Case Study Hematology: The Mouse Strikes again! Lymphocytes with Intracytoplasmic Inclusions

If you read my last blog, you heard the about the story “if You Give a Mouse a Cookie” by Laura Numeroff.4 The curious little mouse has a mind that never rests. As his mind wanders and hops from one thing to another, he keeps discovering more things to check out along the way. Medical laboratory lcientists are a lot like this. We’re a curious bunch, and, in investigations, one thing often leads to the next. Well folks, the mouse has struck again! We were given another cookie in the form of these beautiful cells.

Image 1. Lymphocytes with intracytoplasmic inclusions.

These cells were found by my coworker Liz Marr, MLS(ASCP), and the adventure began! First, we wanted to know what those were, and then we needed to find out more about them, and then, mostly, I wanted to know why in almost 40 years of working in and teaching hematology that I have never before seen this!

The story begins with our case history. We received a CBC from a 71 year old female with a 4 year history of untreated chronic lymphocytic leukemia/ small lymphocytic lymphoma(CLL/SLL). The patient’s recent history included a myocardial infarction(MI) 5 months prior. The patient was found to have leukocytosis (WBC 25.38 x 103/μL) and absolute lymphocytosis (18.25 x 103/μL) with normal hemoglobin and hematocrit (Hgb 13.4 g/dL, Hct 40.8%) and normal platelet count (272 x 103/μL). The differential had 71.9% lymphocytes with many abnormal forms noted. The slide was sent for a pathology review. The pathologist reported “Atypical lymphocytosis consistent with patient’s known chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Filament-like inclusions are present in the cytoplasm which has been previously reported in patients with CLL.”

Image 2. Lymphocytes with crystalline-like inclusions.

A curious tech can’t stop at just that description. If you tell me they are filament-like inclusions, I will have all kinds of questions. What are these filaments made of? Are they crystals, or something else? How common are these? Are these diagnostic of CLL? Are these only seen in CLL? What is their significance? And, of course, the most puzzling question, why have I never seen these before??

CLL is a form of non-Hodgkin lymphoma and is the most common leukemia in the Western world. It is generally a leukemia of older age with a median age at diagnosis around 67-72. The disease is widely variable, with some patients asymptomatic and requiring no treatment for many years, while others have a more rapidly progressive course of disease requiring treatment. About 60% of patients are diagnosed before they exhibit any symptoms. CLL and SLL are considered to be different manifestations of the same disease. In CLL, the abnormal B lymphocytes are found mostly in the peripheral blood and bone marrow, but in SLL, there is lymph node involvement, with abnormal cells mostly found in the lymph nodes. CLL is diagnosed based on absolute B lymphocyte counts ≥5 x 109/L. Flow cytometry typically reveals a distinctive cell immunophenotype with expression of CD19, CD5, CD23, and Κ/λ; and weak expression of CD20, CD79b, and surface immunoglobulin.1

The most recent flow cytometry report on our patient was from one year ago. An 8 color analysis with CD45/SSC gating was performed by LabCorp. The flow revealed an abnormal cell population representing 56% of total cells. Two monoclonal B cell populations were detected with identical phenotypes except for light chain expression. These cells expressed CD45, CD19, CD20, CD22, CD5, and CD23., CD38-. This phenotype was consistent with her previous diagnosis of CLL/SLL.

A literature search revealed only a few articles about intracytoplasmic inclusions in CLL. Cytoplasmic inclusions in lymphomas are uncommon, but have been noted as vacuoles, crystals, and pseudocrystals. These crystalline inclusions represent immunoglobulin(Ig) heavy and light chain that precipitate in the cytoplasm. Using electron microscopy it has been found that theses Ig deposits localize in the rough endoplasmic reticulum (RER).5 When surface Ig can be demonstrated on the B lymphocytes, it has been found to be same as Ig in the inclusions.6

In two published studies that describe these crystal like inclusions, photographs are very similar to the ones we found on our patient.3,5 It is interesting to note that, in these two studies, neither of the subjects was a known CLL patient. The inclusions were noted in the patients’ cells and the peripheral blood was subsequently sent for flow. Phenotypes reported confirmed monoclonal B-cells representing a large percentage of cells. Huang reported monoclonal B-cells which expressed CD45, CD19, CD20, CD22, CD79b, CD5, CD23, CD148 and CD200(hi), with partial expression lambda, and negative for FMC7, CD10, CD11c, CD49d, CD103, CD38, CD25, CD160, IgM, CD81, kappa and Ki67.3 In the Ramlal case study, phenotype was CD5, CD19, CD20, CD23, positive, CD10, FMC7 negative.5 On the basis of flow, along with the CBC results, the patients were diagnosed with CLL.

Of course, while researching this, the little mouse in me kept asking questions and finding more questions to ask. One question that I still had questions about was if these inclusions have any prognostic value. In three recent studies3,5,6 it was indicated that these inclusions can be used to help with diagnosis, but are not prognostic for course of disease. Rodriguez followed a patient with asymptomatic Rai stage 0 CLL. This patient consistently had inclusions noted in lymphocytes for 9 years before any progression of disease was noted.6 In the medical field even if one study reports no prognostic significance, this opinion could change in the future with more studies. Could these crystalline inclusions be used to forecast time to first treatment (TFT) or overall survival?(OS). So far, because of the rarity of these cytoplasmic inclusions, there is no evidence of prognostic value. As well, the mechanism related to their formation and their role in CLL is yet to be determined.

Our case study patient and the various reports found in literature had common flow cytometry immunophenotypes. Patients were all either previously diagnosed with CLL or lymphocytic lymphoma, or were diagnosed at the time of the findings of these inclusions. While these crystalline inclusions alone are not considered diagnostic for CLL, their recognition can be used to assist in a prompt diagnosis of a lymphoproliferative disease. And they are so pretty! What medical laboratory scientist doesn’t love pretty cells? Be like that mouse. Be curious, keep your eyes open, and be on the lookout for these interesting cells in CLL patients, but, more importantly, in patients with lymphocytosis without a known diagnosis of a lymphoproliferative disorder.

References

  1. AJMC, January 7, 2019
  2. Chronic Lymphocytic Leukemia: An Overview of Diagnosis, Prognosis, and Treatment
  3. Huang, Y., Zhang, L. Intracellular rod-like crystals in chronic lymphocyte leukemia. Int J Hematol 112, 267 (2020). https://doi.org/10.1007/s12185-020-02933-7
  4. Numeroff, Laura If You Give a Mouse a Cookie. 1986
  5. Ramlal, B, DiGiuseppe, JA. Intracytoplasmic crystalline inclusions in chronic lymphocytic leukemia. Clin Case Rep. 2019; 7: 1460– 1461. https://doi.org/10.1002/ccr3.2250
  6. Cecilia M. Rodríguez, Carmen Stanganelli, Claudio Bussi, Daniela Arroyo, Darío Sastre, Viviana Heller, Pablo Iribarren & Irma Slavutsky (2018)Intracytoplasmic filamentous inclusions and IGHV rearrangements in a patient with chronic lymphocytic leukemia, Leukemia & Lymphoma, 59:5,1239-1243, DOI: 10.1080/10428194.2017.1370549

-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.

