In parts 1 and 2, we discussed pre-analytical and analytical issues that can be faced when culturing tissue specimens. Part 3, the final part of the Tissue is the issue series, will review analytical and post-analytical issues of tissue culture requests.
The Issue
Let’s consider a case of “culture-negative” endocarditis (1), in which organism was detected during direct observation of the specimen (2); but as you would expect for suspected “culture-negative” endocarditis, the culture does not yield an organism. This can happen for a variety of reasons. Perhaps the culture request was for a routine aerobic culture, but the organism was a strict anaerobe and therefore could not grow. Maybe the patient was on antibiotics, so the organism observed was not viable. Or it is possible that the organism in question is fastidious and requires special media or growth conditions which were not met. Another frequent occurrence is that tissue is sent for pathology, but not for culture. This is more common when malignancy is expected, but the pathological findings suggest an infectious process. These scenarios may seem hopeless, but don’t despair; there is a non-culture alternative that can aid in identifying the causative agent.
The Solution
Microbial (bacterial, fungal, and viral) DNA can be detected from fresh, frozen, or fixed tissue. Simply put, DNA is first extracted from the specimen, of which the microbial DNA of interest (bacterial, fungal, viral) is then amplified via PCR. Broad-range or pathogen-specific PCRs are commonly available from a variety of reference laboratories. If broad-range PCR is performed, then sequencing of the amplicon is required to determine the organism identification. Sequences are queried against a library of known microbial genomes to obtain a match.
Depending on the DNA of interest, different primer sets are utilized. For broad-range bacterial PCR, the 16S ribosomal RNA gene is typically used. Although mycobacteria are bacteria, they require additional gene targets for optimal detection and identification (16S rRNA, rpoB, and hsp65). For fungi, the 28S rRNA and the ITS (internal transcribed spacer; ITS1 and ITS2) genes are used.
The organism burden and specimen type can affect the probability of detecting an organism and obtaining its identification. The likelihood of a positive outcome is proportional to the organism burden. For example, if organism observed in the direct exam (i.e., Gram or acridine stain), then the organism can usually be detected and identified. However, if no organism is observed, then the chances of a positive result is unlikely. Therefore, our protocol is to only send specimens for microbial DNA sequencing on specimens in which organisms were observed in the direct exam. This is true for all specimen types (fresh, frozen, fixed).
It is important to note that fixed specimens may not yield as good results as a fresh or frozen specimen. This is because the process of fixation can degrade the microbial DNA (3). Additionally, because detection of microbial DNA is the basis for pathogen identification, susceptibility results are not going to be available. Treatment options will need to be based on known empiric therapies.
The Conclusion
Microbial DNA sequencing is a viable option for the identification of etiological agents of infection from a variety of sources, such as culture-negative infections. Other uses include slow-growing organisms and organisms that are unidentifiable by traditional methods (4). In my experience, this is a valuable tool that should be considered when culture does not yield a result and a result is necessary to drive clinical decisions.
References
- Tissue is the Issue, Part 1
- Tissue is the Issue, Part 2
- Martinez, R.M. Genes in your tissue: probe identification and sequencing microbial targets from formalin-fixed, paraffin-embedded tissue. Clin. Microbiol. Newslett. 36: 139-147.
-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.
Lablogatory 
very impressive