A 32 year old male with a history of HIV presented with a non-productive cough, pleuritic chest pain, and subjective fevers and chills. The patient’s CD4 count was 23 cells/mL; he was not on highly active anti-retroviral therapy (HAART) or prophylaxis for opportunistic infections. A bronchoscopy was performed out of concern for opportunistic infection; a silver stain of bronchoalveolar lavage fluid from the right middle lobe revealed the following.
Laboratory identification:
Pneumocystis jiroveci (P. carinii) was identified based on morphologic examination of the silver stained fluid. The spherical, slightly dented or concave firm-walled cystic form (4-7 mm in diameter) was identified within the foamy alveolar fluid. Flexible-walled trophozoite forms may also be present in pulmonary fluid but are difficult to recognize. The organism may also be identified by a monoclonal antibody microscopic method of detection (highly sensitive but expensive and may have nonspecific staining) or nucleic acid amplification methods. The organism does not grow in routine culture.
Discussion:
Pneumocystis is an opportunistic pathogen with worldwide distribution that has closer homology to fungi than protozoa, which is why the name was changed from P. carinii to P. jiroveci. Its life cycle is not entirely understood, but there are cyst forms, sporozoite forms, and trophozoite forms.
Infection is acquired by inhalation. It is believed that most people are sub-clinically infected during childhood and that the infection is usually well contained by the immune system. During times of immunocompromise, the organism adheres to type I pneumocytes and replicates extracellularly while bathing in alveolar lining fluid causing the alveolar spaces to fill with foamy material. The organism is not susceptible to common antifungal agents and is treated with trimethoprim-sulfamethoxazole.
-Lauren Pearson, D.O. is a 2nd 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.
Most people who work in a clinical laboratory know a little about ketones or ketone bodies. The two facts that most people know include: 1) when you perform a urinalysis (UA), it includes a semiquantitative ketone result, and 2) high ketones are seen in diabetic ketoacidosis. But what is a ketone, where do they come from, and what are we measuring when we measure ketones?
In the laboratory medicine world, “ketones” refers specifically to acetoacetate (Acac), acetone and beta-hydroxybutyrate (BOHB). When the human body cannot utilize glucose, either because it is not present (fasting, starvation) or because it is present but cannot be used (lack of insulin to get glucose into the cells), the body instead breaks down fatty acids for energy. Fatty acids are mostly made up of long chains of carbons with hydrogens attached, so one of the main products of fat breakdown is 2-carbon acetyl-CoA. When a person is using lots of fats, like when they cannot use glucose, the production of acetyl-CoA exceeds the body’s ability to metabolize it via the Kreb’s cycle and it ties up lots of coenzyme A (CoA) needed for other processes. Thus, the body combines two excess acetyl-CoA into an acetoacetate, freeing up the CoA. The more acetyl-CoA produced from fat breakdown, the more acetoacetate produced. From there, the acetoacetate is converted to BOHB enzymatically or degrades spontaneously to acetone. BOHB is a dead end. Once there, the BOHB simply continues to build up until the production of acetyl-CoA no longer exceeds its utilization capacity. At that point, the BOHB is converted back to acetoacetate and then to acetyl-CoA for the body to be able to utilize it.
The most common form of ketoacidosis is probably diabetic ketoacidosis, in which blood glucose levels are high, but the glucose cannot get into the cells and be used, so fats are broken down for energy. At the height of a ketoacidosis, roughly 70% of the ketones in the body will be in the form of BOHB. This has implications for what we measure and for the monitoring of the treatment of ketoacidotic crises. UA dipstick ketones measure acetoacetate, and some will also detect acetone. None of the available UA methods measure BOHB. Thus, ketones measured in a UA will rise as ketoacidosis occurs, drop at the height of ketoacidosis as they are converted to BOHB, and then rise again as the condition is resolving and BOHB is converted back to Acac. A high Acac will occur both at the beginning and toward the end of the ketoacidosis, and Acac may actually be low at the height of a ketoacidotic crisis. BOHB on the other hand rises as the crisis evolves and drops as the crisis is resolved. The best test for following the resolution of a ketoacidotic crisis is repeat BOHB measurements.
