Microbiology Case Study: A 28 Year Old Female with Perirectal Abscess

Case History

A 28 year old female with a history of Ulcerative Colitis on humira and azathioprine presented with proctitis and a recent perirectal abscess. The patient reported a two week history of progressively worsening pain and swelling in the perianal region. In addition, she reported recent purulence excreted with bowel movements.  On physical exam, the patient was afebrile and negative for rash, oral lesions, joint pain, or abdominal pain. A perirectal abscess was identified and drained. Abscess culture was positive. Patient reported recently engaging in high-risk sexual behavior with multiple male sexual partners often without protection.

Lab Identification

neissgono
Image 1. Kidney-bean shaped gram negative cocci identified on Gram staining of perirectal abscess culture (100x oil immersion).  
neissgono2.png
Image 2. Aerobic growth of flat, gray-white, and moist colonies from perirectal abscess on chocolate agar plate.  

Abscess culture grew kidney-bean shaped gram negative diplococci. Colonies on chocolate agar plate appeared medium sized, flat, gray-brown, and moist. The organism was oxidase positive and identified by MALDI to be Neisseria gonorrhoeae.

Discussion

Neisseria gonorrhoeae is a kidney-bean shaped gram negative diplococci for which humans are the only host. The organism causes gonorrhea, a common sexually transmitted disease, among young people between the ages of 15-24 years. Gonorrhea is spread by sexual contact or through the birth canal. The most common site of infection is the urogenital tract.2 Males commonly present with dysuria with penile discharge.2 Females commonly present asymptomatically or with symptoms such as mild vaginal mucopurulent discharge and severe pelvic pain2. In addition, gonorrhea can cause infections of the anus, conjunctiva, pharynx, ovary and uterus.2 In the neonate, the culprit organism can lead to ophthalmia neonatorum.2 Lastly, gonorrhea causes disseminated disease such as arthritis, endocarditis, meningitis, and skin lesions on extremities.2 CDC currently recommends treating gonorrhea with dual therapy, a single dose of 250 mg intramuscular ceftriaxone and 1g of oral azithromycin.1

Antibiotic resistance in gonorrhea is an increasing public health concern. The World Health Organization has a program that monitors the global antimicrobial resistance of gonorrhea.3 The data from 77 countries between 2009 and 2014 showed that 66% of reporting countries had encountered gonorrhea strains with either resistance or reduced susceptibility to ceftriaxone.3 81% of reporting countries had encountered gonorrhea strains resistant to azithromycin.3 Given these data, it is important to improve gonorrhea prevention and continue to monitor gonorrhea antibiotic resistance at both the national and global levels.

References

  1. Gonorrhea treatment and care. (2017, Oct 31st). Retrieved on March 1st, 2018 from https://www.cdc.gov/std/gonorrhea/treatment.htm
  2. Miller KE. Diagnosis and Treatment of Neisseria gonorrhoeae Am Fam Physician. 2006 May 15:73 (10): 1779-1784.
  3. Wi T, et al. Antimicrobial resistance in Neisseria gonororheae: Global surveillance and a call for international collaborative action. PLoS Med 14(7): e1002344.https://doi.org/10.1371/journal.pmed.1002344

 

-Ting Chen, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

Wojewoda-small

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

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.

Microbiology Case Study: A 27 Year Old Male with Unintended Weight Loss

Case History

A 27 year old African American male presented to the emergency department with confusion and abdominal pain. His past medical history was significant for a 100 pound unintended weight loss and oral candidiasis which prompted a recent diagnosis of HIV. He was prescribed anti-retroviral therapy and antibiotic prophylaxis with which he reported compliance. Currently, he had no fever or chills. An abdominal CT scan showed an enlarged liver & spleen, generalized lymphadenopathy and a small amount of fluid. Significant lab work included anemia with a platelet count of 18,000 TH/cm2, absolute CD4 100 cells/cm2 (reference range: 506-3142 cells/ cm2) and a HIV viral load of 4,871 vc/mL. Given the concern for an infectious process, the infectious disease service was consulted and the patient underwent a thorough infectious work up including lumbar puncture, was started on board spectrum antibiotics and antifungals and was placed in airborne isolation until Mycobacterium tuberculosis could be ruled out.

Laboratory Identification

mac1
Image 1. Direct smear from a stool specimen showed beaded acid fast bacilli (Kinyoun stain, 1000x oil immersion).
mac2
Image 2. Direct smear from a stool specimen showed beaded acid fast bacilli (Kinyoun stain, 1000x oil immersion).

