Microbiology Case Study: Interesting Case of a Cavitary Lung Mass

Case History

A 50 year old male with a significant past medical history of poorly controlled type 2 diabetes mellitus, hypertension, hyperlipidemia, smoking tobacco abuse and obstructive sleep apnea was referred to our institution’s pulmonology clinic for cavitary lung mass. The lung mass was incidentally discovered on chest x-ray and has been clinically stable on serial imaging for over two years; however, a previous extensive laboratory workup including computed tomography (CT) guided biopsy was unrevealing to its etiology. The patient was noted to be largely asymptomatic at his initial office visit; repeat diagnostic workup was ordered. CT chest imaging revealed a 2.8 x 1.9 x 2.0 cm cavitary lung mass in the posterior left lower lobe that was unchanged compared to outside CT imaging from approximately 4 months prior.

Image 1. Cross section (left) and Sagittal (right) views from CT chest without contrast revealed a 2.8 cm transverse by 1.9 cm anteroposterior by 2.0 cm craniocaudal stable mass-like opacity in the left lower lobe superior segment broadly abutting the posterior pleura with a tiny internal focus of cavitation.

Given the chronicity of the lung mass, atypical infection (Nocardia, endemic fungi, mycobacterium) and primary pulmonary cancer were highest on the differential diagnosis. Blood tests including endemic fungal serologies, QuantiFERON-TB Gold, cryptococcal antigen, galactomannan and Fungitell (1-3)-B-d glucan assay were negative. Given the unrevealing non-invasive workup, a repeat CT guided biopsy was performed and core biopsy samples were sent for AFB, fungal and Nocardia cultures as well as for histopathological examination.

Histopathology revealed necrotizing granulomatous inflammation with empty spherules of Coccidioides suggestive of a remote infection of long duration (Images 2, 3). Additionally, no microorganisms were isolated from cultures. Based on these findings, an infectious disease (ID) consult was placed. The patient remained asymptomatic and revealed a long history of residing within areas of the Southwestern United States endemic to Coccidioides species (sp.) during his ID office visit. Repeat Coccidioides complement fixation was positive for IgG (Titer: 1:4) with negative IgM by immunodiffusion testing. Urine Coccidioides antigen tested by quantitative sandwich enzyme immunoassay was negative. These findings likely represent past history of coccidiomycosis and not active infection. Antifungal therapy was deferred due to the patient’s asymptomatic status. The patient was monitored with close clinical follow up and continued serial imaging.

Histopathology Images

Image 2. Hematoxylin and eosin stained sections of formalin fixed paraffin embedded (FFPE) tissue from core biopsy of cavitary lung mass. Necrotizing granulomatous inflammation at 40X (A) and 100X (B) with empty spherules of Coccidioides (C, D) at 600X.
Image 3. Special stains performed of formalin fixed paraffin embedded (FFPE) tissue from core biopsy of cavitary lung mass highlighting empty spherules. Grocott’s methenamine silver stain at 100X (A) and 400X (B). Periodic Acid Schiff for Fungus stain at 600X (C).

Discussion

Coccidioides sp. are dimorphic fungi with a mycelial (saprophytic) phase in the environment and a spherule (parasitic) phase in its host.1 It is the cause of coccidiomycosis, also known as valley fever, desert fever or San Joaquin fever, which has a wide range of clinical presentations from subclinical manifestations (~60%) to an influenza-like illness followed by skin lesions to the most pathological form, disseminated disease.1 It can also cause the development of cavitary lung masses, as described in this case.1 It is endemic to the Southwestern region of the United States where it prefers dry, arid conditions.2 Infections normally occur by inhalation of infective arthroconidia, which have matured from mycelium, following disruption of soil.1 Once inside the host, lungs spherules containing endospores develop (Image 4).1 These spherules rupture releasing the endospores which can continue to develop into spherules to maintain a continuous parasitic cycle or can be exhaled into the environment to continue its saprophytic phase.1

Image 4. High magnification images of hematoxylin and eosin stained sections of formalin fixed paraffin embedded (FFPE) lung tissue revealing multiple spherules containing endospores (left) consistent with active Coccidioides infection and a giant ruptured spherule releasing endospores (right) that will continue to propagate Coccidioides infection.

Two morphologically indistinct species exist (C. immitis and C. posadasii) that can only be definitively identified by molecular methods.3 C. immitis is predominantly found in central and southern California while C. posadasii can be found in other non-Californian southwestern US states and extending into western Texas and down throughout Mexico and South America.3 When cultured, it grows rapidly at both 25°C and 37°C into woolly white colonies that develop alternating barrel-shaped arthroconidia that can be seen on tape prep with lactophenol blue.4

References

  1. Donovan FM, Shubitz L, Powell D, Orbach M, Frelinger J, Galgiani JM. 2019. Early Events in Coccidiomycosis. Clinical Microbiology Reviews, 33, e00112-19, DOI: 10.1128/CMR.00112-19
  2. Hernandez H, Erives VH, Martinez LR. 2019. Coccidioidomycosis: Epidemiology, Fungal Pathogenesis and Therapeutic Development. Current Tropical Medicine Reports, 6, 132-144,  DOI: 10.1007/s40475-019-00184-z
  3. Kirkland TN, Fierer J. 2018. Coccidioides immitis and posadasii; a review of their biology, genomics, pathogenesis, and host immunity, Virulence, 9:1, 1426-1435, DOI: 10.1080/21505594.2018.1509667
  4. Love GL, Ribes JA. 2018. Color Atlas of Mycology, An Illustrated Field Guide Based on Proficiency Testing. College of American Pathologists (CAP), p. 234-235

-John Markantonis is the current Medical Microbiology fellow at UT Southwestern and will be completing his Clinical Pathology residency in 2022. He is also interested in Transfusion Medicine and parasitic diseases.

-Dominick Cavuoti is a Professor at UT Southwestern and specializes in Infectious Diseases Pathology, Medical Microbiology and Cytology.

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

What to Expect When You Don’t Know What You’re Expecting: COVID-19 and Flu Season in the Laboratory

Welcome to October 2020 and a flu season unlike any other. What can we expect? Well, it’s complicated. And if we aren’t sure what to expect, can we still be prepared? Yes (at least for some things)!

From the beginning of the COVID-19 pandemic and throughout the summer of 2020 clinicians and laboratorians have been anxiously wondering what effect global presence of respiratory virus SARS-CoV-2 would have on the 2020-2021 flu season. “Flu season,” the annual, relatively predictable period of increased cases and deaths due to Influenza A and B, occurs during colder, winter months. In the northern hemisphere this is September through March. We have extensive experience tracking the onset and genetic variability of the predominant influenza viruses. We manufacturer flu vaccines based on data of potentially likely influenza strains. Other viruses that cause respiratory symptoms follow similar seasonal patterns. These include common (non-SARS-CoV-2) human coronaviruses, and Respiratory Syncytial Virus (RSV). In short: this is a known, annual occurrence that we can usually prepare to some extent.

So what will that look like this year? During the historic 1918 pandemic influenza, deaths seen during the first winter of the outbreak paled in comparison to those seen the following winter. Even if that kind of terrible scenario doesn’t occur during this pandemic year, it is possible we are facing “perfect storm” of COVID-19 plus influenza resulting in overwhelmed hospitals and depleted testing supplies. [https://www.cidrap.umn.edu/news-perspective/2020/09/fears-perfect-storm-flu-season-nears]

We know that COVID-19 spreads well in enclosed spaces with prolonged person-to-person contact, regardless of climate and temperature, via respiratory secretions. Because of this, there has been widespread adoption of mask wearing, social distancing, and limitations on in-person gathering. Promisingly, these interventions to prevent the spread of COVID-19 seem to be contributing to historically low influenza rates in the Southern Hemisphere! [https://www.cdc.gov/mmwr/volumes/69/wr/mm6937a6.htm] But adoption of these mitigation strategies are not being universally or rigorously followed in all regions and communities. As temperatures drop, we could see more people conducting activity indoors – will this change transmission patterns? Will regions with ongoing COVID-19 outbreaks be more prone to influenza as well? If hospital capacity becomes strained, will criteria for ordering tests change?

