Microbiology Case Study: A 60 Year Old Male with Dysuria

Case Description

A 60 year old Hispanic male with a past medical history significant for chronic pancreatitis, hypertension and cirrhosis was admitted with decompensated cirrhosis. He underwent paracentesis for ascites and subsequently developed a hematoma as a complication of the procedure which required embolization. During his 12-day long hospital stay, he also developed hypoxia due to volume overload that improved with diuresis. A Foley catheter was placed during his hospital stay which was removed prior to discharge. Weeks later, at a follow up appointment with urology, he complained of dysuria, very little urine during voiding and the sensation of incomplete bladder emptying. A clean catch urine culture was performed and grew >100,000 colonies of Escherichia coli. As shown in Table 1, the isolate was resistant to multiple classes of antibiotics including penicillins, cephalosporins, fluoroquinolones, one aminoglycoside (Tobramycin), Trimethoprim/Sulfamethoxazole, aztreonam and carbapenems (Ertapenem/Meropenem) making this isolate multi-drug resistant (MDR). Because of the resistance profile to the carbapenems, molecular testing for carbapenemase genes was performed and the New Delhi metallo-beta-lactamase (NDM-1) gene was detected. The patient was treated with nitrofurantoin for his symptomatic urinary tract infection (UTI).

Table 1. Antimicrobial susceptibility of this isolate of E. coli.

Discussion

Escherichia coli is a gram negative, motile bacillus that is a normal constituent of the gastrointestinal tract and is one of the most common causes of uncomplicated UTI. Antimicrobial susceptibilities are nearly always performed because the isolates of E. coli can vary in resistance. E. coli do not have any intrinsic resistance to antibiotics other than penicillin; however, they can acquire resistance through numerous mechanisms including structural mutations and plasmid-borne genes that encode enzymes to various classes of antibiotics. One such plasmid-encoded enzyme is the NDM, which was identified in our patient’s isolate. Its resistance is the result of bacterial synthesis of a carbapenemase that deactivates carbapenems by breaking down the beta-lactam ring.1 In the United States, K. pneumoniae carbapenemase (KPC) is the most common, but other types carbapenemase enzymes have also been reported.1,2 NDM is uncommonly isolated in E. coli; it is more often identified in other gram negative bacteria including MDR Pseudomonas aeruginosa or Acinetobacter baumannii complex, which can cause, among other things, devastating nosocomial infections within a healthcare setting. Because these enzymes are on mobile elements, a patient can be colonized with one bacterial strain that carries the plasmid with the carbapenemase on it and transfer a copy of that plasmid to another bacterial strain, thereby conferring new carbapenem resistance to the new bacterium (e.g., P. aeruginosa with the NDM on a plasmid shares that plasmid with an E. coli). Carbapenem resistant Enterobacteriaceae (CRE) are of great importance in healthcare. Carbapenem resistance mediated by enzyme activity (e.g., KPC, NDM, OXA-48, etc), typically confers resistance to all beta lactams. Interestingly, NDM enzymes typically do not destroy aztreonam, a monobactam;3 however, it is common for bacteria to have multiple resistance genes, so NDM carrying strains can be resistant to aztreonam. Although these CRE isolates can cause significant morbidity and mortality when found in clinical samples including sputum or blood, luckily for our patient, he had an uncomplicated UTI and nitrofurantoin was susceptible.

-Limin Yang is a PGY-1 resident in Anatomic and Clinical Pathology at University of Texas Southwestern. She has varied interests including anatomic pathology specialties.

-Dominick Cavuoti is a professor of Anatomic and Clinical Pathology at UT Southwestern and active faculty on both Microbiology and Cytology services.

-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.

How to Rock a Risk Assessment

As a lab safety professional, we know performing risk assessments is an integral piece of managing a safety program. In fact, assessing risks and identifying hazards are considered the beginning steps that must be completed when approaching the management of any safety-related area. Risk assessments are the starting point for handling a bloodborne pathogens program, chemical hygiene, personal protective equipment, and many other lab matters. But how can you be sure they have been performed correctly, and how often should they be performed?