Microbiology Case Study: 83 Year Old Male with Bladder Cancer

Case History An 83 year old male with bladder cancer was treated with Mycobacterium bovis Bacillus Calmette-Guérin (BCG), his last treatment occurring 1.5 months prior to presentation. He has a past medical history of chronic obstructive pulmonary disease, hypertension, obstructive sleep apnea, obesity, and diabetes. The patient has been hospitalized four times over the last two months and his symptoms include generalized weakness, malaise, shortness of breath and recurrent fever. He was found to have patchy lung infiltrates and he was diagnosed with pneumonia, COPD exacerbation and symptoms of heart filature. He was treated previously with antibiotics, steroids and fluid management which would temporarily relieve his symptoms. He presents to the hospital again, four days after his last hospital discharge, with generalized weakness, malaise, shortness of breath and recurrent fever. On initial evaluation he was found to be pancytopenic.  

Laboratory Identification

Blood cultures were negative. A bone marrow biopsy was performed for fever of unknown origin and pancytopenia. The biopsy showed non-caseating granulomas which were negative for acid-fast bacilli (AFB) by Ziehl-Neelsen stain and fungal elements by Gomori Methenamine Silver Stain (GMS). A laboratory-develped PCR test for Mycobacterium tuberculosis complex (MTBC) was performed on the bone marrow and was negative. AFB culture of bone marrow was positive for after 30 days of incubation and the organism was confirmed to be acid-fast bacilli by auramine-rhodamine fluorescent dye and Kinyoun stain. A second laboratory-developed test that uses heat shock protein (HSP) 2 and HSP3 to determine species level identification of Mycobacteria identified the organism as M. tuberculosis complex. Due to the patient’s history, further identification was performed at a reference lab using specific oligonucleotides targeting the gyrb DNA sequence polymorphisms which is able to separate different members of the MTBC. The patient’s isolate contained a RD1 deletion which is specific for Mycobacterium bovis bacillus Calmette-Guérin (BCG).

Discussion

Mycobacterium bovis is a slow growing mycobacterium which produces rough, dry colonies on growth solid media. It is one of the species in the MTBC with a natural host of domestic and wild animals. Routine molecular tests will not accurately differentiate between members of the MTBC. For definitive identification of M. bovis, 16S rRNA and gyrB gene sequencing is necessary. Safe handling procedures should be followed prior to molecular testing of MTBC.

Mycobacterium bovis BCG is a live, attenuated strain of Mycobacterium bovis that was created for vaccine and is used in the treatment of superficial bladder cancer. The treatment may cause localized symptoms including hematuria, fever, nausea, and dysuria which are marker of anti-tumor effect. Serious complications occur in <5% of patients with complications including sepsis, pneumonitis, hepatitis, lymphocytic meningitis, bone marrow involvement, and mycotic aneurysms. The cardinal sign of BCG infection is a relapsing fever with drenching night sweats persisting beyond 48 hours. Disseminated infection can occur days to years after the therapy. Clinical suspicion should be high for M. bovis BCG dissemination if there are symptoms and a high grade fever ≥72 hours. Treatment includes a regiment of isoniazid, rifampin and ethambutol. Most isolates of M. bovis are resistant to pyrazinamide.

References

  1. Lamm DL. Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer. Clin Infect Dis 2000; 31 Suppl 3:S86.
  2. Shelley MD, Court JB, Kynaston H, et al. Intravesical Bacillus Calmette-Guerin in Ta and T1 Bladder Cancer. Cochrane Database Syst Rev 2000; :CD001986.
  3. Richter E, Weizenegger M, Rusch-Gerdes S, Niemann S. Evaluation of Genotype MTBC Assay for Differentiation of Clinical Mycobacterium tuberculosis Complex Isolates. Journal of Clinical Microbiology 2003; 41(6): 2672-2675
  4. UpToDate

-Crystal Bockoven, MD is a 4th year anatomic pathology resident at University of Chicago (NorthShore). Crystal has an interest in and will be doing a fellowship in pediatric and perinatal pathology. In her spare time, she enjoys reading, hiking and biking. 

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Microbiology Case Study: 35 Year Old Male with Chest Pain

Case History

A 35 year old man presented to the Emergency Department (ED) with intermittent chest pain for 3-4 days, abdominal pain, fatigue, and lightheadedness over the same time period. Additionally, his family reported symptoms of progressive malaise for about a month, worse over the last week. In the ED, he was found to have ST elevations in the inferior leads of the electrocardiogram, which can be indicative of a heart attack. He was given 325 mg of aspirin and was emergently taken to the catheterization lab. He was found to have multiple complete occlusions in the distal left anterior descending artery (LAD), posterior descending artery (PDA), and posterior left ventricular artery (PLV). He underwent aspiration thrombectomy and the resulting clots were thought to be emboli; segments were sent to pathology for histopathologic evaluation and to microbiology for culture. There was no evidence of underlying plaque. He was admitted for management of ST-elevation myocardial infarction (STEMI). While in the ED, he was found to have white blood cell count of 23,000 and tachycardia to 110 beats per minute. A transthoracic echocardiogram demonstrated thickened aortic valve leaflets with evidence of leaflet destruction, severe aortic insufficiency, and right coronary cusp perforation which are consistent with endocarditis. Blood cultures were obtained and he was started on broad spectrum antibiotics (Vancomycin and Cefepime).

He has a past medical history significant for previous shoulder abscess with Methicillin-resistant Staphylococcus aureus (MRSA) and intravenous drug use (IVDU) (heroin, last use ~6 days prior to admission).

Computed tomography (CT) of his abdomen and pelvis revealed multiple renal infarctions and a splenic infarction (Image 1). In addition, the CT of the brain showed: “Multifocal scattered supratentorial and infratentorial subarachnoid hemorrhages and findings suggestive of evolving ischemic infarct involving the right inferior frontal gyrus, without evidence of hemorrhagic transformation currently. No midline shift or other complication identified.”

Image 1. Computed tomography of the abdomen demonstrating multiple renal infarctions (left, circled) and a splenic infarction (right, circled).

On hospital day 1 (HD1), both sets of initial blood cultures turned positive with gram positive cocci (GPC) in clusters and thrombectomy cultures were also growing GPC in clusters (Image 2). On HD2, the GPC in the thrombectomy culture was identified as Rothia mucilaginosa. GPC growing in the blood cultures were also Rothia mucilaginosa (Image 2). The patient was continued on Vancomycin. Repeat blood cultures were obtained after catheterization on HD0, and HD2, which were negative. On HD2, the pathology of the initial clots showed “fibrinopurulent debris and fibrin plaques with innumerable cocci in clusters” (Image 3).

Image 2. Microscopic and culture morphology of Rothia mucilaginosa. Left: Gram stain from a blood culture demonstrating groups of Gram-positive cocci in small clusters (1000x magfication, oil immersion). Right: Blood agar plate with mucoid light pink-gray colonies.
Image 3. Hematoxylin and eosin stained slide of formalin fixed paraffin embedded tissue of the thrombus removed during the initial emergent catheterization procedure. Sections demonstrate fibrinous material with entrapped white cells and innumerable cocci. Top: 100x magnification; Bottom: 400x magnification.