During COVID-19 laboratories have responded heroically and rapidly to test kit shortages, supply chain issues, and staffing challenges. At this stage (October of 2020) many high-level decisions about SARS-CoV-2 testing, like test platform purchasing and validation or manufacturer test kit allocations, might already be set in stone. So is there anything that can be done to help labs and laboratory workers successfully make it through flu season?

Here are 3 suggestions:

1) Establish testing algorithms and clear sample workflows.

Each facility and laboratory will have their own platforms for testing COVID-19 and other respiratory pathogens. Depending on the service ordering the test, there can be both immediate and downstream consequences for when a test comes back positive, negative, or even when that test result is slower than expected!

An algorithm helps set institutional expectations for what tests are ordered under different scenarios. For example symptomatic patients presenting to a hospital with influenza-like illness (ILI), especially when they will be admitted, should likely have both SARS-CoV-2 and influenza tests ordered simultaneously. But asymptomatic patients being admitted for procedures may only require a SARS-CoV-2 test.

Let’s say your lab has both a SARS-CoV-2 PCR test and SARS-CoV-2 rapid antigen test. But due to risk a false negative, lab and clinical leaders are uncomfortable using only a rapid antigen test to conclusively rule out COVID-19 in patients being admitted to the hospital. Your algorithm could use specify the use of SARS-CoV-2 antigen testing in symptomatic patients to quickly “rule in” potential positives, where antigen-negative patients will also have a PCR test. Algorithm specifics come down to what your institutions stake holders (clinical AND laboratory) need and capacity are. The details of an algorithm will be dependent on your lab test platforms, your available test orders, and may need to be modified to accommodate restricted test allocations.

Along with clinical algorithms, clear workflow for specimens and test types can help laboratory workers get tests where they need to go within the lab. Not all SARS-CoV-2 tests have approval in the instructions for use for, say, nasal swabs. If nasal swab comes to the lab with orders for both influenza and SARS-CoV-2 tests, what is the procedure for informing the floor for an appropriate collection? Or say that your test platforms for different tests live in different areas of the lab. Your workflow may be to set up one test and do a pour off into an aliquot tube so tests can be run at the same time. Or you may have sufficient test collection materials to request a separate sample for each test.

Probably the most important part of developing or reviewing your existing algorithms and laboratory workflow is doing it in connection with others. The purpose is to streamline the entire process from clinical decision making to test performing and reporting and help everyone be on the same page.

2) Communicate to clinical staff frequently about your tests.

Because of the intense interest surrounding COVID-19 laboratory testing, it’s entirely possible that more people have had to learn about previously niche laboratory concepts like “sensitivity vs. specificity” and “PCR vs. antibody vs. antigen tests” than at any previously time in human history! However, it is also likely that many clinicians or administrators in your own institution may know more about a test platform they read about in the news than the COVID-19 test platform that their laboratory performs.

Even at this stage in the pandemic with perhaps more exposure (pun not intended!) then the laboratory has ever had, miscommunication and unclear expectations abound surrounding test performance or turnaround times.

Whenever possible, lab leaders who interact with clinicians and administrators should look for ways to educate on test platforms, testing capacity, and expected test performance (i.e. time to result, comparative sensitivity etc.). This could include asking for time to provide formal updates during monthly meetings, monitoring test statistics (e.g. a test “dashboard”), or just informal reminders about what tests the lab performs during phone calls.

3) Keep the lab staff off the phone.

A critical part of the job of the lab is to provide information and updates on when test results are available. But when the hospital floors or clinics are busiest with patients, often the lab is busiest performing those patients’ tests. A phone call about the status of a respiratory virus test can be undeniably helpful to that patient’s clinical care team! But a dozen such phone calls over the course of a lab worker’s shift, especially under normal lab conditions (e.g. no staff shortages or instrument issues) is a failure of communication and can be detrimental to both lab performance and lab worker wellbeing.

In addition to the need for regular education about testing mentioned above, to help protect your lab staff’s bench time here are some possible ways keep from being overwhelmed with phone calls:

  • In some institutions, passive reminders (for example about hand hygiene or upcoming events) cycle through computer screen savers or on television screens in clinical areas. You could see if a message like “Reminder from the lab: COVID-19 tests are completed in [length of time].” could be put on a rotation.
  • If there is no client service or switchboard for your lab, but people call the lab directly for updates, you could institute a message stop. This is where phone calls routed to the laboratory must listen to a reminder that (for example), “If you are calling for an update of a COVID-19 test, these tests cannot be completed faster than [length of time] after arriving in the lab.”

    While these messages can be undeniably annoying and disruptive for people calling the lab for other reasons (and become less effective over time) if phone calls get out of hand, this option could be considered.
  • A lab instrument going down can result in test backlogs and numerous phone calls to the lab. Some institutions centralize their information in the form of a duty officer (for example in the emergency department). This will be a person who can be informed of actionable information, like test delays due to instrument issues, and who will post and distribute that information to those affected.

There is a lot we don’t know about what’s to come in the COVID-19 pandemic. While we can’t predict the ways the lab may be challenged with the next unforeseen disruption, or even what our flu season testing needs may look like, hopefully we can prepare now to continue to support our patients by helping and supporting our labs.

-Dr. Richard Davis, PhD, D(ABMM), MLS(ASCP)CM is a clinical microbiologist and regional director of microbiology for Providence Health Care in Eastern Washington. A certified medical laboratory scientist, he received his PhD studying the tropical parasite Leishmania. He transitioned back to laboratory medicine (though he still loves parasites!), and completed a clinical microbiology fellowship at the University of Utah/ARUP Laboratories in Utah before accepting his current position. He is a 2020 ASCP 40 Under Forty Honoree.

Microbiology Case Study: A Middle-Aged Woman with Forearm Pain

Case History

A middle-aged female was evaluated for left forearm pain and erythema following a cat bite one-day prior, and was prescribed trimethoprim-sulfamethoxazole for management in the outpatient setting.  She subsequently presented for follow-up where she was noted to have a 3 x 4 cm raised, red, indurated lesion of left arm without any discharge (Image 1).  MRI demonstrated a 6.5 x 2.3 x 2.3 cm abscess within the distal ulnar soft tissues with surrounding cellulitis.  As her pain and erythema were progressively worsening, she was admitted for surgical management.   

Upon admission, a bedside incision found purulent drainage which grew mixed anaerobic gram negative rods.  Blood cultures collected at this same time were negative and remained so for the duration of her hospital course.  Empiric antibiotic therapy was initiated with piperacillin-tazobactam, and the patient underwent formal surgical incision and drainage.  Intraoperative findings were notable for abscess, diffuse and severe tendinopathy, and a thick inflammatory rind surrounding the associated neurovascular bundle.  Intraoperative cultures were obtained and sent to the microbiology laboratory.  The patient’s postoperative course was uneventful, and she was discharged with plans to complete a two week course of amoxicillin-clavulanate.  Follow-up clinic visits demonstrated successful recovery, with a well-healed incision and normal grip strength and range of motion.

Laboratory Identification

Bacterial culture of abscess material collected intraoperatively grew smooth, mucoid colonies on chocolate and blood agars with less than 24 hours of incubation at 35°C in CO2 (Image 2, bacterial isolate). Growth was notably absent on MacConkey agar. Gram stain of the colony revealed tiny, gram negative coccobacilli (Image 2). Biochemical testing determined this organism to be indole, oxidase, and catalase positive.  The organism was definitively identified as Pasteurella multocida by MALDI-TOF MS.

Image 1. Arm lesion prior to incision and drainage.
Image 2. P. multocida growth on Blood and Chocolate agars. Gram stain from a colony revealed small, gram negative coccobacilli (far right).

Discussion

Members of the genus Pasteurella are small, Gram-negative coccobacilli which are able to readily grow on Sheep’s blood agar and chocolate agar, but will typically not grow on MacConkey media.  Infection with these organisms is usually considered to be a zoonosis, with both wild and domestic animals serving as reservoirs.  In animal hosts they can be part of the endogenous flora or pathogens.  P. multocida is the most common member of the genus associated with human infections, which has now been divided into multiple taxonomic subspecies through the use of more discriminatory molecular methods.  Biochemically, P. multocida is positive for catalase, oxidase, indole, and nitrate reduction.

