonephritis and acute abdominal pathology. 

Procedure for taking blood cultures (Department of Health, 2011):

  • Perform hand hygiene and put on gloves and apron.
  • Use both blood culture bottles (aerobic and anaerobic), remove the plastic cap and scrub the bung with a 2% chlorhexidine/70% alcohol wipe (eg Clinell®) for 15 seconds and allow to dry prior to inoculation. 
  • Prior to venepuncture soap and water should be used to clean any visibly soiled skin. The skin must then be decontaminated with a 2% chlorhexidine/70% alcohol applicator (eg Chloraprep® Sepp 0.67 ml) and allowed to dry. Do not re-palpate vein (even with a gloved hand) after decontamination. 
  • After withdrawing the blood, remove and safely discard the needle, and insert the blood into the container with a new sterile needle. There is a risk of contamination of skin organisms on the needle used to withdraw the blood. 
  • When inoculating the blood culture bottles, the anaerobic culture bottle should be inoculated first and then the aerobic culture bottle, so that oxygen trapped in the syringe will not be transferred to the anaerobic bottle. 
  • The volume of blood is the most critical factor in the detection of blood stream infections. Place up to 4ml in the aerobic bottle (priority) and up to 10ml in the anaerobic bottle, but ensure that when using both bottles, the anaerobic bottle is inoculated first. For neonates, one to two millilitres of blood is recommended (Kellogg et al, 2000). However, the sensitivity of neonatal blood cultures is increased if more blood is cultured.
  • Inoculation of the blood into the blood culture bottles should be performed first before inserting blood into other bottles as many of these bottles are not sterile and accidental contamination may occur. 
  • In children with suspected central venous line sepsis, blood for culture may be taken from a peripheral vein stab and also from (all lumen) of the intravascular lines to enable identification of colonisation of the line. In cases of suspected bacterial endocarditis three blood cultures should be taken from separate venepunctures to optimise recovery of bacteria which may be present low in numbers. 
  • Blood sampling for culture from a peripheral cannula: cultures from should only be taken from newly inserted peripheral cannulae if there is no alternative to obtain a blood sample for culture through a separate venepuncture. Strict asepsis must be maintained. The specimen must be clearly labelled indicating that the blood sample was taken from a peripheral cannula, as the risk of contamination is high.
  • Where available a closed system should be used to inoculate blood culture bottles.
  • A poster with detailed illustration of the blood culture technique is available on the Infection Control GOSH Intranet site.

Biopsy material

Specimens such as skin, muscle, kidney, liver, jejunal tissue or brain biopsies are generally obtained by medical staff either under general or local anaesthetic according to the site. A sterile technique is required for all these procedures. 

Biopsy specimens, especially if small or requesting multiple tests, must be discussed with the relevant laboratory personnel in order that:

  • The most appropriate specimen and laboratory tests are undertaken. If the specimen is small it may be necessary to limit the range of tests. 
  • Please ensure there are clear contact details for clinical staff able to prioritise testing if the sample proves to be inadequate.
  • Check if the specimen is to be fixed in formalin. Formalin must not be used if the specimen is for microbiological investigation. In many cases, both histopathological and microbiological/virological analysis will be required and it is critical that separate specimens be sent for these purposes so they are processed and transported appropriately. 

Bronchoalveolar lavage (BAL)

Information TBC 

Buccal swab

See oral fluid (saliva) test

Cerebrospinal fluid (CSF) 

Sampling of cerebrospinal fluid (CSF) is essential for the accurate diagnosis of infectious meningitis and may aid in the diagnosis of encephalitis.

Cerebrospinal fluid is most commonly obtained via a lumbar puncture performed by medical staff. A sterile technique is required as there is a risk of introducing infection itself causing meningitis. Specimens of CSF should be dispatched to the laboratory immediately. 

Outside normal working hours, it is essential that the on-call laboratory staff are contacted when the sample is being transported. It is important not to store the specimen in a refrigerator as this may cause the cells to deteriorate or lyse giving rise to misleading results. 