On HD3, the patient developed 10/10 chest pain with troponin elevation and T-wave inversion. He was taken back to the catheterization lab for another procedure where he was found to have recurrent, complete occlusion of the PDA with unsuccessful recanalization due to the dense thrombus. On HD6, he developed tamponade physiology due to a large pericardial effusion that was drained. Cultures of the pericardial fluid were negative. Given the recurrent embolization events, the patient was transferred to another hospital to undergo aortic valve replacement surgery and coronary artery bypass graft surgery. Cultures taken at the time of the valve replacement surgery were negative and the valve tissue was not sent for pathologic evaluation.  

Discussion

We present an uncommon case of extensive Rothia mucilaginosa sepsis with septic emboli and endocarditis. Rothia mucilaginosa has experienced the scientific name-change game over the last several decades. It was first identified as Micrococcus mucilaginosus, then became Stomatococcus mucilaginosus, was also known as Staphylococcus salivarius before finally arriving to today’s name of Rothia mucilaginosa.1,2 R. mucilaginosa is a normal inhabitant of the oropharynx and is often associated with dental caries.3 R. mucilaginosa can cause invasive infections, typically in patients with compromised immune systems, disrupted mucosal barriers or injection drug use.4

R. mucilaginosa is a facultatively anaerobic, gram positive, non-fastidious coccus that is coagulase negative but with variable catalase positivity. Colony morphology is usually white to gray nonhemolytic colonies with a mucoid appearance. Although the variable catalase reaction may point toward a Streptococcus spp., the Gram stain morphology of clusters helps to rule it out. Although not all strains are mucoid, the classic colony morphology is wet and is due to polysaccharide capsule.

The organism is generally susceptible to antibiotics designed to target gram positive bacteria including, penicillin, ampicillin, cefotaxime, rifampin and vancomycin.4 It is important to note that R. mucilaginosa is not predictably susceptible to clindamycin, trimethoprim-sulfamethoxazole or ciprofloxacin.5 The patient presented in this case received intravenous vancomycin in part due to the extensive disease on presentation, but also because he was at risk for methicillin-resistant Staphylococcus aureus (MRSA) sepsis and had a previously documented abscess from MRSA.

References

  1. Bergan T, Kocur M. 1982. Stomatococcus mucilaginosus gen. nov., sp.nov., ep. Rev., a member of the family Micrococcaceae. Int. J. Syst. Bacteriol. 32:374-377
  2. Collins MD, Hutson RA, Baverud V, Falsen E. 2000. Characterization of a Rothia-like organism from a mouse: description of Rothia nasimurium sp.nov. and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb.nov. Int. J. Syst. Evol. Microbiol. 3:1247-1251.
  3. Trivedi MN, Malhotra P. Rothia prosthetic knee joint infection. 2015. J. Microbiol. Immunol. Infect. 48(4):453-455.
  4. Bruminhent J, Tokarczyk MJ, Jungkind D, DeSimone JA. Rothia mucilaginosa Prosthetic Device Infections: A Case of Prosthetic Valve Endocarditis. J. Clin. Microbiol. 5;15:1629-1632.
  5. Kaasch AJ, Saxler G, Seifert H. 2011. Septic arthritis due to Rothia mucilaginosa. Infection. 39:81-82.

-Doreen Palsgrove, MD is a board certified Anatomic and Clinical Pathologist who joined the faculty at UT Southwestern as an Assistant Professor in 2019. She specializes in head and neck and genitourinary pathology. 

Dominick Cavuoti, DO is a professor of AP and CP at UT Southwestern, specializing in infectious disease pathology, cytology and medical microbiology.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Microbiology Case Study: An 80 Year Old Man with Dyspnea, Fatigue, and Weight Loss

Case History

An 80 year old male was seen by his cardiologist for approximately one month of dyspnea, fatigue, and weight loss. Past medical history was significant for aortic stenosis requiring placement of a bioprosthetic valve and multivessel coronary artery disease 13 years prior. He underwent cardiac catheterization and echocardiography that revealed severe bioprosthetic valve stenosis. The patient was in the process of evaluation for a prosthetic valve replacement when he presented to the emergency room for rapid decline of the previously noted symptoms. Exam upon hospital admission was notable for cardiac murmur, lower extremity edema, mild leukocytosis, and anemia. He had normal dentition and no skin lesions. A pre-operative TEE confirmed severe aortic prosthetic valve stenosis, restricted leaflet motion, thrombus on all three leaflets, and thickening of the periannular aortic root and ascending aorta. Subsequent cardiac CT was concerning for either pseudoaneurysm or paravalvular leak suggestive of an infectious or inflammatory process.

Due to the persistent, mild leukocytosis, blood cultures were obtained on the second day of admission. On hospital day 3, one set of blood cultures flagged positive with Gram-variable rods in the aerobic bottle (Image 1). The patient was empirically started on vancomycin and piperacillin/tazobactam. Repeat blood cultures were obtained on hospital days 4 and 7, both again positive for Gram-variable rods within 2 days of collection. The infectious diseases consult team suspected subacute bacterial endocarditis and changed therapy to ceftriaxone.On hospital day 9, the patient underwent a redo sternotomy for aortic valve replacement and aortic root repair. Intraoperative findings included a large amount of phlegmon on the aortic leaflets, near circumferential aortic annulus tissue destruction and abscess cavity. Culture of the intraoperative specimens was negative for bacterial growth. The anatomic pathology findings revealed fibrinoid vegetations and acute inflammation and reparative changes. The patient was subsequently discharged home in stable condition 20 days after his admission. Interval outpatient clinic visits demonstrate that he is recovering well, including a return to baseline levels of endurance and function.

Laboratory Identification

Gram stain of the positive blood cultures revealed pleomorphic gram variable rods which were arranged in clusters, pairs, short chains, and characteristic rosette patterns (Image 1 and inset). Pinpoint, opaque colonies were visible on blood and chocolate agars after 48-72 hours of incubation at 35°C in CO2 (Image 2). No growth was observed on MacConkey agar. The colonies were catalase-negative, and oxidase- and indole-positive. The recovered organism was definitively identified by MALDI-TOF MS as Cardiobacterium hominis.

Image 1. Gram stain from the positive aerobic blood culture bottles exhibiting gram variable rods (1000X magnification, oil immersion). Organisms were visualized in characteristic “rosette” patterns. Image inset is a magnified view of the rosette arrangement from another field.
Image 2. Growth on blood agar following 48 hours incubation at 35°C in 5% CO2. Small, white, pinpoint colonies were observed on blood and chocolate agars.