Animal bite wounds are often polymicrobial and contain mixtures of both aerobic and anaerobic organisms.  These organisms can be reflective of the oral flora of the biting animal, or of endogenous skin flora of the bite victim.1  While 80-90% of bites per year can be attributed to dogs, an estimated 400,000 cat bites (5-10% of the total) occur in the United States annually.2  While dog bites often manifest as localized crush injuries with contusions and/or lacerations, a majority of such wounds are accessible to irrigation and cleaning which leads to a relatively low infection rate (5-10%).  By contrast, cat bites are often deep, localized puncture wounds which provide excellent environments for the growth of both aerobic and anaerobic bacteria.  While feline bite wounds may appear less severe after cursory inspection, these wounds can be considerably more difficult to clean, resulting in overall infection rates up to 50%.3 

Management of bite wounds includes cleansing, irrigation and debridement.  Importantly, antimicrobial therapy should include coverage for both aerobic and anaerobic bacteria.4  In this case, amoxicillin-clavulanate was utilized with good results, and provides coverage for the most common oral aerobes and anaerobes encountered in animal bite wounds.  Amoxicillin-clavulanate also has activity against beta-lactamase producing bacteria such as Prevotella sp. and Porphyromonas sp. which are oral anaerobes of dogs, cats, and humans.  The use of macrolides should be avoided due to variable activity against Pasteurella multocida.4  As in this case, bite wounds most frequently are encountered on the upper extremities, and Pasteurella sp. is one of the most common isolates recovered from bites from both cats and dogs (50% of dog bites, and 75% of cat bites).2

References

1. Abrahamian FM, Goldstein EJC. 2011. Microbiology of Animal Bite Wound Infections. Clinical Microbiology Reviews 24:231.

2. Bula-Rudas FJ, Olcott JL. 2018. Human and Animal Bites. Pediatrics in Review 39:490.

3. Kannikeswaran N, Kamat D. 2008. Mammalian Bites. Clinical Pediatrics 48:145-148.

4. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJC, Gorbach SL, Hirschmann JV, Kaplan SL, Montoya JG, Wade JC. 2014. Practice Guidelines for the Diagnosis and Management of Skin and Soft Tissue Infections: 2014 Update by the Infectious Diseases Society of America. Clinical Infectious Diseases 59:e10-e52.

 -Francesca Lee, MD, is an associate professor in the Departments of Pathology and Internal Medicine (Infectious Diseases) at UT Southwestern Medical Center.

-Huy Dao, MLS(ASCP)CM graduated from the University of Minnesota and has worked for eight years as medical technologist for eight years.  He is interested in clinical mycology and bacteriology.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Microbiology Case Study: 83 Year Old Female with a Perisplenic Abscess

Case History

An 83 year old female with a past medical history of breast cancer, multiple strokes, dysphagia, hypertension and gastroesophageal reflux disease (GERD) presented to an outside hospital with altered mental status, metabolic encephalopathy, decreased appetite, acute kidney injury, and E. coli/Proteus urinary tract infection (UTI). There, she was diagnosed with a perforated gastric ulcer, which was repaired, with a gastrostomy (G) tube in place. The patient later developed a fever and an aspiration pneumonia, which was treated with ampicillin/sulbactam. A follow up imaging revealed a new gastric perforation along the fundus of the stomach with perisplenic fluid collection, along with a pleural effusion with possible communication with the fluid collection in the stomach. Due to her complex medical history, an additional intervention was not pursued and the family agreed to comfort measures, and the patient was discharged home.

The family presented to our emergency department the same day of discharge, as the patient had not been eating and the family needed assistance in using the G tube. In-house computed tomography (CT) of the abdomen/pelvis again showed an organizing collection near the spleen (Image 1). A medium-sized left pleural effusion with left lower lobe collapse due to the communication with the perforation was observed on CT. The patient received IV fluids and a dose of vancomycin and zosyn in the ED. A vascular and interventional radiology (VIR) consult was recommended for potential drainage of the perisplenic abscess and left pleural effusion.

Image 1. CT of the abdomen demonstrating an organizing collection (black circle, approximately 7 cm x 3 cm) posterior/superior to the spleen near the site of the prior gastric perforation concerning for an abscess.
Image 2. Small gram negative rods on a Gram stain of Burkholderia cenocepacia from a subculture.
Image 3. Culture morphology of Burkholderia cenocepacia on a blood agar plate after overnight incubation: smooth colonies are present (upon prolonged incubation, the colonies turned yellowish color – not shown in image).

VIR drained the perisplenic abscess, which was sent to the microbiology lab for aerobic & anaerobic cultures. The Gram stain revealed many white blood cells (WBC’s) and rare yeast. The culture grew 3+ Burkholderia cepacia complex (Burkholderia cenocepacia) and 3+ Candida glabrata. The Gram stain and colony of the subculture of B. cepacia on a blood agar plate are shown in Images 2 and 3. C. glabrata was also isolated from the urine culture. Susceptibility testing of B. cepacia showed that it was sensitive to both meropenem and trimethoprim-sulfamethoxazole. Vancomycin and zosyn were stopped and the patient was switched to IV trimethoprim sulfamethoxazole for B. cepacia and IV micafungin for C. glabrata.

Due to the recurrent perforation, the G tube could not be used; a jejunostomy (J) tube was placed instead. Feeds were successfully started with the J tube. Trimethoprim-sulfamethoxazole was also given via the J-tube. A follow-up endoscopy showed a normal esophagus, the known perforation in the gastric fundus, and erythematous duodenopathy at the level of the duodenal bulb, the remainder of the duodenum was normal. The patient’s clinical status improved and she was discharged home with the support of home health services.

Discussion

We present an uncommon presentation of Burkholderia cenocepacia, a member of B. cepacia complex, in perisplenic abscess fluid. B. cepacia complex consists of at least 17 closely related species. They are rod-shaped, aerobic, motile Gram-negative bacteria. B. cepacia complex has been well characterized as opportunistic pathogens, particularly in patients with cystic fibrosis and chronic granulomatous disease (CGD). However, they can also infect immunocompetent patients and have been reported to cause endocarditis (specifically in IV drug abusers), pneumonitis, UTIs, osteomyelitis, dermatitis, and other wound infections. In the United States, B. multivorans and B. cenocepacia together account for approximately 80% of B. cepacia complex infections. Burkholderia have been isolated on contaminated hospital equipment and even disinfectants. They present a large problem in nosocomial infections due largely to their ability to survive in aqueous environments.1 They are soil-dwelling bacteria commonly found on plant roots. They are of environmental interest secondary to their antifungal and antinematodal properties as well as the ability to degrade many toxic compounds in agriculture (pesticides, herbicides, preservatives).2 Clinically important Burkholderia species outside of the B. cepacia complex include B. pseudomallei, the causative agent of melioidosis, and B. mallei, the causative agent of glanders.1

Rare case reports have previously documented B. cepacia isolated from splenic abscesses/infections. Most B. cepacia splenic infections occurred secondary to pneumonia or multi-organ involvements in CGD patients. 3, 4, 5 However, one report indicated the B. cepacia-mediated multiple splenic abscesses, in the setting of malignancy and diabetes. 6 While the splenic abscesses in the context of meliodosis, either due to B. pesudomallei or B. mallei infection, have been reported, 7 B. cepacia-mediated splenic infections are rarely encountered.

B. cepcacia complex has intrinsic resistance toseveral antibiotics including penicillins, amoxicillin-clavulanate, ertapenem, polymixin B, Colistin, and fosfomycin.8 B. cepacia complex possesses an inducible β-lactamase, encoded by the gene penA, which can hydrolyze penicillin and use it as a source of carbon. In one study involving 40 bloodstream isolates of B. cepacia in patients without cystic fibrosis, 93% of the isolates were susceptible to ceftazadime and 95% of isolates were susceptible to trimethoprim-sulfamethoxazole.9 Following discussion with our infectious disease colleagues, we believe that the B. cepacia isolate from our patient was likely a nosocomial infection from possible contamination of her G-tube in combination with the gastric perforation.

