Microbiology Case Study: A 24 Year Old Male with Painful Pustules

Case History and Laboratory Findings

A 24 year old male presented to an urgent care clinic with the complaint of painful pustules on his scrotum for the past 4-5 days. He claims that he had not had any recent exposure to sexually transmitted diseases (STD) or to new sexual partners. A month prior to presentation, he was seen for STD evaluation and was treated empirically for suspected exposure.  Testing at that time was negative.  The patient was unsure if these pustules were exacerbated by his work, in which he is in tight, hot spaces doing manual labor. His review of systems was otherwise negative. A swab of one of the pustules was sent for gram stain and culture which showed the following:

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Image 1. Gram stain from a swab illustrating small intracellular Gram negative diplococci (100x, oil immersion).
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Image 2. Chocolate agar showing small to medium, flat, grey-brown, and moist colonies.

MALDI-TOF identified the organism to be Neisseria gonorrhoeae.

Discussion

N. gonorrhoeae is a gram negative diplococci and the second most common cause of sexually transmitted infections in the United States. The diplococci are described as having adjacent flattened sides giving it the appearance of the letter “D”. N. gonorrhoeae is classically described as a fastidious organism, requiring specialized media (Chocolate, modified Thayer-Martin, Martin-Lewis, New York City agar, etc.) and an enhanced CO2 environment in order to grow, though it is also known to grow on blood agar. Colonies are small to medium in size and are described as flat, grey-brown, and moist. Biochemically, N. gonorrhoeae is catalase and oxidase positive, and is a glucose fermenter.

Neisseria gonorrhoeae can infect the epithelium of the urethra, cervix, pharynx, rectum, and conjunctiva. Infection at these sites results in pain, irritation, and purulent discharge. Dissemination to other locations, such as the skin and joints, can also occur. Though dissemination is uncommon, associated symptoms include: skin sores, fever, migratory polyarthritis, tenosynovitis, and pauciarticular septic arthritis.  N. gonorrhoeae infections typically present as acute urethritis with associated discharge.  Infections are symptomatic in 10% of males and upwards of 70% of females, putting females at a higher risk of developing ascending infections, and potentially, pelvic inflammatory disease. Disseminated infections occur much less commonly, happening in 0.5% to 3% of all gonococcal infections. Therapy guidelines recommend treating uncomplicated infections with intramuscular ceftriaxone and oral azithromycin. Treatment of disseminated infections is variable depending on patient symptoms, but can include a combination of ceftriaxone/cefotaxime and azithromycin/doxycycline, with variable route of administration and length of treatment times. Susceptibility testing is limited to testing for beta-lactamase activity, though in cases of suspected resistance, CLSI guidelines are available for further testing.

References

  1. McCormack WM, Stumacher RJ, Johnson K, Donner A. Clinical spectrum of gonococcal infection in women. Lancet 1977; 1:1182.
  2. Workowski KA, Bolan GA, Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep 2015; 64:1.
  3. Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance, 2015. Atlanta, GA: US Department of Health and Human Services; October 2016.

 

-Clayton LaValley, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-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.

Is Your Clinician Concerned About Biotin Interference?

Many clinical laboratorians received questions in the past few months from clinicians about biotin interference on laboratory tests. Although biotin interference is not something new to most clinical chemists, it became more of a concern for clinicians since FDA released a safety communication warning to the public and healthcare professionals that “Biotin May Interfere with Lab Tests” in Nov 2017.

Why does biotin interfere with some laboratory tests?

Immunoassays employed in clinical laboratories often use biotin-streptavidin linkage to separate bound antibody-antigen complex from unbound components.  For example, in a sandwich immunoassay setting, analytes bind to signal antibodies and biotinylated capture antibodies, which are immobilized on streptavidin-coated solid phase via biotin-streptavidin binding. In the excess of exogenous biotin, it interferes the binding of biotinylated antibodies and streptavidin, causing erroneous results.

