Surgical Pathology Case Study: An Enlarging Neck Mass with A Non-Diagnostic FNA

Case History

The patient is a 41 year old male with a history of smoking who presents with a tender, slowly growing mass on the angle of the left mandible for the past 1 to 1.5 years. The patient also complains of otalgia, but no dysphagia or weight loss. A computed tomography (CT) scan was performed, which demonstrated a 3.2 x 2.6 cm enhancing mass in the superficial lobe of the left parotid gland with no significantly enlarged lymph nodes and a patent Stensen’s duct (Image 1). A fine needle aspirate (FNA) was performed that showed acinic and ductal cells, but was not diagnostic. The decision was made to take the patient to surgery in order to perform a parotidectomy.

Image 1. CT scan demonstrating the mass in the left parotid gland (red arrow)

Diagnosis

Received in the surgical pathology laboratory for intraoperative consultation was a 0.3 x 0.2 x 0.2 cm biopsy of the left superficial lobe parotid gland mass. The tissue was frozen, stained and read out as an “acinic cell neoplasm”. Following the frozen diagnosis, the main specimen was received for routine processing, weighing 19.0 gm and measuring 4.0 x 4.0 x 3.0 cm. The specimen was unoriented and entirely inked black. It was serially sectioned revealing a 2.5 x 2.5 x 2.4 cm tan, friable, well-circumscribed mass and surrounding tan-brown, beefy-appearing parotid tissue (Image 2). A second tan-brown nodule measuring 1.5 x 1.0 x 0.8 cm abuts the larger tan mass. Representative sections of the larger mass are submitted in cassettes 1-8, and the smaller nodule is entirely submitted in cassettes 9 and 10.

Image 2. Cut surface of the parotid gland demonstrating the well-circumscribed tan mass

Microscopy demonstrates a low-grade, very well differentiated tumor consistent with an acinic cell carcinoma with complete inked surfaces (i.e. the mass has not been transected and excision appears complete). There is a small focus of capsular “disruption”/parenchymal hemorrhage, which most likely corresponds to the area sampled for intraoperative consultation. In addition, there are two separate benign periparotid lymph nodes.

Discussion

Acinic cell carcinoma (ACC) is a rare tumor of the parotid gland, representing 2 to 4% of all primary parotid gland neoplasms. It is the second most common childhood salivary gland malignancy behind mucoepidermoid carcinoma, but has been found throughout the age range. There is a gender predilection, as ACC is found in females more than males in a 3:2 ratio. One of the first cases of ACC dates back to 1892, in which the tumor was diagnosed as being a “blue dot tumor”, thought to be called this due to the intracytoplasmic zymogen granules.

Clinically, ACC presents as a slowing growing mass in the salivary glands, most commonly in the parotid gland. Other symptoms are not commonly found until late in the diagnosis, and include pain, facial nerve palsy, and nodal disease. There have also been cases of ACC that arise in the minor salivary glands. Unlike minor salivary gland carcinomas that arise in the palate, ACC of the minor salivary glands will mostly be found in the buccal mucosa and upper lip.

Grossly, ACC presents as a round, well-circumscribed to variably encapsulated mass with a rubbery, gray-tan, solid to cystic cut surface, commonly with areas of hemorrhage and necrosis. Histologically, the mass will be composed of acinar type cells with basophilic granular cytoplasm, clear cells with glycogen or mucin, intercalated ducts, non-specific glandular cells and a few mitotic figures. ACC is defined by the World Health Organization as a malignant epithelial neoplasm of the salivary glands in which at least some of the neoplastic cells demonstrate serous acinar cell differentiation, which is characterized by zymogen secretory granules, and can also include salivary ductal cells (Image 3). It is common for sections taken of ACC to show microscopic invasion of the capsule with nests of tumor cells outside the capsule. There are four histologic patterns that were described by Abrams et al in 1965 that are still applicable today: solid, microcystic, papillary cystic and follicular. Immunohistochemical stains, if needed, will be positive for keratin, alpha-1-antichymotrypsin and alpha amylase. It can be difficult to distinguish ACC from normal acini or benign salivary gland tumors (leading to a false negative result) on cytology due to the absence of any hallmark malignancy features such as necrosis and pleomorphism, but centrally placed large nuclei, distinct nucleoli, binucleated cells, and ill-defined cell borders can help make this distinction. The same caution applies to aspirates because if the tumor is cystic, it may be interpreted as being hypocellular and deemed to be a benign salivary cyst.