BOHB is generally measured enzymatically on blood samples. BOHB response is maximal about 3 hours after glucose peaks. For example in a diabetic ketoacidosis, the peak BOHB will occur about 3 hours after the glucose peaks and in a normal patient given a glucose load, the BOHB will be lowest about 3 hours after the glucose peaks. During resolution of ketosis BOHB decreasing by half about every four hours as long as no more ketones are being produced. The test for BOHB is most commonly performed quantitatively using a kit adapted to the open channel on a chemistry analyzer. A point of care analyzer is also now available for BOHB.
Measuring ketones is most commonly used to monitor ketoacidosis, but ketone measurement can also be helpful in the differential diagnosis of some inborn errors of metabolism. For example, in fasting states, ketones should be elevated. If they are not, it can be an indication of disorders in fatty acid metabolism, or ketone metabolism itself. Additionally, in hyperammonemia states, the absence of ketones and acidosis indicates a urea cycle defect. Their presence suggests an organic acid disorder. Thus measuring ketones has multiple uses in medicine.
-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.
If you’re a laboratory director or manager who’s recently stepped into that role, the editors of Lab Medicine want to hear from you. As a new leader, you’re sure to have questions, challenges, and insecurities. Leave your questions in the comments, and it could be discussed in a future podcast.
A 51 year old woman with a significant smoking history presented with 8-9 weeks of fever and cough. Shortly after the beginning of her illness, she developed pleuritic left-sided chest pain and hemoptysis. She was treated with amoxicillin and then prednisone without improvement. She had progressively worse pain and hemoptysis as well as fevers and night sweats, with weight loss. A chest x-ray and CT scan showed a left upper lobe mass- like infiltrate suspicious for a carcinoma. She underwent transbronchial fine needle aspiration biopsy of the lesion which showed the following morphology.
Bronchoalveolar lavage fluid (Pap stain).
The specimen was also sent for fungal culture.
Colony morphology on fungal media.Organism morphology with lactophenol cotton blue scotch tape prep.
Laboratory diagnosis:
Blastomyces dermatidis was identified by microscopy and colony morphology. Septate, delicate hyphae with single, circular-to-pyriform condia on short conidiophores (lollipops) were seen on a scotch tape prep. The colonies appeared waxy and wrinkled, with a cream-tan color. Large, thick-walled yeast with buds attached by a broad base, 8-15 um with double-contoured walls, were demonstrated in tissue. Additionally, this patient had a positive urine antigen test for Blastomyces.
Discussion:
Blastomyces’ natural habitat is unknown but the organism is thought to reside in soil or wood, particularly in the Ohio, Mississippi, and Missouri River valley regions. It takes 2-30 days to grow in the lab. The infectious form is the conidia which are transmitted by inhalation. Common sites of infection include skin, lungs, and bone. The typical presentation in an immunocompetent individual is a pulmonary infection with associated acute or chronic suppurative and granulomatous lesions. Blastomyces infection may also cause osteomyelitis, prostatitis, urethritis, CNS infection, and disseminated infection. Immunocompromised patients may present with disseminated infection with involvement of skin, bone, and multiple organs. Infection may be confirmed by exoantigen testing or by nucleic acid probe testing.
-Lauren Pearson, D.O. is a 2nd 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.
Tandem mass spectrometry (MS/MS) is a methodology with so much versatility that new usages and applications seem to arise daily. MS/MS began as strictly a research tool, but over the last 20+ years it has made its way firmly into the clinical laboratory. The basic start of that transition came roughly 20 years ago when MS/MS assays were developed that allowed multiple intermediates of metabolism to be identified and quantified using a single punch from a dried blood spot. That development with this technology revolutionized newborn screening in the US over the next decade. Since then, more and more clinical uses for MS/MS have been recognized and developed.
Watching the growth of clinical MS/MS assays in hospital labs has been fascinating. As a reflection of this clinical emergence, journal articles containing MS/MS have increased in number over the same time period. For example, in 1998 the first MS/MS article appeared in the journal, Clinical Biochemistry. Between 1998 and 2003, 0 – 2 MS/MS articles were published each year, but from 2004-2007 that number was in the teens. From 2008-2010 more than 25 articles each year contained MS/MS technology, and from 2011 – 2013 that number ranged from 35-55 MS/MS-containing articles per year.
