Tissue is the Issue: Splitting Specimens, Part 1

When we think about infectious disease (ID) and specific syndromes (culture-negative endocarditis, for example), it can be difficult to know the etiology.1 This is because different microorganisms can cause similar symptoms and depending on the specimen submitted to the laboratory for testing, you may need to split your specimen. It may be that the infection is localized to a small area (valve vegetation) and all you get for processing is a small volume of tissue. Another scenario is that you get a sufficient amount of specimen, but you must split the specimen for culture, multiple send out studies, pathology, etc. Or even worse–a small volume specimen that you need to split for multiple diagnostic tests.

We recently had a case of endocarditis. The patient’s blood cultures were negative, and she was going to have her mitral valve replaced. The ID team requested that we send the tissue for broad-range bacterial and fungal sequencing. We can do that- not a problem.

The Issue

As requested, the specimen (mitral valve vegetation) was split once it was received in the laboratory. Half went into the freezer for sequencing requests which we send to a reference laboratory and the other half was processed for bacterial (including mycobacteria) and fungal cultures.

In our experience, if no organisms are observed, then no DNA is detected. Therefore, it is not beneficial to send tissue for sequencing if we do not observe an organism (or something that looks suspicious for an organism) to begin with. In parts 2 and 3 of this series we will go into greater detail of the workflow for examining tissue for infection, but for now we will focus on the processing piece.

No organisms were observed in the direct smears (Gram, fungal, and acid-fast) and all cultures were negative. Because no organisms were observed, we did not send the tissue for sequencing. However, the patient was not improving and ID insisted that we send the tissue for sequencing anyway. As a last ditch effort we decided to homogenize the frozen tissue to see if by chance organism was present.

Long story short: the Gram stain did not reveal organism, but acridine orange2 did. We cultured the tissue and recovered an organism. Moral of the story: specimen processing can be tricky. It is an inherent issue that we must be aware of. How were we to know in which part of the tissue the organisms were? By definition, this is sampling error at its finest.

The Solution

Moving forward, rather than split the specimen prior to processing we have changed our protocol to homogenize the tissue first, then split the specimen. We believe this will eliminate similar scenarios from happening again in the future.

The Conclusion

All specimens are different in their composition. Unlike body fluids, which are easy to vortex and make homogeneous; tissue is more complex. Whatever the specimen, make sure your protocol(s) reduces sampling error.

References

  1. Subedi SJennings ZChen SC. 2017. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart Lung Circ. 26(8):763-771.
  2. Lauer BAReller LBMirrett S. 1981. Comparison of acridine orange and Gram stains for detection of microorganisms in cerebrospinal fluid and other clinical specimens. J Clin Microbiol.14(2):201-5.

 

Martinez Headshot-small 2017

-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.

Blood Bank Case Study: Transfusion Transmitted Malaria

Case Study

A 26 year old African American female with sickle cell anemia presented to a New York emergency room with cough, chest pain, fever and shortness of breath. Laboratory results showed an increased white blood cell count, slightly decreased platelet count and a hemoglobin of 6.2 g/dl. Her reticulocyte count was 7%, considerably below her baseline of 13%. Consulting the patient’s medical records revealed history of stroke as a child and subsequent treatment with chronic blood transfusions. She was admitted to the hospital for acute chest syndrome and aplastic crisis and care was transferred to her hematologist. Two units of RBCs were ordered for transfusion.

The blood bank technologists checked the patient’s blood bank history and noted her blood type was A, Rh(D) positive, with a history of a warm autoantibody and anti-E. The current blood bank sample confirmed the patient was blood type A, RH(D) positive with a negative DAT but the antibody screen was positive. Anti-E was identified. Per request of the hematologist, phenotypically similar units were found and the patient was transfused with 2 units of A RH(negative), C/E/K negative, HgS negative, irradiated blood. The patient’s hemoglobin rose to 8g/dl and she was discharged from the hospital 3 days after transfusion.