Discussion

In 1962, four cases of infective endocarditis (IE) due to a Pasteurella-like organism belonging to CDC Group-IID were reported. Two years later, this group of organisms was reclassified as Cardiobacterium in recognition of their ability to cause endocarditis. Two species, Cardiobacterium hominis and Cardiobacterium valvarum, have been reported to cause IE, with the former being the etiological agent in a vast majority of cases.1 There is a strong association between C. hominis bacteremia and IE, as the organism is rarely recovered from blood cultures outside of this setting. Most cases of C. hominis endocarditis involve the aortic valve, particularly in the presence of pre-existing abnormalities or when a prosthetic valve is in place.2 C. hominis is a member of the normal flora of the nose and throat of ~70% of individuals (1), and endocarditis can be caused by periodontitis or dental procedures without prophylaxis.3

C. hominis is a member of the HACEK group of organisms which also include Haemophilus spp., Aggregatibacter spp., Eikenella corrodens, and Kingella kingae. HACEK organisms exhibit similar manifestations of disease, prognosis, and epidemiology. While over 80% of cases of IE are caused by Gram-positive bacteria (notably staphylococci and oral streptococci), Gram-negative IE is far less frequent, with a majority of cases caused by HACEK organisms (1-3% of all IE cases).4 In general, IE caused by HACEK organisms has an excellent prognosis, but delays in diagnosis and associated complications can lead to poorer outcomes.2 Susceptibility testing of C. hominis is difficult to perform due to its nutritional requirements. Most strains are susceptible to fluoroquinolones, rifampin, tetracycline, and beta-lactams. As beta-lactamase producing isolates have been reported, the current American Heart Association Guidelines recommend the use of a 4-6 week course of ceftriaxone for treatment of HACEK IE; fluoroquinolones may be used in cases where patients cannot tolerate cephalosporin therapy.5

Historically, prolonged blood culture incubation for the recovery of HACEK group organisms has been recommended due to their fastidious nature and slow growth rate. However, modern automated blood culture systems utilize enriched media which readily support their growth and facilitate recovery within a standard 5-day incubation period (average of 3.4 days incubation).6 Additional studies have demonstrated that prolonged incubation times do not significantly enhance the recovery of HACEK organisms and are of little clinical value.7 This case demonstrates many hallmarks of a characteristic description of a HACEK bacterial endocarditis: 1) the patient had a prosthetic valve as a pre-existing risk factor, 2) the subacute presentation caused a delay in recognition of an infectious etiology as contributing to his clinical decline, 3) C. hominis grew in less than 5 days in our automated blood culture system without prolonged incubation, 4) blood culture Gram stain findings were consistent with the MALDI identification of a HACEK group member, and 5) the patient was treated with ceftriaxone and with surgical intervention and has recovered successfully.

References

  1. Malani AN, Aronoff DM, Bradley SF, Kauffman CA.2006. Cardiobacterium hominis endocarditis: two cases and a review of the literature. European Journal of Clinical Microbiology and Infectious Diseases 25:587-595.
  2. Sharara SL, Tayyar R, Kanafani ZA, Kanj SS.2016. HACEK endocarditis: a review. Expert Review of Anti-infective Therapy 14:539-545.
  3. Steinberg JP, Burd EM. 2015. 238 – Other Gram-Negative and Gram-Variable Bacilli, p 2667-2683.e4. In Bennett JE, Dolin R, Blaser MJ (ed), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (Eighth Edition) doi:https://doi.org/10.1016/B978-1-4557-4801-3.00238-1. Elsevier, Philadelphia, PA.
  4. Revest M, Egmann G, Cattoir V, Tattevin P.2016. HACEK endocarditis: state-of-the-art. Expert Review of Anti-infective Therapy 14:523-530.
  5. Baddour Larry M, Wilson Walter R, Bayer Arnold S, Fowler Vance G, Tleyjeh Imad M, Rybak Michael J, Barsic B, Lockhart Peter B, Gewitz Michael H, Levison Matthew E, Bolger Ann F, Steckelberg James M, Baltimore Robert S, Fink Anne M, O’Gara P, Taubert Kathryn A.2015. Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications. Circulation 132:1435-1486.
  6. Petti CA, Bhally HS, Weinstein MP, Joho K, Wakefield T, Reller LB, Carroll KC.2006. Utility of extended blood culture incubation for isolation of Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella organisms: a retrospective multicenter evaluation. Journal of clinical microbiology 44:257-259.
  7. Weinstein MP.2005. Emerging Data Indicating that Extended Incubation of Blood Cultures Has Little Clinical Value. Clinical Infectious Diseases 41:1681-1682.

-Francesca Lee, MD, is an associate professor in the Departments of Pathology and Internal Medicine (Infectious Diseases) at UT Southwestern Medical Center. She serves as Medical Director of the microbiology laboratory and pre-analytical services at Clements University Hospital.

-Julia Sweetnam, MLS(ASCP)CM has worked for six years as medical technologist in the microbiology laboratory at Clements University Hospital. She is interested in antimicrobial susceptibility testing and diagnostic bacteriology.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Microbiology Case Study: 40 Year Old Male with A Diabetic Foot Ulcer

Clinical Presentation and History

The patient is a 40 year old male with a past medical history of type 2 diabetes mellitus with significant neuropathy and hypertension with a past surgical history of right metatarsal osteomyelitis. He presents to hospital with fever, right ear pain, headache, two episodes of diarrhea and redness and blistering to the right 3rd metatarsal. Upon examination he was noted to have a 1 cm ulceration on the right 3rd toe on the dorsal aspect associated with redness and edema. He was therefore assessed as having diabetic foot ulcer with possible osteomyelitis for which blood cultures were performed.

Laboratory Identification

Gram stains performed on the positive blood culture broth showed gram negative rods (Image 1). In our institution initial positive blood cultures are tested by the Verigene System (Luminex Corp., Austin, TX), which allows for rapid identification of common bacterial pathogens causing blood stream infections (Escherichia coli, Klebsiella oxytoca, Klebsiella pneumonia, Pseudomonas aeruginosa, Acinetobacter spp., Citrobacter spp., Enterobacter spp., and Proteus spp.) along with detection of several resistance genes (CTX-M, IMP, KPC, NDM, OXA, VIM) within ~ 3 hours. In this case, no targets on the Verigene panel were detected. Simultaneously, the specimen was plated onto blood, chocolate and MacConkey agars where the organism grew robustly on all three plates (Image 2). The MacConkey agar showed the organism to be a non-lactose fermenter. Once the organism adequately grew on these agar plates, final species identification was performed on the automated MALDI-TOF instrument which showed Salmonella species. To appropriately type the organism, Salmonella latex agglutination testing was performed which identified Salmonella species Group B (Non-typhoidal). Of note, multiple blood cultures from this patient were positive for Salmonella species, Group B.

Image 1. Gram stain of blood culture broth containing gram negative rods.
Image 2. Growth of the organism on chocolate, 5% sheep blood, and MacConkey agars.

Discussion

Salmonella is a gram negative, flagellated facultative anaerobic, non-lactose fermenting bacilli. The taxonomy and nomenclature of salmonella organisms are quite complex however the most widely used classification scheme is the Kauffman-White which is updated yearly by the WHO. Currently, members of the 7 Salmonella subspecies can be serotyped into one of more than 2500 serotypes (serovars) according to antigenically diverse surface structures: somatic O antigens (the carbohydrate component of lipopolysaccharide [LPS]) and flagellar (H) antigens.

