Understanding Investigations III Microbiology
Different methods are available to detect infection. Some are non-specific, such as changes in blood inflammatory markers, and others give specific information about the exact infection. Proof of infection includes direct detection (e.g. microscopy, antigen detection, PCR), detection of an antibody respons ogy) or culture of an organism froma normally sterile site.
Culture of a with of a bacterium (or occasionally a fungus) from blood is usually definitive proof of infection. However, if the culture is of several different organisms, or if low pathogenic bacteria normally found on the skin (such as coagulase-negative Staphylococci) are isolated, then the result should be interpreted with caution. The way the blood sample is taken is crucial—the skin must be thoroughly cleaned with antiseptic and an aseptic technique used. Very small blood samples (<1 mL) reduce the chances of a positive culture. Blood cultures usually take 24– 48 hours to show evidence of infection and so are usually used to confirm a clinical suspicion of infection retrospectively. Cultures will provide information about the sensitivity of the organism, which can be used to rationalize antibiotic therapy. Most modern blood culture systems are able to detect significant bacteraemia in children with a single medium, which is designed specifically for this purpose. Unusual results should always be discussed with a microbiologist.
Serological evidence of infection
Measurement of antibody response to specific infectious agents can be useful. This is important to check for prior immunity (e.g. in an at-risk child vaccinated against hepatitis B) or to confirm prior infection (e.g. cytomegalovirus (CMV)). IgG antibody tends to persist after infection, whereas IgM antibody reflects recent infection. This can be important in the newborn period for distinguishing congenital infection (e.g. syphilis) from maternal infection since IgG antibody readily crosses the placenta. Antibody responses to infection are often described as ‘titres’. The titre is the reciprocal of the highest dilution of the patient’s serum in which antibody was detected, e.g. a titre of 1024 means that antibody was detected in a 1:1024 dilution of serum. The higher the titre, the more the anti-body is present. The anti-streptolysin-O titre (ASOT) is sometimes used as a marker of streptococcal infection in rheumatic fever.
Direct detection methods
Molecular biology techniques can now identify certain organisms, such as viruses, that have traditionally been difficult to culture. These tests can either use immunofluorescence, e.g. to identify respiratory syncytial virus (RSV) in pharyngeal secretions in a child with bronchiolitis, or polymerase chain reaction (PCR) to amplify bacterial or viral DNA using specific primers. PCR methods are available for many important paediatric infectious agents, including herpes simplex virus (HSV), Neisseria meningitidis groups B and C, and HIV. They are particularly useful in confirming infection after antibiotics have already been given and in detecting viral CNS infection.
Lumbar puncture and CSF analysis
Lumbar puncture is usually performed to diagnose or exclude meningitis. It should not be performed if there is evidence of raised intracranial pressure, if the child is haemodynamically unstable (e.g. septic shock) or if there is a low platelet count or coagulopathy. A fine spinal needle with a stylet is passed between the vertebral spines into the subdural space. A few drops of CSF are collected for microbiological examination and for analysis of protein and glucose concentrations. Examination of CSF includes microscopy and culture and may also include other direct detection techniques (e.g. DNA detection with PCR). Normal CSF is usually ‘crystal clear’. If it is cloudy, this suggests infection or bleeding. Fresh blood that clears usually indicates a traumatic tap, but a massive intracranial haemorrhage must be considered if the CSF remains bloodstained. Old blood gives a yellow ‘xanthochromic’ appearance. A manometer can be used to easure the CSF pressure, though this is not routinely performed.
Although these are typical CSF findings for the organisms indicated, partially treated infection and infection with specific microorganisms may result in alternative profiles. For example, meningitis caused by Listeria monocytogenes usually presents with a CSF lymphocytosis.
Fresh urine should be collected into a sterile container from a midstream sample if possible. Urine bags applied over the genitalia may be used in infants, but often become contaminated with perineal bacteria. Catheter specimens and supra pubic aspirate are alternatives.
• Observe the urine—is it cloudy (suggests infection) or clear?
• What is the colour?—pink or red suggests haematuria from the lower urinary tract; brown (‘cola’-coloured) urine suggests renal haematuria or haemolytic disease (haemoglobinuria).
• Smell the urine for ketones and for the smell of infection. Unusual-smelling urine may suggest an inborn error of metabolism.
• Test the urine using commercial dipsticks. This may reveal the following:
• protein—infection, renal damage or nephrotic syndrome
• ketones—diabetic ketoacidosis (DKA)
• white cells or nitrites—suggestive of infection
• These sticks are very sensitive to the presence of blood and may detect haematuria even if the urine looks clear.
• Examine the urine under the microscope for white cells, red cells, casts and the presence of organisms. A sample should also be sent for culture. A pure growth of >105 colony-forming units of a single organism and 50 white cells/mm3 confirms infection. Infection is extremely unlikely in the absence of pyuria.
Immunological in needed to investigate a child with suspected immunodeficiency (severe, recurrent or unusual infections—see Chapter 25) or with an autoimmune disorder such as juvenile rheumatoid arthritis see Chapter 47) or systemic lupus erythematosus or renal disease.
Investigations should only be requested to confirm a clinical diagnosis, or if indicated following a thorough history and examination. Sometimes, they are performed to rule out more serious but less likely conditions. Blindly performing investigations as a ‘fishing’ exercise in the hope of throwing up an abnormality is usually counterproductive, often leading to increased anxiety and further investigations when unexpected results are obtained.