Initially, clinical applications using MS/MS were for limited assays, including newborn screening, confirmatory testing for inborn errors of metabolism (IEM), and for specific drugs, especially the immunosuppressant drugs. Testing quickly grew beyond drugs and toxicology using MS/MS methods, as the versatility of this testing became apparent. Assays began appearing for accurate measurement of Vitamin D, thyroid hormones and steroid hormones, to name only a few. In addition most MS/MS methods are sensitive enough that sample volumes requirements are small, or the assay can be performed using dried blood spots. Also, the ability to multiplex and measure multiple analytes in a single sample added to the utility of this methodology. Examples include 5 steroid hormones, 30+ analytes for IEM diagnosis or 200+ analytes for toxicology screening, all of which can be analyzed in a single sample within a short period of time.
As common as MS/MS assays now are in clinical laboratories, like early PCR technology, they have remained mostly manual tests run in specialized sections of the lab. They have required technical expertise and a love of hands-on, manual bench work. That is beginning to change with the advent of MS/MS instruments for bacterial identification and the entrance of MS/MS into the microbiology lab. This was the first MS/MS developed for a single, dedicated purpose and intended to require minimal manual intervention, either with day-to-day operation, or with maintenance and troubleshooting. This development clearly demonstrated that MS/MS can begin to approach the more plug-n-play type of technology needed for more fast-paced clinical labs. Developments like this will allow MS/MS to be integrated into more automated labs and ensures its future in the clinical laboratory.
-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.
Is $12 million enough to jump start innovation in the fight against antimicrobial resistance? The folks at the British initiative The Longitude Prize hope so. The challenge: create a cheap, fast, and accurate point-of-care analyzer or test kit to rapidly diagnose bacterial infections.
A major hurdle to curbing antimicrobial resistance is diagnostic in nature. Because identification and susceptibility testing can anywhere from two days to several weeks (depending on the causative agent), healthcare providers often need to prescribe antibiotics without knowing this critical information.
The challenge is a daunting one–the test kit would need to identify a broad number of bacterial species and resistance factors. It also needs to be functional all over the world (read: low resource settings).
CLSI has released a new fall webinar schedule. Starting this month, the organization will focus on helping laboratory professionals develop their own individualized quality control plans (IQCPs) and provide them with new ways to deal with antimicrobial susceptibility testing (AST) challenges.
On September 15, 2015, CLSI will host a webinar entitled, Developing Your Laboratory’s IQCP for Antimicrobial Susceptibility Testing. Presented by Susan E. Sharp, PhD, ABMM, FAAM, Director, Regional Microbiology and Molecular Infectious Diseases Laboratories at Kaiser Permanente in Portland, Oregon, USA, and Linda C. Bruno, MA, MT(ASCP), Director, Microbiology and Molecular Pathology Labs at ACL Laboratories in Rosemont, Illinois, USA, this webinar will focus on requirements for IQCP as mandated by the Centers for Medicare & Medicaid Services. It will also focus on the new College of American Pathologists (CAP) requirements for IQCP and how to develop an IQCP for AST.
On October 6, 2015, CLSI will partner with CAP to present Antimicrobial Susceptibility Challenges in regards to the CLSI AST documents and other AST resources that are currently available. Led by Janet A. Hindler, MCLS, MT(ASCP), Sr. Specialist, Clinical Microbiology at UCLA Health System in Los Angeles, California, USA, and Audrey N. Schuetz, MD, MPH, D(ABMM), FCAP, Interim Director, Clinical Microbiology Laboratory; Associate Professor of Pathology and Laboratory Medicine; and Associate Professor of Medicine, Weill Cornell Medical College/NewYork-Presbyterian Hospital from New York, New York, USA, this webinar will cover appropriate agents to report on individual species when isolated from select body sites. There will also be information presented about practical strategies for identifying, confirming, and reporting results for multidrug-resistant bacteria.
