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Information required on request form

It is essential that all sections of the request form are completed.

The tests performed on specimens depend on the patient’s clinical details provided on the form; if these are incomplete then appropriate tests may fail to be performed.  For example, Vibrio cholerae is not routinely examined for in stool specimens, the investigation being dependent on, for example, a relevant travel history.

Specimen collection

Specimens must be taken using steps to minimise contamination and delivered to microbiology as soon as possible.  Specimen containers must be labelled with patient’s name, hospital number or date of birth and specimen type.
Specimen containers must be approved containers only

Staff should wash their hands before and after taking specimens and take appropriate safety precautions.

Swabs (see individual test library)

Bacteriology Turnaround Times

Turnaround time is the time from date of receipt into the laboratory to the issue of the final report. Microbiology aim to achieve the quoted turnaround time for 85% of samples received.

Turnaround time will be extended for samples received after 4.00pm (Mon-Fri), samples received on Saturdays, Sundays or Bank Holidays, samples requesting complex investigations or additional tests and samples which are referred to other laboratories for testing.

Final results are reported electronically by GP Link or to the electronic ward reporting system (CRRS/Review/ICE) as soon as they are available. Some preliminary results are also delivered electronically. Significant results, where early diagnosis would be beneficial to the management of the patient, are telephoned immediately by the Duty Microbiologist.

Further advice on sample collection and types of sample required is available elsewhere on this website or by contacting the laboratory.

Factors Affecting Results

The following are important considerations when submitting samples for bacteriological investigation:

Specimen collection:           Samples and request forms should be labelled with sufficient information to allow identification of the patient and requester. Relevant clinical details should be provided. Unlabelled or mis-labelled samples may not be examined. Incomplete requester detail will result in no report being received. Incomplete request forms or incomplete clinical details may lead to inappropriate tests being performed.

Delay in transport:   Samples for bacteriological investigation should be transported without delay to the laboratory. Delayed transport may affect the viability of pathogens and allow overgrowth of normal flora. Morphological appearance of cells may also be affected.

Transport medium:  Swab samples for bacteriology should be sent in charcoal transport medium. Urines for bacterial culture (not TB) should be sent in a sterile universal container with boric acid (red top). Pus, aspirated fluids and urine for TB culture should be sent in a sterile universal container. Tissues and biopsies should be free from formalin. Use of dry swabs affects the recovery of fastidious organisms. Use of non-sterile containers can allow overgrowth of contaminating organisms. Use of formalin affects the viability of bacteria.

Temperature extremes:      Samples for bacteriology should be kept cool if not transported immediately to the laboratory. High temperature can lead to overgrowth of normal flora and may kill the target organism. Low temperature can affect recovery of some susceptible organisms.

Measurement of uncertainty          Measurement of uncertainty (MU) information is available for users on request from the laboratory.

Additional tests     

Additional requests for bacteriology specimens already received are not permitted unless specifically agreed with the Microbiology clinical staff

Medico legal cases

For medico legal cases i.e.: rape, sexual abuse etc, specific protocols have to be followed in order for any evidence to be accepted as genuine.  In these circumstances please discuss with either GUM consultant, or consultant microbiologist before submitting any specimens.

A chain of evidence must be used for these samples.

Trichomonas vaginalis

T vaginalis is a fragile organism and may not survive transportation on a swab

T vaginalis culture is not routinely performed in the laboratory

Sputum

Sputum samples for C&S

These are collected by expectoration directly into screw capped universal containers.  A good quality specimen is essential i.e. purulent material, salivary samples may be rejected for routine culture. BAL specimens should be clearly marked as such.

Sputum for TB

Three consecutive early morning samples should be sent to the laboratory together with a request form indicating that processing for TB is required

Blood Culture

A set of two 'BacT/Alert' bottles (aerobic/anaerobic) is required for adults and a single bottle for paediatric. When investigating Pyrexia of Unknown Origin or suspected endocarditis, three sets should be taken over a period of time. For example in the case of “sub-acute endocarditis”, three sets should be collected over several hours before a suitable antibiotic regime is started.  For suspected line infections a peripheral culture as well as line culture is recommended (ensure bottle(s) and form are labelled clearly line or peripheral culture). Any relevant clinical details such as foreign travel should be stated.

