Microbiology Case Study: A 27 Year Old Male with Unintended Weight Loss

Case History

A 27 year old African American male presented to the emergency department with confusion and abdominal pain. His past medical history was significant for a 100 pound unintended weight loss and oral candidiasis which prompted a recent diagnosis of HIV. He was prescribed anti-retroviral therapy and antibiotic prophylaxis with which he reported compliance. Currently, he had no fever or chills. An abdominal CT scan showed an enlarged liver & spleen, generalized lymphadenopathy and a small amount of fluid. Significant lab work included anemia with a platelet count of 18,000 TH/cm2, absolute CD4 100 cells/cm2 (reference range: 506-3142 cells/ cm2) and a HIV viral load of 4,871 vc/mL. Given the concern for an infectious process, the infectious disease service was consulted and the patient underwent a thorough infectious work up including lumbar puncture, was started on board spectrum antibiotics and antifungals and was placed in airborne isolation until Mycobacterium tuberculosis could be ruled out.

Laboratory Identification

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Image 1. Direct smear from a stool specimen showed beaded acid fast bacilli (Kinyoun stain, 1000x oil immersion).
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Image 2. Direct smear from a stool specimen showed beaded acid fast bacilli (Kinyoun stain, 1000x oil immersion).

Initial diagnostic testing for bacterial, fungal and viral pathogens was negative. Three concentrated sputum AFB smears as well as a TB PCR were negative. The quantiferon gold TB test was also negative. The physician additionally ordered AFB blood and stool cultures. The direct smear from the stool specimen showed rare, beaded acid fast bacilli in a background of bacteria and yeast normally present in the stool via Kinyoun stain (Images 1 & 2). The specimen was sent to the department of health for additional work up. There was growth after 21 days incubation and Mycobacterium avium complex was identified by high performance liquid chromatography (HPLC).

Discussion

Mycobacterium avium complex (MAC) is a slow growing nontuberculous mycobacteria (NTM) frequently involved in human disease. Historically, it was classified as Runyon group III which are non-chromogens and do not produce pigment regardless of culture conditions. The group encompasses two taxa, M. avium and M. intracellulare. The species M. avium can further be classified into four subspecies: subsp. avium, subsp. silvaticum, subsp. paratuberculosis and subsp. hominissuis. Of interest, M. avium subsp. paratuberculosis can often be seen in association with Crohn’s disease.

In general, MAC organisms have low pathogenicity, but in the setting of those with lung disease (including cystic fibrosis), heavy smokers, immunocompromised patients and those with HIV, it is a well-known cause of disease. Infections with MAC can range from localized mycobacterial lymphadenitis and isolated pulmonary disease to bacteremia with dissemination to almost any organ. The organisms are located in circulating monocytes and further spread most commonly to the lungs, gastrointestinal tract and lymph nodes. In the case of HIV positive patients, MAC is the most common environmental NTM causing disease, especially in those with CD4 counts less than 100 cells/mm3 who are more likely to have disseminated disease.

In order to diagnosis MAC infections, specimens from blood, sputum, lymph nodes and other tissues are preferred. In addition, stool may also be an acceptable alternative in HIV patients if other specimens are negative or unable to be obtained. However, the sensitivity of a direct stool smear is only 32 to 34% making it not a very effective approach to identifying those at risk for disseminated infections. Once the culture has growth, various methods can be used to identify MAC, including phenotypic methods, DNA probe testing, HPLC, pyrosequencing and other forms of PCR & sequencing.

In the case of our patient, he was started on M. tuberculosis therapy: rifabutin, isoniazid, pyrazinamide & ethambutol (RIPE) until TB was ruled out. At that time, he was removed from isolation and switched to a drug regimen that included azithromycin, rifabutin and ethambutol. He showed clinical improvement and his cell counts, renal function and liver enzymes trended to normal ranges.

 

Stempak

-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 Clinical Pathology as well as the Microbiology and Serology Laboratories.  Her interests include infectious disease histology, process and quality improvement and resident education.