It is common practice to send three separate collection tubes of CSF when investigating for evidence of subarachnoid haemorrhage, as the initial part of the sample may be contaminated with blood from outside the sub-arachnoid space. 

If this is performed, it is important to label the tubes as such and specifically request counts on the first and third samples. It is also important to remember that a CSF glucose level (sent separately to chemical pathology) can only be accurately interpreted in conjunction with a simultaneous plasma glucose level.

Ear swabs

No antibiotics or other therapeutic agents should have been in the aural region for about three hours prior to sampling the area as this may inhibit the growth of microorganisms.

  • Perform hand hygiene and put on gloves and apron
  • Place a sterile swab into the outer ear and gently rotate to collect the secretions. 
  • If there is purulent discharge this should be sampled. 
  • For deeper ear swabbing a speculum may be used. Only experienced medical staff should undertake this procedure as damage to the eardrum may occur. 
  • Place swab in transport medium.
  • Remove gloves and apron and perform hand hygiene.

Eye swabs

Please note: a swab with charcoal transport medium should be used for suspected bacterial eye infections. For suspected viral (eg adenovirus) conjunctivitis a viral swab with viral transport medium should be used.

  • Perform hand hygiene and put on gloves and apron.
  • Where possible ask the child to look upwards and gently pull the lower lid down or gently part the eyelids. 
  • Use a sterile swab and gently roll the swab over the conjunctival sac inside the lower lid. Hold the swab parallel to the cornea to avoid injury if the child moves.
  • Place the swab in the specimen container with transport medium.
  • Remove gloves and apron and perform hand hygiene.

Please note: for suspected Chlamydia trachomatis infections a ‘Chlamydia sampling swab’ should be obtained from the virology department. These tests are referred to an external laboratory and are infrequently performed, therefore swabs may not be immediately available and testing may be delayed pending request for swabs. 

  • Perform hand hygiene and put on gloves and apron
  • Clean the eye first with sterile normal saline to obtain a clear view of the conjunctiva. 
  • Using the swab, part the eyelids and gently rub the conjunctival sac of the lower lid to obtain epithelial cells. 
  • Place the swab in the transport medium provided.
  • Remove gloves and apron and perform hand hygiene. 
  • Identification will be by the current molecular test employed at the referral laboratory. 

Fungal samples of hair, nail and skin

Special containers for the collection of fungal samples (eg Dermapak®) can be obtained from the microbiology department.

  • Perform hand hygiene and put on gloves and apron.
  • Samples of infected hair should be removed by plucking the hair with forceps or gloves. The root of the hair is infected not the shaft. 
  • Samples of the whole thickness of the nail or deep scrapings should be obtained. 
  • The skin should be cleaned with an 70% alcohol swab (eg Steret®). Epidermal scales scraped from the active edge of a lesion or the roof of any vesicle should be obtained. 
  • Remove gloves and apron and perform hand hygiene.

Gastric washings (lavage) 

Infants and young children are generally not able to expectorate enough sputum for laboratory analysis. It may, therefore, be necessary to obtain swallowed sputum through gastric washings (lavage), particularly to aid the diagnosis of pulmonary Mycobacterium tuberculosis. The detection of alcohol acid fast bacilli (AAFB) is not adequate alone for diagnosis of Mycobacterium tuberculosis as other environmental AAFBs may be present. Culture of the organism is always performed and may take between six to 12 weeks to confirm diagnosis. Three consecutive early morning specimens should be obtained. There are usually only small numbers of microorganisms present, so as much material as possible should be obtained. As AAFB are often found in tap water sterile water must be used.

Please note: Performing this procedure may cause the child to cough, which can expose the healthcare worker to pathogens. It is therefore essential that appropriate PPE including respiratory protection (FFP2 respirator and a visor) is worn when performing this procedure.

  • Fast the child for at least six hours overnight. 
  • Perform hand hygiene and put on PPE
  • Pass a nasogastric tube (refer to GOSH clinical guideline ‘Nasogastric tube management’).
  • Aspirate the stomach contents and place in a sterile container. 
  • Instil at least 30ml of sterile water down the tube to obtain as much stomach content as possible. 
  • Aspirate the contents back and place in the same container. 
  • Remove the tube, if appropriate. 
  • Remove PPE and perform hand hygiene.