How to collect blood for culture

The blood should be collected before antibiotic therapy is begun and preferably while the patient has a rising temperature.

Strict asepsis must be observed. Remove plastic flip-top from each culture bottle and disinfect with an alcohol pad.  Treat the skin with alcohol.  Allow to dry for at least one minute, collect up to 20mL of blood by venepuncture.  Change needles and dispose the needle into Sharpsafe container.  Inject up to 10mL into each bottle, inoculating the anaerobic bottle first.  Mix gently and label all bottles with the patient's details. Transfer both bar-code strips from the bottles to the front of the request form. DO NOT COVER the bar-code on the bottle with the patient label.

Tuberculosis

In cases where “miliary” tuberculosis is suspected, two sodium citrate tubes (light blue) can be collected for liquid TB culture.

Laboratory Procedure

Blood cultures are examined visually on arrival and thereafter monitored constantly by machine.  If growth is detected, Gram films and subcultures are done to identify the organism and significant results are reported by telephone.

Urine

Mid-stream, clean-catch specimens are obtained to avoid contamination.  For some patients it may be necessary to collect a ‘catheter specimen’ or ‘bag urine’ and this must be clearly stated on the request form.

1. Collection of Mid-stream specimens of urine

The genitalia should be washed thoroughly with soap and water and dried.  The labia should be separated or the foreskin drawn back, far enough to expose the urethral opening.

The first urine passed should NOT be collected.  The middle part of the urine stream should be collected in a sterile container.  This should then be placed in a sterile universal container containing boric acid (red top) and filled to the level indicated.  Screw cap on tightly and mix with boric acid powder thoroughly with the urine.  Boric acid stabilises the bacterial population until the specimen is processed in the laboratory, but the specimen MUST BE SENT TO THE LABORATORY WITHOUT DELAY.

When only a small amount of urine can be obtained, place in a sterile universal container without boric acid.  Again refer to the laboratory immediately, or refrigerate specimen until possible.

Urine which are not received in white top universal will be rejected unless they meet any of the following criteria:

1. Small volume (<5ml) urine samples in any patient cohort.
2. Nephrostomy, urostomy, cystoscopy, ileal conduit samples
3. Any child <5yrs old
4. Schistosoma investigation
5. Supra-pubic aspirate
6. Samples for TB investigation

Please see Urine collection guide:

Urine samples are often collected in attempts to microbiologically confirm the diagnosis of a lower or upper urinary tract infection. There is little value in performing a dipstick on urine samples in the > 65 years age group due to a high false-positive rate caused by the presence of non-pathogenic bacteria. Therefore, we have to rely on culture to produce a result.

When to send urine samples for microbiological culture

Signs and symptoms of UTI: Dysuria, frequency, urgency, suprapubic pain, polyuria.
Signs and symptoms of upper UTI (UUTI): As per UTI, plus loin pain, flank tenderness, fever, other manifestations of systemic inflammatory response.

How to collect urine samples for mycobacterial culture1

• Send mid-stream urine samples if possible, and prior to commencing antimicrobial therapy.
• Fill to the fill line wherever possible
• The container MUST BE LABELLED with the patient’s details and placed in a clear plastic bag accompanied by a completed blue microbiology request form then delivered to the laboratory.

DO NOT USE DIPSTICK IN THE DIAGNOSIS OF UTI ION OLDER PEOPLE >65 YEARS OR CATHETERISED PATIENTS.