Not Your Grandmother’s Hematology

Last month we celebrated Lab Week, to recognize and show appreciation for Medical Laboratory Scientists and Technicians. Lab week is also a time to reminisce, and tell stories of the lab “in the old days.” I have worked with many technologists who have now been in the field for more than 50 years, and some who have worked in the same hospital all that time! Lab techs love to share stories about their experiences over the years, the days without computers, old methodologies, ancient lab equipment and manual testing. Listening to these stories always makes me think about just how far we have come in the field in the last 50- 60 years, and gives me a true appreciation for modern technology. It causes me to reflect on all the changes and developments that enable us to give physicians a wealth of knowledge that was previously unavailable.

During the first half of the 20th century, the complete blood count (CBC) was performed using exclusively manual techniques. Blood cell counts (erythrocytes, leukocytes, thrombocytes) were performed under the microscope using diluted blood samples and a hemocytometer. For each specimen, a technologist spent about 30 minutes at a microscope manually counting the cells and calculating the total count using a mathematical formula. A spectrophotometer was used to perform the hemoglobin by the cyanmethemoglobin method, and a spun hematocrit was performed. Indicies were calculated. A manual smear was made, stained, and cells were counted and differentiated under the microscope. To complete a CBC, all these procedures had to be performed individually, with duplicate testing and applying mathematical calculations, and could take over 2 hours. After all these tests were performed, results were reported on paper and sent to the patient’s doctor or the nursing floor.

In 1953 Wallace Coulter patented the Coulter Principle for counting and sizing microscopic particles. The Coulter Principle can be used for measuring any particles that can be suspended in an electrolyte solution, and has been used in the food and drug industry, in beer making, in the manufacture of construction materials and thousands of other applications. However, probably the most important application has been in the medical field where it has revolutionized the science of hematology. Coulter suspended red blood cells in a solution and, with an electrical current flowing, passed the solution through an aperture. As the cells pass through the current, the impedance between the terminals changes, and this change can be measured as a pulse. The first Coulter Counter measured the number of cells by counting the number of these pulses. The first Model A Coulter Counter was sold in 1956, manufactured in Coulter’s basement in Chicago. The Model A counted red blood cells in a sample in 10 minutes, a marked improvement over manual counting! The Coulter Counter was hailed for its speed, accuracy, and opportunities for reducing human error, tedium and eye strain.

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Image 1. Model A Coulter Counter, 1956. https://www.beckman.com/resources/discover/fundamentals/history-of-flow-cytometry/the-coulter-principle

During the 1960’s, an improved Model B Coulter Counter was developed and Model A and Model B were used to count both leukocytes and erythrocytes. Other Coulter Counter models soon followed, and competitors entered the market with their versions of cell counters. Within a decade, nearly every hospital in the United States had a Coulter Counter, and the new, advanced Coulter Model F was widely used. In 1968 the first fully automated hematology analyzer, The Coulter Counter Model S was introduced, and could perform a seven-parameter CBC. The Model S could perform not only WBC and RBC counts, but also reported Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC).  In 1955 it took one or several technologists 2 hours to perform a CBC, and in 1969 an automated hematology analyzer could analyze a sample in under 2 minutes.

Image 2. “Woman Using a Model F Coulter Counter Cell Counter,” 1969. Beckman Historical Collection, Box 58, Folder 94. Science History Institute. Philadelphia. https://digital.sciencehistory.org/works/736664585.

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Image 2. “Woman Using a Model F Coulter Counter Cell Counter,” 1969. Beckman Historical Collection, Box 58, Folder 94. Science History Institute. Philadelphia. https://digital.sciencehistory.org/works/736664585.

As these improvements and advancements continued, and Coulter patents expired, new manufacturers entered the field. Technicon Instruments Corporation, Ortho Diagnostics, Instrumentation Laboratories and Toa Medical Electronics, (presently Sysmex Corporation) were among the first Coulter competitors. From a simple automated blood cell count, to the first seven-parameter CBC, we saw hematology changing before our eyes. More reliable automated platelet counts were added in the 1970s. In the 1980s we saw the first hematology analyzers that could perform automated differentials and the first automated reticulocyte analyzers. In the late 1990’s, we saw the advent of digital cell images and automated manual differentials.