MRSA screening

Specimens are taken to detect carriage of MRSA either before or on admission or as part of a screening process in the event of cases or an outbreak of MRSA.

As part of the routine admission screen, each patient should have a nose and throat swab taken in the 30 days prior to admission or within the first 24 hours of admission and then at least every 30 days during admission (Coia et al, 2006) (Department of Health, 2014). In addition, if present, any skin lesions and sites of indwelling devices (eg tracheostomy or gastrostomy) should be swabbed. In neonates, the umbilicus should be swabbed.

Please note: Children with epidermolysis bullosa do not need to have a nose or throat swab taken as this may cause mucosal damage. They should still be screened on non-mucosal sites such as hairline, axillae, groin/perineum and sites of indwelling devices.  

Procedure for nose swab
  • Perform hand hygiene and put on gloves and apron
  • If the nose is dry, moisten the swab in sterile 0.9% saline solution beforehand to enhance the collection of material and alleviate discomfort.
  • Insert the swab into the anterior nares and direct it up into the tip of the nose and gently rotate. Both nares should be swabbed using the same swab to obtain adequate material. 
  • Return the swab to the container with the transport medium. 
  • The outside of the nostrils may be rubbed after the procedure to alleviate the unpleasant sensation of swabbing. 
  • Remove gloves and apron and perform hand hygiene.
Procedure for throat swab
  • Place the child in a position with a good light source. This will ensure maximum visibility of the tonsillar bed. 
  • Perform hand hygiene and put on gloves and apron.
  • Either depress the tongue with a spatula or ask the child to say “aahh”. The procedure is likely to cause gagging and the tongue will move to the roof of the mouth. This can prevent accurate sampling, therefore it is important to quickly but gently rub the swab over the tonsillar bed.
  • Care should be taken not to contaminate the swab by contact with the tongue or the oral mucosa on removal. 
  • Return swab to the container with transport medium. 
  • Removes gloves and apron and perform hand hygiene.

Patients that have previously been screened positive for MRSA should, in addition to the nose and throat swabs, also have swabs taken from the following sites:

  • hairline (swab along the outline of scalp hair) 
  • axillae 
  • groin 
  • perineum 
  • any wounds or skin lesions 
  • umbilicus (only in neonates) 
  • insertion sites of devices (eg tracheostomy, gastrostomy, central venous catheter) 

When taking samples from intact skin the swab should be moistened with sterile 0.9% saline solution before sampling as this assists in the transfer of bacteria from the sampling site to swab and can increase the number of microorganisms collected (Perry, 2007).

Molecular bacteriology testing

  • Requests have to be discussed with a Consultant Microbiologist or Clinical Scientist if uncertain of the suitability of a specimen or the availability of a test. 
  • Specimen collection: 
    • Blood should be sent in an EDTA tube. 
    • All other material needs to be sent in a plain sterile container without any transport medium or fluid. 
  • Suitable specimens: 
    • A range of specific or broad range bacterial and fungal PCRs may be performed on specimens from any normally sterile site eg empyema, pericardial fluid, joint aspirate, CSF, BAL, any tissue and pus. 
    • Specific PCRs may be performed on non-sterile sites if the target organism in not normally present e.g. Bordetella pertussis on pernasal swabs or NPAs.  Broad range 16S bacterial PCR testing is usually delayed until after primary culture results are known to be negative. 
  • Positive results will be telephoned to discuss significance.

Nose swabs

Specify on the lab form if this is a routine admission screen for MRSA or for the investigation of a suspected infection. For the collection of nose swabs as part of screening for MRSA refer to the clinical guideline for the control of Meticillin-resistant Staphylococcus aureus (MRSA).

  • Perform hand hygiene and put on gloves and apron
  • If the nose is dry, moisten the swab in sterile 0.9% saline solution beforehand to enhance the collection of material and alleviate discomfort.
  • Insert the swab into the anterior nares and direct it up into the tip of the nose and gently rotate. Both nares should be swabbed using the same swab to obtain adequate material. 
  • Place in transport medium. 
  • For viral investigation same as above and place in viral transport medium. 
  • The outside of the nostrils may be rubbed after the procedure to alleviate the unpleasant sensation of swabbing.
  • Remove gloves and apron and perform hand hygiene. 