Image 3. Photomicrograph demonstrating the zymogen granules within the cytoplasm of the cells

Imaging by ultrasound, CT and magnetic resonance imaging (MRI) can prove to be worrisome as similar with cytology, the scans can demonstrate a mass with benign features, and thus a more favorable diagnosis. On ultrasound, ACC will appear lobular, well-defined, hypoechoic and poorly vascularized. Ultrasound can be useful help to determine the size and location of the mass, as well to help with ultrasound guided fine needle biopsies. On CT, the mass will appear non-specific with limited heterogenous enhancement but can be used to demonstrate the relationship of the mass to the facial nerve, and to identify any distant metastases. On MRI, ACC can have a nonspecific intensity pattern similar to benign salivary gland neoplasms, but low T1 and T2 signals can help suggest vascularity, fibrosis and calcification within the mass. In addition, MRI can help in assessing the parotid gland, stylomastoid foramen, and any possible facial nerve invasion or perineural invasion.

Risk factors for the development of ACC include radiation exposure and familial predisposition. Risk factors for the development of salivary gland tumors, but not necessarily ACC, include radiation exposure, the use of iodine 131 in the treatment of thyroid disease (isotope is concentrated in the salivary glands), and working with materials in certain industries, such as those that use asbestos and rubber manufacturing, metal in the plumbing industries, and woodworking in automobile industries.

Complete surgical excision is considered the primary treatment option, with postoperative radiotherapy in cases of incomplete removal, recurrence, undifferentiated ACC, positive margins, and cervical lymph node metastasis. Removal of the facial nerve may be necessary in T3 and T4 cases, as well as a possible neck dissection. As of now, ACC has been considered chemo-resistant, and treatment with chemotherapy is not suggested. Around 35% of tumors will recur, and that percentage rises to 80-90% if the tumor is incompletely excised. ACC has a 5 year survival rate of 90%, a 10 year survival rate of 88%, and there have even been of cases of recurrence occurring up to 30 years after the initial procedure. If metastasis was to occur, although rare, the spread tends to be more hematogenous than lymphatic, with the most common sites being the lungs and bones.

References

  1. Al-Zaher N, Obeid A, Al-Salam S, Al-Kayyali BS. Acinic cell carcinoma of the salivary glands: a literature review. Hematol Oncol Stem Cell Ther. 2009;2(1):259-64.
  2. Bury D, Dafalla M, Ahmed S, Hellquist H. High grade transformation of salivary gland acinic cell carcinoma with emphasis on histological diagnosis and clinical implication. Pathol Res Pract. 2016;212(11):1059-1063. DOI: 10.1016/j.prp.2016.08.005.
  3. Rosero DS, Alvarez R, Gambó P, et al. Acinic Cell Carcinoma of the Parotid Gland with Four Morphological Features. Iran J Pathol. 2016;11(2):181–185.
  4. Vander Poorten V, Triantafyllou A, Thompson LD, et al. Salivary acinic cell carcinoma: reappraisal and update. Eur Arch Otorhinolaryngol. 2016;273(11):3511-3531. DOI: 10.1007/s00405-015-3855-7
  5. Zahra Aly F. Acinic Cell Carcinoma. Pathology Outlines. http://www.pathologyoutlines.com/topic/salivaryglandsaciniccell.html. Revised April 30, 2019. Accessed August 23, 2019.

-Cory Nash is a board certified Pathologists’ Assistant, specializing in surgical and gross pathology. He currently works as a Pathologists’ Assistant at the University of Chicago Medical Center. His job involves the macroscopic examination, dissection and tissue submission of surgical specimens, ranging from biopsies to multi-organ resections. Cory has a special interest in head and neck pathology, as well as bone and soft tissue pathology. Cory can be followed on twitter at @iplaywithorgans.

