Tag: tissue specimens

Tissue is the Issue: Microorganism Identification (part 3)

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

  1. Tissue is the Issue, Part 1
  2. Tissue is the Issue, Part 2
  3. 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.

 

Martinez Headshot-small 2017

-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.

Tissue is the Issue: Direct Observation of Microorganisms (part 2)

Most laboratories (and clinicians) utilize and rely on microscopic observation as the first step in the detection and identification of microorganisms. In some cases, direct microscopic analysis is used to determine the immediate clinical course of action. For example, if during a surgical procedure infection is suspected, then it is possible for the surgeon to submit a specimen to the laboratory for rapid (STAT) analysis. If polymorphonuclear leukocytes (PMN) and organisms are observed, then the differential diagnosis of infection is confirmed. Assuming proper specimen collection; if no PMN and no organisms are seen, then infection is less likely (true in most cases; there are always exceptions). These direct microscopic observation results thus drive the surgeon’s decision to either remove the infected area or perhaps advises on the use of intraoperative antimicrobials.

In part 1 of this series we discussed the pre-analytical problems associated with tissue culture; specifically, how specimen processing can affect whether or not organism is detected and recovered (1). In part 2 we will consider methods used for the direct observation of microorganisms in tissue specimens. 

The Issue

Let’s take a step back to review our previous “culture-negative” endocarditis case (1). Recall that the blood cultures were negative at first, no organisms were observed on the Gram stain and the culture was also negative of the valve tissue. However, when we evaluated the frozen tissue that was “split” and saved for sequencing, the organism was observed via acridine orange (AO).

AO.png
Image 1. AO of a positive broth culture from a homogenized tissue specimen. The AO stain displays cocci, bacilli, and yeast. The DNA-containing cells fluoresce orange.

 The Solution

AO is a fluorescent dye that intercalates nucleic acids. It is a rapid, inexpensive, and most importantly- it is a sensitive alternate dye that can aid in microorganism detection in a variety of specimen types (2, 3 ,4, 5). AO is more specific than Gram stain (2, 3). Because bacterial DNA is not contained in a nucleus, but freely contained within the cell, the AO-stained cell takes the shape of the organism’s cellular morphology (Image 1). For example, if the organism is Staphylococcus aureus, then the AO would exhibit Gram positive cocci in clusters. Similarly, if the organism in question is Escherichia coli, then Gram negative bacilli would be observed, etc.

Some organisms (Campylobacter, Mycoplasma, etc.) do not stain well with Gram stain and as such they can be difficult to detect in direct specimen observations. The presence of many PMN and no organisms may be a clue that the organism is present in low numbers or that the organism does not stain well with the Gram stain. In cases such as these, the use of AO has proven to be very useful. Additionally, if the morphology of an organism in a Gram stain is difficult to interpret, then AO can also provide a more clear-cut answer. Lastly, the presence of artifact(s) can be problematic when reading Gram stains. “Is that Gram positive cocci or junk?”. Junk usually does not contain DNA and therefore does not fluoresce. Therefore, the information provided by the AO stain can aid in your decision to report cocci or not.

One disadvantage is that a fluorescent microscope is required to visualize the stain. Implementing AO may require the purchase of new equipment as not all laboratories have access to a fluorescent microscope.  Because AO stains nucleic acid, anything with DNA or RNA will stain positive. This is another disadvantage because too much material that is positively stained can make interpreting the stain difficult at times. Another disadvantage is that a Gram stain is still required because the AO only allows the visualization of DNA-containing cells, it does not determine the Gram stain reaction.

Thinking back to our original case, the organism was present in the frozen tissue piece. It was not detected in the direct Gram stain, but rare organisms were noted in the AO. Because the AO was positive, we then reviewed the Gram stain and found rare Gram negative bacilli. This suggests that the organism was initially missed in the Gram stain because of the low abundance present. Bottom line, the AO was positive and we were thus able to provide the clinical team with relevant information.

The Conclusion

There are many stains used to aid in the observation of microorganisms. The AO stain is easy to perform, inexpensive, rapid, sensitive, and versatile. AO can be used on direct specimen smears, isolated colonies, and formalin-fixed paraffin embedded sections. Although there are limitations to using AO, the benefits considerably outweigh the shortcomings. AO is a great tool that laboratories should consider implementing as an alternate method for the direct observation of microorganisms.

References

  1. https://labmedicineblog.com/2018/04/20/tissue-is-the-issue-splitting-specimens-part-1/
  2. Mirrett, S., Lauer, B.A., Miller, G.A., and Reller, B. 1982. Comparison of Acridine Orange, Methylene Blue, and Gram stains for Blood Cultures. J. Clin Microbiol. 15; 4: 562-566.
  3. Lauer, B.A., Reller, B., and Mirrett, S. 1981. Comparison of Acridine Orange and Gram Stains for Detection of Microorganisms in Cerebrospinal Fluid and Other Clinical Specimens. J. Clin Microbiol. 14; 2:201-205.
  4. Martinez, R.M., Bowen, T.R., and Foltzer, M.A. Prosthetic Devise Infections. Diagnostic Microbiology of the Immunocompromised Host. 2016 (Book chapter, chapter 27. Pages 711- 733. ASM Press.
  5. Cooper, J.D., Dometita, D., Hasan, A., Dorion, P., Wolk, D.M. and Martinez, R.M. Orange you glad you checked the buffy coat? Clin. Microbiol. Newslett. 37: 9-13.

 

Martinez Headshot-small 2017

-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.