Owing to assay design, tests that utilize the biotin–streptavidin linkage have different tolerance on biotin interference. There has been recent publications that summarized the tolerance level of biotin on commonly used immunoassays from different manufacturer platforms (1, 2). The recommended daily intake (RDI, 30 µg/day) of biotin do not typically interferes with laboratory testing. However, many over-the-counter dietary supplements may contain biotin much higher than the RDI, and the level used for treatment of multiple sclerosis or some other diseases can be even higher. These levels of biotin can cause either falsely high or falsely low test results.

As high-dose biotin use has been increased among general population for nutraceutical purposes, it requires clinicians’ awareness of biotin interference and communication with laboratories to identify incorrect laboratory results. It may require patients to discontinue dietary supplements containing high dose biotin for a period of time before blood drawn to minimize potential biotin interference with testing.

References

  1. Li D, Radulescu A, Shrestha R, Root M, Karger A, Killeen A, et al. Association of Biotin Ingestion With Performance of Hormone and Nonhormone Assays in Healthy Adults. Jama. 2017;318:1150–1160.
  2. Colon P, Greene D. Biotin Interference in Clinical Immunoassays. J Appl Laboratory Medicine Aacc Publ. 2018;2:941–951.

 

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-Xin Yi, PhD, DABCC, FACB, is a board-certified clinical chemist, currently serving as the Co-director of Clinical Chemistry at Houston Methodist Hospital in Houston, TX and an Assistant Professor of Clinical Pathology and Laboratory Medicine at Weill Cornell Medical College.

Lab Safety for Every Member of the Team

Gretchen had been the lab secretary for six months, and she was getting comfortable in the role. From her office she scheduled meetings for the manager and paid bills, but her job took her into the lab proper at least once every day. She liked that her job allowed her to wear skirts and sandals in the hot summer months-she was glad she didn’t have to follow the dress code that was used in the lab.

Stephan was new to the lab courier team, and his training had to occur quickly since he replaced someone who filled weekend slots often. He was shown the routes to drive, but when trained in the lab area, he was only shown where to pick up and drop off specimens.

Dr. Kane had been the lab medical director for many years. One day she was talking to the histology tech and noticed the use of pictogram labels on secondary chemical containers. She had no idea what they were for, and she asked how long the lab had been using them.

Unfortunately, these scenarios are realistic, and they illustrate a problem that can create deep roots in a laboratory, and those roots can lead to a poor safety culture that will be difficult to manage. If you’re in charge of safety in the lab, it is vital to know who needs safety training, how to give that training, and when to provide it.

The who is important. Does your lab host students for clinical rotations? Do research personnel perform tasks in the department? Administrative personnel and even lab leaders who enter the department should also have safety training on record. Don’t expect pathologists to keep up with the latest safety regulations on their own either, they have many other things on their plates. Even if they are under contract and not truly employees, they should be included with certain safety training offerings. Consider biomedical engineering personnel and maintenance workers- some safety training can prevent accidents and exposures for those important team members as well. Fully train couriers and phlebotomists or anyone else who will process specimens in the lab setting. If you’re just starting to figure out safety training in your lab, make a list of all the different people who may enter the area.

Clearly all of these various people will not need the same level of lab safety training. A courier might need to know about dry ice safety, for example, but that information may mean nothing to the secretary. Be sure to customize the training for the different employees as needed. Nothing will turn people off faster than information they don’t need. If there are changes to safety regulations that require new education, be sure to involve laboratory medical staff. For example, the implementation of the Globally Harmonized System in 2016 or this year’s EPA Generator Improvement Regulations both created major changes with lab safety processes. The lab medical director is responsible for oversight of the lab, and not having knowledge about such major changes can hinder that responsibility and expose the lab to both safety and accreditation issues.

Now that you know who to train and what education is needed for each role, it is time to figure out when and how to provide that lab safety training. Some topics require annual training by OSHA and other agencies, and a computer-based module is usually acceptable. That said, other required training must include live interaction, quizzes, return demonstrations and certificates of completion. It can be a complicated task to figure out which is which, and reviewing the requirements from the source agency (OSHA, DOT, EPA, CAP, etc.) will guide you. Next, it becomes important to know your audience- those who will receive the training. What type of education will work best- a live class, computer modules, webinar, interactive round-table sessions- there may be a need for a combination of these styles.