Today, modern automated cell counters sample blood, and quantify, classify, and describe cell populations. These instruments use optical light scatter, impedance methods based on the Coulter principle or a combination of both optical and impedance methods. Progressive improvement in these instruments has allowed the enumeration and evaluation of blood cells with great accuracy, precision, and speed, at a very low cost per test. The latest descendant of the Model A Coulter Counter, the LH 750, can determine 26 reportable hematological parameters. The Sysmex XN-9100 with four XN analyzers reports 30 parameters and has a throughput of up to 400 CBCs and 75 smears per hour. Today’s analyzers can accomplish more and more routine diagnostics, and the role of the hematology technologist continues to evolve and expand.

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Image 3. Sysmex XN-9100™ Automated Hematology System
https://www.sysmex.com/us/en/Brochures/XN9100ScalableAutomationBrochure_mkt-10-1177_10252017.pdf

This is not your grandmother’s hematology! We’ve truly come a very long way in 60 years. Modern hematology instruments not only perform a CBC, but they give us next generation diagnostics as well. Many give us advanced clinical parameters and other new parameters which provide physicians with additional information about the state of blood cells. We can report out immature granulocytes with every differential, automated nucleated red blood cell counts, immature platelet fractions and fluorescent platelet counts, and report the amount of hemoglobin in reticulocytes and the immature reticulocyte fraction. Future directions of hematology instrumentation include the addition of even more new parameters. In upcoming Hematology blogs I will be presenting case studies that highlight each of these advanced clinical parameters and discuss how physicians can use this new information in making diagnoses.

 

References

  1. Beckman Coulter, Inc. History http://www.fundinguniverse.com/company-histories/beckman-coulter-inc-history/
  2. https://www.beckman.com/resources/discover/fundamentals/history-of-flow-cytometry/the-coulter-principle 
  3. Clinics in laboratory Medicine. Development, history, and future of automated cell counters Green RWachsmann-Hogiu S. Clin Lab Med. 2015 Mar;35(1):1-10. doi: 10.1016/j.cll.2014.11.003. Epub 2015 Jan 5. March 25, Vol 35, Issue 1, p1-10
  4. Cytometry: Journal of Quantitative Cell Science. Wallace H. Coulter: Decades of invention and discovery Paul Robinson First published: 17 April 2013 https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.22296
  5. J.clin.Path. An assessment of the Coulter counter model S P.H.Pinkerton,I.Spence, J.C. Ogilvie, W.A Ronald, Patricia Marchant, and P.K, Ray. 1970,23,68-76 http://jcp.bmj.com/content/jclinpath/23/1/68.full.pdf
  6. SLAS TECHNOLOGY: Translating Life Sciences Innovation. The Coulter Principle: Foundation of an Industry. Marshall Don, Ph.D., Beckman Coulter, Inc.. Volume: 8 issue: 6, page(s): 72-81. Issue published: December 1, 2003 https://doi.org/10.1016/s1535-5535(03)00023-6
  7. Medical Electronic Laboratory Equipment 1967-1968. G.W.A Dummer and J. MacKenzie Robertson. 1967 Pergamon Press

 

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-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Ethical Leadership

Ethical decisions are a part of everyday life, but they are even more prominent when leading a team, a company or an entire organization. Ethics are essential for effective leadership, and leaders are responsible for creating ethical organizations.  Because people make decisions multiple times a day, ensuring that every decision is ethical is an essential skill, especially for leaders. Ethical decisions are often based on intuition without a logical explanation for why a certain solution was chosen. However, to increase the ethics of a situation, logic needs to be applied to the decision-making process.  Logical analysis of a situation creates a deeper understanding of the underlying issues and so improves outcomes. As ethical leaders it is our duty to lead employees and the company towards the best possible outcomes.

There are many logical approaches to ethics and multiple approaches can be used simultaneously to arrive at the best ethical answer. Some analytical approaches to ethics include:

The Utilitarian Approach

This approach relies on the concept that the best ethical decision has the most beneficial consequences for the largest group of people. The four steps of a utilitarian analysis include defining the ethical challenge, identifying those affected by the decision, determine the positive and negative consequences of the decision, and weigh the differences between those consequences.

Kant’s Categorical Imperative

Kant approaches ethical dilemmas based on the belief that people should always be the main focus and never be treated as a means. Kant recommends basing your ethical decision on one simple question: “Would I want everyone else to make the decision I did?” If the answer is no, then it is not the right decision. If the answer is yes, Kant argues that the analysis and decision are correct.