Hematology Case Study: Thrombocytopenia in a 4 Year Old Child

A 4 year old child was brought to the pediatrician by her mother with a complaint of new onset of severe bruising on her legs. The mother could not recall any falls or bumps that would have caused the bruising. On exam, the physician also noted mucosal bleeding in the oral cavity. Questioning revealed that the patient had experienced flu like symptoms several weeks earlier. The physical exam was normal except for the bleeding. There was no family history of bleeding disorders. A CBC was ordered.

Reported CBC Results

WBC, RBC, Hgb, Hct, RBC indicies normal

Platelet count 26 x 103/μL

IPF 22% (reference range IPF% 1.0-7.0%) The physician evaluated the results, noting the normal CBC but decreased platelet count. The above results also show the immature platelet fraction (IPF), an additional Advanced Clinical Parameter reported from the Sysmex XN hematology analyzer. A low platelet count, as seen in this patient, will reflex a fluorescent platelet count (PLT-F). The impedance count (PLT-I) can be falsely increased if small RBCs or fragments are counted as platelets. On the other hand, in an optical platelet count, when measuring platelets by size (PLT-O), large platelets can be missed, giving a falsely low count. In this case there was a low platelet count and an instrument flag for an abnormal platelet scattergram. The PLT-F, on the other hand, uses a platelet specific dye which eliminates interference seen with other methods. The fluorescent dye labels the RNA, and forward scatter is used to determine size while side fluorescence is used to measure RNA content. With gating set based on cell volume and RNA content, the PLT-F can be measured. Therefore, the reflexed and more reliable PLT-F was the reported count.

Figure 1. PLT-F scattergram. The PLT-F channel measures forward scatter (FSC) on the Y axis and side fluorescence (SFL) on the X axis.1

Additionally, when there is an abnormal scattergram or a low platelet count, the IPF% and IPF# are also reported. The immature platelet fraction is a measure of the youngest platelets, or reticulated platelets. These are the first circulating platelets, right out of the bone marrow. An increased IPF indicates an increase in platelet production, yet this child’s platelet count was very low. This suggests that the thrombocytopenia may be due to excessive destruction of platelets; the bone marrow was actively making platelets, but they were being destroyed, causing the low platelet count.

Figure 2. Platelet scattergrams from a healthy individual with a normal IPF (a) and a patient with a high IPF (b). Mature platelets appear as blue dots, green dots represent the IPF with increased cell volume and higher fluorescence intensity compared to mature platelets.1

Diagnosis

Immune Thrombocytopenia- ITP.

Primary immune thrombocytopenia (ITP), formerly known as idiopathic thrombocytopenic purpura or immune thrombocytopenic purpura, is one of the most common bleeding disorders of children. In most cases, it presents with sudden onset of bruising and petechiae in an otherwise healthy child, with normal WBC and hemoglobin. ITP is an autoimmune bleeding disorder in which the immune system makes anti-platelet antibodies which bind to platelets and cause destruction. Even though the exact cause of ITP remains unknown, it is recognized that it can follow a viral infection or live vaccinations. While there are some similarities between pediatric ITP and ITP in adults, in children this tends to be an acute disease which is self-limiting and resolves itself in several weeks, with no treatment. However, in a small number of children, the disorder may progress to a chronic ITP. In contrast to ITP in children, a chronic form is more commonly seen in adults. It is usually a diagnosis of exclusion, does not follow a viral illness and requires treatment.

This patient recovered in a few weeks. One month after the initial episode, her PLT was 174 x 103/μL and her IPF% was 6.0%

Conclusion

An IPF reported with a CBC, in combination with a low platelet count, is fast, inexpensive, and can be extremely beneficial in aiding in a timely diagnosis. As the child’s platelet count recovered, the IPF% returned to normal range. ITP can therefore be monitored with a CBC. Thus, the IPF can be used not only to help diagnose but also as an indicator of remission.