Once you determine your safety training needs and methods, there will be more to consider in order to maintain a steady culture of safety. Conducting regular drills to ensure staff understanding should be added to your calendar. Fire drills, evacuation drills, disaster drills, and hazardous spill drills are just some that can be conducted throughout the year to ensure staff readiness. Consider giving out information on a specific safety topic each month at staff meetings. This reinforcement of the required training will benefit the entire team and the lab safety program.

It takes time and effort to create a solid laboratory safety training program. If you have to start at the beginning, learn your resources and ask for help. If you are taking over a safety program already in place, make sure the on-going training meets regulations, and create a plan to continually raise safety awareness in the laboratory for all whose job may take them into the department. That will create long-lasting value and safety for every member of the team.

 

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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.

Microbiology Case Study: A 56 Year Old Man with Bacteremia

Case History

A 56 year old man with a history of hypertension, asthma and COPD, bladder stones, congenital bladder abnormality, and bladder exstrophy presented to the ED with fatigue, generalized weakness, poor appetite, fever, and nausea. He had intermittent right flank pain and generalized abdominal pain. The patient also reported he had passed bladder stones in the past year.

In the ED he had an elevated peripheral white blood cell count of 12.9 x103/μl with a differential of 81% neutrophils, 11% lymphocytes, and 6% monocytes. His creatinine was elevated at 5.4 mg/dl. CT imaging of the urogenital tract showed hydronephrosis with evidence of infection.

Blood and urine specimens were sent for culture. The urine culture grew 10-50,000 cfu/ml Aerococcus urinae. After 30 hours of incubation, the anaerobic blood culture was positive for small, coccoid-looking gram positive rods (Image 1). The following bacteria grew on solid media under anaerobic conditions after 48 hours of incubation (Image 2).

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Image 1. Gram stain showing small, coccoid-looking Gram-positive rods
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Image 2. Tiny colonies growing on non-selective anaerobic media after 48 hours.

Discussion

The organism growing in the blood culture was identified as Actinotignum schaalii. A. schaalii is a facultative aerobe that prefers enriched media and grows best in anaerobic or 5% CO2 environments. It is a non-hemolytic, non-motile, and non-spore forming Gram-positive rod. A. schaalii is negative for catalase, oxidase, CAMP factor, and nitrate reduction, but is hippurate positive. It is also negative for lactose, mannitol, raffinose, sorbitol, and trehalose acid production.

Actinotignum schaalii was formerly named Actinobaculum schaalii. The Actinotignum genus now consists of A. urinale, A. sanguinis, and A. schaalii. A. schaalii is part of urogenital microbiota in healthy adults and can cause urinary tract infections, especially as it thrives in the humid atmosphere of diapered children or elderly adults. Isolation of this organism is associated with urinary incontinence, bladder cancer, catherization, neurogenic bladder, renal failure, prostate cancer, and chronic renal failure. The presence of A. schaalii in the urogenital tract can progress to bacteremia and rarely it can also cause groin abscesses.

Actinotignum schaalii is an under recognized uropathogen due to its fastidious nature—it is slowly growing and prefers enriched media grown under anaerobic conditions. None of these conditions are met as part of a routine urine culture as urine cultures are often finaled in < 48 hours, they may not use enriched media, and they are incubated under aerobic rather than anaerobic conditions. Because of these factors, A. schaalii rarely grows in urine culture despite it being a known colonizer of the urogenital tract and occasional uropathogen. Instead it is most commonly recovered from blood cultures as a result of urosepsis. When colonies do grow from blood culture, they are very difficult to identify by biochemical methods. A. schaalii is often misidentified by biochemical panels as Arcanobacterium spp. or Gardnerella vaginalis (API Coryne system, bioMerieux), Actinomyces meyeri (Rapid ID32A system, bioMerieux), or Actinomyces israelii (Rapid ANA II system, Remel). In our lab Bruker Biotyper MALDI-TOF MS was able to identify A. schaalii with high confidence. Based on the most current literature, Vitek MS misidentifies A. schaalii as G. vaginalis or A. meyeri. This data is from 2015, so it’s possible the Vitek MS spectra database has been updated and now identifies A. schaalii.