Rawls’ Justice as Fairness

This concept is centered on two principles based in theories of fairness and justice. First, everyone has an equal right to basic liberties, and second, inequalities, both social and economic, have two conditions: 1) everyone has an equal opportunity to qualify for job, and 2) priority should be given to meeting the needs of the less fortunate.

Pragmastism

This approach uses pragmatism as an ethical decision making tool. The process of using the scientific method allows people to come up with ethical solutions, because the hypotheses are tested through dramatic rehearsal. One should come up with a solution to an ethical dilemma and then test it, hypothetically, to see if the solution and its consequences were indeed ethical. What sets this approach apart is the use of emotion and feeling as indicators of unethical decisions.

Altruism

This method to ethical dilemmas focuses on what is best for others and not what is best for oneself.  People helping one another and witnessing leaders make sacrifices to the benefit of their employees or customers has a trickle-down effect on the rest of the staff.

Ethical decisions have a profound impact on others, even when they are not directly involved or affected by the decision. Good character is created when making ethical decisions and that character disintegrates when unethical decisions are made. Everyone has an influence on other people, therefore it is our obligation to others and the world to not only keep our ethical character intact, but to increase it so that it can withstand the tests of our time while encouraging others to do the same.

 

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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.

 

 

 

Microbiology Case Study: A 51 Year Old Male with Swelling in His Finger

Case History

A 51-year-old man was hospitalized for swelling of his left middle finger. He was in a normal state of health until 6 months prior, when he sustained an injury to his left third finger from a catfish while fishing in Florida in brackish water. The patient claimed he had caught a catfish and was stabbed by one of the spines of the fish in the middle portion of his finger. It became infected and he was prescribed a couple different courses of antibiotics, the details of which are not entirely clear. About one week leading up to his hospitalization, the finger became more swollen, and he had prominent lymphangitic streaking up the left arm as high as the upper bicep, with pain. He did not have any fevers, chills, or night sweats. Initial blood cultures taken were negative. He was admitted for three days on vancomycin/cipro, with resolution of the lymphangitic streaking but ongoing prominence of the swollen left third finger. MRI showed prominent tenosynovitis of the left 3rd finger tendon sheath, though the second, fourth, and fifth digits were also involved. Surgical debridement was recommended at the time but he preferred to pursue medical management. He was started on cipro/doxy/clarithro empirically at the time.

The infection was refractory to medical management. About 3 weeks later, his left third finger “burst open” with a draining sinus tract and purulent discharge (Image 1). He still denied any systemic symptoms such as fever, chills, night sweats. He was seen by infectious disease and orthopedic surgery and subsequently consented to radical tenosynovectomy for suspected mycobacterial chronic tenosynovitis.

mycomar1
Image 1. Infected third finger.

Tissue biopsy was sent for surgical pathology and culture. Histopathologically, the lesion showed chronic lymphohistiocytic and granulomatous inflammation; no microorganisms were identified on special stains. However, AFB culture was positive in the LJ slant and speciation was confirmed (Image 2).

mycomar2
Figure 2. Bright yellow colonies after light exposure on LJ slant media.

Discussion

Mycobacterium marinum was identified at a reference laboratory. Although infection with M. marinum is uncommon, the epidemiology of M. marinum disease is distinctive from other non-tuberculous mycobacterial species. The natural habitat of M. marinum is aquatic, and can be found in both fresh and salt water, including marine organisms, swimming pools, and fish tanks. The annual incidence is estimated at 0.27 cases per 100,000 adult patients.1 The infection is typically limited to the skin, mostly involving limbs, with rare spread to deeper structures reported.

M. marinum causes cutaneous disease as a consequence of exposure to water, usually in the context of a minor abrasion, laceration, puncture wound, or bite wound. Skin infections can occur from putting one’s hand into a contaminated fish tank, resulting in the condition called fish tank granuloma.

Histopathology often demonstrates suppurative granulomatous inflammation. In the microbiology laboratory, M. marinum is a photochromogen, meaning it produces pigment when cultured and exposed to light. Culture growth is optimal at 32°C over 7-14 days. Therefore, cooler extremities, particularly hands, are affected more often than central areas. Providers should be aware that M. marinum may cause a positive tuberculin skin test.2

M. marinum is a slow growing organism that is readily detected by acid fast smear and culture techniques. Confirmation of the presence or absence of mycobacteria in clinical specimens has traditionally required culture. However, traditional methods of identifying mycobacterial isolates to the species level are slow, based upon growth characteristics on solid media and subsequent biochemical tests, requiring additional weeks for subcultures. Newer techniques include MALDI-TOF MS, 16S ribosomal DNA sequencing, PCR-restriction length polymorphism analysis (PRA), and high-performance liquid chromatography (HPLC).