Completing the request form

• Clinical details MUST state:
• Symptoms with date of onset
• Any antimicrobial therapy
• Request for microscopy, culture & sensitivity
• Plus (if required): 
• Chlamydia/gonorrhoea
• Acid fast bacilli/TB- send 3 consecutive early morning urine Samples in white topped containers only
• Schistosomiasis- send midday urine sample in a white-topped Universal tube

Reference
1/PHE (2019). Investigation of urine. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/770688/B_41i8.7.pdf
2/ PHE (2017). Investigation of specimens other than blood for parasites https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/622944/B_31i5.1.pdf
 

b.    Catheter specimens of urine
In most circumstances, such specimens should only be taken when the patient is systemically unwell, e.g. pyrexial. Urine should be collected directly via the catheter tubing via the aspiration port.  Swab port with 70% alcohol swab allow to dry, and then aspirate urine using needle and syringe (taking care to avoid sharps injuries).  DO NOT collect urine from the catheter bag.

c.     Culture of urine for Mycobacterium tuberculosis
The first 30ml or so of the first morning specimen of urine passed by the patient should be obtained by a clean catch technique on each of three successive days.  The urine should be transferred to a plain sterile screw capped sterile universal container (DO NOT USE BORIC ACID CONTAINER), filling it to the shoulder.  The lid should be screwed on tightly. The request form and specimen should be clearly marked according to the day of collection; e.g. EMSU 1st day for TB culture.

Faeces

Plastic disposable screw capped containers with enclosed spoon are used.  Two spoonfuls will suffice for most microbiological investigations, but don’t fill more than 1/3rd.  Special care should be taken with fluid stools to ensure that the container is securely closed.  Rectal swabs should only be sent for multiresistant/ CPE organism screen as a negative routine culture result is not as reliable.

a.    Faecal culture
Specimens from the community and all “admissions” units are routinely examined for Salmonella, Shigella, Campylobacter and Verotoxogenic E coli 0157.  Further investigations will be guided by clinical details. All specimens from adults are tested for Clostridium difficile toxin (see below).

b.     Parasitology investigations
Specimens are not routinely examined for parasites.  Where indicated please request parasitology on the form and give supporting clinical details.  As excretion of parasites may be intermittent three separate stool specimens may be required and it is essential that they are collected on different days.  If amoebic dysentery suspected, a “hot stool” specimen is required. For the detection of S. haematobium;
A complete urine sample collected between 10am – 2pm should be submitted.

c.    Threadworm investigation
An anal swab in saline is now preferred over a sellotape slide.  The anus should be swabbed, and the swab then placed in a sterile universal container half filled with normal saline.

d.    Clostridium difficile
Clostridium difficile may produce a wide spectrum of diarrhoeal illness through to life threatening pseudomembranous colitis.  Infection is almost always associated with antibiotic use (a significant percentage of cases arise after stopping the antibiotics).  C. difficile is a predominantly hospital associated organism, but with shorter hospital stays more cases are likely to arise in the community.  All stools from adult patients are routinely tested for the toxin of Clostridium difficile (CDT).
Only samples which take the shape of the container will be tested for C-diff in line with national guidance.

e.    Viral diarrhoea
For investigation of viral causes of gastroenteritis in children under 5, (Rotavirus/Adenovirus), submit a stool specimen as described above requesting viral investigation.

f.    CPE/CRO screening
Rectal swab or faeces required for Molecular method. This is for GEH and SWUFT only. Please see details in Virology section.
 

Fungal Infections of the Body Surface
These are diagnosed by direct microscopy and culture, the latter being more sensitive.

Skin scrapings, nail parings or depilated hairs should be sent in a small sheet of clean paper, preferably black, folded three times, marked with the patient's details, and attached to the request form.  If it has to be sent from outside the hospital, the form and attached packet of material should be put in an envelope. For thrush, throat swabs may be used as for Bacteriology.

Systemic Fungal infections
Please contact the microbiologist for advice on diagnosis and treatment.

Antibiotic dosing / levels (please see the UHCW Trust intranet)

NB. All antibiotics are now analysed by the Biochemistry department. Please use biochemistry request forms, clearly marking the antibiotic assay(s) required. Please also use the standard biochemistry clotted tube and if other biochemistry tests are required, a separate sample tube is not required.
 

