Microbiology Case Study: A 7 Month Old Female with Fever and Seizure-Like Episodes

Case History

A 7 month old female presented to the emergency department (ED) due to fever and seizure-like episodes. Her mother reported the child had been persistently febrile for 5 days (Tmax 103.9°F) with rhinorrhea, fussiness and decreased oral intake. The patient experienced 3 seizure-like episodes on the day of admission, which the mother described as periods of “shaking” with eyes rolling back. The child was unresponsive during these episodes, which lasted 1 to 2 minutes each. The child had been taken to her pediatrician the day prior to presentation to the ED where she was given a shot of ceftriaxone for presumed otitis media. The child received a chest x-ray, influenza testing, and blood and urine cultures were collected. She also had a lumbar puncture performed and the cerebral spinal fluid (CSF) was sent for chemistries, bacterial culture and polymerase chain reaction (PCR) testing for meningitis/encephalitis pathogens. She was started on IV ceftriaxone.

Laboratory Testing

The child’s white blood cell count from peripheral blood was 7.1 TH/cm2 and chest x-ray, urinalysis and flu testing were unremarkable. The CSF was clear and colorless with 7 WBC/cm2, glucose of 57 mg/dL and protein of 21 mg/dL. The cytospin Gram stain identified no organisms. The meningitis/encephalitis panel detected the presence of human herpesvirus 6 (HHV-6).

Discussion

Human herpesvirus 6 is a member of the Herpesviridae family and was the sixth herpes virus identified. Structurally, HHV-6 possesses a double stranded DNA genome and is enveloped. Clinically, it is the etiologic agent of roseola infantum (exanthum subitum) in infants and toddlers. Primary infection occurs in early childhood and those infected can be asymptomatic or have a non-specific febrile illness while only the minority present with the characteristic red macular rash prominent on the trunk and extremities, lymphadenopathy and high fevers. HHV-6 is highly neurotropic and as such causes viral encephalitis with 5-15% of children experiencing febrile seizures as a result of this illness. HHV-6 is highly prevalent with a greater than ninety percent seropervalence rate. HHV-6 establishes latency in T lymphocytes and can reactivate & cause disease, especially in immunocompromised patients such as those recipients of stem cell or solid organ transplants.

Traditional laboratory methods of identification for HHV-6 were challenging as viral culture, while once the gold standard for active disease, is not practical for most labs and is no longer used in routine diagnostics. PCR from serum, plasma or CSF has become the preferred test as there are now FDA-cleared, commercial platforms that are easy to use, allow for rapid turnaround time and in the case of multiplex PCR panels, the ability to target multiple pathogens from one test. Serology, while helpful in the diagnosis of primary infections, may not be provide conclusive results in a timely manner and is of limited utility in reactivation. Other less commonly used methods include immunohistochemistry, in situ hybridization and electron microscopy.

The prognosis for patients infected with HHV-6 is generally good with self-limited illness not requiring treatment. Rarely, multi-organ involvement can occur and HHV-6 infection in immunosuppressed patients can be a major cause of morbidity and mortality. There is no antiviral therapy licensed for the treatment of reactivated disease in this setting, but approaches using ganciclovir and valganciclovir have been proposed.

In the case of our patient, her blood, urine and CSF cultures were negative and her antibiotics were stopped after cultures were no growth at 24 hours. She required no treatment other than supportive care with acetaminophen for fever control. Prior to discharge, she developed a fine rash on her face, the back of her neck and trunk that was characteristic of an HHV-6 rash. This case demonstrates the utility of multiplex PCR testing in providing rapid identification of pathogenic organisms allowing for real time diagnosis and the limiting of unnecessary treatment.

 

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-Eric Tillotson, MD, is a second year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center.

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

 

 

Poll: Differentials on Patients with CLL

In a new paper on Lab Medicine, Gulati et al discuss counting smudge cells as lymphocytes on patients with confirmed or suspected chronic lymphocytic leukemia. Check out the paper and take our poll.