OSHA gives simple guidance on basic assessment of risk in the workplace. “The employer shall assess the workplace to determine if hazards are present, or are likely to be present, which necessitate the use of personal protective equipment (PPE).” They further state, “the employer shall verify that the required workplace hazard assessment has been performed through a written certification that identifies the workplace evaluated; the person certifying that the evaluation has been performed; the dates(s) of the hazard assessment; and which identifies the document as a certification of hazard assessment.”

OSHA’s Bloodborne Pathogens standard requires that labs perform an exposure risk determination for each employee. Labs must assess exposure risk levels by job classification, and then assess exposure risk for tasks performed in the department. The Hazard Communication standard explains the need for chemical hazard determination. There are many types of risk assessments that must be performed, and regulations stipulate that they must be reviewed and updated (if necessary) every year. Things change in a dynamic department like the laboratory, and understanding the changing risks of harm can be key to keeping staff safe.

The four basic steps included in a risk assessment are hazard identification, identifying those at risk, choosing control measures, and reviewing the findings. It may sound easy, but lab hazards can come in many forms (physical, mental, chemical, biological, etc.), so walk around the department to look for those you may have missed. Review incident records as well to see what harm is occurring in the lab. Next, determine what employees may be harmed and how. Consider those who work in the lab each day and those that are just passing through the area. An evaluation of the risks follows. If the risk cannot be removed, decide what controls (engineering, administrative, PPE) need to be in place. Finally, review each risk assessment on a regular basis. Things change in the lab, and with change may come new hazard risks – or even the reduction of potential harm if risks have been reduced via elimination or substitution. Again, examine these assessments at least annually or whenever major changes occur in a particular lab safety arena.

For many laboratories, the advent of the COVID-19 pandemic brought new testing platforms and procedures to the department, and this testing had to be implemented quickly. Is there a way to use risk assessments to help introduce new processes safely? Absolutely! The use of a standard form to assess the potential hazards of new or updated processes and/or equipment is actually a high quality finishing touch on an overall assessment program, and unfortunately, it is something that is often missing in many labs.

Considering the tasks, sample handling, equipment, reagents, and overall biosafety of the new process, choose the likelihood of hazard incidents (rare, possible, likely, etc.), and classify the consequences of each occurrence (minor, moderate, major, etc.). Use a matrix to calculate the overall level of risk for the new procedure or equipment. For example, if a new COVID-19 test platform requires opening samples, the likelihood and risk of exposure may be designated as “high” or even “very high.” Next, determine the controls that should be put in place to decrease the exposure opportunities. For example, if centrifuge rotor covers, a biological safety cabinet, and a surgical mask is added to the normal lab PPE, the overall risk of that testing process is reduced. Document the decision and keep those records available for any future reviews that may be needed. Once the assessment is complete, complete the appraisal of the new process with a quick safety audit. Look for additional biohazards encountered, chemical safety, electrical safety, and even potential waste handling issues. If you couple that safety analysis with the risk assessment, you are doing an above-average job at circumventing hazards in the department. (For examples, go to https://www.aphl.org/programs/preparedness/Documents/APHL%20Risk%20Assessment%20Best%20Practices%20and%20Examples.pdf)

Keeping staff safe from exposures and injuries in the laboratory is a massive and time-consuming task, but it is required by our regulatory agencies and it needs to be a top priority. When used properly and completely, a risk assessments can be a powerful tools that begins your look into safety hazards and then closes the loop to avert them. Having an awareness and control of departmental hazards is one way to rock safety in the laboratory.

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.

Overview of Laboratory Tests for Cytomegalovirus

Introduction

Cytomegalovirus (CMV) is considered the most important pathogen in transplant recipient patients as it can cause significant morbidity and mortality. Anti-CMV treatments have proven to be effective but are not without adverse side effects. Thus, there is a strong need for sensitive and reliable tests to diagnose and monitor active CMV infection. Several testing methodologies are available in today’s clinical laboratories to evaluate a patient’s CMV status: viral culture, serology, histopathology, pp65 antigenemia, and quantitative PCR. In this post, we will review the advantages and limitations of these tests.