M. marinum infection responds slowly to appropriate antibiotic therapy. Infected patients may require treatment for 2 weeks or up to 18 months. M. marinum infection sequelae include persistent ulceration, draining sinuses, or septic arthritis.

References

      1. Akram SM, Bhimji SS. Mycobacterium Marinum. 2017. StatPearls.
      2. Lewis et al. Fish tank exposure and cutaneous infections due to Mycobacterium marinum: tuberculin skin testing, treatment, and prevention. CID. 2003;37(3):390-7.

 

-Thomas Rogers, DO 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.

Chemistry Case Study: Hypercalcemia in Sarcoidosis

Our patient is a 47-year-old female with a history of type II diabetes mellitus, hypertension, pancreatic insufficiency, systemic sarcoidosis with lung and liver involvement. She was admitted into the ED for severe hypercalcemia, hypokalemia and hypomagnesemia. Her total calcium concentration was at 15.1 mg/dL (ref range: 8.3-10.2 mg/dL, critical: >13.0) at admission and albumin was low. Further testing revealed 25-hydroxy vitamin D (25(OH)D) of 23.2 ng/mL, which is considered insufficient, and decreased PTH of < 15 pg/mL (ref range: 15 – 65). From these results, primary hyperparathyroidism was ruled out. PTH related peptide (PTHrp) was tested given her sarcoidosis history.

PTHrp is produced by some cancers, especially kidney, breast and lung cancers, and as well as lymphoma and leukemia. It has the same N-terminal and binds to the same receptor as PTH, therefore sharing some functions of PTH. In patients with hypercalcemia associated with malignancy, PTHrP may be evaluated. There are also case reported sarcoidosis-related hypercalcemia due to production of PTHrp. In the case, PTHrp was normal at 0.4 pmol/L (ref range: < 2.0).

Further tests showed that 1, 25-dihydroxyvitamin D (DHVD) was elevated at a concentration of 93.3 pg/mL (ref: 18.0 – 78.0). In the presence of decreased 25(OH)D, this result suggested that the 1-alpha-hydroxylase could be the cause of hypercalcemia. DHVD is the active form of vitamin D. It promotes intestinal calcium absorption and, in concert with PTH, skeletal calcium deposition. 25(OH)D converts to DHVD via 1-alpha-hydroxylase, which is almost exclusively expressed in the kidney, but can also be found in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage commonly seen in sarcoidosis and other granulomatous diseases. DHVD produced in extrarenal tissues is PTH-independent, and moreover, elevated calcium induced by extrarenal DHVD can inhibit PTH production via calcium-sensing receptor (CaSR) on parathyroid cells.

Sarcoidosis is a multisystem inflammatory disease of unknown etiology manifests as granulomas found predominantly in the lungs and lymph nodes. Hypercalcemia is seen in about 10-13% of patients. Overproduction of 1-alpha-hydroxylase and production of PTHrp can both contribute to the hypercalcemia in some patients with sarcoidosis. In this case, PTHrp was normal and elevated 1-alpha-hydroxylase was found to be the cause of hypercalcemia.

In addition to treatment of the underlying disorder, treatment of hypercalcemia in sarcoidosis is aimed at reducing intestinal calcium absorption and DHVD synthesis. Besides dietary interventions, glucocorticoids and bisphosphonates have also been used successfully to treat hypercalcemia in sarcoidosis:

  • Glucocorticoids: inhibit DHVD synthesis by the activated mononuclear cells (major contribution), inhibit intestinal calcium absorption and osteoclast activity
  • Bisphosphonates: inhibit the resorption of bone by osteoclasts

 

References

  1. https://emedicine.medscape.com/article/301914-overview
  2. https://www.uptodate.com/contents/hypercalcemia-in-granulomatous-diseases
  3. https://www.uptodate.com/contents/diagnostic-approach-to-hypercalcemia
  4. https://www.ncbi.nlm.nih.gov/books/NBK109831/

 

Huang

-Rongrong Huang, PhD is a first year clinical chemistry fellow at Houston Methodist Hospital. Her interests include general clinical chemistry, genetic biochemistry and applications of mass spectrometry in clinical laboratories.