Possible viral infection may be investigated by molecular methods such as PCR, or serological investigations such as immunoassay. Techniques often change rapidly; therefore if there is doubt about test specimen requirements please contact the laboratory (Ext. 25461 or 25468). If advice on clinical management or investigation strategy is required contact the duty Virologist (Ext. 25349 or 25471). Alternatively you may email ClinicalVirology@uhcw.nhs.uk.

Clinical Details

Precise clinical information is very important for determining which test to perform; vague information is unhelpful and will delay processing of samples.

Clinical details should include (where appropriate):

1. Concise clinical summary of symptoms
2. Significant past medical history
3. Date of onset of illness,
4. Contact with other infected individuals and date of contact
5. Previous vaccination
6. Travel history

Specimen requirements according to syndrome

Infections of the skin and mucous membranes

Herpes Simplex (HSV), Varicella Zoster (VZV), and Enterovirus

PCR is the test of choice to determine viral causes of infection of the skin or mucous membranes. For a vesicular rash, where skin infection caused by HSV, VZV or enterovirus is considered, a swab should be used to sample the opened lesion and placed in a sterile universal container. If the lesion is dry, pre-moisten the swab with sterile saline before swabbing in the normal way. If moist, gather as much exudate as possible on the swab. Please use sterile swab. DO NOT SEND CHARCOAL SWABS. Please notify Virology if VZV is suspected so that appropriate infection control measures can be implemented.

Measles

Measles PCR can be performed for inpatients, but this is usually limited to infection control risks (including outbreaks), and resolving diagnostic conundrums only. Please contact the duty Virologist or email ClinicalVirology@uhcw.nhs.uk to arrange. Measles is a notifiable infection and any suspected/confirmed cases should be notified to the local health protection unit.

Respiratory infections including COVID-19

Viral respiratory infection

If infection with a respiratory virus is suspected, please send either a dry sterile nose or throat swab in a universal container for PCR. DO NOT SEND CHARCOAL SWABS. NPAs, BALs and sputum samples can also be tested.. If rapid or urgent COVID-19 testing is required, please contact the duty Virologist or email ClinicalVirology@uhcw.nhs.uk. Where an atypical viral pneumonitis is suspected e.g. CMV, HSV, VZV, contact the duty Virologist to discuss and arrange testing.

Atypical bacterial pneumonia

If atypical bacterial pneumonia is suspected in a patient, please submit urine for Legionella urinary antigen testing. Please note, complement fixation testing is no longer available for the investigation of atypical pathogens as it has been withdrawn by our reference lab providers. Please contact Virology or Microbiology to discuss investigation of atypical pathogens.

Gastroenteritis

Investigations of sporadic viral gastroenteritis including Adeno/Rotavirus in children less than 90 days old and outbreaks; Norovirus (winter vomiting disease) diagnosis is largely restricted to outbreak investigations, in all age groups where clinically indicated in outbreaks. Faeces samples or vomit specimens can be used. Swabs from faeces or vomit will not be accepted.

Genitourinary infections caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC) and Herpes Simplex (HSV)

For CT and GC, self-taken vaginal swabs, urethral, rectal, eye and throat swabs are accepted; please use the ‘dry’ swabs. For endocervical swabs please use the ‘wet’ swabs i.e. break into BD viper diluent. Urine samples should be aliquoted into a BD urine preservative tube. For genital HSV, use the ‘wet’ swabs i.e. break into BD viper diluent.

Viral infections of the eye

Excess pus should be removed. The eyelid must be inverted and the swab pressed firmly along the inside of the lower lid. Diagnosis is dependent on obtaining a satisfactory number of cells. Place swab into a sterile universal. DO NOT SEND CHARCOAL SWABS.

Congenital CMV

The best sample type to send is urine or saliva, preferably within the first three weeks of life, for CMV PCR. Where congenital CMV is suspected after three weeks, submit urine, and if after 12 months, send serum only for CMV IgG. Investigations carried out after three weeks of age may require confirmation by testing the Guthrie card. Please contact the duty Virologist or email ClinicalVirology@uhcw.nhs.uk if further advice is required.

