A 25 year old Caucasian male with no significant past medical history presented to the emergency department (ED) with a several day history of persistent fevers, headache (pain 10/10), dizziness and neck pain. He also reported facial and hand numbness and difficulty focusing at work. His laboratory values at that time showed a normal white blood cell count (5.3 TH/cm2), normal hemoglobin (13.8 g/dL), slightly decreased platelet count (142,000 TH/cm2) and slightly elevated liver enzymes. A computed tomography (CT) of his head showed no abnormalities, and a lumbar puncture was performed that was suggestive of viral meningitis (92% lymphocytes). After obtaining blood cultures, the patient received a dose of vancomycin and ceftriaxone and was discharged home.
Two days later he returned to the ED with complaints of worsening neck pain, photophobia, decreased appetite, and fevers reaching 105°F. He reported fevers of such intensity that he resorted to soaking himself in ice baths. On further questioning, he reported working in a microbiology lab that handles cytomegalovirus (CMV) and attenuated mycobacterium, but was unaware of any exposures or sick contacts. He has 2 dogs he rescued (one with a history of heartworm), 3 cats (one with a history of tapeworms) and a mouse. Infectious disease was consulted and a thorough workup was initiated, which included repeat blood cultures and testing for hepatitis, human immunodeficiency virus (HIV), respiratory pathogens and syphilis. He was started on cefepime initially and then later changed to meropenem and levofloxacin.
Figure 1. Gram stain from a positive blood culture illustrating small Gram negative coccobacilli (100x, oil immersion).
The microbiology laboratory reported the blood cultures collected during his second hospital visit were positive with small gram negative coccobacilli after approximately 60 hours on the automated instrument (Figure 1). No growth was noted after 24 hours incubation at 35°C in 5% CO2 on standard media. After 48 hours, small white colonies grew on sheep blood and chocolate agars but failed to grow on MacConkey agar. Biochemical tests revealed the organism was positive for catalase, oxidase and urease. In accordance with the suspected agents of bioterrorism manual, the culture was sent to the State Department of Health for further classification. The organism was identified by PCR as Brucella spp. Subsequently, the Centers for Disease Control and Prevention (CDC) performed species level PCR and identified the isolate as B. canis.
On further questioning, the patient denied consuming unpasteurized milk products but reported recently adopting a pregnant dog from a local shelter, who had subsequently delivered stillborn puppies of which the patient had been in close proximity. At this point, the patient’s antibiotics were switched to a 6 week course of oral doxycycline and rifampin. On follow up visits, he was doing well and symptom free. Unfortunately, the dog also tested positive for B. canis and had to be euthanized.
Brucella spp. are common zoonoses among wildlife and domestic animals including cattle (B. abortus), pigs (B. suis), goats (B. melitensis) and dogs (B. canis) who are usually asymptomatic carriers. While rare in the United States due to vaccination of livestock, Brucella spp. is considered endemic in areas of the Middle East, Central and South America and the Indian subcontinent. Symptoms of infection generally occur during an infectious abortion in which the placenta, fetal tissues and secretions contain high levels of the bacteria which can survive in the environment under various conditions for long periods of time. Humans are usually infected due to consumption of unpasteurized milk and cheeses. High risk professions such as veterinarians and slaughterhouse workers can also be infected by direct contract with contaminated materials or inhalation of aerosolized particles. Symptoms generally appear 1 to 2 weeks after infection with remittent/undulant fever the characteristic feature of the illness, in addition to arthralgias, fatigue, weight loss and hepatomegaly.
Laboratory identification of Brucella spp. is the gold standard but can be challenging as it is a slow growing organism and can infect personnel leading to laboratory acquired infections (LAI). When small Gram negative coccobacilli are identified that fail to grow on MacConkey agar, this should alert the laboratory worker of a potential agent of bioterrorism and work up should be performed in appropriate biosafety cabinets. Brucella spp. grows as small, smooth white colonies that appear after 24 to 48 hours incubation. It is catalase, oxidase and urease positive. Automated systems and MALDI-TOF mass spectrometry are not terribly reliable or recommended for identification of this organism due potential aerosolization events. When Brucella spp. is suspected, the level A clinical laboratory (a sentinel lab) should notify and send samples to a Level B/C lab (state health department) for confirmation. Subsequently, confirmed isolates can be forwarded to a level D lab (CDC) for speciation.
While overall, the mortality for Brucella spp. is very low, significant morbidity can result with long term non-specific symptoms and cardiac and osteoarticular complications. Good outcomes result when acute presentations are treated with combined regimens of antibiotics. The World Health Organization (WHO) recommends the use of oral doxycycline and rifampin for 6 to 8 weeks. Susceptibility testing is not recommended as resistance is rare and the concern for laboratory safety. In the case of laboratory exposures, prophylaxis with doxycycline and rifampin for 3 weeks is recommended for high risk workers. In the case of low risk employees, temperature monitoring for 6 months and serologic testing at defined time points is standard as the incubation period for Brucella can be this long.