Urine Luck: The Diagnostic Value of Urine Dipstick Testing

A major challenge to providing diagnostic laboratory services in resource-limited settings, like the refugee camps discussed in last month’s post, is lack of infrastructure. Without running water, electricity, and even access to good phlebotomy supplies, specimen collection and preparation can be difficult, let alone the actual testing. I’ve found that in many instances, going back to the basics can often help determine a means of providing useful laboratory results. This post focuses on the humble, yet powerful, urine dipstick.

The urine dipstick is lightweight, easily portable, requires no special handling or storage and most have long shelf lives. These characteristics make the dipstick a great tool for use in the field. The specimen–simply urine–is also easy to collect and requires no special preparation. Dipstick testing can serve as a screening tool for some diseases and a diagnostic test for others. Urine dipstick measures pH, specific gravity, nitrites, leukocyte esterase, peroxidase activity, glucose, ketone, bilirubin, protein, and urobilinogen all performed within about a minute.

Urine is slightly acidic fluid and its pH is maintained essentially by the kidney. Any acid-base imbalance affects urinary pH. Urinary pH levels are helpful in the evaluation of nephrolithiasis, infection, and renal tubular acidosis. Kidney’s ability to concentrate urine is readily assessed by measuring the specific gravity of urine and the measurement generally correlates with urine osmolality.

Both nitrites and leukocyte esterase are used to evaluate urinary tract infection (UTI). A specific group of bacteria with reductase enzyme reduces nutritional nitrates in urine to nitrites which is detectable by urine dipstick testing. Some bacteria are not capable of converting nitrates to nitrites and therefore patients with UTI could still be negative for nitrite. Patients on a nitrate-deficient diet could be negative for nitrite despite the presence of bacteria with reductase enzyme in urine. In addition, the conversion of nitrate to nitrite requires time as well as at least 10,000 bacteria in milliliter of urine for the chemical reaction on the pad to occur. Thus, first morning urine is a specimen of choice for nitrite test. Outdated dipstick or a dipstick exposed to air could also cause a false positive reaction for nitrite. In the context of these limitations, nitrite test is only specific (92-100%) for bacteria capable of converting nitrate to nitrates and has very poor sensitivity (19-48%).

Leukocyte esterase is an enzyme produced by neutrophils. This enzyme is released from lysed neutrophils. The presence of esterase enzyme in the urine may imply UTI. However, white blood cells could present in the urine secondary to bacterial and viral infections, or because of other conditions such as tumor in the bladder. Unlike nitrite, leukocyte esterase is somewhat sensitive (72-97%) but not as specific (41-86%).

The presence of glucose or ketone in the urine is not normal. Glucose is detected in the urine when the blood glucose level is greater or equal to 180 mg/dL. In this level of glucose in blood, the kidney readily overwhelms its ability to re-absorb by filtering excess glucose. The presence of ketone in urine is suggestive of poorly managed blood glucose level, starvation, and prolonged fasting. When used combined, both measurements can be used to identify and monitor diabetic patients.

Detectable protein (only detects albumin, not other proteins) in urine is indicative of renal disease. The normal protein level in urine is less than 150 mg/dL and is below the threshold a urine dipstick can detect. Significant proteinuria with 96% sensitivity and 87% specificity is detected when urine protein level exceeds 300 mg/dL. Because of its insensitivity to microalbuminuria, dipstick test has limited clinical utility in screening diabetic patients.

Urine dipstick is used to identify the peroxidase activity of red blood cells, not for the presence of intact erythrocytes. Therefore, urine dipstick alone cannot be used to diagnose hematuria unless combined with microscopy finding. Hematuria is defined by American Urological Association the presence of at least 3 red blood cells per high power field. Positive peroxidase activity in the absence of red blood cells under microscope could mean myoglobinuria (e.g. caused by rhabdomyolysis), hemoglobinuria (e.g. caused by hemolytic condition and infections), or false positives. False-positives could arise from urine contamination with oxidizing agents, semen in the urine, blood contamination from hemorrhoids or vaginal bleeding, or from foods and medications such as beets, hydroxocobalamin, and phenazopyridine.