Nontyphoidal salmonellae are a major cause of diarrhea worldwide. In the United States, non-typhoidal salmonellosis is one of the leading causes of foodborne disease. Salmonella enteritidis and Salmonella typhimurium are among the most frequently isolated organisms. Salmonella is most commonly associated with ingestion of contaminated poultry, eggs, and milk products. Salmonella gastroenteritis typically occur within 8 to 72 hours following exposure, however lower bacterial doses can prolong the incubation period. Although Salmonella typically causes diarrheal diseases including gastroenteritis and enteric fever, however there are rare instances where hematogenous involvement leads to bacteremia, osteomyelitis or endovascular infections.

In this case the source of Salmonella-related bacteremia is still a mystery. The presumed source was osteomyelitis, but the patient’s subsequent toe amputation revealed minimal osteomyelitis and rare fungal organisms.

References

  1. Procop, Gary W. et al (2017). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 7th edition. Philadelphia, PA.
  2. Hohmann, Elizabeth L. (2018). Nontyphoidal salmonella: Gastrointestinal infection and carriage. Uptodate.com. Retrieved on November 14, 2019. https://www-uptodate.com/contents/nontyphoidal-salmonella-gastrointestinal-infection-and-carriage

-Anna-Lee Clarke-Brodber, MD is a 3rd year AP/CP resident at University of Chicago (NorthShore). Academically, Anna-Lee has a particular interest in Cytopathology. In her spare time she enjoys hanging out with her family.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Microbiology Case Study: A 24 year old with Sore Throat and Difficulty Breathing

Case History

A 24 year old male with a past medical history of recurrent streptococcal pharyngitis presents to the emergency department with a sore throat and dyspnea. His symptoms began three days prior and included left-sided upper neck and lower jaw pain and odynophagia. The patient’s evaluation demonstrated tachycardia, cervical lymphadenopathy, and a small left tonsillar abscess. Labs were significant for an elevated WBC count but blood cultures, Group A streptococcal and mononucleosis screens were negative. The patient was admitted for pain management and treated with a combination of IV ampicillin/sulbactam (amp/sulb) and steroids. He improved with treatment and was discharged the following day on oral amoxicillin/clavulanic acid (amox/clav). Nine days later, the patient re-presented with similar complaints. The tonsillar abscess had increased in size to 2cm. Labs were significant for leukocytosis and a now positive Group A streptococcal screen. 2mL of pus was aspirated from the lesion but no cultures were ordered. The patient’s status again improved, and he was discharged home again on oral amox/clav. The patient returned the following day and was placed on IV amp/sulb and admitted for imaging and symptom management. A neck CT with contrast revealed a now 3cm tonsillar abscess with reactive cervical lymphadenopathy (Image 1). A throat culture was collected; however, no beta-hemolytic streptococci were recovered after 48 hours of incubation. Incision and drainage of the abscess was performed at bedside, recovering an additional 10 mL of purulence that was sent to the microbiology laboratory for aerobic and anaerobic culture. The patient improved on IV amp/sulb and was switched to high dose amox/clav on day 15.  

Laboratory Identification

Gram stain of the aspirated purulence revealed many WBCs and a mixture of gram positive rods and cocci (Image 2). The aerobic culture grew a heavy amount of tiny, weakly beta-hemolytic colonies on blood agar. Smears of these colonies revealed Gram-positive coryneform rods. Biochemical testing determined the growth to be catalase-negative and MALDI-TOF MS definitively identified the organism as Arcanobacterium haemolyticum. The anaerobic culture grew oral flora.

Image 1. Computed tomography of the neck in a 24 year old male who presents with difficulty breathing. Area of large tonsillar abscess (yellow circle).
Image 2. Gram stain demonstrating small, pleomorphic gram positive rods in a background of neutrophils and Gram-positive cocci in pairs or short chains. (1000x magnification, oil immersion)
Image 3. A. haemolyticum isolate after 48 hours of incubation. The weak beta-hemolysis was not readily apparent using room (reflected) light. Placing the plate on a lightbox revealed beta-hemolysis.
Image 4. Streptococcus agalactiae exhibiting synergetic hemolysis with a beta-lysin producing strain of S. aureus (CAMP reaction, top). A. haemolyticum inhibits hemolysis by S. aureus in a CAMP-test set up (CAMP inhibition, middle). A. haemolyticum exhibits synergistic hemolysis with S. agalactiae. (Reverse CAMP, bottom).

Discussion

A. haemolyticum is an infrequently isolated  gram positive rod which is an etiologic agent of non-streptococcal pharyngitis diagnosed predominantly in adolescents or young adults. The diagnosis of A. haemolyticum can be challenging because itis often clinically indistinguishable from cases caused by beta-hemolytic streptococci. Most patients exhibit some degree of cervical lymphadenopathy, and a scarlatiniform rash can be present in up to 50% of cases. From a laboratory perspective, A. haemolyticum is slowly growing and weakly beta hemolytic after 24-48 hours on media containing sheep blood (including SBA and Strep Selective agars routinely used for screening throat cultures). The beta-hemolytic activity of A. haemoltyicum is attributed to expression of arcanolysin, a cholesterol-dependent cytolysin. Interestingly, arcanolysin more robustly binds to rabbit and human erythrocytes than those from sheep,1 which may explain the organism’s weak beta hemolysis on routine media.  In this setting, the organism can be missed or dismissed as commensal flora without careful observation. Conversely, if beta-hemolysis is observed, the colony morphology and catalase non-reactivity can lead to misidentification as beta-hemolytic streptococci in the absence of a Gram stain or other determinative methods (i.e. MALDI-TOF MS).

The beta hemolysis of this patient’s A. haemolyticum isolate is difficult to appreciate in reflected (room) light, and was best observed after 48 hours using transduced light from a light box (Image 3). A. haemolyticum displays CAMP inhibition due to the production of phospholipase D which inhibits the hemolytic activity of beta-lysin produced by S. aureus (Image 4) and is reverse-CAMP positive when perpendicular to Group B streptococci which can aid in identification.2

Erythromycin is the drug of choice for treatment of A. haemolyticum, further highlighting the need for definitive identification of this organism in settings of pharyngitis. The use of penicillin for treatment of A. haemolyticum pharyngitis can result in treatment failure, possibly due to invasion of host cells, thus establishing a reservoir,3 or due to a penicillin-tolerant phenotype.4 It is unclear in this case if source control or decreased susceptibility necessitated the multiple courses of antibiotics utilized. Fortunately, the patient’s symptoms resolved on high dose amoxicillin/clavulanic acid following thorough incision and drainage. He subsequently returned for an outpatient tonsillectomy.