Initial diagnostic testing for bacterial, fungal and viral pathogens was negative. Three concentrated sputum AFB smears as well as a TB PCR were negative. The quantiferon gold TB test was also negative. The physician additionally ordered AFB blood and stool cultures. The direct smear from the stool specimen showed rare, beaded acid fast bacilli in a background of bacteria and yeast normally present in the stool via Kinyoun stain (Images 1 & 2). The specimen was sent to the department of health for additional work up. There was growth after 21 days incubation and Mycobacterium avium complex was identified by high performance liquid chromatography (HPLC).

Discussion

Mycobacterium avium complex (MAC) is a slow growing nontuberculous mycobacteria (NTM) frequently involved in human disease. Historically, it was classified as Runyon group III which are non-chromogens and do not produce pigment regardless of culture conditions. The group encompasses two taxa, M. avium and M. intracellulare. The species M. avium can further be classified into four subspecies: subsp. avium, subsp. silvaticum, subsp. paratuberculosis and subsp. hominissuis. Of interest, M. avium subsp. paratuberculosis can often be seen in association with Crohn’s disease.

In general, MAC organisms have low pathogenicity, but in the setting of those with lung disease (including cystic fibrosis), heavy smokers, immunocompromised patients and those with HIV, it is a well-known cause of disease. Infections with MAC can range from localized mycobacterial lymphadenitis and isolated pulmonary disease to bacteremia with dissemination to almost any organ. The organisms are located in circulating monocytes and further spread most commonly to the lungs, gastrointestinal tract and lymph nodes. In the case of HIV positive patients, MAC is the most common environmental NTM causing disease, especially in those with CD4 counts less than 100 cells/mm3 who are more likely to have disseminated disease.

In order to diagnosis MAC infections, specimens from blood, sputum, lymph nodes and other tissues are preferred. In addition, stool may also be an acceptable alternative in HIV patients if other specimens are negative or unable to be obtained. However, the sensitivity of a direct stool smear is only 32 to 34% making it not a very effective approach to identifying those at risk for disseminated infections. Once the culture has growth, various methods can be used to identify MAC, including phenotypic methods, DNA probe testing, HPLC, pyrosequencing and other forms of PCR & sequencing.

In the case of our patient, he was started on M. tuberculosis therapy: rifabutin, isoniazid, pyrazinamide & ethambutol (RIPE) until TB was ruled out. At that time, he was removed from isolation and switched to a drug regimen that included azithromycin, rifabutin and ethambutol. He showed clinical improvement and his cell counts, renal function and liver enzymes trended to normal ranges.

 

Stempak

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the Director of Clinical Pathology as well as the Microbiology and Serology Laboratories.  Her interests include infectious disease histology, process and quality improvement and resident education.

Microbiology Case Study: An Elderly Woman with a Cough Develops Skin Nodules

Case History

Our patient is an 83 year old female with previous history of arterial hypertension, atrial flutter and chronic obstructive pulmonary disease who presented with dry cough (~2 weeks), fever (102ºF), and cutaneous ulcerated plaques with elevated borders on forearm, foot, leg, and neck. Chest radiographs and chest CT scan showed numerous bilateral nodular consolidations compatible with pneumonia. Additionally, mild leukocytosis (14,200 cells /mm3) and hypohemoglobinemia (10.9 mg/dl) were documented. A skin biopsy was taken from the forearm lesion. Periodic acid–Schiff (PAS) and Grocott-Gomori’s (or Gömöri) methenamine silver (GMS) stains identified rare budding yeast (PAS, Image 1). Acid-fast bacilli (AFB) and Gram stains were negative for mycobacteria and bacteria, respectively.

blasto
Image 1. Budding yeast with a thick double cell wall were identified microscopically surrounded by prominent supurative granulomatous reaction (PAS, 100x).

Discussion

A diagnosis of disseminated blastomycosis was made based broad based budding yeast seen on PAS stain (Image 1).