Nasopharyngeal aspirate (NPA)

A nasopharyngeal aspirate (NPA) is required for the diagnosis of viral respiratory infections such as influenza, parainfluenza, metapneumovirus and respiratory syncytial virus (RSV). It is also the preferred type of specimen for the diagnosis of Bordetella pertussis infections by PCR and culture.

Please note: Remember that performing this procedure may generate droplets and/or aerosols, which can expose healthcare workers to these pathogens. It is therefore essential that appropriate PPE (gloves, apron, FFP2 respirator and a visor) is worn when performing this procedure. 

  • Perform hand hygiene and put on PPE
  • Attach a mucus trap to the suction system and the appropriately sized catheter, leaving the wrapper on the suction catheter. 
  • Turn on the suction and adjust the pressure. 
  • Without applying suction, insert the catheter into the nose, directed posteriorly and towards the opening of the external ear.
  • Please note: The depth of insertion necessary to reach the posterior pharynx is equivalent to the distance between the anterior naris and external opening of the ear. When you feel a resistance you have reached the posterior nasopharynx.
  • Apply suction and slowly withdraw the catheter using a rotating movement. The catheter should remain in the nasopharynx for no longer than 10 seconds. The mucus trap should be kept upright. 
  • If necessary rinse the catheter with a small volume of sterile 0.9% saline solution to ensure adequate specimen volume. 
  • Disconnect suction and seal mucus trap with the supplied lid. Ensure a tight fit of the lid to prevent leakage. 
  • Remove PPE and perform hand hygiene.

© Image courtesy of NHS Pathology

Please note: NPA specimen must not be sent to the laboratory via the pneumatic tube delivery system (‘chute’) due to the risk of leakage. They must be transported to the laboratory by a porter.

Oral fluid (saliva) test

This test is used to confirm suspected cases of measles, mumps and rubella. Oral fluid test kits are available from the microbiology laboratory. 

Please note: Ideally the sample should be taken as soon as possible after the onset of the first symptoms.

  • Perform hand hygiene and put on gloves and apron
  • Place the child in a position with a good light source.
  • Remove pink sponge swab from clear tube
  • Ask the child to open the mouth wide.
  • Swab the area between the cheek and gum by rubbing the pink sponge swab all along the gums and teeth (if present) for one to 2 minutes.
  • Replace pink swab in clear tube.
  • Remove gloves and apron and perform hand hygiene.

© Image courtesy of Public Health England

Pernasal swabs

This investigation is most commonly used to diagnose whooping cough (Bordetella pertussis). Specimen for polymerase chain reaction (PCR) can be taken at any age if there is a clinical diagnosis. If acute infection is suspected, the pernasal swab will also be cultured, but must be taken to the laboratory immediately after collection.

A nasopharyngeal aspirate (NPA) is the preferred type of specimen for the diagnosis of Bordetella pertussis. Pernasal swabs may be sent, but it must be ensured that only special thin wire swabs for pernasal specimen collection are used (eg Dryswab™) which are available from the microbiology laboratory. Do not use charcoal swabs. When obtaining a pernasal swab the nurse must be proficient in the procedure and ensure suction, oxygen and resuscitation equipment is easily available as the procedure may induce paroxysmal coughing and/or vomiting. The child should be held securely during the procedure and be observed carefully during and immediately after the procedure. 

© Image courtesy of Gloucestershire Hospitals NHS Foundation Trust

Please note: Remember that performing this procedure may generate droplets and/or aerosols, which can expose healthcare workers to this pathogen. It is therefore essential that appropriate PPE (gloves, apron, FFP2 respirator and a visor) is worn when performing this procedure. 