References

  1. Jost BH, Lucas EA, Billington SJ, Ratner AJ, McGee DJ. 2011. Arcanolysin is a cholesterol-dependent cytolysin of the human pathogen Arcanobacterium haemolyticum. BMC Microbiology 11:239.
  2. Kang H, Park G, Kim H, Chang K. 2016. Haemolytic differential identification of Arcanobacterium haemolyticum isolated from a patient with diabetic foot ulcers. JMM Case Reports.
  3. Österlund A. 1995. Are Penicillin Treatment Failures in Arcanobacterium haemolyticum Pharyngotonsillitis Caused by Intracellularly Residing Bacteria? Scandinavian Journal of Infectious Diseases 27:131-134.
  4. Nyman M, Danek G, Thore M. 1990. Penicillin Tolerance in Arcanobacterium haemolyticum. The Journal of Infectious Diseases 161:261-265.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern in the Department of Pathology and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Microbiology Case Study: Skin and Soft Tissue Infection Caused by an Unusual Bacterium

Case History

A 20 year old female with no significant past medical history presented with a painful pruritic rash on the bilateral inner thighs that had been persistent for one month. Prior to presentation, she had been treated with oral and topical antihistamines, topical steroids, valacyclovir, and partial courses of doxycycline and cephalexin without improvement. Physical examination was notable for diffuse erythema and dermal edema of the bilateral medial thighs with superimposed exophytic papules with dark, necrotic cores, the largest of which measured 1 cm in diameter (Image 1). Punch biopsy of the lesions was taken and sent for histology. A sample from necrotic tissue was sent to microbiology laboratory for gram stain and cultures.

Laboratory diagnosis

Gram stain showed gram positive cocci in clusters. After 32 hours of incubation, tissue cultures grew white, β-hemolytic colonies which were catalase positive, coagulase negative, and pyrrolidonylarylamidase (PYR) positive. The organism was identified as Staphylococcus lugdunensis by MALDI-TOFmass spectrometry. Histology revealed eosinophilic inclusions consistent with molluscum bodies as well as inflammatory infiltrate (Image 2). Brown and Hopps stain on tissue showed Gram-positive cocci is small clusters (Image 3). A diagnosis of molluscum contagiosum superinfected with Staphylococcus lugdunensis was made based on laboratory and histologic findings.

Image 1. Lesions on left medial thigh (left) and right medial thigh (right).
Image 2. Molluscum bodies
Image 3. Brown and Hopps stain on tissue showing gram positive cocci

Discussion

S. lugdunensis is a coagulase-negative staphylococcus first isolated in 1988 that was initially thought to be a commensal skin organism but has been shown to cause skin and soft tissue infections (SSTIs), bacteremia, endocarditis, prosthetic joint infections, and osteomyelitis,2 with a virulence more similar to S. aureus than to that of other coagulase-negative staphylococci. SSTIs are one of the more common manifestations of S. lugdunensis infection; one analysis of 229 S. lugdunensis clinical isolates demonstrated that 55.4% were associated with SSTIs.3 The spectrum of S. lugdunensis-related SSTIs includes folliculitis, pustulosis, cellulitis, abscesses, and rarer secondary infection of molluscum contagiosum and hidradenitis suppurativa.5 Molluscum superinfection itself is a rare phenomenon, and when it occurs, the superinfecting agent is most often S. aureus.1 Our case suggests that S. lugdunensis should also be considered as a potential causative agent of molluscum superinfection. There is growing recognition that S. lugdunensis is a virulent pathogen that should not be disregarded as a contaminant if found on culture. Importantly, when compared with S. aureus, S. lugdunensis has a more limited resistance profile; methicillin resistance is still uncommon, and 74.6% of isolates in one recent study were penicillin susceptible.4 Awareness of this more favorable resistance profile can facilitate selection of narrower-spectrum antibiotic therapies for S. lugdunensis infections.

In our case, patient received one dose of vancomycin and metronidazole in the emergency department and was then started on cefazolin for cellulitis. After wound culture identified S. lugdunensis, the patient was discharged on cefadroxil 1g twice daily for 10 days. On follow up, the rash had resolved.

References

  1. Berger EM, Orlow SJ, Patel RR, Schaffer JV. Experience With Molluscum Contagiosum and Associated Inflammatory Reactions in a Pediatric Dermatology Practice: The Bump That Rashes. Arch Dermatol. 2012;148(11):1257–1264. doi:10.1001/archdermatol.2012.2414
  2. Douiri N, Hansmann Y, Lefebvre N, Riegel P, Martin M, Baldeyrou M, Christmann D, Prevost G, Argemi X. Staphylococcus lugdunensis: a virulent pathogen causing bone and joint infections. Clinical Microbiology and Infection, 2016;22(8):747-748. doi:10.1016/j.cmi.2016.05.031
  3. Herchline TE, Ayers LW. Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection. J Clin Microbiol. 1991;29(3):419–421.
  4. Taha L, Stegger M, Söderquist B. Staphylococcus lugdunensis: antimicrobial susceptibility and optimal treatment options. Eur J Clin Microbiol Infect Dis. 2019;38(8):1449–1455. doi:10.1007/s10096-019-03571-6
  5. Zaaroura H, Geffen Y, Bergman R, Avitan‐Hersh E. Clinical and microbiological properties of Staphylococcus lugdunensis skin infections. J Dermatol, 2018;45: 994-999. doi:10.1111/1346-8138.14496

-Ansa Mehreen, MD. 1st year AP/CP resident at University of Chicago hospital program based at Evanston Hospital. Her academic interests include gastrointestinal pathology.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Microbiology Case Study: A 27 Year Old Man with Lumbar Pain

Clinical History

A 27 year old male with past medical history of mixed connective tissue disease was transferred from an outside hospital where he had initially presented for 6 days of progressively worsening lumbar pain preceded by 1 day of fevers up to 104 F, as well as chills and rigors. Also noted was pain and swelling in his left thumb and a mild rash present on the hands and feet. It is notable that the patient has multiple pets, including a rat.

Workup at the outside hospital included and MRI showing facet arthrosis and effusions at L4-L5 with mass effect on the S1 nerve root. Neurosurgery recommended biopsy of the lumbar spine by Interventional Radiology.

Laboratory Findings

Two sets of blood cultures at the outside hospital showed no growth at 5 days. Two sets of blood cultures obtained at our institution were positive at 15 and 29 hours, with a smear showing gram negative bacilli. The blood culture media was tested by nucleic acid hybridization and no targets were detected. When subcultured, there was only significant growth on the blood plate, with very small gray-green colonies that were not well appreciated

Image 1. Gram stain showing gram negative bacilli.
Image 2. Blood agar plate showing difficult to discern growth of very small grayish colonies.

A sample was sent to a reference lab for testing, which identified Streptobacillus moniliformis.