A. schaalii is routinely susceptible to all β-lactams but requires an extended duration of antimicrobial treatment compared to other uropathogens. It is resistant to trimethoprim/sulfamethoxazole and quinolones, both of which are commonly prescribed for urinary tract infection. Therefore, patients with A. schaalii are at risk for recurrent urinary tract infections due to inappropriate or inadequate antibiotic treatment.

Our patient was prescribed ciprofloxacin for presumed urinary tract infection prior to his bacteremia. He was switched to ceftriaxone upon hospital admission. His bacteremia was cleared and the patient was discharged 5 days later on an oral β-lactam antibiotic. 

References

  1. Lotte, R., L. Lotte, and R. Ruimy. “Actinotignum schaalii (formerly Actinobaculum schaalii): a newly recognized pathogen—review of the literature.” Clinical Microbiology and Infection1 (2016): 28-36. Le
  2. Le Brun, Cécile, et al. “Urinary tract infection caused by Actinobaculum schaalii: a urosepsis pathogen that should not be underestimated.” JMM Case Reports2 (2015).
  3. Manual of Clinical Microbiology, 11th edition

 

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

Essential Diagnostics List – Ready, Set, Go!

In a recent post I introduced the WHO’s draft of the Essential Diagnostics List (the EDL). The EDL is a catalog of in vitro diagnostics (IVD) designed to complement the Essential Medicines List. The EDL is not necessary meant to be a global list, but something to be adopted and adapted by each country, and tests added, subtracted, or prioritized based on each country’s disease burden. While the draft is still in development, at least one country is already on track to have adopted a country specific EDL by the end of the year!

India is working towards being the first country to have an EDL by the end of 2018. It so happens that the draft of the EDL was announced while India was in the process of rolling out a Free Diagnostics Initiative (FDI) in 29 of its states. The goal of the FDI is to “ensure the availability of a minimum set of diagnostics appropriate to the level of care to reduce out of pocket expenditure on diagnostics and to encourage appropriate treatment of based on accurate diagnosis”. Similar to the EDL, the FDI plans for different IVDs at different levels of laboratories, from community healthcare centers to reference laboratories. The development of an EDL seems like a natural product of India’s FDI. Talk about perfect timing!

The Indian Council on Medical Research (ICMR), comprised of clinicians, microbiologists, and medical device industry leaders, has convened to adapt the EDL to India’s infection patterns and diseases. They plan to have their national EDL ready to present by the beginning of 2019. The ICMR intends that an Indian EDL will optimize utilization of the Indian EML. “The objective is to test and treat rather than treat and test” states Dr. Kamina Walia of the ICMR. The ICMR also realizes that in order for diagnostics to be affordable, the country’s laboratory infrastructure will need to be strengthened, including building laboratory capacity where none currently exists.

It is so exciting to see the EDL already under consideration by a nation. It’s even more exciting to hear medical experts speak about how laboratory infrastructure should be strengthened, and to know that medical device industry leaders are coming to the table. It’s going to be fun to watch this develop over the next decade.

Do you want to be involved in the EDL project? There is time! The WHO is accepting applications for IVDs to be added to the second edition of the EDL, which will be released in 2019. The deadline for submissions is September 15, 2019. Instructions can be found here.

 

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Sarah Riley, PhD, DABCC, is an Assistant Professor of Pediatrics and Pathology and Immunology at Washington University in St. Louis School of Medicine. She is passionate about bringing the lab out of the basement and into the forefront of global health.  