Ten days after discharge the patient returned to the emergency room with symptoms including aching muscles, fever and chills. A delayed transfusion reaction was suspected. A type and screen was immediately sent to the blood bank. The post transfusion type and screen remained positive for anti-E, DAT was negative. No additional antibodies were identified. However, a CBC sent to the lab at the same time revealed malarial parasites on the peripheral smear. The patient was consulted for a more complete medical history and reported that she had never traveled outside of the country. A pathology review was ordered and the patient was started on treatment for Plasmodium falciparum.

plasfal1

Discussion

Red Blood cell transfusions can be life saving for patients with sickle cells anemia. These patients are frequently transfused by either simple transfusion of red cell units or by exchange transfusion. Because of this, alloimmunization is reported to occur in 20% to 40% of sickle cell patients.1 Blood bank technologists are very diligent in adhering to strict procedures and follow a standard of practice aimed to prevent transfusion reactions. While preventing immune transfusion reactions may be the most forefront in our minds when transfusing the alloimmunized patient, it is important to consider transfusion transmitted diseases as a potential complication of blood transfusions.

Malaria is caused by a red blood cell parasite of any of the Plasmodium species. Mosquito transmitted infection is transmitted to humans through the bite of an infected mosquito. Transfusion-transmitted malaria is an accidental Plasmodium infection caused by a blood transfusion from a malaria infected donor to a recipient.

Donors, especially those from malarial endemic countries who may have partial immunity, may have very low subclinical levels of Plasmodium in their blood for years. Even these very low levels of parasites are sufficient to transmit malaria to a recipient of a blood donation. Though very rare, transfusion-transmitted malaria remains a serious concern for transfusion recipients. These transfusion-transmitted malaria cases can cause high percent parisitemia because the transfused blood releases malarial parasites directly into the recipient’s blood stream.

Blood is considered a medication in the United States, and, as such, is closely regulated by the FDA. Blood banks test a sample of blood from each donation to identify any potential infectious agents. Blood donations in the US are carefully screened for 8 infectious diseases, but malaria remains one infectious disease for which there is no FDA-approved screening test available. For this reason, screening is accomplished solely by donor questioning.2 A donor is deferred from donating if they have had possible exposure to malaria or have had a malarial infection. Deferral is 12 months after travel to an endemic region, and 3 years after living in an endemic region. In addition, a donor is deferred from donating for 3 years after recovering from malaria. It is important, therefore, for careful screening to take place by questionnaire and in person, to make sure that the potential donor understands and responds appropriately to questions concerning travel and past infection.

Malaria was eliminated from the United States in the early 1950’s. Currently, about 1700 cases of malaria are reported in the US each year, almost all of them in recent travelers to endemic areas. From 1963-2015, there have been 97 cases of accidental transfusion-transmitted malaria reported in the United States. The estimated incidence of transfusion-transmitted malaria is less than 1 case in 1 million units.4 Approximately two thirds of these cases could have been prevented if the implicated donors had been deferred according to the above established guidelines.3 While the risk of catching a virus or any other blood-borne infection from a blood transfusion is very low, a blood supply with zero risk of transmitting infectious disease may be unattainable. With that being said, the blood supply in the United Sates today is the safest it has ever been and continues to become safer as screening tests are added and improved. Careful screening of donors according to the recommended exclusion guidelines remains the best way to prevent transfusion-transmitted malaria.

References

  1. LabQ, Clinical laboratory 2014 No.8, Transfusion Medicine. Jeanne E. Hendrickson, MD, Christopher Tormey, MD, Department of Laboratory Medicine, Yale University School of Medicine
  2. Technical Manual, editor Mark K. Fung-18th edition, AABB. 2014. P 201-202
  3. https://www.cdc.gov/malaria/about/facts.html. Accessed April 2018
  4. The New England Journal of Medicine. Transfusion-Transmitted Malaria in the United States from 1963 through 1999. Mary Mungai, MD, Gary Tegtmeier, Ph.D., Mary Chamberland, M.D., M.P.H., June 28, 2001. Accessed April 2018
  5. Malaria Journal. A systematic review of transfusion-transmitted malaria in non-endemic areas. 2018; 17: 36. Published online 2018 Jan 16. doi: 1186/s12936-018-2181-0. Accessed April 2018
  6. http://www.aabb.org/advocacy/regulatorygovernment/donoreligibility/malaria/Pages/default.aspx