Discussion

In the United States, Streptobacillus moniliformis is the most common causative agent of rat bite fever, a relatively rare infectious systemic illness. This syndrome is also caused by infection with Spirillum minus, primarily seen in Asia, and rarely by Streptobacillus notomytis.1,2

S. moniliformis is a fastidious pleomorphic gram negative rod which grows slowly; in cases where there is clinical suspicion for rat bite fever, cultures can be held up to seven days. It is part of the normal nasal and oropharyngeal flora of rodents, with carriage rates of up to 100% in some rat populations. It can be found in oral, nasal, and conjunctival secretions, as well as urine, and can be transmitted to humans via bites, scratches, or oral contact. This includes kissing a pet rat or ingesting food or water that is contaminated with rat secretions.1,2

S. minus is a gram negative spirochete. It cannot be cultured on synthetic media, but may be visualized by Giemsa or Wright stain, or by using dark-field microscopy. Transmission of S. minus is similar to that of Steptobacillus spp., but has not been documented to be associated with contaminated food or water. (1, 2)

The clinical presentation of rat bite fever ranges from a mild case with only a flu-like illness to cases of severe sepsis. In untreated cases, the mortality rate is approximately 10 to 13 percent. Initial symptoms typically begin within 7 days of exposure for S. moniliformis and in 1 to 3 weeks for S. minus. These initial symptoms can include fever, myalgias, vomiting, pharyngitis, headache, and migratory arthralgias. If exposure was through a wound, it is typically resolved by the time symptoms develop, although it may reappear with ulceration, edema, and regional adenopathy in cases caused by S. minus. Cases associated with ingestion may have more severe vomiting and increased likelihood of developing pharyngitis.1,2

Symptoms may further develop to include a maculopapular rash on the extremities, and asymmetric polyarthralgias. The rash is seen most commonly on the extensor surfaces but can involve the palms and soles. Spontaneous remission may occur, but without treatment the fever can show a relapsing course, and the arthritis may last for several years. Possible complications include bacteremia, endocarditis, myocarditis, pneumonia, abscesses, septic arthritis, osteomyelitis, multiorgan failure, fulminant sepsis and death.1,2

Rat bite fever is usually diagnosed empirically based on consistent symptoms and history of rat exposure, because it is difficult or impossible to culture the causative organisms in the lab and there is no serologic test available. 16S rRNA sequencing can be used for definitive diagnosis, but only for certain sample types, and it is not always available.1,2

The empiric treatment of choice is penicillin, with the dose and duration being dependent on the clinical presentation. Ceftriaxone is also commonly used due to better ease of use in the outpatient setting, and tetracyclines are used in patients with beta-lactam allergies. For uncomplicated cases, most patients are treated for a total of 14 days; initially with IV antibiotics, and then transitioned to oral agents after 5 to 7 days if there is sufficient clinical improvement.1,2

Also remarkable in the patient’s history was a note indicating that they had been known to share drinks with his pets and instances of the pet rat licking the patient’s face. Initial treatment was vancomycin and cefepime, with vancomycin discontinued after the gram stain results. The patient was discharged before a definitive identification of the organism was made, with a plan for 6 weeks of outpatient treatment with ceftriaxone via infusion.

References

1.  King, Katherine Yudeh, MD, PhD “Rat Bite Fever.” UpToDate, Wolters Kluwer, 1 Jun. 2020. https://www.cdc.gov/rat-bite-fever/index.html

2.  “Rat-bite Fever (RBF).” Centers for Disease Control and Prevention, 1 Jun. 2020. https://www.uptodate.com/contents/rat-bite-fever?search=rat%20bite%20fever&source=search_result&selectedTitle=1~17&usage_type=default&display_rank=1

-Tom Koster, DO is a 1st 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 Associate Professor at the University of Vermont.

Microbiology Case Study: Elderly woman with Ear Pain

An elderly woman with a past medical history significant for end-stage renal disease status post deceased donor kidney transplant in 2018 (on immunosuppression), type 2 diabetes mellitus, and recurrent urinary tract infections presented to nephrology clinic with right ear pain and rash of three weeks. She was otherwise in her usual state of health. On physical exam, there were exophytic itchy papules with hemorrhagic crust and ulcerations on the ear (Image 1) and arm. A few of these papules showed central umbilication (Image 2). Erosions were also present on the upper back, face, neck, and forearms. Patient was referred to dermatology with concern for disseminated infection versus neoplasia. Complete blood count showed mildly elevated white cells. Serologies, cultures, imaging, lumbar puncture, and biopsy were performed.

Blood studies revealed a Cryptococcus antigen titer of 1:4096 along with a CSF antigen titer of 1:2048. Additionally, the CSF gram stain demonstrated yeast and cultures grew Cryptococcus neoformans. Opening pressure was normal and protein was slightly elevated to 53 mg/mL with 36 nucleated cells with a differential including 64% lymphocytes. Biopsy culture of the left cheek was positive for C. neoformans and a left forearm biopsy showed nodular aggregates of encapsulated yeasts, surrounded by relatively sparse lymphohistiocytic inflammation (Images 3-4). A CT of the chest showed innumerable pulmonary nodules concerning for infection.

Image 1. Erythematous and crusted pink plaque with ulceration on the pinna of the ear.
Image 2. Pink, domed papule on the arm with central umbilication and crust.
Image 3. Hematoxylin and eosin stain of the arm biopsy with Cryptococcus, low magnification. Note the loss of epidermis (left-hand side) and underlying foamy stroma with numerous yeasts within the dermis.
Image 4. Hematoxylin and eosin stain of Cryptococcus, high magnification. The yeasts show variable size and some demonstrate a halo of pale staining capsule. There is no significant inflammation in the background parenchyma.

Discussion

Cryptococcus is an encapsulated basidiomycetous fungus typically found in soil and pigeon droppings.1 Two species comprise the majority of Cryptococcus infections: C. neoformans and C. gatti. C. neoformans is most commonly seen in immunosuppressed patients, particularly in the setting of AIDS.2 C. gatti infections may be seen in more immunocompetent patients and appears to be more geographically restricted to the tropics and Pacific Northwest.3 C. neoformans infections can present as lung disease associated with symptoms of fever, shortness of breath, or cough and characteristically may spread to the central nervous system to cause meningitis.4 Lumbar puncture may show significantly elevated opening pressures.5 Other features of disseminated Cryptococcus infections include rash, endocarditis, ocular lesions, or multiorgan failure.6

This case is a somewhat unusual presentation of disseminated Cryptococcus infection characterized only by skin findings without clinical features of pulmonary or CNS infection. Approximately 15% of patients with disseminated infection may show cutaneous findings but primary cutaneous cryptococcosis is rare.7 Cryptococcal skin findings are quite varied, but may present similar to molluscum contagiosum, as dome shaped papules with central umbilication.7,8 On microscopy, small variably sized round yeasts without hyphae are characteristic. These yeasts may show a clear or pale staining halo representing the capsule and are highlighted well on Grocott’s Methenamine Silver or Periodic Acid-Schiff stains. Histology may demonstrate innumerable extracellular yeasts accompanied by foamy stroma and minimal inflammation or more granulomatous tissue reaction with necrosis, ulceration, and mixed inflammation. In conclusion, disseminated Cryptococcus must be considered in the context of new skin findings in an immunocompromised patient even if typical pulmonary or CNS findings are not identified.