Viral culture

Viral culture is performed most commonly by the shell vial assay (also known as rapid culture), in which a cell line (usually human fibroblast cells) is inoculated with patient sample by centrifugation. The virus is then detected by either direct or indirect fluorescent monoclonal antibody, providing results within 1-3 days. The centrifugation step greatly improves turnaround time when compared to traditional tube cell culture technique, which may take 2-3 weeks before a result can be reported as negative.

Culturing CMV has been largely replaced by newer methodologies like quantitative PCR and CMV antigenemia. This is due to relatively weaker sensitivity for diagnosing CMV infection compared to newer tests, as well as slower turnaround time. Viral cultures of urine, oral secretions, and stool are not recommended due to poor specificity; however, for diagnosis of congenital CMV, viral culture of urine or saliva samples is an acceptable alternative if PCR is not available.

Serology

CMV serostatus is an important metric to evaluate prior to patients receiving a hematopoietic or solid organ transplant. Serologic testing is done primarily via enzyme immunoassays and indirect immunofluorescence assays. These tests check for presence of anti-CMV immunoglobulin (Ig)M and IgG to provide evidence of recent or past infection. Outside of establishing serostatus (primarily in organ donors and recipients), serologic testing for CMV is not recommended in diagnosing or monitoring active CMV infection.

CMV IgM antibodies can be detected within the first two weeks of symptom development and can be present for another 4-6 months. IgG antibodies can be detected 2-3 weeks after symptoms develop, and remain present lifelong. These antibody measurements are particularly useful in determining risk of CMV acquisition in seronegative patients (negative for IgM and IgG) at time of transplantation. IgG titers can also be measured every 2-4 weeks to assess for CMV reactivation in seropositive patients. Since CMV IgG persistently remains in circulation, testing for it has a higher specificity compared to IgM, and thus is the preferred immunoglobulin to test for in establishing serostatus. Serologic tests can be falsely positive if patients have recently received IVIG or blood products, so testing on pretransfusion samples are preferred if possible.

Histopathology

Under the microscope, cells infected with CMV can express certain viral cytopathic effects. These infected cells classically show cytoplasmic and nuclear inclusions (owl eye nuclei) with cytoplasmic and nuclear enlargement. Additionally, immunohistochemistry (IHC) can stain antibodies specifically for CMV proteins to highlight infected cells, making histologic examination quicker and improving diagnostic sensitivity.

Histopathology can be useful in identifying tissue-invasive disease, such as CMV colitis or pneumonitis. Cases in which PCR testing is negative does not necessarily exclude tissue-invasive disease; thus, the diagnosis of tissue-invasive disease relies on histologic examination (with or without IHC) or possibly viral culture. On the other hand, a negative histologic result does not exclude tissue-invasive disease, possibly due to inadequate sampling, and shows the potential for weak diagnostic sensitivity.

pp65 antigenemia

CMV antigenemia testing uses indirect immunofluorescence to identify pp65 antigen, a CMV-specific matrix protein, in peripheral blood polymorphonuclear leukocytes. Whole blood specimens are lysed and then the leukocytes are cytocentrifuged onto a glass slide. Monoclonal antibodies to pp65 are applied, followed by a secondary FITC-labeled antibody. The slide is then read using a fluorescence microscope for homogenous yellow-green polylobate nuclear staining, indicating presence of CMV antigen-positive leukocytes. Studies have suggested that a higher number of positive cells correlates with an increased risk of developing active disease. The sensitivity of antigenemia testing is higher than that of viral culture and offers a turnaround time within several hours.

This test has been utilized since the 1980s, but has seen less use recently due to the increasing popularity of quantitative PCR. Antigenemia testing is labor intensive, and requires experienced and trained personnel to interpret the results (which can be somewhat subjective). This test also must be performed on whole blood specimens within 6-8 hours of collection due to decreasing sensitivity over time, which limits transportability of specimens. Additionally, It is not recommended to be run on patients with absolute neutrophil counts below 1000/mm3, due to decreased sensitivity. Despite these limitations, CMV antigenemia testing is still considered a viable choice for diagnosing and monitoring CMV infection, especially when viral load testing is not available.