 

Socha-small

-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: A 79 Year Old Male with Rheumatic Heart Disease

Case History

The patient is a 79 y/o male with past medical history of rheumatic heart disease, permanent atrial fibrillation, mechanical aortic and mitral valves (2004), status post single chamber pace maker for bradycardia (2010), and prostate adenocarcinoma treated in 2000. He had new MRI compatible pace maker placed on Oct 19, 2017. During follow-up he was noted to have a hematoma over the incision site. He had a revision done on Nov 3, 2017. At that time, the blood from the incision site was sent for culture. 

Laboratory Identification

Gram stain showed moderate amount of polys with no bacteria seen. The isolate was a gram-negative rod that was identified on the MALDI-ToF as Burkholderia multivorans.

 

burkmult1
Image 1: Semi-mucoid, yellow-grey colonies on Chocolate agar and on Blood agar plates.

Discussion

The Burkholderia genus appears as gram-negative medium-sized straight rods, with the exception being B. mallei which is a coccobacillus. The will grow on blood, chocolate, and MacConkey agar. Oxidative-fermentative-base-polymyxin B-bacitracin-lactose (OFPBL) agar can be used to isolate B. cepacia and Ashdown medium can be used to isolate B. pseudomallei. They are non-lactose fermenters on MacConkey, but B. cepacia can turn into a dark pink to red due to oxidation of lactose after 4-7 days.

B. multivorans is a species within the Burkholderia genus, which are normal to plant, soil, and water, but not normally considered common human flora. Formerly of the Pseudomonas genus, B. cepacia, B. mallei, and B. pseudomallei are the most commonly seen as infections in humans. Further, B. cepacia and B. mallei are not typically human pathogens in a healthy human host. Because of the rarity of this genus to infect humans, their pathogenicity is not well known; but, importantly, they are intrinsically resistant to many antibiotics and can thus be associated with hospital acquired infections.

Of this genus, very little literature is present on B. multivorans specifically, and of the literature that does exist, most of it is in relation to cystic fibrosis patients. Taxonomic advances has shown that B. cepacia complex is a cluster of nine closesly related genomic species or genomorvars (1).  B. multivorans represents genomorvar II. Hospital acquired clinical infections from this complex (but perhaps not specifically from this particular genomorvar) has been seen following catheterization, cystoscopy, heart surgery, and with contaminated ventriculoatrial shunt (2). B. multivorans biochemically is oxidase positive, catalase positive, lipase positive, nitrate-reducing, urease positive, resistant to colistin, and can grow at 42C (3, 4).

A recent comparative genomic study showed that B. multivorans is a highly evolutionarily preserved genome with genomic characteristics from the environment and isolated from cystic fibrosis patients to be similar, and that isolates from different continents are also similar (5). Further, a murine model for pulmonary infections showed that B. multivorans could persist in the host by establishing an intracellular presence within macrophages, which could explain the persistence of this pathogen in cystic fibrosis patients (6). Importantly though, due to the conserved and common genomic structure, there rests a possibility for potential vaccination for cystic fibrosis patients against B. multivorans.

The patient was prescribed a single dose of oral Bactrim and then advised to come into the hospital for admission for IV antibiotics. IV ceftazidime was started with pending blood cultures, which are negative at the time of this documentation.

References:

  1. Coenye T. et al. Taxonomy and identification of the Burkholderia cepacia complex. J Clin Microbiol 2001;39:3427-3436.
  2. Pallent LJ. et al. Pseudomonas cepacia as contaminant and infective age. J Hosp Infect 1983;4:9-13.
  3. Henry DA. et al. Phenotypic methods for determining genomovar status of Burkholderia cepacia complex. J Clin Microbiol 2001;39:1073-1078.
  4. Vandamme P. et al. Occurrence of multiple genomovars of Burkholderia cepacia in patients with cystic fibrosis and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 1997;47:1188-1200.
  5. Peeters C. et al. Comparative genomics of Burkholderia multivorans, a ubiquitous pathogen with a highly conserved genomic structure. PLoS One. 2017, 21; 12 (4): e0176191.
  6. Chu KK. et al. Persistence of Burkholderia multivorans with the Pulmonary Macrophage in the Murine Lung. Infect Immun 2004; 72 (10): 6142-6147.