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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.

Microbiology Case Study: An Elderly Woman with a Cough Develops Skin Nodules

Case History

Our patient is an 83 year old female with previous history of arterial hypertension, atrial flutter and chronic obstructive pulmonary disease who presented with dry cough (~2 weeks), fever (102ºF), and cutaneous ulcerated plaques with elevated borders on forearm, foot, leg, and neck. Chest radiographs and chest CT scan showed numerous bilateral nodular consolidations compatible with pneumonia. Additionally, mild leukocytosis (14,200 cells /mm3) and hypohemoglobinemia (10.9 mg/dl) were documented. A skin biopsy was taken from the forearm lesion. Periodic acid–Schiff (PAS) and Grocott-Gomori’s (or Gömöri) methenamine silver (GMS) stains identified rare budding yeast (PAS, Image 1). Acid-fast bacilli (AFB) and Gram stains were negative for mycobacteria and bacteria, respectively.

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Image 1. Budding yeast with a thick double cell wall were identified microscopically surrounded by prominent supurative granulomatous reaction (PAS, 100x).

Discussion

A diagnosis of disseminated blastomycosis was made based broad based budding yeast seen on PAS stain (Image 1).

Blastomycosis infection most commonly affects persons living in the Mississippi and Ohio River valleys, Great Lakes Region of the United States, and southern Canadian provinces. It is a fungal infection that can cause asymptomatic infection, isolated pulmonary disease, or serious and potentially fatal disseminated disease. B. dermatitidis can infect every organ of the body giving great variety of clinical manifestations, which is the reason why it is known as “the great pretender.” More than half of infected patients are asymptomatic. Symptomatic patients generally present with pulmonary symptoms, and the development of disseminated disease after hematogenous spread is common (~25 to 40% of symptomatic cases). The most common extra-pulmonary locations are: skin, bone, genitourinary tract, and central nervous system (CNS). Unlike histoplasmosis, most cases of blastomycosis are seen in immunocompetent patients, although immunocompromised patients may be at higher risk to develop severe forms of the disease.

Blastomyces is a thermally dimorphic fungus that grows as a yeast in the body and as filamentous fungi with septate hyphae in the environment. Recent phylogenic analysis has divided the Blastomyces genus into 2 species, B dermatitidis and B gilchristi . Culture of B. dermatitidis from the environment is extremely difficult, and much of what we know is conjecture from a few documented outbreaks, of which several occurred in wooded areas near waterways. These investigations found that exposure to dust clouds associated with construction or crop harvesting were the only identified risk factors for infection. Blastomycosis infection occurs through aerosolization of conidia from the environment causing respiratory infection or less commonly through direct inoculation into cutaneous abrasions. Once in the host, the conidia transform into yeast. The specific proteins expressed during the yeast phase allow the evasion of phagocytic and CD4+ cells.

Laboratory diagnosis

The most expedient method to diagnose blastomycosis remains examination of stained fluid or tissue specimens. Yeast are 8-15 µm in size with broad based buds of 4-5 µm and have a characteristic refractory double cell wall. Fluid can be stained with 10% potassium hydroxide plus calcofluor white, whereas formalin fixed paraffin embedded tissue samples can be stained with GMS or PAS. B. dermatitidis yeast can be difficult to visualize with Gram or hematoxylin and eosin (H&E) stains, but if found, the characteristic broad-based budding pattern of yeast can lead to presumptive diagnosis before culture and non-culture based diagnostic test results are available.

Culture of B. dermatitidis provides a definitive diagnosis of pulmonary and extra-pulmonary disease. B. dermatitidis grows well on routine fungal media such as Sabouraud dextrose agar, potato dextrose agar, and brain–heart infusion media. The yeast phase is inhibited by media containing cyclohexamide. Culture typically demonstrates growth in as little as 4-7 days. Colonies will initially appear yeast-like, but then develop white cottony aerial mycelium and turn tan with age. Mature growth is achieved around day 14 and the reverse of the colony is a tan color. At 25-30°C, B. dermatitidis forms septate hyphae with round or pear-shaped conidia attached to the hyphae by short or long conidiophores. This gives the characteristic appearance of “lollipops.” Scedosporium spp. and Chrysosporium spp. are common confounders because they make similar structures. Definitive identification of Blastomyces sp. can be made by conversion of the mold phase to the yeast phase by incubation at 37°C. An alternative to conversion is using a DNA probe assay.