Viral infections of the central nervous system e.g. meningitis, encephalitis

CSF will be tested for viruses by PCR where this is indicated by the cell count or requested by the clinician. PCR testing for viral pathogens such as HSV, VZV, enterovirus, and parechovirus, and bacterial pathogens such as meningococcus or pneumococcus are available as reference laboratory tests. An expanded panel of pathogens including CMV, EBV and JC virus can be requested if clinically indicated. Please ensure a separate CSF sample is sent specifically for virology if possible. If enterovirus is suspected a throat swab and stool sample may also be sent for PCR.

If Lyme disease, syphilis, toxoplasmosis, cryptococcus and leptospirosis are being considered, send CSF with a paired blood sample. Negative blood serology for the above infections essentially rules out neurological disease.

Blood borne virus infections – HIV, HBV, HCV

To diagnose potential blood borne virus infection, please send serum (clotted blood) for HIV/HBV/HCV serology. Molecular methods (i.e. PCR) are not appropriate for the routine diagnosis of blood borne virus, except in the case of hepatitis C where it is used to diagnose active infection following positive serology. In patients who have been diagnosed with a blood borne infection by serology, viral load monitoring may be indicated to monitor response to treatment. Please send 3 x EDTA bloods. Note, a minimum of 1.5 mL of plasma is required.

Routine monitoring of immunosuppressed patients

Certain patient groups (e.g. renal transplant, haematology/oncology) are vulnerable to reactivation of viruses such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus. These can cause considerable morbidity and mortality, therefore blood samples should be monitored for the presence of these viruses during periods of immunosuppression. Please send EDTA blood to determine viral loads.

Other specimens

Aspirated fluids, biopsy specimens, products of conception and other fluids - see under Bacteriology.

Guidance on Test Usage

A wide array of serological tests are available, and their correct utilisation is highly dependent on clinical information. There is not the scope to discuss every test, but highlighted below are some common investigations. Please ring for advice if in doubt.

Antenatal Infectious Diseases Screening

CWPS supports the Infectious Diseases in Pregnancy Screening Programme by providing the laboratory component of the screening pathway for pregnant women in Coventry and Warwickshire. The UK National Screening Committee (NSC) policy for the IDPS programme is to offer and recommend screening to all eligible women for HIV, hepatitis B and syphilis. This is to enable early detection and treatment for infections in pregnancy in order to significantly reduce the risk of vertical transmission. A secondary benefit is the identification of women with these conditions who can be offered appropriate care for their own health needs.

Laboratory testing is provided in-house and results are reported within 8 days (IDSP standard) with positive screens notified directly to the local screening teams.

Varicella-zoster – Management of chicken pox/zoster contacts

Varicella may occasionally produce severe disease in non-immune contacts. Following assessment, such contacts should be offered post-exposure prophylaxis (PEP) either in the form of aciclovir, or varicella zoster immunoglobulin (VZIG) to attenuate the disease course, provided that it is administered within the appropriate time period following contact. Due to a severe national shortage of VZIG, PHE have implemented restrictions on the use of this product. Currently, VZIG is only indicated for:

1. Non-immune pregnant women <20 weeks gestation.
2. Neonates whose mothers develop chickenpox (but not shingles) in the period 7 days before to 7 days after delivery.
3. VZV antibody-negative infants under 1 year who have remained in hospital since birth who are born before 28 weeks gestation OR weighed less than 1000g at birth
4. VZV antibody negative infants who have severe congenital or other underlying condition that require prolonged intensive or special care during the first year of life.
5. VZV susceptible neonates exposed to chickenpox or shingles (other than in the mother) in the first 7 days of life.