 

-Jeff Covington, MD, PhD, is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

Wojewoda-small

-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: A Middle-Aged Man with Malaise, Shaking, and Chills

Case history

A middle-aged male presented to the hospital emergency room with the complaints of malaise, shaking and chills for the last two days. He denied any runny nose, cough, abdominal pain, nausea, vomiting, headache or known sick contacts. His past medical history was significant for alcohol use disorder. Imaging of the abdomen revealed an ill-defined region of decreased attenuation in the right lobe of the liver measuring 4.8 x 4.7 x 2.2 cm. The Gram stain of the abscess showed 4+ WBCs (PMNs) and 4+ gram negative rods with a very large capsule surrounding them (Image 1).  The organisms grew very mucoid colonies on 5% sheep blood, chocolate, and MacConkey agars (Image 2).  A string test performed on the mucoid bacterial colonies was >5 mm (Image 3).

kleb1
Image 1. Gram stain of abscess showing 4+ WBCs and 4+ GNR with large capsule.
kleb2
Image 2. Cultures showed mucoid colonies on the chocolate and MacConkey agars.
kleb3
Image 3. A string test was performed on the mucoid colonies and was positive (mucoid capsule “string” > 5mm).

Discussion

The organism was identified as Klebsiella pneumoniae by MALDI-TOF MS.  Based on the mucoid capsule and positive string test, this organism was further identified as hypermucoviscous K. pneumoniae.

Hypermucoviscous K. pneumoniae is a relatively newly recognized hypervirulent variant of K. pneumoniae. It was first described in the Asian Pacific rim and is now increasingly recognized in Western countries. Defining clinical features include serious, life-threatening community-acquired infection in younger healthy hosts, an unusual feature for enteric gram negative bacilli in the non-immunocompromised population. It can cause a variety of diseases including, but not limited to liver abscess, pneumonia, meningitis, osteomyelitis, necrotizing fasciitis and endophthalmitis.

Intestinal colonization, appears to be a critical step leading to infection. It is seen mostly in Asians, raising the issue of a genetic predisposition vs. geospecific strain acquisition.  The increased virulence might be due to the ability to more efficiently acquire iron and perhaps an increase in capsule production, which confers the hypermucoviscous phenotype to the organism. The vehicles for acquisition and subsequent colonization appear to be food and water, person-to-person transmission (e.g., close contacts such as family members or sexual partners) or animal-to-person transmission (e.g., between pets and their owners).

To date, most strains of hypermucoviscous K. pneumoniae have been very susceptible to antimicrobials except ampicillin.  However, in recent literature, propensity for hypermucoviscous Klebsiella pneumoniae to become multi-, extreme or pandrug-resistant, including the acquisition of extended-spectrum β-lactamases (ESBL) and carbapenemases has been reported. Since hypermucoviscous K. pneumoniae strains often cause abscesses, source control is a major aspect of the overall management plan and a need to drain abscesses and closed space infections is essential for optimal outcome.

References

  1. Alyssa S. Shon, Rajinder P.S. Bajwa and Thomas A. Russo; Hypervirulent (hypermucoviscous) Klebsiella pneumonia: A new and dangerous breed; Virulence 4:2, 107–118; February 15, 2013; 2013 Landes Bioscience
  2. Bonnie C Prokesch, Michael TeKippe, Jiwoong Kim, Prithvi Raj, Erin McElvania TeKippe, David E Greenberg; Primary osteomyelitis caused by hypervirulent Klebsiella pneumonia; The Lancet Infectious Diseases , Volume 16 , Issue 9 , e190 – e195

 

MA

-Muhammad Ahmad, MD is a 2nd year anatomic and clinical pathology resident at University of Chicago (NorthShore) program based at Evanston Hospital, Evanston, IL. His academic interests include breast pathology and cytopathology.