References

  1. Sorrell TC, Ellis DH. Ecology of Cryptococcus neoformans. Rev Iberoam Micol. 1997 Jun;14(2):42-3.
  2. Bratton EW, El Husseini N, Chastain CA, Lee MS, Poole C, Sturmer T, et al. Comparison and temporal trends of three groups with cryptococcosis: HIV-infected, solid organ transplant, and HIV-negative/non-transplant. PloS One. 2012;7(8):e43582
  3. MacDougall L, Fyfe M, Romney M, Starr M, Galanis E. Risk factors for Cryptococcus gattii infection, British Columbia, Canada. Emerg Infect Dis. 2011 Feb;17(2):193-9.
  4. Sabiiti W, May RC. Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans. Future microbiol. 2012 Nov;7(11):1297-313.
  5. Abassi M, Boulware DR, Rhein J. Cryptococcal Meningitis: Diagnosis and Management Update. Curr Trop Med Rep. 2015;2(2):90–99. doi:10.1007/s40475-015-0046-y
  6. Clark RA, Greer D, Atkinson W, Valainis GT, Hyslop N. Spectrum of Cryptococcus neoformans infection in 68 patients infected with human immunodeficiency virus. Rev Infect Dis. 1990 Sep-Oct;12(5):768-77.
  7. Srivastava GN, Tilak R, Yadav J, Bansal M. Cutaneous Cryptococcus: marker for disseminated infection. BMJ Case Rep. 2015;2015:bcr2015210898. Published 2015 Jul 21. doi:10.1136/bcr-2015-210898
  8. Akram SM, Koirala J. Cutaneous Cryptococcus (Cryptococcosis) [Updated 2019 Aug 4]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK448148/

-Dr. Stanton Miller is a second year AP/CP resident at UT Southwestern Medical Center who is interested in Dermatopathology.

-Dr. IJ Frame is a board-certified Clinical Pathologist who is completing his Medical Microbiology fellowship at UT Southwestern Medical Center.

-Dr. Dominick Cavuoti is a professor of AP and CP at UT Southwestern, specializing in infectious disease pathology, cytology and medical microbiology.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Microbiology Case Study: A 53 Year Old Man with an Aortic Valve Mass

Case History

The patient is a 53 year old male with a past medical history of chronic obstructive pulmonary disease, who presented to the emergency department with one day of right calf pain, worse with weight-bearing, with radiating paresthesias, and a pale, cold right foot. He also endorsed a history of intermittent fatigue and knee pain. The patient works on farm and has an 82 pack year smoking history. He was diagnosed with a right popliteal artery occlusion and started on IV heparin. A transthoracic echocardiogram revealed an aortic valve mass.

Five days after admission he underwent a thromboembolectomy of the occluded vessel. A further six days later he underwent a procedure to excise the aortic valve mass, but ended up having an aortic valve replacement. Cardiothoracic surgery described a friable mass with a large base, consistent with a vegetation that might be seen in infective endocarditis, and sent the mass for surgical pathology and aerobic and anaerobic cultures. He was started on empiric vancomycin by infectious disease.

The patient denied recent fevers, chills, sweats, weight loss or changes in appetite, cough, chest pain, abdominal pain, nausea, vomiting, diarrhea, constipation, painful urination, rash, or unusual bone or joint pain. Doxycycline and rifampin were added for further coverage.

Laboratory Identification

Blood cultures drawn on admission showed no growth at 5 days. Initial gram stain of tissue from the mass showed many gram positive beaded rods. Anaerobic cultures of this tissue grew a single colony of Micrococcus and a single colony of a gram positive bacilli, which also grew aerobically and was identified by Mayo as Corynebacterium spp., not jeikeium. Serology for Bartonella was negative. Q fever serology showed elevated titers of phase I Ab IgG only.

Surgical pathology of the aortic valve mass is shown below:

Image 1. H&E stain of the removed aortic valve, demonstrating foamy macrophages. Not shown are abundant necrotic debris and dystrophic calcifications. Photo taken by Jessica Crothers, MD.
Image 2. Periodic Acid-Schiff (PAS) stain of the area depicted in Figure 2. The foamy macrophages are diffusely PAS positive. Photo taken by Jessica Crothers, MD.

GMS, B&B, AFB, and Fite staining were negative for definitive organisms.

A second set of blood cultures showed no growth and 5 days, and AFB cultures were negative, and second Q fever serology showed a decrease in the titer of phase I Ab IgG.

A PCR of residual heart valve tissue identified Tropheryma whipplei.

Discussion

The diagnosis of Tropheryma whipplei is made by T whipplei PCR, PAS stain, or T whipplei immunohistochemical staining.1 As these are all non-routine tests for a microbiology work-up, the diagnosis depends on high clinical suspicion.

Most often seen in the gut, T whipplei infection classically manifests as arthralgias, abdominal pain, weight loss, and diarrhea.2 However, it is also a rare source of culture-negative endocarditis, as seen in the case above.

The organism is a gram positive bacillus that is common in the environment and found in the saliva of up to 35% of healthy hosts.3 Furthermore, IgG antibodies to T whipplei have been detected in the blood of up to 70% of healthy individuals.4 Microscopically, there is minimal inflammatory response to this organism. Because of this and the classic symptoms, the characteristic foamy macrophages were initially thought to be indicative of a metabolic disorder.5 Once discovered and studied, it was found to most commonly affect males of European descent, which with the minimal inflammatory response to the organism led to the postulation that this population may have a heritable immunodeficiency.6 Others have suggested that the organism itself may have a role in down-regulating the immune response.7 A variety of immunologic disturbances have been associated with the disease, including down-regulation/absence of MHC II molecules and general dysfunction of monocytes/macrophages.1,7,8

Treatment varies by extent of disease, but it generally includes ceftriaxone or penicillin G followed by an extended course of TMP-SMX.1

In the case of our patient, he demonstrated symptomatic improvement after aortic valve repair followed by four weeks of ceftriaxone, with a plan to transition to TMP-SMX for one year.

References

  1. Apstein, MD, and T Schneider. “Whipple’s Disease.” UpToDate, Wolters Kluwer, 28 June 2019. Accessed 23 March 2020: https://www.uptodate.com/contents/whipples-disease?search=whipples%20disease%20children&source=search_result&selectedTitle=4~70&usage_type=default&display_rank=4#H703772001
  2. Durand DV, Lecomte C, Cathébras P. “Whipple disease. Clinical review of 52 cases.” Medicine (Baltimore). 1997;76(3):170.
  3. Street S, Donoghue HD, Neild GH. “Tropheryma whippelii DNA in saliva of healthy people.” Lancet. 1999;354(9185):1178.
  4. Raoult D, Birg ML, La Scola B, et al. “Cultivation of the bacillus of Whipple’s disease.” N Engl J Med. 2000;342(9):620.
  5. Whipple GH. “A hitherto undescribed disease characterized anatomically by deposits of fat and fatty acids in the intestinal and mesenteric lymphatic tissues.” Bull. Johns Hopkins Hosp. 1907; 18:382–391.
  6. Fenollar F, Puéchal X, Raoult D. “Whipple’s Disease.” N Engl J Med. 2007;356(1):55.
  7. Ectors NL, Geboes KJ, De Vos RM, et al. “Whipple’s disease: a histological, immunocytochemical, and electron microscopic study of the small intestinal epithelium.” J Pathol. 1994;172(1):73.
  8. Moos V, Schmidt C, Geelhaar A, et al. “Impaired immune functions of monocytes and macrophages in Whipple’s disease.” Gastroenterology. 2010;138(1):210. 

-Frederick Eyerer, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

-Thomas Koster, DO is a 1st 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 Associate Professor at the University of Vermont.