References

  1. Jorgensen, J. H., Pfaller, M. A., & Carroll, K. C. (2015). Manual of clinical microbiology. Washington, DC, DC: ASM Press.
  2. Kenyon College Department of Biology. (2011, April 22). Burkholderia cepacia. Retrieved September 21, 2020, from https://microbewiki.kenyon.edu/index.php/MicrobeWiki
  3. Clegg HW, Ephros M, Newburger PE. Pseudomonas cepacia pneumonia in chronic granulomatous disease. Pediatr Infect Dis. 1986 Jan-Feb;5(1):111. PMID: 3945563.
  4. Sirinavin, Sayomporn MD*; Techasaensiri, Chonnamet MD*; Pakakasama, Samart MD*; Vorachit, Malai DSc; Pornkul, Rattanaporn MD; Wacharasin, Rames MD Hemophagocytic Syndrome and Burkholderia cepacia Splenic Microabscesses in a Child With Chronic Granulomatous Disease, The Pediatric Infectious Disease Journal: September 2004 – Volume 23 – Issue 9 – p 882-884 doi: 10.1097/01.inf.0000137565.23501.03
  5. Bottone EJ, Douglas SD, Rausen AR, Keusch GT. Association of Pseudomonas cepacia with chronic granulomatous disease. J Clin Microbiol. 1975 May;1(5):425-8. doi: 10.1128/JCM.1.5.425-428.1975. PMID: 1176612; PMCID: PMC275137.
  6. Jayawardena, M. N., Chandrasiri, N. S., Wijekoon, S., Madanayake, P., Corea, E., Ranasinghe, D. D., & Lamahewage, N. D. (2017). Burkholderia cepacia; an unusual cause of multiple splenic abscesses : A case report. Sri Lankan Journal of Infectious Diseases, 7(2), 123. doi:10.4038/sljid.v7i2.8146
  7. Chen, H., Hu, Z., Fang, Y., Lu, X., Li, L., Li, Y, Mao, X, Qian, L. (2018). Splenic abscess caused by Burkholderia pseudomallei. Medicine, 97(26). doi:10.1097/md.0000000000011208
  8. Patel, J. B., Weinstein, M. P., Eliopoulos, G.M., Jenkins, S.G., Lewis, J.S., Limbago, B., Mathers, A., Mazzulli, T., Patel, R., Richter, S.S., Satlin, M., Swenson, J.M., Traczewski, M.M., Turnidge, J.D. & Zimmer, B.L. (2017). Performance standards for antimicrobial susceptibility testing. Wayne, PA: Clinical and Laboratory Standards Institute.
  9. Bressler A.M., Kaye K.S., LiPuma, J.J., Alexander, B.D., Moore, C.M., Reller, L.B. & Woods, C.W. Risk factors for Burkholderia cepacia complex bacteremia among intensive care unit patients without cystic fibrosis: A case-control study. Infect Control Hosp Epidemiol 2007; 28(8):951-8 doi : http://dx.doi.org/10.1086/519177

-J. Stephen Stalls, MD is a PGY-II pathology resident at the East Carolina University Department of Pathology and Laboratory Medicine. He plans to pursue hematopathology and molecular pathology fellowships, but also greatly enjoys his time in the microbiology lab. Outside of work, he enjoys playing the drums and going to concerts.

-Phyu Thwe, PhD, MLS(ASCP)CM is Technical Consultant/Technical Director of Clinical Microbiology Laboratory at Vidant Medical Center, Greenville, NC.

Breakpoint Breakdown

Working bacteriology benches in the clinical microbiology laboratory often comes with its fair share of questions about the susceptibility of patient isolates. In training, we are taught about breakpoints – clinically essential values which determine if an organism is susceptible, resistant, or somewhere in-between for a given drug. These values are readily accessible to us in guidance documents (e.g., CLSI M100, FDA website), and are programmed into instruments to allow for automated interpretation. But, have you ever wondered how these values are derived? Raw microbiological and pharmacological data, patient outcomes, and regulatory considerations must be examined through multiple lenses, and by many different entities, before these values ever make it to the printed page and used clinically. Here, we will highlight some of the “moving parts” of breakpoint determination an effort to demystify this process and gain a better understanding and appreciation of the clinical application of these life-saving measurements. To help, I’ve recruited two of our outstanding infectious disease pharmacists from UT Southwestern Medical Center to enhance this discussion. It’s my hope we will all learn something in the process!

Breakpoint Breakdown – The ABC’s of MICs and ECVs, plus Pharmacology 101!

A breakpoint represents a defined antibiotic concentration or zone of inhibition diameter that serves as a gatekeeper for antimicrobial use. This value categorizes organisms as susceptible, susceptible-dose dependent, intermediate, resistant, or nonsusceptible to various antimicrobials. As such, is an indispensable component of appropriate and effective antimicrobial prescribing.1 Breakpoints are set through a rigorous examination of data by various national and international organizations which we will discuss in a later post. Determining the optimal value at which a breakpoint should be set is multifactorial, requiring a multidisciplinary approach to incorporate data from bench and bedside. Now, if that introduction sounded daunting, don’t panic! We’re going start at the beginning with basic biological measurements of susceptibility that are needed to begin to establish a breakpoint. For those not currently working in microbiology (or if it’s been a while), a basic understanding of susceptibility testing mechanisms is necessary and we will briefly review here. Bacteria will be the focus for this discussion, but many of these concepts are also broadly applicable to fungi as well.

Determination of susceptibility to an antibiotic can be evaluated by examining the response of a bacterial isolate to antimicrobial exposure. In the laboratory, this is usually achieved through dilution, whereby an isolate may grow at some drug concentrations, and growth is inhibited at others. Dilution of the antimicrobial can come either through directly applying the antibiotic at defined concentrations uniformly to growth media (i.e. broth dilution, agar dilution), or utilizing a diffusion gradient through media when an antibiotic is applied at a single source (i.e. disk diffusion) (Image 1). Application of defined antibiotic concentrations to the growth media allows for a minimal inhibitory concentration (MIC) to be determined, while a diffusion gradient allows for a zone of inhibition to be measured (ZOI). An MIC is the lowest concentration of antimicrobial that inhibits organism growth of an isolate. This value is unique to the isolate that is being tested. However, when establishing a broad measurement which will encompass all isolates of that species (or group of species) such as a breakpoint, it’s easy to imagine that significantly more data is needed.

Image 1. Three methods of antimicrobial susceptibility testing. Kirby-Bauer disk diffusion generates a zone of inhibition – the diameter of that zone is measured and correlated with MIC data to establish breakpoints. Broth and agar dilution are two dilution methods which directly generate an MIC as an endpoint; the lowest concentration of antibiotic in which growth is inhibited. In the broth microdilution experiment, isolate 1 has an MIC of 4μg/mL. In the agar dilution experiment, isolate 2 has an MIC of 1μg/mL.

Thus, a first step in setting an optimal breakpoint begins with an antimicrobial’s in vitro activity against an organism. A descriptive summary of the MIC range across a given species helps define the MIC distribution. This analysis usually includes MICs from hundreds of tested isolates! This MIC distribution helps to define epidemiologic cutoff values (ECV or ECOFF). Like a breakpoint, these values separate the MIC distribution into bacterial populations that are either wild-type, and those with resistance. The wild-type MIC distribution aims to exclude outlier MICs that may represent organisms with acquired resistance (either through mutation or acquisition of resistance determinants) reflected by elevated MICs.  An isolate in the population with an MIC above the ECV is likely to have acquired resistance, whereas an isolate with an MIC lower than the ECV likely originates from the wild-type distribution and lacks mechanisms of acquired resistance or reduced susceptibility.2 So great, we have an experimental MIC value which separates wild-type organisms from ones that have acquired resistance, why not just stop there and call it a breakpoint? The answer is the ECV does not account for host responses, clinical outcomes, site of infection, pharmacokinetics/pharmacodynamics, dosing, and a number of other important variables which go into establishing a clinical breakpoint. Thus, using ECVs for clinical decision making is challenging.