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

Microbiology Case Study: A 28 Year Old Female with Cough.

Case History

A 28 y/o female with a past medical history of chronic eosinophilic pneumonia, chronic persistent asthma, and elevated IgE status post Xolair therapy presented with a cough. She is a former smoker and a former IV drug user. She has been having a productive cough since March and has not improved despite multiple courses of antibiotic therapy. She coughs mostly in the morning and describes her sputum as thick and greenish. She does not have any associated fevers and does not feel that her rescue inhalers help much. She was given a course of doxycycline for 10 days, and sputum was sent for culture.

Laboratory Identification

pasmul1
Image 1: Gram stain showed many polys, moderate mixed gram positive and gram negative organisms. Sputum culture was reported out as mixed gram negatives.
pasmul2
Image 2: Chocolate and blood agar plates of the mixed gram positive and gram negative organisms.

One of the gram negative rods was identified by the MALDI-ToF as Pasteurella multocida.

Discussion

The genus Pasteurella consists of multiple identified species with the one most commonly seen in the clinical setting as Pasteurella multocida. As a genus, they are typically gram-negative straight bacilli that are nonmotile, oxidase-positive, catalase-positive, nitrate reducing, and ferment glucose. They will grow on blood and on chocolate agars, but importantly will not grow on MacConkey. Their colony morphology on blood agar is generally convex, smooth, and nonhemolytic.

Infections with Pasteurella are classically associated with animal bites, such as from a dog or cat. However, prior cases in the literature have shown that pulmonary infection with Pasteurella can be associated with other chronic pulmonary diseases such as COPD (1). The choice for using doxycycline is supported in the literature and was specifically discussed in a prior case with improvement (2).

References:

  1. Klein NC. et al. Pasteurella multocida pneumonia. Semin Respir Infect 1997; 12 (1): 54-56.
  2. Bhat S. et al. A case of lower respiratory tract infection with canine-associated Pasteurella canis in a patient with chronic obstructive pulmonary disease. J Clin Diagn Res 2015; 9 (8): DD03-DD04.

 

-Jeff Covington, MD, PhD, is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

Wojewoda-small

-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.

A Candida Comeback?

Hello again everyone! And special thanks to the readers who read, commented, shared, and reached out to me from my last post “A Serious Aside,” talking about physician burnout and health worker suicide. Numerous people had so much to say in support of this topic—and it’s well deserved—sharing their personal stories and relating their own connects, so I truly appreciate it.

This time, how about something different? In the past few months, I’ve been working through my clinical rotations at a major community hospital in New York City, in the Bronx. A CDC-sponsored screensaver image keeps appearing at terminals throughout floors, services, and clinics; and it directly addresses healthcare professionals to monitor hygiene practices to eliminate Candida infections. I’ll have to admit—innocuous stuff—I’ve been seeing health-message PSA-like screensavers at work for years, about a myriad of topics. Who hasn’t seen those? “Keep beds out of the hallways,” “Protect you and your patients from MRSA,” “Make sure lab requisitions are filled out properly…” the list is endless. But having seen my aforementioned screensaver about Candida one too many times, I had to find out what this was about. You might have thought that, since I spent time working in an HIV clinic, this was a simple PSA for those patients otherwise immunocompromised. Right? Nope.

This particular PSA from the CDC warns about Candida auris, a true blue (or pinkish gold, rather) member of everyone’s favorite budding, germ-tube positive, yeast family. C. auris has been in literature for roughly the past decade. Having etiologic origins in southeast Asia and spreading west through the Middle-East, all throughout Africa, and even the UK, this bug has caught the eyes of epidemiologists around the world. Two years ago, the CDC1 and Public health England2 issued warnings about this pathogen, its multi-drug resistance, and its virulence in healthcare-associated infections. Last fall, the NY State Department of Health published their official update for guiding clinicians and laboratory staff.3 In this report, they discussed infection control, prevention, and detection limitations.

canaur1
Image 1. CDC screensaver on hospital computers. Due to increased incidence of reported cases, epidemiologic data suggest prevention measures would benefit patients. C. auris is associated with healthcare-related infections and can live in the environment for an extended period of time.
canaur2
Image 2. NY State Department of Health, Report on C. auris. Informing clinicans and laboratory staff about epidemiology, prevention, detection in the laboratory, and associated implications of limitations and multidrug resistance.