Quantitative PCR

Quantitative real-team polymerase chain reaction (PCR) is the most commonly used method to monitor patients at risk for CMV disease and response to therapy, as well as for diagnosing active CMV infection. The advantages of using a quantitative PCR assay include increased sensitivity over antigenemia testing, quick turnaround time, flexibility of using whole blood or plasma specimens for up to 3-4 days at room temperature, and the availability of an international reference standard published by the World Health Organization (WHO).

Several assays from Roche, Abbott, and Qiagen are available and FDA-approved. The targets of these assays vary, with some targeting polymerase and other targeting CMV major immediate early gene. These assays are all calibrated with the WHO international standard, which was developed in 2010 to help standardize viral load values among different labs when results are reported in international units/mL. The goal of this international standard is to decrease the interlaboratory variability of viral load, and determine the appropriate cut-offs for determining clinical CMV disease. There is still improvement to be made in this area, as variability still exists between labs.

Conclusion

There are several tests to determine the CMV status of patients. Some of these tests are suited for particular goals, such as serology for determining serostatus prior to organ transplantation, or histology and IHC to diagnose tissue-specific CMV disease. For diagnosis and monitoring of general CMV disease, the test of choice in most laboratories is quantitative PCR, which offers automated, quick and sensitive results. Antigenemia, while dated and labor intensive, is still an acceptable alternative when PCR is neither available nor cost-effective for smaller labs. Both of these testing methods are preferred over viral culture, which has poorer diagnostic sensitivity and relatively longer turnaround time.

Despite the numerous advantages quantitative PCR has, there is still variability in viral load counts among laboratories. This is due to varying DNA extraction techniques, gene targets used by PCR, and specimen types used. There is still a lot of work to be done in standardizing testing in regards to not just CMV, but also other viral pathogens like Epstein-Barr virus, BK virus, adenovirus and HHV6. Updated standards and increased use of standardized assays will hopefully decrease this variability between labs to improve testing results and in turn, improve patient care.

References

  1. https://www.uptodate.com/contents/overview-of-diagnostic-tests-for-cytomegalovirus-infection#H104411749
  2. https://www.uptodate.com/contents/congenital-cytomegalovirus-infection-clinical-features-and-diagnosis?topicRef=8305&source=related_link#H9542666
  3. Kotton CN, Kumar D, Caliendo AM, et al. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation. 2013;96(4):333-60.
  4. Hayden RT, Sun Y, Tang L, et al. Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards. J Clin Microbiol. 2017;55(2):423-430.
  5. Kotton CN, Kumar D, Caliendo AM, et al. The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation. Transplantation. 2018;102(6):900-931.

-David Joseph, MD is a 2nd year anatomic and clinical pathology resident at Houston Methodist Hospital in Houston, TX. He is planning on pursuing a fellowship in forensic pathology after completing residency. His interests outside of work include photography, playing bass guitar and video games, making (and eating) homemade ice cream, and biking.

2019 Call for Abstracts

The Abstract Submission site is open for ASCP 2019. Last year, ASCP had a record number of submissions and we aim to bring in even more this year. ASCP’s selection process is known as highly competitive, and as a result, presenters receive close attention from big-name faculty and industry contacts. Some have even gained immediate funding opportunities to expand their research.

Abstracts can be submitted until 11:59pm CST on March 20th. Submit your abstract HERE.

Call for Abstracts 2019

History of Generations: Baby Boomers

One of most well-known names of generations, besides perhaps Millennials, are the Baby Boomers. The Baby Boomers generation is currently the largest generation. Of all the generations, they cover the largest span of time (those born from 1946 to 1964). In large parts of the world, there was a big surge in births after the Second World War. That war had a significant influence on their values, perceptions, attitude, and approach to work.