References

  1. Sysmex America, 2019. www.sysmex.com/us. Used with permission
  2. Arshi Naz et al. Importance of Immature platelet Fraction as a predictor of immune thrombocytopenic purpura. Pak J Med Sci 2016 Vol 32 No 3:575-579
  3. Briggs,C. Assessment of an immature plateletfraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004Jul;126(1):93-9
  4. Sysmex White Paper. The role of the ImmaturePlatelet Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us
  5. D-Orazio, JA, Neely, J, Farhoudi,N. ITP in children: pathophysiology and current treatment approaches.J Pediatr Hematol Oncol.2013 Jan;35(1): 1-13

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Microbiology Case Study: A 35 Year Old Female with Post-Op Drainage

Case History

A 35 year old female with a history of BRCA-positive breast cancer (status-post right radical mastectomy February 2018) underwent prophylactic left mastectomy and revision of right mastectomy. She received prophylactic clindamycin and cefazolin. She was discharged on post-op day 5 with bacitracin and 3 weeks of cefadroxil. She initially healed well. On post-op day 43 she noted drainage from the left incision site. At presentation she was afebrile.  There was a 3.0 x 2.0 cm area of induration and erythema on the right lateral aspect of her abdominal incision with seropurulent drainage. Incision and drainage was performed in office and a swab of the fluid was sent to microbiology. The initial gram stain and cultures were negative for bacteria. The patient was placed on sulfamethoxazole-trimethoprim and levofloxacin. At follow up 7 days later, another abscess was medial to the prior site was incised and drained. A swab of the fluid was sent to microbiology for bacterial and fungal cultures.

Laboratory Identification

Fungal cultures grew at 36 hours on potato-flake agar. Gram stain revealed gram-variable bacilli. Growth on 7H10 agar produced colonies at 72 hours, and Kinyoun staining was positive for acid-fast bacilli. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) at 72 hours identified Mycobacterium abscessus complex.

Image 1. Growth on 7H10 agar.
Image 2. Gram stain from 7H10 agar showing gram variable bacilli.
Image 3. Kinyoun stain showing acid-fast bacilli.

Discussion

M. abscessus complex is a group of rapidly-growing, nontuberculous mycobacteria. As such they are acid-fast bacilli that grow within 7 days when transferred from solid media to solid media. The subspecies are M. abscessus abscessus, M. abscessus massiliense, and M. abscessus bolletii. The complex is known to cause progressive pulmonary disease in patients with underlying structural lung diseases. It has been estimated to comprise up to 13% of all mycobacterial pulmonary infections. It has also been implicated in skin and soft tissue infections (SSTIs) following surgical procedures or environmental exposure (i.e. spas). SSTIs can also occur by seeding from disseminated disease. Rarer manifestations include central nervous system (CNS) and ocular involvement. Identification is by culture and molecular techniques. It is classically resistant to many drug classes with limited consensus on appropriate therapy. It can harbor the erm gene, which confers inducible erythromycin resistance. Clarithromycin, amikacin, and cefoxitin tend to have the lowest rates of resistance. Long-term multidrug regimens are recommended, based on susceptibility testing. Changes to initial therapy are usually required due to side effects or lack of efficacy. Surgical therapy is often required, when possible. Mortality post therapy is approximately 15%.

At two-week follow up, the wound had no purulent drainage or erythema. The plan was for prolonged three-drug therapy tailored to susceptibility data.

  1. Griffith DE. Rapidly growing mycobacterial infections: Mycobacteria abscessus, chelonae, and fortuitum. Von Reyn CF and B A, eds. UpToDate. Waltham, MA: UpToDate Inc. https://www.uptodate.com (Accessed on May 21, 2019.)
  2. Lee MR, Sheng WH, Hung CC, Yu CJ, Lee LN, Hsueh PR. Mycobacterium abscessus Complex Infections in Humans. Emerg Infect Dis. 2015;21(9):1638–1646.
  3. Novosad SA, Beekmann SE, Polgreen PM, et al. Treatment of Mycobacterium abscessus Infection. Emerging Infectious Diseases. 2016;22(3):511-514.

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

-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.

The Fundamental Attribution Error

As laboratory safety professionals, we know that an important part of the job is the ability to coach other lab team members when unsafe situations are observed. To coach someone is to confront a coworker about an issue for the sake of safety-theirs, yours, or that of a patient. Those coworkers may be fellow lab employees, supervisors, managers, or even physicians. The word “confront” might sound strong, particularly to those who may be uncomfortable with these types of encounters, but this coaching is an important and valuable skill. 