So what’s so scary about C. auris? The two most challenging features of this emerging pathogen are its multi-drug resistance and its relatively difficult identification.

This yeast has been shown to show resistance to many antifungal/antimicrobial agents including fluconazole, voriconazole, amphotericin-B, echinocandins, and even flucytosine. Even more concerning is that nearly half of the C. auris strains collected in research done in Asia, Africa, and South America demonstrated multi-drug resistance patterns to two or more combination therapies. These are most of our first-line standard of care therapies for invasive candidiasis in patients!

canaur3.png
Image 3. UpToDate recommendations summary for candidemia and invasive candida infections.

There are various other recommendations regarding therapies to C. auris specifically, as its potential for resistance are known, but infection control along with empiric therapy seem to be the current standard.

The major risk factors for C. auris infections include the relative status of individual patients: intensive care, acute renal failure, immunocompromised status, localized or systemic infections, and colonization. Simply being hospitalized is an associated risk. On my current service of patients I’m part of a nephrology/medicine team. There are several chronic infection, ESRD, immunocompromised, or otherwise applicable patients to these risk stratifications. No wonder we’ve got those screensavers!

Concerns for identifying C. auris take us back to the lab. Detecting this bug is not as simple as a couple microscopic morphologies and a yeast API strip—sorry to my old mycology professors. C. auris based on chemical tests like these can produce confounding results. Even VITEK identification (unless you’re running Vitek 2 with Biomerieux software) or culture growth can yield non-specifics like C. haemulonii or Saccharomyces cerevisiae. C. auris has a very high salt and temperature tolerance, and with no particular morphologic identifiable features, it remains a challenging identification. It can be grown on dulcitol agar or CHROMagar, but you do not get clear results. What’s the way to get the ID then? Ultimately MALDI-TOF, PCR, and molecular testing is the answer. There are already available C. auris sequences you can obtain for in-house validation if you’re using MALDI already. And when it comes to susceptibility, fear not: as far as I’ve been able to read E-Tests still work.

canaur4.png
Image 4. Definitely not a definitive CHROMagar result.

I was very impressed with MALDI when I was working in Chicago, and a community hospital I was with just finished validating when I left for medical school. I am glad to see it again with this emergent pathogen, and it definitely demonstrates the next wave of instrumentation. Extremely rapid and very accurate.

The variable drug susceptibility, virulence, and ability to thrive in the environment actively threaten those with long inpatient stays. This microorganism is treated with standard precautions and infection control measures. Currently NY leads the nation by far in purported cases of C. auris. So … please wash your hands. A lot. I know I am.

Thanks! See you next time!

References

  1. Centers for Disease Control and Prevention. Clinical Alert to U.S. Healthcare Facilities – Global Emergence of Invasive Infections Caused by the Multidrug-Resistant Yeast Candida auris. https://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-alert.html
  2. Public Health England. Candida auris identified in England. https://www.gov.uk/government/publications/candida-auris-emergence-in-england/candida-auris-identified-in-england
  3. NY State Department of Health https://www.health.ny.gov/diseases/communicable/c_auris/docs/c_auris_update_for_lab_staff.pdf

 

ckanakisheadshot_small

Constantine E. Kanakis MSc, MLS (ASCP)CM graduated from Loyola University Chicago with a BS in Molecular Biology and Bioethics and then Rush University with an MS in Medical Laboratory Science. He is currently a medical student at the American University of the Caribbean and actively involved with local public health.