Blastomycosis infection most commonly affects persons living in the Mississippi and Ohio River valleys, Great Lakes Region of the United States, and southern Canadian provinces. It is a fungal infection that can cause asymptomatic infection, isolated pulmonary disease, or serious and potentially fatal disseminated disease. B. dermatitidis can infect every organ of the body giving great variety of clinical manifestations, which is the reason why it is known as “the great pretender.” More than half of infected patients are asymptomatic. Symptomatic patients generally present with pulmonary symptoms, and the development of disseminated disease after hematogenous spread is common (~25 to 40% of symptomatic cases). The most common extra-pulmonary locations are: skin, bone, genitourinary tract, and central nervous system (CNS). Unlike histoplasmosis, most cases of blastomycosis are seen in immunocompetent patients, although immunocompromised patients may be at higher risk to develop severe forms of the disease.

Blastomyces is a thermally dimorphic fungus that grows as a yeast in the body and as filamentous fungi with septate hyphae in the environment. Recent phylogenic analysis has divided the Blastomyces genus into 2 species, B dermatitidis and B gilchristi . Culture of B. dermatitidis from the environment is extremely difficult, and much of what we know is conjecture from a few documented outbreaks, of which several occurred in wooded areas near waterways. These investigations found that exposure to dust clouds associated with construction or crop harvesting were the only identified risk factors for infection. Blastomycosis infection occurs through aerosolization of conidia from the environment causing respiratory infection or less commonly through direct inoculation into cutaneous abrasions. Once in the host, the conidia transform into yeast. The specific proteins expressed during the yeast phase allow the evasion of phagocytic and CD4+ cells.

Laboratory diagnosis

The most expedient method to diagnose blastomycosis remains examination of stained fluid or tissue specimens. Yeast are 8-15 µm in size with broad based buds of 4-5 µm and have a characteristic refractory double cell wall. Fluid can be stained with 10% potassium hydroxide plus calcofluor white, whereas formalin fixed paraffin embedded tissue samples can be stained with GMS or PAS. B. dermatitidis yeast can be difficult to visualize with Gram or hematoxylin and eosin (H&E) stains, but if found, the characteristic broad-based budding pattern of yeast can lead to presumptive diagnosis before culture and non-culture based diagnostic test results are available.

Culture of B. dermatitidis provides a definitive diagnosis of pulmonary and extra-pulmonary disease. B. dermatitidis grows well on routine fungal media such as Sabouraud dextrose agar, potato dextrose agar, and brain–heart infusion media. The yeast phase is inhibited by media containing cyclohexamide. Culture typically demonstrates growth in as little as 4-7 days. Colonies will initially appear yeast-like, but then develop white cottony aerial mycelium and turn tan with age. Mature growth is achieved around day 14 and the reverse of the colony is a tan color. At 25-30°C, B. dermatitidis forms septate hyphae with round or pear-shaped conidia attached to the hyphae by short or long conidiophores. This gives the characteristic appearance of “lollipops.” Scedosporium spp. and Chrysosporium spp. are common confounders because they make similar structures. Definitive identification of Blastomyces sp. can be made by conversion of the mold phase to the yeast phase by incubation at 37°C. An alternative to conversion is using a DNA probe assay.

 

References

  1. Medically Important Fungi, 5th edition
  2. Principles and Practices of Infectious Disease, 7th edition

Julio picture

-Julio Diaz-Perez, MD is a 1st year anatomic and clinical pathology resident at University of Chicago (NorthShore).

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.

Microbiology Case Study: 42 Year Old Female with HPV

Case History

A 42 y/o female G2P2002 patient presented to her Ob/Gyn for Colposcopy for monitoring of persistent High-Risk HPV. She was originally found positive for HPV in 2015, but has had never had a Pap with a squamous intraepithelial lesion, abnormalities on colposcopy, or dysplasia seen on endocervical curettage. Additionally, she endorsed a complaint of vague diffuse pelvic/lower abdominal pain for approximately the last 2 months. She states that the pain is mild and comes and goes and is not associated with anything in particular. She has noticed some clear to gray-white discharge now and then since she first noticed the pain, but nothing that really worried her. Pt denies changes in bowel or bladder habits, denies nausea, fever, or chills. Pt has been in a monogamous relationship with her partner for the last 12 years. She had a Mirana IUD placed 4 years prior, without complication, and has not had menses since placement. Prior to that, the patient had normal, regular cycles. She has 2 children with the same father, both were delivered by spontaneous vaginal delivery without complications. She has mild anxiety and depression for which she is treated, but no other medical problems. There is no surgical history. She has 1-2 glasses of red wine every week, denies tobacco use, and denies illicit drug use.