One of the major aspects that make Baby Boomers stand out from previous generations is that this was really the first generation in which women started to work outside the home in large numbers, at least in the Western Hemisphere. This has a major influence on the home and work environment. In the United States, the children of Baby Boomers often had a latchkey around their neck so that they could go home after school without their parents being there.

Baby Boomers played a large role in shaping today’s society; they used music as a political tool, they increased focus environmental conservation, they were involved with the civil rights and women’s rights movements, and they are politically informed and outspoken. It is also the first generation in which both divorce and homosexuality became accepted. Overall, this generation is known for optimism, adaptability, having a strong work ethic, and being team-oriented.

Even though technology did not become part of daily life until Generation X, Baby Boomers witnessed enormous technological milestones, such as the first orbit around earth, landing a man on the moon, and the creation of the first nuclear power plant. All these events set the stage for later advances, and Baby Boomers are typically interested in learning how to use technology, although it does not come as natural to them as future generations. They also have tend to work longer and retire later in age, mainly because they link their self-worth to their job. In other words, their work ethic becomes their “worth ethic.” Knowing this when working with them is important, as they appreciate recognition in forms of awards, title changes, and public acknowledgement for their contributions.

Because this generation spans such a long time (and because some Boomers had children later in life due to second and third marriages), Baby Boomers are parents to both Generation X and Generation Y.  There is a lot to learn from this generation, so next time you work with one ask for some of their insights and understanding. This generation makes great mentors, especially because they are likely to have children of mentee age.

 

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-Lotte Mulder earned her Master’s of Education from the Harvard Graduate School of Education in 2013, where she focused on Leadership and Group Development. She’s currently working toward a PhD in Organizational Leadership. At ASCP, Lotte designs and facilitates the ASCP Leadership Institute, an online leadership certificate program. She has also built ASCP’s first patient ambassador program, called Patient Champions, which leverages patient stories as they relate to the value of the lab.

 


 

Here is an interesting fact: there are two sub-sets of Baby Boomers. The first ones are the “Save-The-World Revolutionaries” of the ’60s and ’70s. The second set of Boomers are the career climbers, the yuppies, of the ’70s/’80s. The most profound characteristic of a Baby Boomer is their work ethic. They identify with their job, profession, or their career. So much so, that this generation has remained in the workforce beyond the age of 70.

In a lot of ways, I’m the typical Baby Boomer woman. I married the first time just before I was 20 years old. Divorced in my early 30’s and moved forward in my career because that’s what the “Boomer Women” did. They worked inside and outside of the home.

As a laboratory professional that left the bench many decades ago, and now working in the field of Organizational Leadership and Development, I am approaching the age of 70. I’m starting to realize my retirement day is closer than I’d like.    Like others of my generation, this concerns me because I am defined by my career! The thought of not working left me searching for my identity so much that I started seeing a therapist last year. I was, and am fortunate to work for an incredible organization that doesn’t judge one by their age. They look at the skills and competencies one brings to the table. I’m consciously working on succession planning so that my institutional knowledge remains with the organization and its people. It also helps to have two gifted professionals who wanted to learn from me and grow. Then it takes a manager like mine who supports me through this often painful process. I am blessed with that kind of support. Sometimes the work ethic equals “worth ethic” in the body, mind, and spirit of a Baby Boomer, which is something to keep in mind when working with this generation.

 

 

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-Catherine Stakenas, MA, is the Senior Director of Organizational Leadership and Development and Performance Management at ASCP. She is certified in the use and interpretation of 28 self-assessment instruments and has designed and taught masters and doctoral level students.  

Microbiology Case Study: A 45 Year Old Woman with Breast Abscess

Case History

Our patient is a 45-year-old female who presents to the Emergency department with breast pain. She was diagnosed with granulomatous mastitis 3 months prior. She was treated with 3 weeks of steroids, but they were stopped when the mass was unchanged and the patient was experiencing increasing breast tenderness. Since then she and has undergone several procedures to drain her right breast abscess, the most recent being five days prior. The woman has been treated with sequential courses of sulfamethoxazole–trimethoprim and metronidazole without improvement. On this visit, the abscess was again drained and sent to the microbiology laboratory for culture. The Gram stain showed no bacteria and 3+ polymorphonuclear cells. After 48 hours incubation there was scant growth on the blood agar plate and no growth on the chocolate, MacConkey or CNA plates. The colonies growing on the blood plate were tiny, white, and lipophilic (Image 1).