-Melissa Brents, MD, is a 4rd year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center.
-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.
Key to successful delivery of an online course (or as in our case, a blended model of online and traditional), along with achievement of the learning objectives, is the learner experience. I’ll never forget the feelings of trepidation I had on our first day with our inaugural class, piloting this new model of curriculum delivery with our bacteriology course.
Our lesson plan requires that the students prepare for class by studying the online lecture material as homework, prior to the next day’s laboratory section. Our students were excited about starting our program and eager to learn, yet some were hesitant. I remember one student stating that they “might not be so sure about this new format.” After all, we hadn’t tried it before, and to be frank, it was scary. I remember thinking to myself, “What are we going to do if they do not study the online content? What if they do not prepare for class? What if they dislike this format? What will we do if they flunk their first exam?
Fortunately that was not the case, and our student’s performance in our program has been and continues to be highly successful.
Alex, a student in our current class put it this way:
“It is worth noting that this is not your typical college course. The program here really emphasizes the “reverse classroom” technique. For those unfamiliar, this term means that one will read about the lesson the night before and come to class the next day and perform a laboratory assignment based on that reading.
I came into the program experiencing nothing like this before, so I wasn’t sure how this learning strategy would work for me. After completing our didactic schedule, many of my peers would agree with me that this learning technique is fantastic and is very beneficial to the overall learning experience.
However, to maximize this benefit, time management is vital. Simply reading the lesson at the last minute does not cut it. Whether it helps you to take notes as you go, doing a re-read, or fill out a study guide, this style of learning is a classic example of getting out what you put into it.”
I loved hearing our student reflect that “you get out of it what you put into it.” To me, that is the ultimate goal of education, to prepare our students to be able to think critically and self-direct their learning. In this regard, our inaugural class was a success.
-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.
A 71 year old man presented with a dehisced corneal wound status post corneal transplant.
Corneal scrapings from the ulcer were submitted for interpretation and two separate organisms were isolated from the blood agar plates. The first was a non-motile gram negative rod that grew on 5% horse blood agar and MaConkeys agar. The organism was oxidase negative and spot indole negative and was identified as Klebsiella pneumoniae.
The second organism grew in bright yellow-pigmented colonies on 5% horse blood agar (Image 1) but did not grow on MacConkeys. The organism was oxidase positive and spot indole positive and was identified as Chryseobacterium indologenes by MALDI.
Chryseobacterium indologenes, previously known as Flavobacterium indologenes is a yellow pigmented, gram-negative filamentous, non-motile rod that is a non-glucose fermenter and can be found in soil, plants, foodstuffs and water sources including those found in hospitals. It produces a water-insoluble pigment, flexirubin which gives it its characteristic color. It was first isolated from a clinical specimen in 1983 however there have been more recent reports of bacteremia related to C. indologenes related to use of indwelling devices, such as a catheters.
-Agnes Balla, MD is a 3rd year anatomic and clinical pathology resident at the University of Vermont Medical Center.
-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.
Well, even though the groundhog has predicted another 6 weeks of winter, it’s not too early to start thinking about those summer pests – mosquitos – and the diseases they bring with them. Zika virus, in particular. Even though the winter weather has dulled our sensitivity to this emerging threat, it remains a significant problem. The virus is now circulating in 65 countries, mostly in the Americas (1). Currently, the CDC has issued travel alerts to areas where Zika is spreading including Miami-Dade, Florida, Puerto Rico, American Samoa, and the US Virgin Islands (2). Laboratory professionals should be aware of Zika virus because the diagnosis of Zika Virus Disease (ZKD) relies on the laboratory, and many healthcare professionals require guidance from the laboratory on how to proceed with diagnostic testing.
Zika is a flavivirus borne by the Aedes mosquito. Symptoms of ZKD usually last 2-7 days and include mild fever, skin rash, conjunctivitis, muscle and joint pain, malaise or headache. There is scientific consensus that Zika virus infection during pregnancy is the cause of congenital brain abnormalities of the fetus, including microcephaly. This devastating effect of the virus sets it apart from the other Aedes-borne viruses dengue, chikungunya, and yellow fever.
Diagnosis of Zika virus relies on laboratory testing, and yet, there are no FDA approved assays for Zika virus currently available. There are however a number of assays that have been given approval for emergency use, including: Real-Time PCR, MAC-ELISA, and a plaque neutralization reduction test. In the United States, these tests are available from the CDC and some state health labs. An algorithm describing the appropriate use of these tests can be found here. Unfortunately, in developing countries where Zika is endemic, access to the appropriate diagnostic test can be very difficult.