Coaching your peers is no easy task, and it takes practice to be able to do it well. I recently walked into a laboratory that was unfamiliar to me, and I saw a technologist working at the bench with no lab coat, no gloves, and no face protection. At first I thought, “that would never happen in a one of my labs,” and then, “the lab safety culture here is terrible.”

I learned I was wrong on both counts, and the incident reminded me of the necessity to stop and think before forming an opinion or even speaking about a lab safety issue. I provide training often about how to coach staff who are acting unsafely while in the lab, and I have learned that how a coaching moment will go depends largely on what is in the head of the coach before he or she speaks. It is important to remember that if someone acts in a manner that displeases or disappoints you, there are several possible sources of influence acting on that person.

Psychologists have coined it the “Fundamental Attribution Error.” Humans who are disappointed usually think the other person has committed the wrong intentionally or because they are not intelligent. Neither of these conclusions is ever correct, and that thought process usually leads to a coaching session that will not be successful.

Take the scenario I mentioned above, for example. What is your gut reaction when you see someone working in a lab without PPE? Maybe that lab tech just found out a relative had passed away and they were waiting for someone to relieve them, or maybe there were no lab coats or gloves available in their size. The possibilities are endless, so you need to train yourself to be calm first and to ask questions to learn what is really happening without making assumptions. It’s more difficult to do than one would think.

The success of a safety coaching moment is determined in your head before you even speak. You have the power to make it a positive event. It is true that some people just will not accept it well no matter what we do (a reminder to ourselves to always be ready to accept coaching), but by and large a successful event starts in the mind of the person who is coaching for safety.

When you see a lab safety problem, it is vital that you confront the person. However, before you do so, ask yourself, “why would a rational person behave this way? What am I not seeing here?” If you start with that, your coaching for safety will be much more successful, and you will see a positive change in your overall lab safety culture.

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

The X-games of PCR

This is not your Mom’s PCR. These new kids on the block are making PCR extremely fast. PCR (Polymerase Chain Reaction) technology won the Nobel Prize for allowing molecular research to advance much more rapidly (for an interesting read on the quirky Laureate who gave up science to go surfing, read more here: Wikipedia ). It has become the most commonly used work horse of most molecular diagnostic assays, usually in the form of real-time PCR. It is used for a variety of purposes from detecting bacteria and viruses, identity testing for forensics and bone marrow engraftment, cancer mutation analysis, and even sequencing by synthesis used by Illumina for massively parallel sequencing.

This technique is still limited by requiring highly trained technologists to perform DNA extraction, time-consuming processing, and the time of real-time PCR itself. Overall, this process takes about a 5-8 hours. While this is much faster than in the past, it would be unacceptable for use in the point-of-care (POC).

But why would DNA testing need to be POC? The term sounds like an oxymoron in a field where many results have a 2-month turnaround time. There are certain circumstances where molecular testing would impact patient care. For instance, a doctor testing a patient in their office for a sexually transmitted infection would want to know if they have gonorrhea/ chlamydia so they could prescribe proper antibiotics. Similarly, POC molecular testing could be applied in a bioterrorism incident to test samples for an infectious agent. Or POC testing would benefit low-resource areas internationally where HIV testing could be used to manage anti-retroviral therapy in patients many miles from a laboratory.

For PCR as a test to be useful at the POC setting, it would have to provide a result within 10-15 minutes and be performed as a waived test. Two recent examples the demonstrate how this is possible have been highlighted at recent conferences of the American Association of Clinical Chemistry, which I just got back from: Extreme PCR1 and Laser-PCR.2

Extreme PCR refers to a technique of rapidly cycling the temperature of PCR reactions. The reaction occurs in a thin slide that evenly distributes the reagents, temperature and is clear to permit easy reading of fluorescence measurements (Figure 1). DNA Polymerase enzyme and primers to amplify the target DNA are added at much higher concentrations than normal (20x).

Figure 1. Thin reaction chamber for ultra-fast PCR.