  • Perform hand hygiene and put on PPE.
  • Place the child in a good light to facilitate observation 
  • Use a swab suitable for pernasal specimen collection (eg Dryswab™). Holding the head upwards, pass the swab along the floor of the nasal cavity to the posterior wall of the nasopharynx. 
  • Gently rotate and withdraw the swab and place back in the tube. 
  • Remove PPE and perform hand hygiene.
  • Dispatch specimen to the laboratory immediately to ensure maximum chance of growth of the organism. 

Sputum

Good quality sputum samples are essential for accurate microbiological diagnosis of pneumonia, but also acute tracheitis and bronchitis. Sputum cultures are routinely used for patients with chronic and often progressive suppurative lung diseases such as Cystic Fibrosis and Primary Ciliary Dyskinesia. However, samples contaminated with oropharyngeal secretions and saliva are difficult to interpret and can be misleading.

Please note: Remember that performing this procedure may generate droplets and/or aerosols, which can expose healthcare workers to these pathogens. It is therefore essential that appropriate PPE (gloves, apron, FFP2 respirator and a visor) is worn when performing this procedure. 

  • Perform hand hygiene and put on PPE.
  • Encourage the child to cough especially after sleep and expectorate into a plain sterile container. 
  • Alternatively a sputum sample may be obtained from nasopharyngeal, oropharyngeal or tracheal suction using a sputum trap. 
  • Chest physiotherapy may help to facilitate expectoration. 
  • Ensure the material obtained is sputum and not saliva. 
  • Remove PPE and perform hand hygiene

Stool samples

In children, stool specimen are frequently collected to identify parasites and microorganisms that cause diarrhoea. Please specify whether the sample is a routine (admission) screening sample or an investigation for suspected intestinal infection. If viral gastroenteritis (eg norovirus, rotavirus) is suspected, the stool specimen should be sent to the virology laboratory. To exclude a bacterial cause a second stool specimen can be sent to the microbiology laboratory. 

  • Perform hand hygiene and put on gloves and apron
  • Continent children should be asked to urinate first, flush the toilet and then defecate into a clean potty or a pulp bed pan that is placed on the toilet.
  • In nappy wearing children a urine bag can be applied to prevent contamination of the stool with urine.
  • The specimen can then be obtained from the nappy or potty/bedpan. 
  • Using the scoop attached to the inside of the lid of the specimen container place faecal material into the container. 
  • Where diarrhoea is present, a small piece of non-absorbent material lining the nappy can be used to prevent material soaking into the nappy. 
  • Examine the sample for consistency, odour and blood and record observations to monitor changes. 
  • If segments of tapeworm are seen, send these to the laboratory. Tapeworm segments can vary from the size of rice grains to a ribbon shape, 2cm long. 
  • For the identification of Enterobius vermicularis (threadworm/pin worm) material should be obtained first thing in the morning on awakening by using a clear adhesive tape (eg Sellotape®) slide. Place the sticky side of a strip of tape over the anal region to obtain the material and stick the tape smoothly onto a glass slide. The worms and/or ova can then be identified under the microscope. Threadworm lay their ova on the perianal skin at night and therefore will not be seen in a faecal specimen. 
  • Where acute amoebic dysentery is suspected, the specimen of stool must be freshly dispatched to the laboratory. The parasite causing amoebic dysentery exists in a free-living motile form and in the form of non-motile cysts. Both forms are characteristic in their fresh state but the motile form cannot be identified when dead. ‘Hot faeces’ should be discussed with the laboratory prior to collection. 
  • Remove gloves and apron and perform hand hygiene.

Please note: a stool specimen is more suitable than a rectal swab, as it is more likely to contain microorganisms. 

Throat swabs

Specify on the laboratory form if this is a routine admission screen for meticillin-resistant staphylococcus aureus (MRSA) or a swab for the investigation of a suspected infection. For the collection of throat swabs as part of screening for MRSA refer to the clinical guideline for the control of Meticillin-resistant Staphylococcus aureus (MRSA). 