Pelvic exam revealed a benign appearing cervix that was not painful to touch or motion. There was a clear to white mild discharge that was suspected to be normal vaginal secretions. IUD strings were noted. Colposcopy revealed an easily appreciated transformational zone without any obvious lesions. A routine endocervical curettage (ECC) was performed followed by observed increased clear discharge from the cervical os. ECC was sent for routine pathology:

actinomyces1
Actinomyces, H&E, 20x
actinomyces2
Actinomyces, H&E, 40x

Discussion

Actinomycosis is an infection by a species within the Actinomyces genus, generally seen in dental and other oropharyngeal abscess formations. However, rare occurrences of pelvic Actinomycosis can be seen in women with intrauterine devices in place. Pelvic infections can result in cervicitis and endometritis and progress into abscess formation within the fallopian tubes and the ovaries along with salphigitis. The more profound disease consisting of abscess formation generally presents with fever, specific lower abdominal tenderness, and elevated WBCs, thus can mimic acute appendicitis, ovarian torsion, or ectopic pregnancy (1). The first case reported in the literature was in 1967 (2).

Three main species of Actinomyces have been found to be associated with IUD-associated pelvic infection: A. naeslundii, A. odontolyticus (3), and A. hongkongensis (4). All of these species are obligate to facultative anaerobes, catalase negative, and nitrate reducing. A sub-species group of A. naeslundii, however, can be catalase positive and is CAMP test-positive. All members of A. naeslundii are urease positive while A. odontolyticus and A. hongkongensis are urease negative.

References

  1. Joshi et al. Pelvic Actinomycosis: a Rare Entity Presenting as Tubo-ovarian Abscess. Arch Gynecol Obstet. 2010, 281:305-306.
  2. Brenner et al. Pelvic Actinomycosis in the Presence of an Endocervical Contraceptive Device. Obstet Gynecol. 1967, 29: 71-73.
  3. Woo et al. Diagnosis of Pelvic Actinomycosis by 16S ribosomal RNA Gene Sequencing and its Clinical Significance. Diagnostic Microbiology and Infectious Disease. 2002; 43: 113-118.
  4. Flynn et al. Identification by 16S rRNA Gene Sequencing of an Actinomyces hogkongensis Isolate Recovered from a Patient with Pelvic Actinomycosis. J. Clin. Microbiol. 2013, 51(8):2721. DOI: 10.1128/JCM.00509-13.

 

-Jeff Covington, MD, PhD, is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

Wojewoda-small

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

 

Microbiology Case Study: A 36 Year Old Man with Symptoms of Bowel Obstruction

Case History  

A 36 year old Caucasian male presented to the hospital with symptoms of a bowel obstruction.  His past medical history was significant for a gunshot wound to the abdomen followed by the development of colon cancer with metastasis to the liver. Recently, he had an intestinal stent placed in order to relieve an obstruction from tumoral growth. During the current admission, he was taken to the operating room for a diverting colostomy and two days later developed a fever of 101.1°F and increasing abdominal pain. Due to a concern for sepsis, blood cultures were collected and piperacillin-tazobactam was added to his antibiotic therapy regimen which already included vancomycin and ciprofloxacin.

 

Laboratory Identification

aero1
Image 1. Gram stain from the blood bottle showing many gram negative rods (100x oil immersion).
aero2
Image 2. The organism grew as mucoid colonies on blood, chocolate and MacConkey agars after 24 hours of incubation at 35°C in 5% CO2.

 

aero3
Image 3. The organism exhibited beta hemolysis on blood agar when held up to a light source.

The blood culture signaled positive after less than 24 hours on the automated instrument and Gram stain revealed gram negative rods (Image 1). The organism grew as mucoid colonies on blood, chocolate and MacConkey agars after 24 hours of incubation at 35°C in 5% CO2 (Image 2). When the blood agar plate was examined with a light source, the organism clearly illustrated beta hemolysis (Image 3). Rapid biochemical testing was positive for catalase, oxidase and indole. The Vitek II identified the isolate as Aeromonas veronii bv. sobria.

Discussion 

Aeromonas spp. are facultative anaerobic gram negative rods. They are inhabitants of aquatic ecosystems and as such can cause wound infections in people exposed to polluted lakes or brackish water with fresh breaks in their skin. Additionally, gastroenteritis is common with Aeromonas spp. and are often acquired through ingestion of unpurified water or to a lesser extent by consumption of contaminated meats, fresh produce or dairy products. Extraintestinal infections, including sepsis and meningitis, can result by spread from GI sources or wound infections. Interesting, medicinal leeches, used in the post-operative setting to increase blood flow to the surgical site, are colonized with Aeromonas spp. (most commonly Aeromonas veronii bv. sobria) and can result in systemic infections in the patient.