Now that we have considered some aspects of the microbiological side of the equation, let’s switch gears and look at the host and other factors not addressed by an ECV. Pharmacokinetic (PK) and pharmacodynamic (PD) parameters are key to assessing the clinical applicability of a breakpoint. PK parameters represent how the body handles the antimicrobial, including absorption, distribution, metabolism and elimination, whereas PD parameters represent the effect between the drug and bug. Taken together, PK/PD parameters represent the relationship between drug concentration (PK) and antimicrobial effect (PD) over time. Different antimicrobials have distinct PK and PD characteristics, thus several PK/PD indices are utilized to determine optimal target concentrations or exposures that improve antibacterial efficacy. Common PK/PD indices are the percent of time the free drug concentration remains above an organism’s MIC (fT > MIC), the ratio of free max drug concentration (or peak) to MIC (fCmax/MIC), and the ratio of free drug exposure (area under the curve, AUC) over a 24-hour period to MIC (fAUC/MIC) (Image 2).3

Image 2. Common PK/PD Indices

For example, β-lactam antimicrobials require 40-60% fT > MIC for maximum antibacterial efficacy; however, the exact fT > MIC (or other exposure) required for optimal efficacy varies between different β-lactam antimicrobials and bacterial species. Importantly, the desired antimicrobial exposure should be obtainable based on the established breakpoint, meaning the β-lactam of interest should achieve 40-60% fT > breakpoint. Various strategies are employed to optimize PK/PD parameters relative to a given breakpoint (more on that to come in Part 2 of this series). These target exposures are calculated from data for each bug-drug combination.

Of note, the antimicrobial exposure in relation to MIC is often based on antimicrobial blood concentrations. However, this is obviously not always the site of infection. The same bug-drug combination can have multiple breakpoints specific to infection site. For example, central nervous system (CNS) infections may have lower established breakpoints compared with non-CNS infections. The use of a lower breakpoint improves PK/PD target attainment in areas where there may be low drug concentrations, such as the CNS. For CNS-specific breakpoints, only organisms with MICs within the lower range of the MIC distribution will be deemed susceptible. With a lower MIC, the PK/PD exposure may be more obtainable given the potential poor drug penetration to the site of infection.

Outcomes from clinical data are often deemed the most important factor used to determine breakpoints. Treatment successes or failures of an antimicrobial against a specific MIC may provide validity to an established breakpoint or support revision. For example, the piperacillin-tazobactam breakpoint for Pseudomonas aeruginosa, was lowered from 64/4 mcg/mL to 16/4 mcg/mL due to an increase in mortality observed in patients who had organisms with MICs 32 or 64 mcg/mL.4 Unfortunately, clinical outcome data is often influenced by other confounders beyond antimicrobial therapy and the organism’s MIC, such as source control and other therapeutic interventions. Furthermore, clinical data for “resistant” organisms may not be available, limiting the assessment of the antimicrobial against organisms with high MICs.

Finally, it is important to remember that breakpoints are not set in stone and change regularly as more data become available. Common reasons for breakpoint revisions include: 1) new PK/PD data suggesting the breakpoint is too low or high based on antimicrobial exposure, 2) identification of novel resistance mechanisms, and 3) new clinical data to suggest poor correlation of clinical response with the established breakpoint. Furthermore, microbiological methods may become more accurate and ultimately affect quantification of the MIC.5

In summary, establishing a breakpoint is not a straightforward process and requires an aggregate of information. We are really only scratching the surface of the very complex process here, but hope that this sheds some light on where these important values originate. Various types of data; microbiological, pharmacological, in vitro, in vivo, they all create the story. However, a complete picture is often not available at the time of breakpoint creation, resulting in the need to constantly review and update breakpoints as more data becomes available.

Next post we will discuss how breakpoints are used at the bedside.

References

  1. Clinical and Laboratory Standards Institute. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters, 5th Edition (M23).
  2. Turnidge J, Paterson DL. Setting and revising antibacterial susceptibility breakpoints. Clin Microbiol Rev. 2007 Jul;20(3):391-408.
  3. Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis. 1998 Jan;26(1):1-10; quiz 11-2. doi: 10.1086/516284.
  4. Tam V, et al. Outcomes of bacteremia due to Pseudomonas aeruginosa with reduced susceptibility to piperacillin-tazobactam: implications on the appropriateness of the resistance breakpoint. Clin Infect Dis. 2008 Mar 15;46(6):862-7.
  5. Humphries RM, Abbott A, Hindler JA. Understanding and Addressing CLSI Breakpoint Revisions: a Primer for Clinical Laboratories. J Clin Microbiol. 2019 May 24;57(6):e00203-19.

-Marguerite Monogue, PharmD is an infectious diseases pharmacy specialist and assistant professor at the University of Texas Southwestern Medical Center in Dallas, Texas. She is interested in antimicrobial pharmacokinetics/pharmacodynamics and multi-drug resistant Gram-negative bacteria.

-James Sanders, PharmD, PhD, is an infectious diseases pharmacy specialist and assistant professor at the University of Texas Southwestern Medical Center in Dallas, Texas. His academic and research interests are focused on multi-drug resistant Gram-negative bacteria, surgical site infections and HIV pharmacotherapy.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Microbiology Case Study: 83 Year Old Male with Bladder Cancer

Case History An 83 year old male with bladder cancer was treated with Mycobacterium bovis Bacillus Calmette-Guérin (BCG), his last treatment occurring 1.5 months prior to presentation. He has a past medical history of chronic obstructive pulmonary disease, hypertension, obstructive sleep apnea, obesity, and diabetes. The patient has been hospitalized four times over the last two months and his symptoms include generalized weakness, malaise, shortness of breath and recurrent fever. He was found to have patchy lung infiltrates and he was diagnosed with pneumonia, COPD exacerbation and symptoms of heart filature. He was treated previously with antibiotics, steroids and fluid management which would temporarily relieve his symptoms. He presents to the hospital again, four days after his last hospital discharge, with generalized weakness, malaise, shortness of breath and recurrent fever. On initial evaluation he was found to be pancytopenic.  

Laboratory Identification

Blood cultures were negative. A bone marrow biopsy was performed for fever of unknown origin and pancytopenia. The biopsy showed non-caseating granulomas which were negative for acid-fast bacilli (AFB) by Ziehl-Neelsen stain and fungal elements by Gomori Methenamine Silver Stain (GMS). A laboratory-develped PCR test for Mycobacterium tuberculosis complex (MTBC) was performed on the bone marrow and was negative. AFB culture of bone marrow was positive for after 30 days of incubation and the organism was confirmed to be acid-fast bacilli by auramine-rhodamine fluorescent dye and Kinyoun stain. A second laboratory-developed test that uses heat shock protein (HSP) 2 and HSP3 to determine species level identification of Mycobacteria identified the organism as M. tuberculosis complex. Due to the patient’s history, further identification was performed at a reference lab using specific oligonucleotides targeting the gyrb DNA sequence polymorphisms which is able to separate different members of the MTBC. The patient’s isolate contained a RD1 deletion which is specific for Mycobacterium bovis bacillus Calmette-Guérin (BCG).

Discussion

Mycobacterium bovis is a slow growing mycobacterium which produces rough, dry colonies on growth solid media. It is one of the species in the MTBC with a natural host of domestic and wild animals. Routine molecular tests will not accurately differentiate between members of the MTBC. For definitive identification of M. bovis, 16S rRNA and gyrB gene sequencing is necessary. Safe handling procedures should be followed prior to molecular testing of MTBC.

Mycobacterium bovis BCG is a live, attenuated strain of Mycobacterium bovis that was created for vaccine and is used in the treatment of superficial bladder cancer. The treatment may cause localized symptoms including hematuria, fever, nausea, and dysuria which are marker of anti-tumor effect. Serious complications occur in <5% of patients with complications including sepsis, pneumonitis, hepatitis, lymphocytic meningitis, bone marrow involvement, and mycotic aneurysms. The cardinal sign of BCG infection is a relapsing fever with drenching night sweats persisting beyond 48 hours. Disseminated infection can occur days to years after the therapy. Clinical suspicion should be high for M. bovis BCG dissemination if there are symptoms and a high grade fever ≥72 hours. Treatment includes a regiment of isoniazid, rifampin and ethambutol. Most isolates of M. bovis are resistant to pyrazinamide.

References

  1. Lamm DL. Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer. Clin Infect Dis 2000; 31 Suppl 3:S86.
  2. Shelley MD, Court JB, Kynaston H, et al. Intravesical Bacillus Calmette-Guerin in Ta and T1 Bladder Cancer. Cochrane Database Syst Rev 2000; :CD001986.
  3. Richter E, Weizenegger M, Rusch-Gerdes S, Niemann S. Evaluation of Genotype MTBC Assay for Differentiation of Clinical Mycobacterium tuberculosis Complex Isolates. Journal of Clinical Microbiology 2003; 41(6): 2672-2675
  4. UpToDate

-Crystal Bockoven, MD is a 4th year anatomic pathology resident at University of Chicago (NorthShore). Crystal has an interest in and will be doing a fellowship in pediatric and perinatal pathology. In her spare time, she enjoys reading, hiking and biking. 

