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Inside the Lab Microbiology and immunology, Biochemistry and molecular biology, Point of care testing, Training and education

Spreading the Word: Molecular Diagnostics for Infectious Disease

Molecular tests support infectious disease diagnosis by detecting specific organisms – but they aren’t “magic bullets” for pathogen detection. Although tempting, broad-range assays aren’t always the best option for detecting multiple bacteria in a single sample, so pathologists must work with clinical colleagues to select and interpret the most appropriate tests.

Molecular diagnostics have an increasingly important role to play across all areas of pathology, but their importance in infectious disease cannot be underestimated. Thanks to a simple, widely available technique – the polymerase chain reaction (PCR) – molecular techniques now serve a variety of applications in routine clinical laboratories, with real-time PCR allowing the rapid detection of various infectious microorganisms.

But are these cutting-edge assays too easy? Often, pathologists perform them even when there is no prior evidence of infection. It’s true that there are many circumstances where molecular diagnostics can be valuable, but we need to educate our surgical pathologists on what the tests can do – and when they might not be the best option.

As our clinical colleagues become more familiar with the molecular diagnostic tests that are now widely available, surgical pathologists can expect to receive more frequent requests for them. But we cannot blindly follow these requests; we are also diagnosticians and need to be prepared to turn down tests that aren’t suitable or suggest more appropriate alternatives. Pathologists are physicians who are ultimately responsible for the tissue we work on, so we must play an active role in making these decisions. And to do so, we need to fully understand the applications and limitations of molecular diagnostic assays so that we can use them properly.

Target-specific or broad-range?

In the context of infectious disease, the strength of molecular diagnostic tools lies in identifying specific organisms. For example, a lymph node may be consistent with cat-scratch disease (CSD), but the pathologist wants to rule out any other potential causes. A PCR test for Bartonella henselae, the specific bacterium that causes CSD, would be entirely suitable – and a conclusive result would either rule in or rule out CSD.

The use of 16S ribosomal RNA gene sequences has opened new doors in terms of detecting multiple bacterial species in a single sample.

However, now that we can increasingly use broad-range assays that can detect multiple organisms simultaneously, things aren’t so straightforward. The use of 16S ribosomal RNA gene sequences – present and highly conserved in nearly all bacteria – has opened new doors in terms of detecting multiple bacterial species in a single sample. The molecular amplification method can be applied to a number of different samples, including cerebrospinal fluid and both fresh and formalin-fixed surgical pathology specimens. Because it’s essentially a PCR test, once you’ve amplified the 16S gene in a sample, you can sequence it to identify the specific bacteria that are present.

This broad-range test is ideal in a number of scenarios. For example, if a patient has received antibiotics, the bacteria may not grow in standard microbiology culture – which means that the surgical pathologist might be the only one who can provide insight by using the 16S test. This is particularly important for patients who have diseases such as infective endocarditis. These patients will almost always receive broad-spectrum antibiotics before surgery, so the 16S test can help determine which bacteria are present and therefore which specific antibiotics to use.

Figure 1. Numerous Gram positive cocci in a case where no tissue was obtained for culture (4X and 100X magnification). This is an example where a broad range bacterial assay (16S rRNA gene PCR and sequencing) would be appropriate because there is an abundance of organisms and an identification cannot be made by morphology alone.

No magic bullet

But don’t mistake the test for a magical, detect-all assay. The first thing to bear in mind is that a 16S test is only for bacteria; it won’t detect parasites, viruses, or fungi. And when a sample doesn’t show signs of any organisms, there’s little reason to carry out the test, because the sensitivity plummets if you don’t see anything that looks like inflammation or the presence of organisms. When a case that does require a 16S test arises, it’s important to remember that the results will only be as good as the assay. Factors such as the extraction method, the gene region targeted, and the database used for analysis can all impact their accuracy.