  • Perform hand hygiene and put on gloves and apron.
  • Place the child in a position with a good light source. This will ensure maximum visibility of the tonsillar bed. 
  • Either depress the tongue with a spatula or ask the child to say “aahh”. The procedure is likely to cause gagging and the tongue will move to the roof of the mouth. This can prevent accurate sampling it is therefore important to quickly but gently rub the swab over the tonsillar bed or area where there is exudate or a lesion. 
  • Care should be taken not to contaminate the swab by contact with the tongue or the oral mucosa on removal. 
  • Return swab to the container with transport medium. 
  • For viral investigation same as above and place in viral transport medium. 
  • Remove gloves and apron and perform hand hygiene.

Urine 

Bedside urine testing for the presence of blood, protein and other analytes is usually undertaken with reagent strips the results of which indicate that further laboratory investigation is required (Cook,1995). 

Most urine samples sent to the microbiology laboratory are for bacteriological investigation. The same collection techniques also apply to samples sent for virological investigation.

Urine samples should be dispatched to the laboratory as soon as possible or no more than four hours if kept at room temperature or up to 24 hours if kept at four degrees centigrade to avoid overgrowth of organisms and misleading results (Griffiths, 1995).

All methods used to collect urine samples can result in contamination with bacteria from outside the bladder. This can lead to an inaccurate diagnosis, involve unnecessary treatment or require a sample to be repeated which has implications for patient care and cost-effectiveness (Lewis, 1998).

Normal social hygiene, such as washing the genitalia with soap and water and drying thoroughly is considered sufficient to minimise contamination from the skin prior to collection of the specimen. Assess the clinical and psychosocial needs of the child as to whether cleaning the genitalia is necessary. The healthcare worker must be sensitive to the cultural issues surrounding touching intimate parts of the body.

The most popular non-invasive method to obtain a urine specimen is the midstream or ‘clean catch’ specimen. NICE (2007) defines this method as the gold standard. In pre-continent infants obtaining a midstream or ‘clean catch’ specimen can be challenging. 

For infants aged one to 12 months, the ‘Quick-wee’ method can be considered to increase the voiding and success rate of a ‘clean-catch’ urine (Kaufman et al, 2017). This method uses gentle cutaneous suprapubic stimulation with gauze soaked in cold 0.9% saline to trigger faster voiding.

© Image courtesy of Bill Reid, Royal Children’s Hospital, Parkville, Australia

Midstream or ‘clean catch’ specimen
  • Perform hand hygiene and put on gloves and apron.
  • Ensure that the child’s/young person’s genitalia have been washed with soap and water and dried thoroughly. Ask the child/young person to wash hands with soap and water.
  • In the female, encourage separation of the labia whilst passing urine.
  • In the male, encourage retraction of the prepuce, if appropriate, whilst passing urine.
  • Ask the child/young person to void a small amount of urine into the toilet first
  • Then ask the child/young person to urinate 10-20 ml directly into a plain sterile specimen container.
  • Instruct the child/young person that the remaining urine can passed into the toilet.
  • Place the lid securely on the specimen container. Wipe the outside of the container with a sanitising wipe, label the container and place it in polythene transport bag.
  • Remove gloves and apron and perform hand hygiene.
Pad or bag specimen 

Urine collection pads or urine collection bags are often used for incontinent or non-toilet trained children, but are more susceptible to contamination due to close contact with the anogenital area. NICE (2007) suggests urine collection pads as the next best option to clean catch. When using urine collection pads, manufacturer’s instructions should be followed. Cotton-wool balls, gauze- or sanitary pads should not be used (NICE, 2007).