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Microbiology Case Study: 35 Year Old Male with Chest Pain

Case History

A 35 year old man presented to the Emergency Department (ED) with intermittent chest pain for 3-4 days, abdominal pain, fatigue, and lightheadedness over the same time period. Additionally, his family reported symptoms of progressive malaise for about a month, worse over the last week. In the ED, he was found to have ST elevations in the inferior leads of the electrocardiogram, which can be indicative of a heart attack. He was given 325 mg of aspirin and was emergently taken to the catheterization lab. He was found to have multiple complete occlusions in the distal left anterior descending artery (LAD), posterior descending artery (PDA), and posterior left ventricular artery (PLV). He underwent aspiration thrombectomy and the resulting clots were thought to be emboli; segments were sent to pathology for histopathologic evaluation and to microbiology for culture. There was no evidence of underlying plaque. He was admitted for management of ST-elevation myocardial infarction (STEMI). While in the ED, he was found to have white blood cell count of 23,000 and tachycardia to 110 beats per minute. A transthoracic echocardiogram demonstrated thickened aortic valve leaflets with evidence of leaflet destruction, severe aortic insufficiency, and right coronary cusp perforation which are consistent with endocarditis. Blood cultures were obtained and he was started on broad spectrum antibiotics (Vancomycin and Cefepime).

He has a past medical history significant for previous shoulder abscess with Methicillin-resistant Staphylococcus aureus (MRSA) and intravenous drug use (IVDU) (heroin, last use ~6 days prior to admission).

Computed tomography (CT) of his abdomen and pelvis revealed multiple renal infarctions and a splenic infarction (Image 1). In addition, the CT of the brain showed: “Multifocal scattered supratentorial and infratentorial subarachnoid hemorrhages and findings suggestive of evolving ischemic infarct involving the right inferior frontal gyrus, without evidence of hemorrhagic transformation currently. No midline shift or other complication identified.”

Image 1. Computed tomography of the abdomen demonstrating multiple renal infarctions (left, circled) and a splenic infarction (right, circled).

On hospital day 1 (HD1), both sets of initial blood cultures turned positive with gram positive cocci (GPC) in clusters and thrombectomy cultures were also growing GPC in clusters (Image 2). On HD2, the GPC in the thrombectomy culture was identified as Rothia mucilaginosa. GPC growing in the blood cultures were also Rothia mucilaginosa (Image 2). The patient was continued on Vancomycin. Repeat blood cultures were obtained after catheterization on HD0, and HD2, which were negative. On HD2, the pathology of the initial clots showed “fibrinopurulent debris and fibrin plaques with innumerable cocci in clusters” (Image 3).

Image 2. Microscopic and culture morphology of Rothia mucilaginosa. Left: Gram stain from a blood culture demonstrating groups of Gram-positive cocci in small clusters (1000x magfication, oil immersion). Right: Blood agar plate with mucoid light pink-gray colonies.
Image 3. Hematoxylin and eosin stained slide of formalin fixed paraffin embedded tissue of the thrombus removed during the initial emergent catheterization procedure. Sections demonstrate fibrinous material with entrapped white cells and innumerable cocci. Top: 100x magnification; Bottom: 400x magnification.

On HD3, the patient developed 10/10 chest pain with troponin elevation and T-wave inversion. He was taken back to the catheterization lab for another procedure where he was found to have recurrent, complete occlusion of the PDA with unsuccessful recanalization due to the dense thrombus. On HD6, he developed tamponade physiology due to a large pericardial effusion that was drained. Cultures of the pericardial fluid were negative. Given the recurrent embolization events, the patient was transferred to another hospital to undergo aortic valve replacement surgery and coronary artery bypass graft surgery. Cultures taken at the time of the valve replacement surgery were negative and the valve tissue was not sent for pathologic evaluation.  

Discussion

We present an uncommon case of extensive Rothia mucilaginosa sepsis with septic emboli and endocarditis. Rothia mucilaginosa has experienced the scientific name-change game over the last several decades. It was first identified as Micrococcus mucilaginosus, then became Stomatococcus mucilaginosus, was also known as Staphylococcus salivarius before finally arriving to today’s name of Rothia mucilaginosa.1,2 R. mucilaginosa is a normal inhabitant of the oropharynx and is often associated with dental caries.3 R. mucilaginosa can cause invasive infections, typically in patients with compromised immune systems, disrupted mucosal barriers or injection drug use.4

R. mucilaginosa is a facultatively anaerobic, gram positive, non-fastidious coccus that is coagulase negative but with variable catalase positivity. Colony morphology is usually white to gray nonhemolytic colonies with a mucoid appearance. Although the variable catalase reaction may point toward a Streptococcus spp., the Gram stain morphology of clusters helps to rule it out. Although not all strains are mucoid, the classic colony morphology is wet and is due to polysaccharide capsule.

The organism is generally susceptible to antibiotics designed to target gram positive bacteria including, penicillin, ampicillin, cefotaxime, rifampin and vancomycin.4 It is important to note that R. mucilaginosa is not predictably susceptible to clindamycin, trimethoprim-sulfamethoxazole or ciprofloxacin.5 The patient presented in this case received intravenous vancomycin in part due to the extensive disease on presentation, but also because he was at risk for methicillin-resistant Staphylococcus aureus (MRSA) sepsis and had a previously documented abscess from MRSA.

References

  1. Bergan T, Kocur M. 1982. Stomatococcus mucilaginosus gen. nov., sp.nov., ep. Rev., a member of the family Micrococcaceae. Int. J. Syst. Bacteriol. 32:374-377
  2. Collins MD, Hutson RA, Baverud V, Falsen E. 2000. Characterization of a Rothia-like organism from a mouse: description of Rothia nasimurium sp.nov. and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb.nov. Int. J. Syst. Evol. Microbiol. 3:1247-1251.
  3. Trivedi MN, Malhotra P. Rothia prosthetic knee joint infection. 2015. J. Microbiol. Immunol. Infect. 48(4):453-455.
  4. Bruminhent J, Tokarczyk MJ, Jungkind D, DeSimone JA. Rothia mucilaginosa Prosthetic Device Infections: A Case of Prosthetic Valve Endocarditis. J. Clin. Microbiol. 5;15:1629-1632.
  5. Kaasch AJ, Saxler G, Seifert H. 2011. Septic arthritis due to Rothia mucilaginosa. Infection. 39:81-82.

-Doreen Palsgrove, MD is a board certified Anatomic and Clinical Pathologist who joined the faculty at UT Southwestern as an Assistant Professor in 2019. She specializes in head and neck and genitourinary pathology. 

Dominick Cavuoti, DO is a professor of AP and CP at UT Southwestern, specializing in infectious disease pathology, cytology and medical microbiology.

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

Microbiology Case Study: An 80 Year Old Man with Dyspnea, Fatigue, and Weight Loss

Case History

An 80 year old male was seen by his cardiologist for approximately one month of dyspnea, fatigue, and weight loss. Past medical history was significant for aortic stenosis requiring placement of a bioprosthetic valve and multivessel coronary artery disease 13 years prior. He underwent cardiac catheterization and echocardiography that revealed severe bioprosthetic valve stenosis. The patient was in the process of evaluation for a prosthetic valve replacement when he presented to the emergency room for rapid decline of the previously noted symptoms. Exam upon hospital admission was notable for cardiac murmur, lower extremity edema, mild leukocytosis, and anemia. He had normal dentition and no skin lesions. A pre-operative TEE confirmed severe aortic prosthetic valve stenosis, restricted leaflet motion, thrombus on all three leaflets, and thickening of the periannular aortic root and ascending aorta. Subsequent cardiac CT was concerning for either pseudoaneurysm or paravalvular leak suggestive of an infectious or inflammatory process.