This flies in the face of traditional PCR chemistry dogma as specificity would plummet and normal DNA could be amplified instead of target DNA. This would create a false positive. However, let’s think about what is actually happening with non-specific reactions. Primers are designed to match one region of DNA, which is very unique within the whole genome. However, the genome is so large that some segment may look very similar and be different in just 1 or 2 of the 20 base pairs that a primer matches. A primer could bind to this alternate region but less efficiently. So, the binding would be weaker and take more time to occur.

Therefore, by speeding up the cycling time to just a few seconds, only the most specific interactions can take place and non-specific binding is offset (Figure 2)!

Figure 2. Fluorescence from a dye that fluoresces when bound to double stranded DNA, which is increasing here within seconds (high point represents when the reaction temperature cools and dsDNA anneals, then low points represent heating to high temperatures).

Laser PCR does not report the use of increased reagents like Extreme PCR (it may be proprietary), but they boast a very innovative method to quickly heat and cool PCR reactions. GNA Biosciences use gold nanoparticles with many DNA adapters attached (Watch the video below for a great visual explanation!).

These adapters are short sequences of DNA that bring the target DNA and primers together to amplify the target DNA sequence. Then as the name implies, a laser zaps the gold beads and heats them up in a very localized area that releases the DNA strands. The released DNA binds another gold particle, replicates, rinses, and repeats. The laser energy thus heats the gold in a small area that allows for quick heating and cooling within a matter of seconds.

These new PCR methods are very interesting and can have a big impact on changing how molecular pathology advances are brought to the patient. On a scientific note, I hope you found them as fascinating as I did!

References

  1. Myrick JT, Pryor RJ, Palais RA, Ison SJ, Sanford L, Dwight ZL, et al. Integrated extreme real-time PCR and high-speed melting analysis in 52 to 87 seconds. Clin Chem 2019;65:263–71.
  2. CLN Stat. A Celebration of Innovation. AACC’s first disruptive technology award to recognize three breakthrough diagnostics. https://www.aacc.org/publications/cln/cln-stat/2018/july/10/a-celebration-of-innovation
  3. G. Mike Makrigiorgos. Extreme PCR Meets High-Speed Melting: A Step Closer to Molecular Diagnostics “While You Wait” Clin Chem 2019.

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

Microbiology Case Study: A 55 Year Old Male with Altered Mental Status

Case History

A 55 year old male presented to the emergency department (ED) with altered mental status (AMS). His past medical history includes stage 4 pancreatic cancer with known invasion into the distal splenic vein. Currently undergoing chemotherapy, his last infusion was one week prior to presentation. On physical exam, the patient is a cachectic male with dry mucous membranes, scleral icterus, hypotension (79/37), hypothermia (35o C), tachypnea (Respiratory Rate of 20/min) and tachycardia (pulse up to 130s). Initial labs were ordered including blood cultures and were significant for hypoglycemia (40mg/dL), pancytopenia, mild liver function test abnormalities, an ammonia level of 80 µmol/L and lactate of 11.2 mmol/L. It was concluded that the AMS resulted as a consequence of hypoglycemia. However, there was also concern for intracranial pathology vs. stroke vs. metastatic disease. Brain imaging showed no obvious lesion or mass. The cause of the hypotension was uncertain but could be a result of volume depletion or sepsis. Empiric vancomycin and cefepime were initiated in the ED. However, the patient decompensated, developing mid-abdominal and periumbilical ecchymoses suspicious for a retroperitoneal hemorrhage. Despite aggressive therapy, he expired in the ED.

Laboratory Identification

An anaerobic blood culture bottle flagged positive at 5 hours incubation. A gram stain showed large, boxy, gram positive rods without spores (Image 1). Brucella blood agar was inoculated and incubated anaerobically.  Following overnight incubation, the surface of the plate showed a subtle film of growth covering the plate and detectable hemolysis (Image 2). No discreet colonies were identified. A catalase test was performed and was negative. Definitive identification of Clostridium septicum was obtained by MALDI-TOF.