Another caveat to the test is that we don’t live in a sterile world; bacteria are all around us. That’s why it’s important to familiarize ourselves with all of the ways that exogenous DNA can be introduced to tissue samples, so that we can account for any contamination. The journey that a sample takes from tissue biopsy to glass slide is long and convoluted. From the cutting block in the grossing room, to the reagents used during tissue processing, to the staining process when transferring a paraffin ribbon section onto a slide, exogenous DNA contamination is inevitable at some point.

The risk of contamination is simply a side effect of having a highly sensitive assay that can detect every single bacterium.

The risk of contamination is simply a side effect of having a highly sensitive assay that can detect every single bacterium – and there’s not much that can be done to prevent it. Ultimately, contamination means that the 16S test will detect bacteria with no clinical correlation to the patient. To combat this, it’s important to know what you do – and don’t – expect to find; for example, the presence of a species commonly found on skin has probably been introduced during the histologic process, rather than being an infective agent.

Consider the pathologist who notices bacteria in tissue and completes a Gram stain that indicates the presence of Gram positive cocci. A 16S sequencing assay that detects Gram negative bacilli is more likely to be showing contamination than the infection itself, because these are such different types of organism. We must ensure that there’s a correlation between what the pathologist sees and what the test actually detects, and our clinical colleagues are on hand to help with the process of deciding whether a result makes sense for a particular patient.

Although we can’t completely eliminate the possibility of contamination, we can control a few things to minimize the introduction of exogenous DNA (and false-positive results). For instance, if you know that you might want to carry out molecular testing on a specific sample, freeze a small piece of fresh tissue to serve as an ideal specimen, rather than using a formalin-fixed, paraffin-embedded block. This is sometimes easier said than done, but if a fairly large tissue sample comes into the frozen section lab, why not take a small piece and set it aside in case molecular testing is needed? This is much better than putting the tissue through a lengthy process that risks exposing it to numerous environmental organisms. Pathologists can also work with treating physicians to ensure that any tissue that might be used for molecular testing is sent without any type of media, as this could contain unwanted DNA.

Figure 2. Whipple disease in the brain showing a cluster of macrophages with deeply-positive cytoplasmic inclusions on PAS-D stain (40X magnification). If PCR confirmation is desired, the preferred test would be a Tropheryma whipplei PCR rather than a broad range assay, since a specific organism is suspected.

Curbing temptation

Pathologists are often faced with the difficult question of whether to select a broad-range or a target-specific PCR assay. If you’re looking at a tissue and suspect that a certain bacterium is present, it’s always better to order the target-specific PCR test, because it will be more sensitive and more specific than a 16S test. Despite this, there is often a temptation to opt for the broad-range test; it’s easy to think, “Why would I order a test that only gives me one answer, when I could order a different test that gives me every possible answer?” This is why it’s crucial to understand that, although the 16S test can detect more organisms, it’s more likely to produce false-positive and false-negative results – the former due to contamination and the latter because it’s less sensitive than a target-specific assay.

It’s vital that pathologists have detailed conversations with the labs performing molecular diagnostic tests to ensure that everyone involved fully understands their capabilities.

One of the main limitations for molecular diagnostics in the US is that there are currently no FDA-approved tests. As a result, the test you’ll find at the University of Washington may differ in sensitivity and specificity to the test you’ll find at Mayo Clinic. The situation in Europe is similar – there are no CE-marked tests at the moment, and it’s likely that it will be a few years before either region achieves standardization. Until that point, it’s vital that pathologists have detailed conversations with the labs performing molecular diagnostic tests to ensure that everyone involved fully understands their capabilities.

As molecular tests are used more routinely for the diagnosis of infectious disease in clinical laboratories, a growing number of patients will have their pathology specimens tested this way. In general, this is extremely positive – after all, a new addition to the pathologist’s toolbox can only be a good thing. But, as with any new tool, the key is to know its strengths and limitations so that we can use it correctly. By playing a more active role in the clinical team and taking responsibility for test selection, we can better help our clinical colleagues interpret results and fulfill the great potential of molecular diagnostics.

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About the Author
Bobbi Pritt

Director of the Clinical Parasitology Laboratory in Mayo Clinic’s Department of Laboratory Medicine and Pathology, Rochester, Minnesota, USA.

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