In the laboratory, Aeromonas spp. grow readily from stool, wound and blood sources on commonly used media and exhibits beta hemolysis on blood agar. In addition, Aeromonas spp. will grow on CIN agar (at room temperature as well as incubator temperature) as colonies with a pink center surrounded by a white apron and are indistinguishable from Yersinia spp. Aeromonas spp. is positive for catalase, oxidase and indole by rapid testing. In most cases, identification of Aeromonas spp. to the complex level can be accomplished by biochemical testing (esculin, VP), automated instrumentation or MALDI-TOF mass spectrometry. The three clinically relevant complexes include: A. hydrophila complex, A. caviae complex and A. veronii complex.

With regards to susceptibility testing for Aeromonas spp., the CLSI M45, 3rd edition provides guidelines for the three complexes discussed above. Third or fourth generation cephalosporins, fluoroquinolones and trimethoprim-sulfamethoxazole are recommended as antibiotics for primary testing for isolates from extraintestinal sites. Aeromonas spp. are uniformly resistant to ampicillin, amoxicillin-clavulanate and cefazolin and many strains may possess various inducible beta lactamases.

In the case of our patient, with the laboratory identification of Aeromonas veronii bv. sobria, his gram negative coverage was switched to ciprofloxacin for a 10 day course. His PICC was removed and on further imaging studies he was found to have a large abdominal abscess which required surgical drainage. Subsequent cultures from the abdominal abscess were negative.

 

Stempak

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the director of the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement, and resident education.

Tissue is the Issue: Splitting Specimens, Part 1

When we think about infectious disease (ID) and specific syndromes (culture-negative endocarditis, for example), it can be difficult to know the etiology.1 This is because different microorganisms can cause similar symptoms and depending on the specimen submitted to the laboratory for testing, you may need to split your specimen. It may be that the infection is localized to a small area (valve vegetation) and all you get for processing is a small volume of tissue. Another scenario is that you get a sufficient amount of specimen, but you must split the specimen for culture, multiple send out studies, pathology, etc. Or even worse–a small volume specimen that you need to split for multiple diagnostic tests.

We recently had a case of endocarditis. The patient’s blood cultures were negative, and she was going to have her mitral valve replaced. The ID team requested that we send the tissue for broad-range bacterial and fungal sequencing. We can do that- not a problem.

The Issue

As requested, the specimen (mitral valve vegetation) was split once it was received in the laboratory. Half went into the freezer for sequencing requests which we send to a reference laboratory and the other half was processed for bacterial (including mycobacteria) and fungal cultures.

In our experience, if no organisms are observed, then no DNA is detected. Therefore, it is not beneficial to send tissue for sequencing if we do not observe an organism (or something that looks suspicious for an organism) to begin with. In parts 2 and 3 of this series we will go into greater detail of the workflow for examining tissue for infection, but for now we will focus on the processing piece.

No organisms were observed in the direct smears (Gram, fungal, and acid-fast) and all cultures were negative. Because no organisms were observed, we did not send the tissue for sequencing. However, the patient was not improving and ID insisted that we send the tissue for sequencing anyway. As a last ditch effort we decided to homogenize the frozen tissue to see if by chance organism was present.

Long story short: the Gram stain did not reveal organism, but acridine orange2 did. We cultured the tissue and recovered an organism. Moral of the story: specimen processing can be tricky. It is an inherent issue that we must be aware of. How were we to know in which part of the tissue the organisms were? By definition, this is sampling error at its finest.

The Solution

Moving forward, rather than split the specimen prior to processing we have changed our protocol to homogenize the tissue first, then split the specimen. We believe this will eliminate similar scenarios from happening again in the future.

The Conclusion

All specimens are different in their composition. Unlike body fluids, which are easy to vortex and make homogeneous; tissue is more complex. Whatever the specimen, make sure your protocol(s) reduces sampling error.

References

  1. Subedi SJennings ZChen SC. 2017. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart Lung Circ. 26(8):763-771.
  2. Lauer BAReller LBMirrett 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):201-5.

 

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.