Lastly, on November 19, 2015, CLSI will host a webinar called Finding Value in Your AST IQCP: Improving Accuracy and Timeliness of AST Reports. This webinar will focus on what AST results should be confirmed before reporting and will cover processes that can be implemented to identify the most common AST reporting errors. Join A. Beth Prouse, MS, MT(ASCP), Clinical Microbiologist, at Peninsula Regional Medical Center in Salisbury, Maryland, USA, and Janet Hindler as they present on these topics as well as describe ways to improve AST report turnaround time.
Register for these webinars on the CLSI website at www.clsi.org/webinars. Member discounts apply to all CLSI-hosted webinars (September 15 and November 19).
A recent episode of much-needed filing in my office uncovered some prior contributions I had written for the Journal of the Kentucky Medical Association as part of its editorial board. One of these, written over a decade ago, resonated with me. The editorial represented what at the time I had termed a “daily devotional.” How timely that I should discover and reread this as it came on the heels of several (and not uncommon) frustrating days in the office.
The article highlighted a beautiful mosaic in the lobby of the hospital where I had my practice for 20 years. It featured Maimonide’s Prayer. Maimonide was a 12th century physician and philosopher. Here is a copy of the script:
Almighty Father of Mercy, I begin once more my daily work, Grant that I may be able to devote myself, Body and soul, to Thy children who suffer from pain. In all my efforts to heal the sick may I be filled with love for my fellow man.
One needn’t be particularly religious to understand and appreciate the very simple meaning of this prayer. It reminds us, that as laboratory professionals, we, as part of the healthcare team, ultimately need to remember that our personal daily devotion is to patient care. It is good from time to time to have a moment of philosophical repose.
I believe the next time daily events are extremely exasperating, when frustrations of practice threaten to overshadow my day, I shall have a copy of Maimonide’s Prayer close by to provide a bit of realignment and re-commitment to this professional purpose.
-Dr. Burns was a private practice pathologist, and Medical Director for the Jewish Hospital Healthcare System in Louisville, KY. for 20 years. She has practiced both surgical and clinical pathology and has been an Assistant Clinical Professor at the University of Louisville. She is currently available for consulting in Patient Blood Management and Transfusion Medicine. You can reach her at cburnspbm@gmail.com.
An 80 year old female presented to the emergency department complaining of a productive cough, three episodes of bright red blood stained sputum, persistent night sweats, fever, chills, and weight loss. Chest imaging revealed extensive centrilobular ground glass opacities, multiple pleural based nodules, and a cavitary lesion. The patient was exposed to Mycobacterium tuberculosis as a child when her father was treated for an active infection. Direct smears were negative; respiratory cultures were performed and an AFB culture bottle flagged positive with the following gram stain and culture morphology.
Poor-staining, beaded Gram positive bacilli in clumps and cords.
Laboratory identification:
The organism was auramine fluorescent stain positive from the broth. The AFB culture bottle was sub-cultured to agar based medium in addition to Lowenstein-Jensen medium, which yielded buff colored colonies with a dry bread-crumb like appearance, raising concern for Mycobacterium tuberculosis. Species identification was confirmed by DNA probe.
Discussion:
Definitive diagnosis of Mycobacterium tuberculosis is based on microscopy, culture, and/or PCR. The organisms are typically acid fast, straight or slightly curved rods that occur singly or in small clumps in clinical specimens. They may grow as twisted rope-like colonies called serpentine cords in liquid medium and take up to 2-4 weeks to grow in culture. They are obligate aerobes.
TB is transmitted by inhalation of bacilli in contaminated respiratory droplets. In an immunocompetent host, primary, latent, or reactivation pulmonary infection may occur. Reactivation disease and disseminated (military) disease is possible in immunocompromised patients including those with HIV, transplant recipients, and the elderly. TB also causes meningitis, pleurisy, and spinal infections.
The virulence of the organism is likely multifactorial and is believed to be related to its ability to survive within macrophages. It is treated with multi-drug antimicrobial therapy.
-Lauren Pearson, D.O. is a 2nd 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.