Microbiology Case Study: A 62 Year Old Male with Productive Cough

Case History

62 year old male from Louisiana with a medical history of COPD presents with fever, productive cough, weight loss, and a red nodule on the left hand. Chest x-ray shows interstitial and lobar infiltrates. Patient reports no recent travel history. Mycobacterial culture of the sputum is positive for an organism. MALDI-ToF of the sputum culture confirms the result.

Lab Identification

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Image 1. Kinyoun stained long bacilli with a banded, ladder like pattern isolated from sputum culture.
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Image 2. Bright lemon yellow colonies growing on LJ slant after exposure to light on the right compared with unpigmented control colonies on the left.
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Image 3. Bright yellow colonies growing on 7H11 media after exposure to light on the right compared with unpigmented control colonies on the left.

Mycobacterium kansasii grows on 7H11 media and Lowenstein-Jensen (LJ) slants. The colonies appear smooth or rough and unpigmented when isolated in the dark. Upon exposure to light, colonies turn bright lemon yellow due to enhanced b-carotene production, which makes M. kansasii a photochromogen. M. kansasii stains positive with acid-fast stains. On Kinyoun stain, it shows long bacilli with a banded, ladder like pattern. M. kansasii is positive for nitrate reduction, catalase, urease, and tween hydrolysis. It is negative for niacin and pyrazinamindase. The identification of M. kansasii is confirmed by MALDI or DNA probe of an isolate.

Discussion

Mycobacterium kansasii is a slow growing photochromogen, an organism that grows unpigmented colonies in the dark but produces a bright lemon yellow pigment upon exposed to light3. It is also an acid-fast positive long bacillus that causes TB-like chronic pneumonia, which is the second most common non-TB mycobacterial infection after MAC in the AIDS population. There are five genotypes of Mycobacterium kansasii. Genotypes I and II infect humans, with I being the most prevalent. Because environmental sources of M. kansasii are rarely identified, isolation of M. kansasii from a culture is never considered a contaminant.

M. kansasii is usually found in the tap water in cities endemic for the infection. In the U.S., it is most prevalent in the central and southern states including Louisiana, Illinois, Texas, and Florida1. Internationally, it is most prevalent in Israel, Korea, France, Japan, Portugal, with the highest incidence in England, Wales, and among South American gold miners.

The majority of patients with M. kansasii infection have an underlying pulmonary disease such as COPD, bronchiectasis, or TB infection3. The clinical manifestation of M. kansasii infection includes a primarily unilateral cavitary infiltrate in the lungs without pleural effusions. Patients are generally older compared with those infected with TB. They present with productive cough, weight loss, fever, night sweats, and dyspnea. Symptoms are usually less severe but more chronic compared with those of TB pneumonia. M. kansasii can also present as cutaneous lesions similar to sporothrichosis. Lesions include nodules, pustules, red plaques, and ulcers1.

M. kansasii infection is diagnosed by chest X-ray or chest CT, positive respiratory culture, and clinical exclusion of other diagnoses2. The criteria for a positive culture include either two consecutive positive sputum cultures, one positive culture from bronchoscopy specimens, or one positive culture with compatible clinical symptoms2. The treatment of M. kansasii infection depends on the resistance of the organism to rifampin. Rifampin-sensitive organisms are treated with at least three drugs including rifampin, ethambutol, isoniazid, and pyridoxine1. Rifampin-resistant organisms are treated with three drugs including clarithromycin or azithromycin, moxifloxacin, ethambutol, sulfamethoxazole, or streptomycin1. Due to drug interaction, rifampin is contra-indicated among HIV patients taking protease inhibitors and nonnucleoside reverse transcriptase inhibitors. Because rifampin increases the metabolism of these HIV medications, it can lead to HIV drug resistance among these patients.