The presence of detectable conjugated bilirubin and/or urobilinogen is suggestive of liver disease, in vivo hemolysis, and/or biliary obstruction. Low concentration of urobilinogen in urine is normal. Bilirubin is converted to urobilinogen by bacteria in the intestine.

The chemical analysis of urine provides valuable information about the function of multiple organs or systems within a very short analytical time. If the limitations and inferences of the chemical reactions are properly addressed during interpretation, the diagnostic utility of urine chemical test is high. In situations where access to laboratory testing is low, the dipstick can provide clues to aid diagnosis.

 

-Merih Tewelde, PhD, contributed to this post.

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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.  

 

IRBs and Public Health Pathology

Hello again! Welcome back to my latest check-in following my progress with Zika risk reduction and public health outreach. Partnering with the Sint Maarten Ministry of Health through my medical school has provided amazing resources to take a look at social determinants of risk under the purview of public health, integrating both medical sciences and community service.

Early on in this project, I discussed the early stages in conceiving and planning these public health works in my first post “An Arbovirus Abroad.” This of course seemed like the perfect name for the proposal my team and I authored at the end of our first semester together. Done under an elective service credit, our full Internal Review Board (IRB) proposal for research within the community was called “An Arbovirus Abroad: a Service Learning Project Exploring Public Health Outreach, Social Determinants of Health, and Partnerships with Local Government to Address Zika Virus Knowledge and Community Outcomes.” The goals were to strengthen our partnership with local government offices as we aligned our efforts with reducing infectious risk and addressing community knowledge and attitudes regarding Zika.

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Figure 1. Title Page of original IRB/Research Project Proposal under G. Jackson, Ph.D., Assistant Dean, Community Affairs and Service Learning at AUC School of Medicine

After we secured IRB approval, we began work quickly. Holding meetings with the Ministry’s representative consultant for their office of Collective Prevention Services (i.e. vector control) and scheduling the remaining work for the semester. With five new members of the “Z-Pack” we established a loose timeline with our advisor. Our new goal: integrate what we learned last semester and bring it to a conclusive change within the community.

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Figure 2. Title card from initial briefing meeting with members of the Z-Pack, including coordinating partner from the Ministry of Health (CPS office) Mr. G. Davelaar.

This integration of knowledge from literature review/research, evidence-based best practices, and forward moving progress are all things those of us in the medical laboratory profession are quite familiar with. Getting IRB approval for a lab-centric project is quite involved and requires meticulous proof and substantial support to posit any claim to the benefit/risk ratio involved with human or animal subjects. I remember from my own graduate and undergraduate research that without heaps of evidence, you will be hard pressed to continue in any direction. While public health is a different science, the basis on evidence-based research is still present. During our initial assessments, literature reviews, and brainstorming, the “Z-Pack” went through hundreds of scientific articles covering everything from infection control precedents, to social behavioral change, and even the use of media and fear to illicit change.

Laboratory scientists know the impact of their work, though it may not always be the most evident to the general public. The near 70% of diagnostic information that comes from our work, and the virtual entirety of neoplastic diagnoses rely heavily on our training, skill, and certified competency in evidence-based practices. ASCP has a long-standing mission of advocacy for patients in the way its members and affiliates represent the profession at large. I believe that having those years of experience under my belt and those letters behind my name give me a head start when executing translational research. Going from raw data, analyzing it, and bringing it to life is something we all inherently train to do—and do well!

So, up to date, my team has secured two measures to contribute to our research. First, we gave an educational presentation to a community after-school program in one of Sint Maarten’s endemic regions. We had tailored a wonderful presentation I discussed in a previous post which caught the eye of the Ministry of Health and has spread to numerous places around the island under their sponsorship. With the same success, we managed to reach school-aged children in an engaging way about Zika, their health, and source reduction. Our second event is slated for this weekend where we have partnered with the Muslim student-interest group (MSA) on campus to go with them on their routine visit to a local mosque on a school-sponsored student service day we call “Community Action Day.” While the MSA students engage with their local community, the “Z-Pack” will conduct a two-part effort: to conduct a grounds-inspection for source/vector control around the mosque, and deliver a presentation for both children and adults regarding Zika prevention behavior.