Due to the persistent, mild leukocytosis, blood cultures were obtained on the second day of admission. On hospital day 3, one set of blood cultures flagged positive with Gram-variable rods in the aerobic bottle (Image 1). The patient was empirically started on vancomycin and piperacillin/tazobactam. Repeat blood cultures were obtained on hospital days 4 and 7, both again positive for Gram-variable rods within 2 days of collection. The infectious diseases consult team suspected subacute bacterial endocarditis and changed therapy to ceftriaxone.On hospital day 9, the patient underwent a redo sternotomy for aortic valve replacement and aortic root repair. Intraoperative findings included a large amount of phlegmon on the aortic leaflets, near circumferential aortic annulus tissue destruction and abscess cavity. Culture of the intraoperative specimens was negative for bacterial growth. The anatomic pathology findings revealed fibrinoid vegetations and acute inflammation and reparative changes. The patient was subsequently discharged home in stable condition 20 days after his admission. Interval outpatient clinic visits demonstrate that he is recovering well, including a return to baseline levels of endurance and function.

Laboratory Identification

Gram stain of the positive blood cultures revealed pleomorphic gram variable rods which were arranged in clusters, pairs, short chains, and characteristic rosette patterns (Image 1 and inset). Pinpoint, opaque colonies were visible on blood and chocolate agars after 48-72 hours of incubation at 35°C in CO2 (Image 2). No growth was observed on MacConkey agar. The colonies were catalase-negative, and oxidase- and indole-positive. The recovered organism was definitively identified by MALDI-TOF MS as Cardiobacterium hominis.

Image 1. Gram stain from the positive aerobic blood culture bottles exhibiting gram variable rods (1000X magnification, oil immersion). Organisms were visualized in characteristic “rosette” patterns. Image inset is a magnified view of the rosette arrangement from another field.
Image 2. Growth on blood agar following 48 hours incubation at 35°C in 5% CO2. Small, white, pinpoint colonies were observed on blood and chocolate agars.

Discussion

In 1962, four cases of infective endocarditis (IE) due to a Pasteurella-like organism belonging to CDC Group-IID were reported. Two years later, this group of organisms was reclassified as Cardiobacterium in recognition of their ability to cause endocarditis. Two species, Cardiobacterium hominis and Cardiobacterium valvarum, have been reported to cause IE, with the former being the etiological agent in a vast majority of cases.1 There is a strong association between C. hominis bacteremia and IE, as the organism is rarely recovered from blood cultures outside of this setting. Most cases of C. hominis endocarditis involve the aortic valve, particularly in the presence of pre-existing abnormalities or when a prosthetic valve is in place.2 C. hominis is a member of the normal flora of the nose and throat of ~70% of individuals (1), and endocarditis can be caused by periodontitis or dental procedures without prophylaxis.3

C. hominis is a member of the HACEK group of organisms which also include Haemophilus spp., Aggregatibacter spp., Eikenella corrodens, and Kingella kingae. HACEK organisms exhibit similar manifestations of disease, prognosis, and epidemiology. While over 80% of cases of IE are caused by Gram-positive bacteria (notably staphylococci and oral streptococci), Gram-negative IE is far less frequent, with a majority of cases caused by HACEK organisms (1-3% of all IE cases).4 In general, IE caused by HACEK organisms has an excellent prognosis, but delays in diagnosis and associated complications can lead to poorer outcomes.2 Susceptibility testing of C. hominis is difficult to perform due to its nutritional requirements. Most strains are susceptible to fluoroquinolones, rifampin, tetracycline, and beta-lactams. As beta-lactamase producing isolates have been reported, the current American Heart Association Guidelines recommend the use of a 4-6 week course of ceftriaxone for treatment of HACEK IE; fluoroquinolones may be used in cases where patients cannot tolerate cephalosporin therapy.5

Historically, prolonged blood culture incubation for the recovery of HACEK group organisms has been recommended due to their fastidious nature and slow growth rate. However, modern automated blood culture systems utilize enriched media which readily support their growth and facilitate recovery within a standard 5-day incubation period (average of 3.4 days incubation).6 Additional studies have demonstrated that prolonged incubation times do not significantly enhance the recovery of HACEK organisms and are of little clinical value.7 This case demonstrates many hallmarks of a characteristic description of a HACEK bacterial endocarditis: 1) the patient had a prosthetic valve as a pre-existing risk factor, 2) the subacute presentation caused a delay in recognition of an infectious etiology as contributing to his clinical decline, 3) C. hominis grew in less than 5 days in our automated blood culture system without prolonged incubation, 4) blood culture Gram stain findings were consistent with the MALDI identification of a HACEK group member, and 5) the patient was treated with ceftriaxone and with surgical intervention and has recovered successfully.

References

  1. Malani AN, Aronoff DM, Bradley SF, Kauffman CA.2006. Cardiobacterium hominis endocarditis: two cases and a review of the literature. European Journal of Clinical Microbiology and Infectious Diseases 25:587-595.
  2. Sharara SL, Tayyar R, Kanafani ZA, Kanj SS.2016. HACEK endocarditis: a review. Expert Review of Anti-infective Therapy 14:539-545.
  3. Steinberg JP, Burd EM. 2015. 238 – Other Gram-Negative and Gram-Variable Bacilli, p 2667-2683.e4. In Bennett JE, Dolin R, Blaser MJ (ed), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (Eighth Edition) doi:https://doi.org/10.1016/B978-1-4557-4801-3.00238-1. Elsevier, Philadelphia, PA.
  4. Revest M, Egmann G, Cattoir V, Tattevin P.2016. HACEK endocarditis: state-of-the-art. Expert Review of Anti-infective Therapy 14:523-530.
  5. Baddour Larry M, Wilson Walter R, Bayer Arnold S, Fowler Vance G, Tleyjeh Imad M, Rybak Michael J, Barsic B, Lockhart Peter B, Gewitz Michael H, Levison Matthew E, Bolger Ann F, Steckelberg James M, Baltimore Robert S, Fink Anne M, O’Gara P, Taubert Kathryn A.2015. Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications. Circulation 132:1435-1486.
  6. Petti CA, Bhally HS, Weinstein MP, Joho K, Wakefield T, Reller LB, Carroll KC.2006. Utility of extended blood culture incubation for isolation of Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella organisms: a retrospective multicenter evaluation. Journal of clinical microbiology 44:257-259.
  7. Weinstein MP.2005. Emerging Data Indicating that Extended Incubation of Blood Cultures Has Little Clinical Value. Clinical Infectious Diseases 41:1681-1682.

-Francesca Lee, MD, is an associate professor in the Departments of Pathology and Internal Medicine (Infectious Diseases) at UT Southwestern Medical Center. She serves as Medical Director of the microbiology laboratory and pre-analytical services at Clements University Hospital.

-Julia Sweetnam, MLS(ASCP)CM has worked for six years as medical technologist in the microbiology laboratory at Clements University Hospital. She is interested in antimicrobial susceptibility testing and diagnostic bacteriology.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Microbiology Case Study: 40 Year Old Male with A Diabetic Foot Ulcer

Clinical Presentation and History

The patient is a 40 year old male with a past medical history of type 2 diabetes mellitus with significant neuropathy and hypertension with a past surgical history of right metatarsal osteomyelitis. He presents to hospital with fever, right ear pain, headache, two episodes of diarrhea and redness and blistering to the right 3rd metatarsal. Upon examination he was noted to have a 1 cm ulceration on the right 3rd toe on the dorsal aspect associated with redness and edema. He was therefore assessed as having diabetic foot ulcer with possible osteomyelitis for which blood cultures were performed.

Laboratory Identification

Gram stains performed on the positive blood culture broth showed gram negative rods (Image 1). In our institution initial positive blood cultures are tested by the Verigene System (Luminex Corp., Austin, TX), which allows for rapid identification of common bacterial pathogens causing blood stream infections (Escherichia coli, Klebsiella oxytoca, Klebsiella pneumonia, Pseudomonas aeruginosa, Acinetobacter spp., Citrobacter spp., Enterobacter spp., and Proteus spp.) along with detection of several resistance genes (CTX-M, IMP, KPC, NDM, OXA, VIM) within ~ 3 hours. In this case, no targets on the Verigene panel were detected. Simultaneously, the specimen was plated onto blood, chocolate and MacConkey agars where the organism grew robustly on all three plates (Image 2). The MacConkey agar showed the organism to be a non-lactose fermenter. Once the organism adequately grew on these agar plates, final species identification was performed on the automated MALDI-TOF instrument which showed Salmonella species. To appropriately type the organism, Salmonella latex agglutination testing was performed which identified Salmonella species Group B (Non-typhoidal). Of note, multiple blood cultures from this patient were positive for Salmonella species, Group B.

