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.
When I started my career in laboratory medicine, we tested any fluid that was handed to us, for any analyte requested by the doctor. We did this for a number of reasons that we thought were good ones including that the doctor is a medical professional who knows what he wants and needs, and that the test results will help diagnose and treat the patient. We were trying to be helpful. Along the way though, laboratory professionals have come to understand that testing like this may not provide accurate results and may be doing more harm than good.
Now days, CLIA has clearly mandated that if the manufacturer of an FDA-approved assay system has not validated that system for a specific fluid type, the lab must perform that validation before testing and reporting results on that fluid type.
This is sometimes a hard rule to explain to the medical staff who have been trained in medical school to order such things as amylase on peritoneal fluid to look for pancreatic injury, or glucose on nasal fluid drainage when a CSF leak is suspected. And these doctors often have literature references for what they wish to have measured, although in general the references are not recent. I have a three-pronged approach to the explanation I give doctors as to why I won’t analyze the sample they sent me.
First, and probably most importantly, I cannot guarantee the accuracy of the result. Matrix effects are real and a test designed for serum will not perform the same on urine. Similarly, a test designed for serum and urine will not perform the same on a pharmacy preparation or an ascitic fluid sample. The result I provide if I test that sample could very well be wrong.
Secondly, I have no way to interpret the results of the test on an un-validated fluid type. There are no established reference intervals that allow us to determine the meaning of the result we’re providing. For example, who knows how much glucose is normally present in nasal drainage? I would assume no one knows, because why would you measure it in normal nasal drainage, and for that matter, what constitutes normal nasal drainage? Thus if I test that unknown sample for the analyte requested, I’m providing a possibly inaccurate result that is uninterpretable. And the physician is going to treat the patient on the basis of that result. In most cases, the physician changes his or her mind at this point in the discussion.
However, if that isn’t enough, I bring out the big guns. The agencies under which the lab operates forbid me from analyzing this sample for this analyte unless I validate the sample fluid type in my lab using the stringent validation criteria described in CLIA. This validation would take a considerable amount of time and resources and enough patient samples to set a reference interval.
If a doctor would still like to be able to order that test on that sample type after the discussion, I request that the doctor be involved in the validation process. First of all, I will want to know that enough of these tests will subsequently be ordered that the time and effort spent to validate the test will be worth it. In addition, the doctor will need to collect sufficient numbers of that sample type to allow us to perform an adequate validation and reference interval study. Also, QC material with the same matrix as the fluid type will need to be used and may have to be made in-house as it’s generally not commercially available. Biannual proficiency testing for that fluid type will need to be performed also, with internal PT developed for it. All of these considerations mean that the number of body fluid types and analytes we have validated is small, but we do not analyze un-validated fluid types. We will often try to locate a reference lab who does analyze them for a doctor. When that fails we will try to help the doctor find an answer to his medical question through use of other, legitimate tests.
-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 your physician orders a blood test, can you get your blood drawn at home or at the office? Most likely, that answer is “no.” Iggbo, a company based in Virginia, wants to change that. They’ve taken a page out of the Uber/Lyft playbook to provide on-demand phlebotomy services. To read more about it, check out this article on Dark Daily.
Do you need to know the percentage of Salmonella Typhi resistant to nalidixic acid in California in 2001? A resource now exists that can give you that answer.
The Centers for Disease Control (CDC) has released a tool called National Antimicrobial Resistance Monitoring System (NARMS) Now: Human Data, and it allows users to access antimicrobial resistance data based on year and geographical region. The interactive site tracks resistance for four bacteria that cause foodborne illness: Salmonella, Shigella, Campylobacter, and E. coli O157.
Lablogatory 