 

References

  1. Akram SM, Bhimji SS. Mycobacterium Kansasii. StatPearls. 2018. https://www.ncbi.nlm.nih.gov/books/NBK430906/
  2. Johnston JC, Chiang L, Elwood K. Mycobacterium Microbiol Spectrum. 2017;5(1):TNMI7-0011-2016. doi:10.1128/microbiolspec.TNMI7-0011-2016.
  3. Meraz A, Raheem S. Pulmonary Mycobacterium Kansasii Infection –A Tale of a Right Upper Lobe Cavitary Lesion [abstract]. Journal of Hospital Medicine. 2015; 10 (suppl 2). https://www.shmabstracts.com/abstract/pulmonary-mycobacterium-kansasii-infection-a-tale-of-a-right-upper-lobe-cavitary-lesion/. Accessed March 12, 2018.

 

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

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

Microbiology Case Study: A 50 Year Old Man with Altered Mental Status

Case Histor

A 50 year old man presented to the emergency room with altered mental status, fever, and hypotension. His medical history was significant for quadriplegia, chronic indwelling urinary catheter with frequent urinary tract infections, and diabetes mellitus. The patient was intubated and started on vasopressors for presumed septic shock. CT scan of the abdomen showed dilated loops of bowel with possible ileus and pneumoperitoneum around the sigmoid colon.

Laboratory Identification

  • Clostridium difficile Fecal PCR: Negative
  • Fecal Bacterial Pathogen PCR: Negative for Salmonella spp., Shigella or Enteroinvasive E. coli spp., Campylobacter spp., or Shiga toxin producing genes
  • Urine culture: 2 strains of Pseudomonas aeruginosa and Klebsiella pneumoniae

Blood culture: At 41 hours, two of four blood culture bottles became positive (one aerobic bottle in each set). Gram stain showed budding yeast with pseudohyphae (Image 1). Growth of white, smooth colonies with foot-like projections was observed on agar (Image 2). MALDI-TOF analysis confirmed identification of the organism as Candida albicans.

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Image 1: Gram stain of blood culture bottle demonstrating yeast form (left) and pseudohyphae (right).
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Image 2: Colony morphology on chocolate agar demonstrating smooth white colonies with surrounding “feet.”

Discussion

Candida spp. are the most common yeast cultured from clinical specimens. Candida albicans is characterized macroscopically by growth of smooth, white colonies with surrounding “feet” which represent projections of pseudohyphae. The pseudohyphae are distinguished microscopically from true hyphae by constriction of the cells where they meet (with true hyphae, the cell walls will remain parallel). When incubated at 37 C for 2 hours C. albicans will produce germ tubes (extensions from the yeast cell representing an attempt at forming true hyphae).

Candida spp. are normal flora found in the gastrointestinal tract, mucous membranes, and skin. Invasive candidiasis is typically an opportunistic infection with the patient’s own, endogenous flora (although nosocomial spread also occurs). Although it can be normal skin flora, the growth of Candida spp. from a blood culture should be presumed to be pathogenic; treatment should be initiated, and attempts made to identify the source. Typically, invasive candidiasis arises from one of three sources: 1) colonization and biofilm formation on an indwelling intravenous catheter 2) dissemination from a deep nidus of infection (often urinary tract) or 3) translocation from the gastrointestinal tract. Patients in intensive care, those who are immunocompromised, at extremes of age, those who have been on broad spectrum antibiotics, and those with GI tract perforation or anastamotic leaks post-operatively are at greatest risk for developing invasive candidiasis.

C. albicans has historically been the most common species of yeast causing invasive disease. However, non-albicans species (C. glabrata, C. parapsilosis, C. tropicalis and C. krusei) now cause almost 50% of invasive candidiasis. This changing epidemiology is relevant because the different species demonstrate different susceptibility profiles to antifungal agents, including azoles and echinocandins. C. auris is another recently emerging species that shows resistance to multiple antifungal agents, and has been described as causing outbreaks of healthcare-associated infections.

Although the patient’s chronic indwelling urinary catheter raised suspicion for a urinary source of the candidemia, the urine culture failed to support that theory. Given the findings of the CT abdomen in this patient, translocation from the GI tract is favored as the source of the candidemia. The patient’s history of treatment with antibiotics for repeated urinary tract infections, however, may have placed him at greater risk for developing invasive candidiasis.

-Alison Krywanczyk, MD is a 4th year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-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.