 

References

  1. Medically Important Fungi, 5th edition
  2. Principles and Practices of Infectious Disease, 7th edition

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-Julio Diaz-Perez, MD is a 1st year anatomic and clinical pathology resident at University of Chicago (NorthShore).

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

Modern Radiation Safety in the Laboratory

In the “old days” in the clinical laboratory, the main sources of potential radioactive substances were found in the Radioimmunoassay (RIA) department. Techs who worked in this specialized testing area handled reagents which often were radioactive isotopes. The materials were used to label specific antigens which would compete with unlabeled antigen from patient samples. This method would allow the determination of high-quality quantitative diagnostic values. In the early 1990s, radio-immunoassays were commonly used to perform thyroid testing, narcotics assays, and a variety of hormone level analyses. Unfortunately, the use of such isotopes for testing was costly, difficult to automate, and their use was potentially hazardous to staff. Eventually this major testing method was replaced by ELISA testing, chemiluminescence, and other techniques, but some labs still do utilize RIA analysis today.

In the Anatomical Pathology areas, there has been potential radiation exposure from certain specimens in the past, and newer techniques have introduced other sources into the lab as well. Tissues (such as thyroid gland sections) are not typically removed from patients when treated with radioactive dyes, but it can occur. Good communication to the lab from surgery staff is important so that no one is unnecessarily exposed. Sentinel lymph node biopsies are sometimes infused with radioactive tracer dyes. Pathology staff may also receive radioactive seeds used to treat prostate cancer. Usually these seeds have decayed sufficiently and are inert, but that may not always be true. Again, clear communication about these samples is important. Other radioactive seeds are now used for breast tumor localization, and these do arrive in the lab while radioactive, and they must be handled and stored with care.

The best protection from radiation exposure is distance, duration, and barriers. Being away from a radiation sources isn’t always possible, but working with them for short periods and using some form of barrier protection will help. The types of radioactive material handled in labs today generally emit low levels of energy, and the use of Standard Precautions offers sufficient protection. Gloves, lab coats and face protection will provide the necessary protective barriers when handling these standard materials (Note: items like thyroid tissue that have been infused with Iodine-125 contain above-normal levels of energy and should be treated with extra care).

The College of American Pathologists (CAP) updated its regulations last year regarding radiation safety in the laboratory. Some of the standards were moved from the Anatomic Pathology checklist, and some are new. When asked, the CAP has stated that these standards do not apply to laboratories that handle low-level radiation samples such as sentinel lymph node biopsies.

First, the regulations require radiation safety handling policies and procedures which are maintained in a radiation safety manual. This manual can be paper or electronic, and it does not need to be separate from other lab safety policies. The policies should need to spell out who in the lab is authorized and restricted from handling radionuclides. Specific procedures should also be maintained to describe what actions to follow in the event of a radionuclide leak or damage to radioactive seeds. All radioactive materials and supplies should be inspected to ensure that there is no leakage or compromise that could expose staff unnecessarily.

The updated standards also require workplace radiation decontamination procedures, and labs that perform this type of work must keep records that document the effectiveness of the decontamination processes. Laboratories that handle radioactive substances must post radiation warning signs to communicate to others the potential dangers present, and all laboratory and medical staff must have comprehensive training prior to handling radioactive substances. Lastly, the CAP checklist now requires that if radioactive substances are handled in the lab, a laboratory representative must participate as a member of an institutional radiation safety committee.

Many things have changed in the laboratory setting over the past decades, and the regulations keep changing in an effort to stay current. The bottom line for radiation safety regulations in the lab is that staff need to be aware of what radioactive substances they may become exposed to, so they need to know safe handling processes as well as emergency response procedures. In the real world of lab medicine, radioactive substances do not glow, so lab staff may not be aware of the dangers when they enter the department. If the proper communication and practices are in place, however, everyone can maintain the minimum radiation exposure levels needed to live long and safe lives.

 

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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.