If an individual at high risk of severe VZV infection is exposed to chickenpox, contact the duty Virologist or Consultant Virologist to carry out a risk assessment, and arrange antibody testing/issue of PEP (if appropriate). Note, in general if a pregnant contact gives a definitive history of chicken pox, then she may be reassured, as she will be immune. For other pregnant contacts and immunocompromised patients antibody levels need to be measured (approx. two thirds of patients who give no history of Varicella will be shown to be immune on serological testing).

To ensure timely reporting of results: -

1. Ring laboratory and inform Clinical Scientist or senior BMS that blood is being sent for Varicella antibodies. Give full details of the nature and date of contact. Alternatively clear details should be written on the request form.
2. Please provide a contact number for result to be telephoned to the following day (necessary for weekends/bank holidays) Results for a blood sample received Friday will be available Saturday morning. The patient’s contact telephone number is useful.

Where an individual presents late following contact and there is insufficient time to test sera VZIG may still be issued.

VZIG is obtained from PHE Colindale, and issue with be co-ordinated by the Clinical Virology team if deemed clinically appropriate.

Hepatitis Serology

Requesting ‘Hepatitis serology’ is insufficient as it does not provide adequate information to guide testing. There are a large number of tests available and often differing tests for each virus. Please provide:

1. Full clinical information including risk factors, results of LFTs, vaccination history. The laboratory can then decide on the relevant tests.

2. Additionally request specific tests as appropriate.

SARS-CoV-2 Antibody Testing

At present, CWPS offers testing for antibodies to SARS-CoV-2 nucleocapsid and spike protein.

The clinical utility of SARS-CoV-2 anti-nucleocapsid (anti-N) is unclear and is generally utilised to establish whether an individual has been infected with SARS-CoV-2. Absence of antibody cannot infer that a person has not been naturally infected but presence shows they have. Virus specific antibody responses are prone to wane over time, and consequently protection against outcomes such as infection, transmission risk, or severe disease may also wane over time. Note, this assay does not test for antibody acquired through the currently approved SARS-CoV-2 vaccines.

Internal verification of our SARS-CoV-2 anti-N found that maximal assay sensitivity was achieved for samples ≥ 28 days post PCR positive result at 93.82% (95% CI 89.79-95.18). Thus, a positive result anti-N suggests that the patient was infected with SARS-CoV-2 at least 4 weeks ago but it may be longer than that. This is all that can be inferred. Repeat testing of seronegative individuals may be required if sample was collected <28 days before positive PCR result. No inference to immunity can be gained from this test and people who are anti-N positive may be re-infected with the virus.

SARS-CoV-2 anti-spike (anti-S) can be used to investigate previous exposure to SARS-CoV-2 virus, or vaccination with any of the currently licensed SARS-CoV-2 vaccines which are based upon spike-protein. It is not possible to distinguish between exposure to SARS-CoV-2 or vaccination on the basis of anti-S status alone. 

Investigation of Genitourinary Infection

The laboratory offers open access to chlamydia investigation. Chlamydia collection kits are available from the laboratory on request (Extension 25468).

Current methodology uses nucleic acid amplification techniques for the detection of DNA. Whilst all tests are subject to specimen quality, this is especially so with chlamydia. Chlamydia are intra-cellular pathogens, and good quality cellular material is required as opposed to pus.

It is recommended that all patients who are positive for Chlamydia are referred to the Genito- urinary Medicine Clinic.

Female genital tract

Chlamydia primarily infects the endocervix, and hence an endocervical swab is required. Excess mucous/pus should be removed from the cervix prior to taking the endocervical swab for chlamydia.Self-taken vaginal swabs may be used for Chlamydia screening but in symptomatic patients full examination and endocervical swabs are recommended.

Male genital tract

Infection in males produces a urethritis, although the symptoms may be mistaken for a urinary tract infection. In sexually active men with sterile pyuria, chlamydia urethritis should be considered a possibility.  For men collect either:

1. Chlamydia urethral swab

Or

2. First catch urine. The first 20 mL of urine voided should be placed in a sterile universal container, and preferably aliquoted into a urine preservative tube. The form should be clearly marked for chlamydia investigation. Patients should not have passed urine for at least two hours before the test.