Diagnostics: Species identification

2019-08-28

The first step in microbiological diagnostics is to determine whether the patient carries a pathogen that can explain the observed symptoms, and if so identify what species and sometimes subtype the infecting pathogen belongs to. For infections caused by bacteria, the gold standard method has, since the beginning of microbiological diagnostics, been culture on agar plates followed by microscopy and further subcultivation. New rapid diagnostic methods challenge this old and slow method – some more successfully than others.

Culture-based methods

Most bacterial pathogens can be grown on petri-dishes on one or several different culture media, often made solid by adding agar. A biological sample can be inoculated on or into the medium and is incubated in 35-37 °C for at least 16 hours. For anaerobic bacteria, the plates are placed in a container from which oxygen is removed. On a solid medium, bacteria will typically appear as separated colonies, spots of growth on top of the agar surface that ensure an enriched isolate for further analysis. These bacteria can then be stained with a Gram stain and examined under a microscope to determine if they are Gram-positive or Gram-negative, and if they are for example rods or cocci.

After this first examination, the bacteria can then be further examined by for example testing which substances they can ferment as nutrient sources or other discriminating biochemical characteristics. Some of the specific biochemical tests can be performed rapidly on a benchtop, whereas the fermentation tests generally require another 16 hours incubation. Correctly selected, the combination of these tests make it possible to identify a bacterium by its phenotype (externally observable characteristics). The process takes time and requires not only a basic microbiology laboratory but also access to trained microbiologists and laboratory staff that can perform the procedures and interpret the results.

Today, many of these processes can be automated, and a variety of systems and levels of automation are available on the market. This makes the process less work-intensive and results easier to interpret as machines provide the probable species identification. However, automation does not significantly reduce the turn-around time from sample to answer.

Mass spectrometry

The most recent development that was rapidly adopted to become a standard method in many clinical laboratories was the introduction of mass spectrometry, or MALDI-TOF for bacterial diagnostics. These instruments use high-powered lasers to disintegrate cells of the cultured specimen and analyzes the mass spectrum of different molecules present. The data retrieved from the assay forms a fingerprint which then can be compared to a pre-existing library of data from known bacteria to determine the bacterial species. This technology is inexpensive to run, accurate and greatly reduces the time required for results – from a day or to two to minutes. However, investment cost is high and due to the reliance of an external database, the quality of the results are only as good as the database itself. Thus, newly discovered or rare pathogens such as some zoonotic agents may be mischaracterized or not detected at all.

Serology

Serology commonly refers to the use of antibodies to detect the presence of bacteria or products of bacteria in a sample. The antibodies may, for example, be able to detect surface molecules of bacteria or virulence factors such as toxins. This makes it possible to subtype strains, for example to discern between a “normal” E. coli or the highly pathogenic varieties such as EHEC, VTEC or STEC. Antibody reactions often form the basis of rapid, easy-to-use diagnostics like the malaria RDT test as they are easy to adapt to a paper-based medium, making the tests cheap and easy to use.

Nucleic acid amplification tests (NAAT)

The above described methods determine the identity of the bacterium based on its phenotype. But the phenotype is largely determined by the genotype – the genetic material that carries the information that results in the phenotype. For some bacteria that are difficult or even impossible to culture, or grow slowly like Mycobacterium tuberculosis, genotypic tests are the only realistic alternative to identification. They are also particularly useful for epidemiology, tracing the spread of specific strains in the community or in a hospital outbreak.

Common for most of these tests is that a portion of the genetic material that is unique to the bacteria of interest is copied several times, or amplified, via a polymerase chain reaction. The amplified genetic material can then be detected with electrophoresis or by coloring with fluorescent probes. With qPCR, also called real time PCR, the amount of original genetic material can be estimated, giving an indication of bacterial load.

The NAAT tests come in many different shapes, from labor-intensive, fully customizable methods requiring a laboratory for genetic analysis, to easy-to-use cartridge-based systems where the operator transfers the sample to a cartridge, presses a button and waits for the result. The most easy-to-use methods can even be used at the point of care, where the primary patient sample, like blood, urine or sputum, is added straight into the cartridge.

Advantages and disadvantages

A trained microbiologist can, with limited laboratory resources provide a good microbiological diagnosis from a clinical sample using culture based methods. But as shorter time to results is increasingly seen as important for rational use of antibiotics, automation and more high-technology based diagnostic methods have become increasingly sought after. However, as man is replaced or complemented by machine, versatility and adaptability generally decreases. Still, access to advanced bacterial diagnostics is most common in high income countries, and a major barrier to expanding diagnostic access is training of microbiologists.

High-end technology make analyzing samples generally faster and easier, but trade this off by requiring for example a stable power supply, regular maintenance from a qualified service technician, dust-free environment or stable climate. The choice of method can also limit the question that can be answered. In this case, the trained microbiologist can most often answer the question “which, if any, bacterium is present in this sample?”. But the more specialized, easy to use rapid methods like for example GeneXpert may only be able to answer the question “does this sample contain a typical M. tuberculosis with one of the five most common markers of rifampicin resistance?”

The limited questions that can be answered by some of these rapid tests limit their usefulness, especially in detecting emerging or mutating pathogens. Therefore, the old-school, low tech culture based methods are still necessary at least on a national reference laboratory level to be able to identify those pathogens that are too different to be detected by the more rapid methods. Still, in many contexts, the rapid and easy to use tests certainly are needed both for reducing misuse of antibiotics and saving lives by guiding appropriate antibiotic therapy.