Urine collection pads:
  • Perform hand hygiene and put on gloves and apron.
  • Remove nappy and clean perineum or prepuce of the infant with soap and water. Do not apply any creams.
  • Place the urine collection pad across vulva or penis in a lengthwise fashion.
  • Remove the adhesive backing from the pad and secure to nappy.
  • Change urine collection pad every 30-45 minutes and also when the child has passed stool, to reduce the risk of contamination with skin-or faecal flora.
  • Once the child has passed urine remove the nappy with the urine collection pad in it.
  • Lay the pad down wet side up on an appropriate clean surface.
  • Take sterile 5 ml syringe and place the tip on the pad. Extract the urine by pulling up the plunger. 
  • Repeat until required amount of urine is obtained.
  • Empty syringe into a plain sterile container.
  • Remove gloves and apron and perform hand hygiene.
Urine collection bags:
  • Select the correct size sterile urine bag to avoid leakage or contamination with faeces. 
  • Perform hand hygiene and put on gloves and apron.
  • Remove nappy and clean the perineum or prepuce of the infant with soap and water.
  • Dry area thoroughly and do not apply any creams.
  • Remove the protective backing from the bag, then
  • For the female, place the bag over the vulva, starting from the perineum and working upwards, pressing the adhesive to perineum and symphysis.
  • For the male, insert penis and scrotum into the opening of the bag and press adhesive to perineum and symphysis.
  • Cut a hole in the diaper and pull the urine bag through the opening.
  • Once the child has passed urine, perform hand hygiene, put on gloves and remove the bag.
  • Hold the bag over a sterile urine specimen container and cut off the tip of the bottom corner of the bag. Empty the urine into a plain sterile container.
  • Wash the genitalia after the procedure to prevent soreness of the skin. 
  • Remove gloves and apron and perform hand hygiene.
Suprapubic aspirate

Collection of urine by a suprapubic aspirate should be considered when a sterile sample is required. Ultrasound guidance should be used to indicate the presence of urine in the bladder before a suprapubic aspirate is attempted (NICE, 2007). 

Catheter specimen 

This is collected from the self-sealing bung of the urinary drainage tubing in a child who is already catheterised. Do not disconnect the closed drainage system as infection may be introduced (Loveday et al, 2014) or take the sample from the urinary drainage bag as the specimen may be contaminated. 

  • Perform hand hygiene and put on gloves and apron.
  • Using an aseptic non-touch technique, clean the catheter sampling site with 2% chlorhexidine/70% alcohol wipe (eg Clinell®) and allow to dry. 
  • Collect the urine using appropriate sterile equipment appropriate to access port i.e. either using a sterile syringe and needle and inserting the needle into the bung at an angle of 45 degrees; this will minimise penetration of the wall of the tubing and subsequent needle stick injury or a needle-less system. 
  • Gently withdraw the urine into the syringe. 
  • Remove the needle and syringe, wipe the area with a 2% chlorhexidine/70% alcohol wipe (eg Clinell®) and allow to dry. The rubber bung will self-seal. 
  • Place the urine in a plain sterile container. 
  • Discard the needle and syringe into a sharps container. 
  • Remove gloves and apron and perform hand hygiene.
Obtaining urine from a Mitroffanof stoma 

The specimen should be obtained by a nurse who is familiar with the Mitroffanof operation and the specific anatomy of the area on the child. 

  • The specimen should ideally be taken in conjunction with normal bladder emptying. 
  • A new sterile catheter of the child’s normal catheter size should be used. 
  • Perform hand hygiene and put on gloves and apron
  • Clean the stoma with soap and water and dry. 
  • Gently insert the lubricated sterile catheter into the stoma and collect the urine into a plain sterile container. A water-soluble lubricant should be used. 
  • Ensure the bladder is completely empty before withdrawing the catheter.  
  • Wipe the area dry with a tissue. 
  • Remove gloves and apron and perform hand hygiene.

Vaginal/Vulval swabs  

Taking a vaginal or vulval swab is a sensitive procedure and the medical context, as well as the potential sexual activity of the girl needs to be considered to decide if a swab is clinically indicated.

The procedure should be explained to the girl and the parents/carers. It should be done by a, if preferred, female HCW proficient in the procedure and usually in the presence of a parent/carer in a relaxed and private space. In some circumstances young people should be given the choice whether or not they would like to have a parent or carer present. Please refer to the GOSH ‘Chaperoning Infants, Children & Young People Policy’ if the presence of a chaperone should be considered.