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Image 1. Small, white, lipophilic colonies growing on blood agar plate at 48 h incubation.  There was no growth of this organism on chocolate or MacConkey agars this time point.

Discussion

The organism was identified as Corynebacterium kroppenstedtii.

Colonies on the blood agar plate were identified as Corynebacterium kroppenstedtii using MALDI-TOF mass spectrometry for identification. C. kroppenstedtii is catalase positive, non-motile and a facultative anaerobe. It grows better on 5% sheep blood agar than chocolate agar, as is the case for many Corynebacterium spp. Corynebacterium come in two varieties, lipophilic such as Corynebacterium jeikeium, and luxuriantly growing, such as Corynebacterium straitum. C. kroppenstedtii is part of the former lipophilic group, forming small colonies after extended incubation.  Lipids such as Tween-80 can added to the media to improve growth of lipophilic Corynebacterium such as C. kroppenstedtii, but clinically this is not routinely performed. When viewed on a gram stain, the bacteria are rod-shaped gram positive diptheroids with typical coryneform morphology. Both MALDI-TOF and 16S rRNA sequencing can accurately identify C. kroppenstedtii to the species level.

C. kroppenstedtii is a relatively newly recognized species within the Corynebacterium genus. It was first described in a case series of young Polynesian women with histological evidence of lobar mastitis, from which C. kroppenstedtii was identified from >40% of the patients’ abscesses. Since that time, isolation of C. kroppenstedtii has been clinically associated with breast abscesses and granulomatis mastitis. C. kroppenstedtii is a highly lipophilic bacterium. Its cell wall lacks many mycolic acids, which may explain its propensity to grow in lipid-rich sites such as mammary glands. C. kroppenstedtii typically affects women of reproductive age and can be difficult to diagnose due to the slow growing nature of the lipophilic organism and the relatively few organisms present in abscess specimens.

Prior to identification by MALDI-TOF MS and 16s rRNA sequencing this patient’s culture would have been reported as rare or 1+ “dipthroid,” “coryneform,” or “Corynebacterium spp.” Without knowing the clinical significance of this organism, the culture results could easily be dismissed as contaminating skin flora.

It is very difficult to treat C. kroppenstedtii in abscesses, with the most effective treatment requiring both surgical drainage of the abscess and long term antibiotic use. It is fairly difficult to get antibiotics to the site of infection, so antibiotics that test as susceptible in the laboratory may not eradicate the pathogen. Our patient’s isolate of C. kroppenstedtii was susceptible to ciprofloxacin, clindamycin, doxycycline, and intermediate to penicillin. She remains on ciprofloxacin therapy, but has ongoing right breast tenderness. She had another surgical drainage of her breast abscess a week after this case, and the culture also grew 1+ C. kroppenstedtii with 3+ PMN seen on Gram stain, so her infection has not yet been resolved.
References

  1. Tauch, Andreas, et al. “A Microbiological and Clinical Review on Corynebacterium Kroppenstedtii.” International Journal of Infectious Diseases, vol. 48, 2016, pp. 33–39., doi:10.1016/j.ijid.2016.04.023. ScienceDirect.
  2. Johnson, Matthew G., et al. “The brief case: recurrent granulomatous mastitis due to Corynebacterium kroppenstedtii.” Journal of clinical microbiology 54.8 (2016): 1938-1941.
  3. Paviour, Sue, et al. “Corynebacterium species isolated from patients with mastitis.” Clinical Infectious Diseases 35.11 (2002): 1434-1440.

 

CW

-Carolyn Wiest, MT(ASCP) graduated from Michigan State University with a BS in molecular genetics and is a medical technologist at NorthShore University HealthSystem.  Her interests are in microbiology and molecular diagnostics. 

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