Diagnostic Tests: Tips from the Dermatologist #5

This is the fifth in a series of short articles with the aim of informing colleagues in practice about diagnostic tests commonly done on our dermatological patients. This article will focus on sample collection techniques and the usefulness of carrying out a culture and sensitivity (C&S) test.

Bacterial C&S is an important part of diagnosis and management of various infectious diseases in veterinary medicine. Ideal antibiotic therapy is then based on determination of the aetiological agent and its relevant antibiotic sensitivity. The effectiveness of individual antibiotics varies with the location of the infection, the ability of the antibiotic to reach the site of infection, and the ability of the bacteria to resist or inactivate the antibiotic.

Empiric treatment is often started before laboratory microbiological reports are available when treatment should not be delayed due to the seriousness of the disease. If patients have recurrent skin/ear infection or they do not respond to the empiric treatment chosen it's critical to know which antibiotics will be effective against the specific microorganisms causing the problem.

C&S technique

Successful collection of specimens will depend on the following:

1 - Collection at the appropriate time

  • Whenever possible, collect all culture specimens prior to administration of any antimicrobial agents

2 - Use of the correct technique

  • Use precautions for collecting and handling all specimens
  • Wash hands to prevent contamination
  • Wear gloves to prevent cross infection
  • Avoid contamination of the swab with indigenous flora
  • If necessary, remove surface exudate by wiping with sterile saline or 70% alcohol
  • If collecting a sample from a pustule, open it with a sterile needle
  • When sampling the ear: place the swab into the external ear canal and rotate gently once. Sampling the middle ear is a more complicated procedure and requires general anaesthesia and a myringotomy

3 - Use of the correct equipment

  • Swab vs. tissue - Swabs are convenient but inferior to tissue culture
  • Use swabs with transport medium
  • Sample both ears in presence of bilateral otitis. Do not assume that the same microorganisms are present in both ears. If skin lesions look different sample at least two sites
  • All specimens must be appropriately labelled. When both ears or multiple skin sites are sampled label each swab

Quick transportation to the laboratory without delay

  • Arrange for all specimens to be delivered to the laboratory as soon as possible after collection
  • Specimens for bacterial culture should be transported at room temperature. If transport is delayed the specimens should be refrigerated

Sample

In house or commercial laboratory?

Although there are numerous commercial diagnostic laboratories offering a range of microbiological services, some colleagues may choose to perform bacterial cultures of samples within their practices for various reasons. However, in exploiting the benefits associated with in house culture procedures, there are laboratory quality and biosafety practices that should not be overlooked. For example, laboratory-acquired infections are an important concern in diagnostic and research laboratories.

Most commercial diagnostic laboratories work under strict quality control systems with experienced personnel to provide consistent and accurate results. Although not insurmountable, there can be challenges in developing a similar degree of quality control and laboratory biosafety in a veterinary clinic.

How is a Culture and Sensitivity Test Done?

After the swab is received in the laboratory the microbiologist will spread a sample of the infective material on to a plate of nutrient substance (usually blood agar) and allow whatever species of bacteria were present in the infected area to grow.

When a sufficient population of bacteria has grown on the plate, the microbiologist will identify the species of bacteria by examining the colony characteristic (colour, texture, growth pattern, etc.) and/or carrying out Gram-staining, microscopic examination, metabolic requirement "footprints" and even DNA sequencing. Then the bacterial population sensitivity to a range of antibiotics will be determined. Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection. Hopefully the in vitro results should be helpful in vivo in treating the infection. Testing for antibiotic sensitivity is often done by the Kirby-Bauer method. Small disks containing antibiotics are placed onto a plate upon which bacteria are growing. If the bacteria are sensitive to the antibiotic, a clear ring, or zone of inhibition, is seen around the disk indicating poor growth. Other methods to test antimicrobial susceptibility include the E-test (also based on antibiotic diffusion). Agar and Broth dilution methods for Minimum Inhibitory Concentration determination are carried out in modern laboratories and this is nowadays considered the best test for evaluating antibiotic sensitivity.

What is the goal of testing?

The goal of antimicrobial susceptibility testing is to predict the in vivo success or failure of antibiotic therapy. Tests are performed in vitro, and measure the growth response of an isolated organism to particular drugs. The tests are performed under standardized conditions so that the results are reproducible. The test results should be used to guide antibiotic choice.

The results of antimicrobial susceptibility testing should be combined with clinical information and experience when selecting the most appropriate antibiotic for your patient. Normally laboratories would report the result as:

Sensitive

'The "sensitive" category implies that isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the recommended dosage is used for the site of infection.' (CLSI definition)

Resistant

'The "resistant" category implies that isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules, and/or that demonstrate zone diameters that fall in the range where specific microbial resistance mechanisms (e.g. beta-lactamases) are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.' (CLSI definition).

Note that this definition says nothing about the chances of clinical success; in fact predicting clinical outcome based on susceptibility testing and the use of drugs shown to be in the susceptible or resistant category is imprecise. This imprecision is due to the effect of host responses, site of infection, toxin production by bacteria that is independent of antimicrobial susceptibility, the presence or absence of biofilm, drug pharmacodynamics and other factors.

Intermediate

'The "intermediate" category includes isolates with antimicrobial MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates. The intermediate category implies clinical efficacy in body sites where the drugs are physiologically concentrated (e.g. quinolones and beta-lactams in urine) or when a higher than normal dosage of a drug can be used (e.g. betalactams). This category also includes a buffer zone, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmaco-toxicity margins.' (CLSI definition)