Vulval swab for investigation of simple vaginal discharge:

  • The girl should lie on an examination couch with her legs in a frog-like position.
  • Gentle separation and retraction of the labia should allow visualisation of the external genitalia, introitus and hymen.
  • Discharge can pool in the posterior fourchette and a swab can be taken from this area. Do not insert the swab into the vagina.
  • In very young girls a thin cotton-tipped wire swab (eg Dryswab™, same as for pernasal swabs) can be used.
  • In older girls, a routine charcoal swab can be used.
  • If a viral infection is suspected, a viral swab with viral transport medium should be used.
  • Place the swab into the supplied transport medium.

In the case of suspected or actual sexual abuse:

  • Prior to any examination being undertaken, the named safeguarding doctor must be contacted for advice and support.
  • The named safeguarding doctor will advise on the appropriate investigations, as well as the appropriateness of a forensic procedure.
  • Do not clean the area (unless clinically indicated) as identification of semen or sexually transmitted diseases may be required for evidence.
  • Local child protection protocols must be adhered to. 
  • GOSH Social Work Service should be informed or out-of-hours the Clinical Site Practitioners.

Vesicular fluid for herpes polymerase chain reaction (PCR)

This test may be necessary to confirm a suspected diagnosis of herpes simplex or varicella zoster (chickenpox or shingles).
Vesicle fluid may be collected into a syringe or onto a swab and placed in viral transport medium for testing by PCR.

Collection in a syringe

Explain to the child and parent that the procedure is usually pain-free as the needle only penetrates the vesicle not the skin.
Obtain a sterile syringe and needle, sterile swab and viral transport medium. 

  • Perform hand hygiene and put on gloves and apron.
  • Pierce the top of the vesicle with a sterile needle and if there is sufficient fluid draw up the exudate into a syringe. Keep the needle flush to the skin to prevent accidental stabbing in case the child moves. Then draw up some viral transport medium into the syringe and flush the medium plus vesicle fluid back into the bottle of viral transport medium. Dispose of the needle in a sharps bin. 
  • Send the sample with a request for Varicella zoster and Herpes simplex PCRs. 
  • Place a sterile dressing over the vesicle until dry. 
  • Remove gloves and apron and perform hand hygiene.
Collection on a swab

If the vesicle is already ruptured and moist or dry, a positive diagnosis may be reached from a swab which has been vigorously rubbed over the dry base and sent in viral transport medium. If fluid is easy to collect the vesicle may be punctured and fluid collected on a swab which is then placed in the viral transport medium.

Wound swabs

Interpretation of results must be in conjunction with clinical signs. In the absence of clinical signs of infection wound swabs will provide little if any useful information and simply reflect colonisation (Gilchrist, 1996).

  • Obtain the specimen prior to any dressing or cleaning procedure of the wound. This will maximise the material obtained and prevent killing of the organism by the use of antiseptics. 
  • Perform hand hygiene and put on gloves and apron.
  • Use a sterile swab and gently rotate on the area to collect exudate from the wound and place into transport medium. 
  • Where there is pus collect as much as possible in a sterile syringe and put in a plain sterile container (do not use a swab) and send to the laboratory. 
  • For detection of Mycobacterium tuberculosis, pus collected neat into a pot or tissue biopsy is preferred, however a calcium alginate swab can be used. The alginate swab gradually dissolves maximising the isolation of the organism as the number of organisms is usually small.  
  • Remove gloves and apron and perform hand hygiene.

References

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Loveday, H.P., Wilson, J.A., Pratt, R.J., Golsorkhi, M., Tingle, A., Bak, A., Browne, J., Prieto, J., Wilcox, M. (2014) epic 3: National Evidence-Based Guidelines for Preventing Healthcare-Associated Infections in NHS Hospitals in England . Journal of Hospital Infection 86S1 S1-S70

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Document control information

Lead Author(s)

Barbara Brekle, Deputy Lead Nurse, Infection Prevention and Control

Additional Author(s)

John Hartley, Consultant Microbiologist and Director of Infection Prevention and Control

Document owner(s)

John Hartley, Consultant Microbiologist and Director of Infection Prevention and Control

Approved by

Guideline Approval Group

Reviewing and Versioning

First introduced: 
01 February 2003
Date approved: 
28 June 2017
Review schedule: 
Three years
Next review: 
28 June 2020
Document version: 
6.0
Previous version: 
5.0