Image 1. Gram stain of blood culture broth containing gram negative rods.
Image 2. Growth of the organism on chocolate, 5% sheep blood, and MacConkey agars.

Discussion

Salmonella is a gram negative, flagellated facultative anaerobic, non-lactose fermenting bacilli. The taxonomy and nomenclature of salmonella organisms are quite complex however the most widely used classification scheme is the Kauffman-White which is updated yearly by the WHO. Currently, members of the 7 Salmonella subspecies can be serotyped into one of more than 2500 serotypes (serovars) according to antigenically diverse surface structures: somatic O antigens (the carbohydrate component of lipopolysaccharide [LPS]) and flagellar (H) antigens.

Nontyphoidal salmonellae are a major cause of diarrhea worldwide. In the United States, non-typhoidal salmonellosis is one of the leading causes of foodborne disease. Salmonella enteritidis and Salmonella typhimurium are among the most frequently isolated organisms. Salmonella is most commonly associated with ingestion of contaminated poultry, eggs, and milk products. Salmonella gastroenteritis typically occur within 8 to 72 hours following exposure, however lower bacterial doses can prolong the incubation period. Although Salmonella typically causes diarrheal diseases including gastroenteritis and enteric fever, however there are rare instances where hematogenous involvement leads to bacteremia, osteomyelitis or endovascular infections.

In this case the source of Salmonella-related bacteremia is still a mystery. The presumed source was osteomyelitis, but the patient’s subsequent toe amputation revealed minimal osteomyelitis and rare fungal organisms.

References

  1. Procop, Gary W. et al (2017). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 7th edition. Philadelphia, PA.
  2. Hohmann, Elizabeth L. (2018). Nontyphoidal salmonella: Gastrointestinal infection and carriage. Uptodate.com. Retrieved on November 14, 2019. https://www-uptodate.com/contents/nontyphoidal-salmonella-gastrointestinal-infection-and-carriage

-Anna-Lee Clarke-Brodber, MD is a 3rd year AP/CP resident at University of Chicago (NorthShore). Academically, Anna-Lee has a particular interest in Cytopathology. In her spare time she enjoys hanging out with her family.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Microbiology Case Study: A 24 year old with Sore Throat and Difficulty Breathing

Case History

A 24 year old male with a past medical history of recurrent streptococcal pharyngitis presents to the emergency department with a sore throat and dyspnea. His symptoms began three days prior and included left-sided upper neck and lower jaw pain and odynophagia. The patient’s evaluation demonstrated tachycardia, cervical lymphadenopathy, and a small left tonsillar abscess. Labs were significant for an elevated WBC count but blood cultures, Group A streptococcal and mononucleosis screens were negative. The patient was admitted for pain management and treated with a combination of IV ampicillin/sulbactam (amp/sulb) and steroids. He improved with treatment and was discharged the following day on oral amoxicillin/clavulanic acid (amox/clav). Nine days later, the patient re-presented with similar complaints. The tonsillar abscess had increased in size to 2cm. Labs were significant for leukocytosis and a now positive Group A streptococcal screen. 2mL of pus was aspirated from the lesion but no cultures were ordered. The patient’s status again improved, and he was discharged home again on oral amox/clav. The patient returned the following day and was placed on IV amp/sulb and admitted for imaging and symptom management. A neck CT with contrast revealed a now 3cm tonsillar abscess with reactive cervical lymphadenopathy (Image 1). A throat culture was collected; however, no beta-hemolytic streptococci were recovered after 48 hours of incubation. Incision and drainage of the abscess was performed at bedside, recovering an additional 10 mL of purulence that was sent to the microbiology laboratory for aerobic and anaerobic culture. The patient improved on IV amp/sulb and was switched to high dose amox/clav on day 15.  

Laboratory Identification

Gram stain of the aspirated purulence revealed many WBCs and a mixture of gram positive rods and cocci (Image 2). The aerobic culture grew a heavy amount of tiny, weakly beta-hemolytic colonies on blood agar. Smears of these colonies revealed Gram-positive coryneform rods. Biochemical testing determined the growth to be catalase-negative and MALDI-TOF MS definitively identified the organism as Arcanobacterium haemolyticum. The anaerobic culture grew oral flora.

Image 1. Computed tomography of the neck in a 24 year old male who presents with difficulty breathing. Area of large tonsillar abscess (yellow circle).
Image 2. Gram stain demonstrating small, pleomorphic gram positive rods in a background of neutrophils and Gram-positive cocci in pairs or short chains. (1000x magnification, oil immersion)
Image 3. A. haemolyticum isolate after 48 hours of incubation. The weak beta-hemolysis was not readily apparent using room (reflected) light. Placing the plate on a lightbox revealed beta-hemolysis.
Image 4. Streptococcus agalactiae exhibiting synergetic hemolysis with a beta-lysin producing strain of S. aureus (CAMP reaction, top). A. haemolyticum inhibits hemolysis by S. aureus in a CAMP-test set up (CAMP inhibition, middle). A. haemolyticum exhibits synergistic hemolysis with S. agalactiae. (Reverse CAMP, bottom).

Discussion

A. haemolyticum is an infrequently isolated  gram positive rod which is an etiologic agent of non-streptococcal pharyngitis diagnosed predominantly in adolescents or young adults. The diagnosis of A. haemolyticum can be challenging because itis often clinically indistinguishable from cases caused by beta-hemolytic streptococci. Most patients exhibit some degree of cervical lymphadenopathy, and a scarlatiniform rash can be present in up to 50% of cases. From a laboratory perspective, A. haemolyticum is slowly growing and weakly beta hemolytic after 24-48 hours on media containing sheep blood (including SBA and Strep Selective agars routinely used for screening throat cultures). The beta-hemolytic activity of A. haemoltyicum is attributed to expression of arcanolysin, a cholesterol-dependent cytolysin. Interestingly, arcanolysin more robustly binds to rabbit and human erythrocytes than those from sheep,1 which may explain the organism’s weak beta hemolysis on routine media.  In this setting, the organism can be missed or dismissed as commensal flora without careful observation. Conversely, if beta-hemolysis is observed, the colony morphology and catalase non-reactivity can lead to misidentification as beta-hemolytic streptococci in the absence of a Gram stain or other determinative methods (i.e. MALDI-TOF MS).

The beta hemolysis of this patient’s A. haemolyticum isolate is difficult to appreciate in reflected (room) light, and was best observed after 48 hours using transduced light from a light box (Image 3). A. haemolyticum displays CAMP inhibition due to the production of phospholipase D which inhibits the hemolytic activity of beta-lysin produced by S. aureus (Image 4) and is reverse-CAMP positive when perpendicular to Group B streptococci which can aid in identification.2

Erythromycin is the drug of choice for treatment of A. haemolyticum, further highlighting the need for definitive identification of this organism in settings of pharyngitis. The use of penicillin for treatment of A. haemolyticum pharyngitis can result in treatment failure, possibly due to invasion of host cells, thus establishing a reservoir,3 or due to a penicillin-tolerant phenotype.4 It is unclear in this case if source control or decreased susceptibility necessitated the multiple courses of antibiotics utilized. Fortunately, the patient’s symptoms resolved on high dose amoxicillin/clavulanic acid following thorough incision and drainage. He subsequently returned for an outpatient tonsillectomy.

References

  1. Jost BH, Lucas EA, Billington SJ, Ratner AJ, McGee DJ. 2011. Arcanolysin is a cholesterol-dependent cytolysin of the human pathogen Arcanobacterium haemolyticum. BMC Microbiology 11:239.
  2. Kang H, Park G, Kim H, Chang K. 2016. Haemolytic differential identification of Arcanobacterium haemolyticum isolated from a patient with diabetic foot ulcers. JMM Case Reports.
  3. Österlund A. 1995. Are Penicillin Treatment Failures in Arcanobacterium haemolyticum Pharyngotonsillitis Caused by Intracellularly Residing Bacteria? Scandinavian Journal of Infectious Diseases 27:131-134.
  4. Nyman M, Danek G, Thore M. 1990. Penicillin Tolerance in Arcanobacterium haemolyticum. The Journal of Infectious Diseases 161:261-265.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern in the Department of Pathology and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

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