How do those two events connect with my theme of evidence-based lab scientists? Well, one of my engagements when at Northwestern Medicine was to teach a course discussing transfusion protocols and laboratory information to clinical nursing staff. Presenting information, or teaching people, new ways to think about their environment at work or home is a part of being interdisciplinary. I was able to speak with medical jargon to the clinical staff, but with the children I have to use my ability in translating medical knowledge to understandable facts while also keeping the audience interested. My team proved in our last school-aged project, that when children are engaged and enthusiastic about something they have learned, they will take those messages home with them and hopefully contribute to a positive outcome. As for the second example, what could be more directly appropriate for lab folks to understand here: a surprise inspection! Sure, no one’s losing any accreditation points here, but the fact remains that we all have experience from one side or another making sure that things are up to code on pre-determined conditions and protocols. We have an SOP from the Ministry regarding the items of inspections as they relate to source control, so translating them to a new site should prove interesting.

I’ll close this post off with an interesting piece recently posted by Ms. Susan M. Lehman, MA, MT (ASCP)SM where she discussed learner (i.e. student) experiences. She talked briefly about how online curriculums and other lab-skills courses may rely on more independent learning, changing the expectations of students. One of her students summarized it positively saying, “you get what you put into it.” That’s what I think about the service elective my work is associated with. It could be simple directed readings with great discussions, but what my “Z-Pack” team has and the skills we each bring to it have made the project and its partnerships exciting.

Thanks for reading!

 

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Constantine E. Kanakis MSc, MLS (ASCP)CM graduated from Loyola University Chicago with a BS in Molecular Biology and Bioethics and then Rush University with an MS in Medical Laboratory Science. He is currently a medical student at the American University of the Caribbean and actively involved with local public health.

Microbiology Case Study: 81 Year Old Man with Prior History of Bladder Cancer

Case history

An 81 year old man with prior history of bladder cancer treated with Bacillus Calmette–Guérin  (BCG) presented with dysuria. Three urine samples were sent for AFB culture.

Laboratory Workup

The urine samples were plated on 7H11 solid media and Middlebrook liquid media. Colonies grew in the liquid media in five days and the solid media in 6 days (Figure 1). The colonies were subcultured to 7H11 solid media and Lowenstein-Jensen (LJ) media.  At this time, the colonies were probed for Mycobacterium tuberculosis complex, M. avium complex and M. gordonae, all of which were negative.  The organism grew on all of the subcultured media within one day. This fulfilled the criteria for rapidly- growing Mycobacterium species given the organism had grown in less than 7 days on subculture from solid media to solid media.  The specimen was sent out to our reference laboratory for further speciation and was identified as Mycobacterium chelonae.

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Figure 1: 7H11 media with non-pigmented white colonies.

 

Discussion

Rapidly growing mycobacteria include many species, but the main clinically relevant species are M. fortuitum, M. chelonae, and M. abscessus. These organisms are widely distributed in nature and can survive nutritional deprivation and extreme temperatures. They have been isolated from soil, dust, natural surfaces, water, wild animals and domestic animals.  Risk factors for infection include patients with immunosuppression, organ transplant and autoimmune disorders. Immunocompetent patients are also at risk if they have had trauma or invasive medical procedures.  M. chelonae may cause a spectrum of human disease. The most common manifestations are cutaneous infection, osteomyelitis and catheter infections. Nosocomial outbreaks of M. chelonae have been reported and linked to various water sources, including water-based solutions, distilled water, tap water and ice.  Rapidly growing mycobacterium are generally resistant to the classic antituberculosis drugs (rifampin, ethambutol and isoniazid). M. chelonae is usually sensitive to aminoglycosides, however treatment should be determined by antibiotic susceptibility testing.  In our patient, we had expected the colonies to be M. bovis because of the patient’s history of BCG treatment which is a live attenuated strain of M. bovis. Cystitis induce by M. chelonae is a rare clinical manifestation. We believed this is a true infection, as opposed a contaminated patient sample, given the patient’s symptoms in conjunction with all three urine samples being positive for M. chelonae.