Impediments to accurate diagnosis of ZKD in developing countries include lack of education and access to quality laboratories that offer the right test. Lack of education encompasses not only transmission and prevention of the virus, but also who should seek medical attention and when and who should be tested and when. In many countries where visiting a medical professional is a financial burden to a family, it is less likely that a family will seek medical attention for a disease that has such mild symptoms symptoms as ZKD. While most cases of ZKD don’t require medical attention beyond comfort care, if patients don’t report to clinics or health centers, it is difficult to track and confirm cases if no one presents with a suspected case! Also, there is a need for consensus about what should be called a suspected ZKD case and then how to proceed with confirmatory testing. Some countries, including Brazil, the respective Ministries of Health have issued definitions of a suspected Zika case. The Brazilian definition includes: “patients who present with pruriginous maculopapular exanthema accompanied by two or more of the following signs and symptoms: fever, conjunctival hyperemia without secretion and pruritus, polyathralgia, and periarticular edema” (3). Suspected cases can be confirmed with diagnostic testing, but this is another challenge. The easiest and least expensive test for a clinic in the developing world is a Ig-M based rapid diagnostic test. There are several of these available commercially, mostly from European markets. However, these demonstrate significant cross-reactivity with other flaviruses such as dengue and chikungunya, which are also endemic in areas where Zika is now circulating. The most appropriate tests – RT-PCR, MAC-ELISAs, and plaque reduction tests – are only available in national laboratories it at all. The combination of lack of patients reporting Zika-like symptoms, lack of consensus of what constitutes a suspected case, and limited availability of confirmatory testing means that there is a significant likelihood that the number of Zika cases in many developing countries are underreported.
In January 2016, the WHO presented the Strategic Response Framework and Joint Operations Plan in response to the growing Zika virus epidemic. In October 2016, a quarterly update was released that described the goals and scope of the plan through December 2017. The plan is Strategic Response Plan comprised of four areas: 1) Detection, 2) Prevention, 3) Care and Support, and 4) Research. $10.9 million are dedicated to the detection arm of the strategic plan, which in addition to laboratory testing and diagnosis includes assessment and implementation of preparedness measures, and surveillance and monitoring in it’s scope. $41.2 million are dedicated to the research arm of the plan, which includes the “fast track and scale up of research development and availability of diagnostic tests.”
Hopefully in the next year we will see not only new diagnostic testing, but also medical interventions such as vaccines. In the meantime, it is important that we as laboratory professionals continue to be apprised of available testing, to educate our healthcare partners on the use of lab testing for ZKD, and to support research and development of Zika diagnostics.
–Sarah Riley, PhD, DABCC, is passionate about bringing the lab out of the basement and into the forefront of global health.
A 14 year-old-boy with Cystic Fibrosis had a respiratory culture collected at his routine clinic visit. It grew abundant mixed respiratory flora, and rare Gram-negative coccobacilli. This organism grew as non-lactose fermenting colonies on MacConkey agar (Figure 1) in approximately 36 hours and was oxidase and catalase positive. The isolate was identified by MALDI-TOF MS with a score of 2.39, which is acceptable for species-level identification.
Our isolate was identified as Bordetella bronchiseptica. Bordetella spp. are small Gram-negative rods that often appear as coccobacilli. Like other Bordetella spp., our isolate was catalase positive. Oxidase results vary across the genus, but B. bronchiseptica is oxidase positive. Some Bordetella spp. including B. pertussis and B. parapertussis, are very sensitive to metabolites and toxic substances found in many types of microbiological media. For the best chance of recovering these fastidious Bordetella spp. in culture, specialized agar such as Regan-Lowe, Bordet-Gengou, or Stainer-Scholte medium along with extended incubation periods are used. Due to the difficulty of culturing B. pertussis and B. parapertussis, these days culture is only performed at large reference laboratories or public health facilities. Testing by PCR is the current clinical practice and has greatly improved the sensitivity of detection for these fastidious organisms. In contrast, other Bordetella spp. including B. bronchiseptica are routinely recovered in culture using standard laboratory methods.
The most clinically relevant Bordetella spp. in humans are B. pertussis, the infectious agent of whooping cough, and B. parapertussis, which causes a similar illness to B. pertussis but generally symptoms are less severe. B. bronchiseptica causes respiratory infections in many animals including cats and dogs, and is the infectious agent of kennel cough. B. bronchiseptica can cause respiratory infection in humans who acquire the bacterium primarily from their infected pets. Human infection is rare, and most likely to occur in immunocompromised patients such as those with poorly controlled HIV or Cystic Fibrosis.
B. bronchiseptica produces a β-lactamase making the organism resistant to penicillin and many cephalosporins. Most strains are resistant to trimethoprim-sulfamethoxazole as well. In contrast, strains of B. bronchiseptica are generally susceptible to β-lactam/ β-lactamase inhibitor combinations, quinolones, aminoglycosides, and tetracycline.
Our patient was not having an exacerbation at the time of specimen collection, so he continues to do well. We expect to find B. bronchiseptica in his future sputum specimens, but the pathogenicity of B. bronchiseptica in such a low amount compared to respiratory flora is unclear.
–Erin McElvania TeKippe, PhD, D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.