Image 1. Gram stain from the anaerobic blood culture bottle that flagged as positive following 5 hours of incubation. Boxy, large gram positive rods without spores are observed. Oil immersion photomicrograph (x100 objective).
Image 2. Brucella agar plate following 24 hours of incubation under anaerobic conditions at 35oC. Significant bacterial growth in a haze and detectable Beta hemolysis can be observed. No discreet colonies can be identified. Identification by MALDI-TOF was Clostridium septicum.

Discussion

Clostridium septicum is an anaerobic gram positive bacillus that can produce spores; however, spores are not frequently seen, especially in nutrient-rich environments. Spores, when present, are typically oval and located subterminally. Infection by C. septicum was once thought to be extremely rare, but improvements in anaerobic laboratory techniques have allowed for the discovery of the true potential of this agent. C. septicum is one of several bacteria that can cause myonecrosis (i.e., gas gangrene). Infections are typically seen in settings of immunodeficiency, trauma, surgery, malignancy, skin infections/burns, and septic abortions. The colon may promote the growth of C. septicum better than other anatomic sites due to its anaerobic conditions. As one of the more aggressive etiologies of gas gangreneC. septicum infection progresses very rapidly, with a mortality rate of approximately 79% in adults, typically occurring within 48 hours of infection. Symptoms of infection include pain, described as a heaviness or pressure that is disproportionate to physical findings, tachycardia, and hypotension. Tissue necrosis then causes edema and ischemia resulting in metabolic acidosis, fever, and renal failure. The carbon dioxide and hydrogen produced during the growth of the organism move through tissue planes, causing their separation, producing features characteristic of palpable emphysema (i.e., crepitus). This also results in a magenta-bronze skin discoloration and bulla filled with a foul-smelling serosanguinous fluid.

Four toxins have been isolated from C. septicum: the lethal alpha toxin, DNase beta-toxin, hyaluronidase gamma toxin, and the thiol-activated/septicolysin delta toxin. Alpha toxin causes intravascular hemolysis and tissue necrosis and is well known as the primary virulence factor of C. septicum

C. septicum derived gas gangrene has shown strong correlations with increased levels of malignancy. Patients with C. septicum infections may have an occult colon cancer or a tumor that has metastasized to the colon. C. septicum bacteremia is also associated with typhlitis (defined as inflammation of the cecum that can extend proximally into the terminal ileum or distally into the ascending colon), which can develop in patients with hematologic malignancy receiving chemotherapy. Because the organism may be harbored in the gastrointestinal tract, the organism may gain access to the bloodstream through the ileocecal region.

Therapy includes antibiotics and surgical debridement (with occasional amputation). For antibiotic selection, typical anaerobic coverage includes piperacillin/tazobactam, ampicillin/sulbactam, metronidazole or meropenem. Vancomycin is also effective. Susceptibility testing is not typically performed; moreover, the CLSI makes an annual antibiogram which can be used as a guide. 

Key points

  • C. septicum often has swarming growth that covers the plate surface.
  • Spontaneous myonecrosis with C. septicum bacteremia can be an indicator of possible occult colonic malignancy.
  • C. septicum can be associated with typhlitis in neutropenic patients with hematologic malignancy undergoing chemotherapy.

 References

  1. Smith-Slatas CL, Bourque M, and Salazar JC (2006). Clostridium septicum infections in children: a case report and review of the literature. Pediatrics 117(4): e796-e805.
  2. Alpern, RJ and Dowell, VR (1969). “Clostridium septicum infections and malignancy”. JAMA. 209: 385–388.
  3. Ballard, J, Crabtree, J, Roe, BA, and Tweten, RK (1995). “The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin”. Infection and Immunity. 63 (1):340–344.
  4. Sidhu JS, Mandal A, Virk J, and Gayam V (2019). “Early detection of colon cancer following incidental finding of Clostridium septicum bacteremia”. J Investig Med High Impact Case Rep. Jan-Dec;7:2324709619832050.
  5. Srivastava I, Aldape MJ, Bryant AE, and Stevens DL. (2017). “Spontaneous C. septicum gas gangrene: A literature review” Anaerobe. Dec;48:165-171

Xiang Xu, MD, PhD and Dominick Cavuoti, DO contributed to this case.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. he has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.