 

-Jill Miller, 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 Assistant Professor at the University of Vermont.

Laying a Foundation for a Blended Curriculum Model

In the late 1990s—early 2000s—we were faced with a critical shortage of graduates in clinical/medical laboratory science, and we started evaluating the benefits of having our own program. Once we made the decision to seriously consider implementing our own program, I pitched a proposal for a blended model of curriculum delivery. My proposal was accepted by the Mayo Clinic School of Health Sciences as a pilot program to be supported by our newly created education technology center. Since our program and the education technology center were both new, we certainly experienced some growing pains. Also, had I known we’d have to reconstruct major elements of our online content every time we upgraded our content management system, I might have thought twice about it!

In the end, it was all worth it because today, we have an outstanding program built upon a solid foundation of both traditional and online content delivery that leverages our staff infrastructure and can be effectively managed and maintained over time.

In the curricular model developed for our academic program in Medical Laboratory Science (MLS), the didactic component is provided in an e-learning platform (Blackboard Learn) and is underscored by Transactional Distance Theory (Moore, 1991), in which the three modalities of learner interaction with content, instructor, and fellow students are integrated into the online module. Each lesson plan includes a laboratory module taught by traditional methods of interaction between the instructor and student in a classroom setting. A constructivist learning environment is facilitated, and each lesson plan is closely anchored in the context of the work the student will perform upon employment.

Here is a simple diagram of our curricular model:

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The following learning theories define our program curricular model:

  1. Transactional Distance Theory (e-learning theory): The online lesson plan includes learner-content interaction, learner-learner interaction, and learner-instructor interaction.
  2. Constructivism: The roles of both the teacher and student are redefined in this educational model. The teacher moves away from the traditional role of “sage on stage” to that of a “facilitator” of the student’s acquisition of knowledge. The student becomes a more active learner in this model, moving away from the traditional role of passive learner.
  3. Anchored Learning:
  • Information is taught in the context of how the learner will apply it once he or she is working.
  • The online homework lesson correlates with a hands-on laboratory lesson designed to reinforce the e-learning content.
  1. Reversing the Lecture-Homework Paradigm (Moses, 2002): The traditionally taught lecture is provided as an online homework assignment.

Embracing technology has provided a means by which we can improve our teaching methods and promote change in our education infrastructure. More than half of our didactic courses in our MLS Program apply the new education strategy I learned about as “reversing the lecture-homework paradigm” (more commonly known as “flipping the classroom”). Instead of going to lecture, our students complete web-supported didactic modules asynchronously as “homework” assignments, allowing more classroom time for laboratory instruction.

By providing more hands-on laboratory lessons, we are giving our students the opportunity to practice laboratory procedures and apply new learning material in a way that corresponds more closely with what they will do for a living after they graduate. Instead of giving the typical “one-directional lecture” with limited opportunity for dialogue, our instructors are able to spend more time with our students, teaching practical applications of the content, answering questions, and helping problem-solve.

References

  1. Moore, M. G. (1991). Editorial: Distance education theory. American Journal of Distance Education, 5(3), 1-6.
  2. Moses, G. A. (2002). e-Technology must enable big education goals. Proceedings of the 2002 e-Technologies in Engineering Education (eTEE) Conference, Switzerland, Vol. P01, Article 20, 142-145.

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-Susan M. Lehman, MA, MT(ASCP)SM graduated from the University of Wisconsin-Madison in 1983 with a BS in medical technology. She is program director for the Medical Laboratory Science Program and course director for Clinical Microbiology I and II; her areas of interest include distance education and education methodology.