An 11 month old female with no significant past medical history was admitted with a fever of 104 degrees Fahrenheit, nausea and vomiting for 3 days (now resolved), watery diarrhea 4-5 times/day (resolved), and a new onset of acute pharyngitis/bilateral cervical adenitis. ER staff was concerned for a bacterial superinfection. She appeared sick with pale skin, but vital signs were stable, and labs were unremarkable except for an elevated CRP (15.7) and an absolute monocytosis (though no elevation in total WBCs). Exam showed a hyperemic pharynx without exudates, and no lymph nodes larger than 1 cm. A CT neck shows bilateral cervical adenitis, left greater than right, with some suggestion of necrotic nodes, as well as a likely left 3rd or 4th branchial cleft cyst. Blood cultures were drawn, and they turned up positive in a matter of hours, with the gram stain and plate morphology seen below:
Gram stain of positive blood culture broth showing Gram positive cocci in chainsLarge zones of beta-hemolysis around colonies growing on 5% sheep blood agar
Laboratory Identification:
Gram positive cocci in chains were seen, with small, glossy, gray-white, translucent colonies on blood agar having a wide zone of surrounding beta hemolysis. Catalase testing was negative, PYR testing was positive, and latex agglutination testing for Lancefield antigens was positive for Group A. MALDI-TOF confirmed the presumptive identification of Streptococcus pyogenes.
Discussion:
S. pyogenes (aka Group A Streptococcus [GAS]) is a ubiquitous gram positive cocci that causes a wide range of disease in humans. It is the leading cause of acute pharyngitis, particularly in children aged 5-15, although 15-25% of school aged children are asymptomatically colonized. S. pyogenes can also cause cellulitis, impetigo, necrotizing fasciitis, scarlet fever, toxic shock syndrome, otitis media, osteomyelitis, pneumonia, or even rarely meningitis/brain abscess. It additionally causes several serious post-infectious sequelae, particularly in untreated cases, including acute rheumatic fever with potential rheumatic heart disease, and poststreptococcal glomerulonephritis.
S. pyogenes has numerous virulence factors, most importantly the M protein, of which there are nearly 80 variants. M protein binds fibrinogen, inhibits complement binding, and prevents phagocytosis. As it is the major antigenic target of antibodies formed following infection, immunity is conferred only to the infecting strain, and none of the others, which complicates vaccine development efforts. Streptolysin O is responsible for the organism’s b-hemolysis on blood agar, and is also immunogenic, causing elevated Anti-Streptolysin O antibodies (ASO), which can be useful in diagnosing a recent infection, rheumatic fever, or poststreptococcal glomerulonephritis.
GAS pharyngitis is uncommon in children less than 3 years old, as is the bacteremia seen in the patient, which was presumably secondary to the severity of the throat infection. A later CT scan showed an enlarging abscess in the left lateral neck, corresponding to the earlier supposed branchial cleft cyst, despite IV clindamycin which was begun in the ER. Ceftriaxone was added, and later changed to Piperacillin/Tazobactam to complete a seven day course, though future blood cultures were negative. The additional antimicrobial coverage was due to the concern for other infectious microorganisms, as S. pyogenes is universally susceptible to penicillin.
Interestingly, this patient also developed a severe absolute neutropenia, with her ANC dropping from 7,100 at admission to 300 two days later, and then to 60 after two further days. The hematology/oncology service was consulted, and they determined that this likely represented a reaction to the infection rather than a more sinister bone marrow pathology. Several more days into therapy her ANC did begin to recover.
References:
Henningham A, Barnett TC, Maamary PG, Walker MJ. 2012. Pathogenesis of group A streptococcal infections. Discovery medicine 13:329-342.
Cunningham MW. 2000. Pathogenesis of group A streptococcal infections. Clinical microbiology reviews 13:470-511.
Red Book 2015
Journal of Clinical Microbiology, 10th editionCunningham MW. 2000. Pathogenesis of group A streptococcal infections. Clinical microbiology reviews 13:470-511.
-Paul Yell, M.D. is a 2nd year anatomic and clinical pathology resident at the University of Texas Southwestern Medical Center.
-Erin McElvania TeKippe, Ph.D., 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.
Lablogatory 