Microbiology Case Study: A 59 Year Old Female with Fevers, Weakness, and Altered Mental Status

Case History

A 59 year old African American female presented to the emergency department with fevers, weakness, fatigue and altered mental status. Her past medical history was significant for hypertension, diabetes mellitus (type 2) with end stage renal disease and a recent cerebrovascular accident the month prior. Her surgical history included a mitral valve repair surgery three years ago and a renal transplant two years ago. Current medications included prednisone, mycophenolate and tacrolimus immunosuppressive agents. Physical examination was unremarkable except for a temperature of 101°F and she was oriented to person, place and time. Pertinent labs included a WBC count of 13.2 TH/cm2, microcytic anemia, and a creatinine of 1.51 mg/dL. Due to previous cardiac surgery, a transesophageal echocardiograph (TEE) was performed and showed a large vegetation (1.6 x 1.5 cm) on the mitral valve.  A diagnosis of endocarditis was made and the patient was started on broad-spectrum antibiotics & taken to surgery for a mitral valve replacement. Multiple blood cultures were negative to this point. Portions of the mitral valve were submitted to surgical pathology and the microbiology laboratory for bacterial, fungal and AFB cultures.

Laboratory identification

Surgical pathology received an aggregate of tan-yellow, fibrous tissue fragments (3.1 x 1.5 x 1.1 cm). Histologic assessment showed a confluent mass containing abundant narrow, septate hyphae consistent with a fungal infection (Image 1). No definitive pigment was identified. Grocott’s methenamine silver (GMS) stain also highlighted the narrow hyphae with numerous septations (Image 2). In the microbiology laboratory, a darkly pigmented mold grew after 5 days of incubation on Sabouraud dextrose agar (Image 3). Lactophenol cotton blue prep showed pigmented, curved conidia with 2-3 transverse septations consistent with Curvularia spp (Image 4). All blood cultures were finalized as no growth after 5 days. Fungitell was found to be greater than 500 pg/ml and Aspergillus galactomannan was negative (<0.5).

curve1.jpg
Image 1. Sections of mitral valve tissue showed a confluent mass of abundant hyphal elements (H&E, 4x).
curve2.jpg
Image 2. Special stains of the fungal mass highlighted narrow hyphae with numerous septations and acute angle branching (GMS, 4x).
curve3.png
Image 3. A darkly pigmented mold grew of Sabouraud dextrose agar after 5 days of incubation at 25°C.
curve4.jpg
Image 4. Many pigmented, curved conidia with multiple transverse septations were seen (lactophenol cotton blue prep, low power).

Discussion

Curvularia spp. belong to a heterogeneous group of dematiaceous or black molds. The presence of pigment in this category of molds is due to melanin in the hyphae. Dematiaceous molds are ubiquitous in nature and can occasionally cause human infections.  These molds have a characteristic dark appearance on fungal media that is often dark gray, brown or black in color. In addition, when the reverse of the plate or slant is examined, the under surface is also pigmented. Based on their growth rate, the dematiaceous fungi are divided into the fast growers, such as Curvularia, Bipolaris and Alternaria spp., which are mature in 5-7 days. The second group is slow growers that take between 7-25 days to fully mature. Examples of slow growers include Phialophora, Exophila/Wangiella, Cladosporium, Fonsecaea and Rhinocladiella spp.

Most commonly, dematiaceous molds infections usually present as phaeophyphomycosis, chromoblastomycosis or mycetomas. These three entities are cutaneous or subcutaneous mycoses that are obtained by traumatic implantation but vary from one another based on clinical features and histologic features of the mold in tissues. They are most frequently cause infection in male agricultural workers in rural areas of tropical or subtropical climates.  These infections are indolent in nature but can lead to significant morbidity over time, as they are difficult to treat effectively.

In addition to the above superficial infections, Curvularia spp. has also be known to cause keratitis, sinusitis and wound infections. In immunosuppressed individuals, disseminated infections with spread to the lungs and brain have been documented. Endocarditis due to Curvularia spp. is quite rare with very few cases previously reported in the literature. On those documented, Curvularia spp. infections tend to have a predilection for prosthetic heart valves or occur after cardiac surgery. Diagnosis of infective endocarditis is difficult as symptoms are indolent and blood cultures do not have a high yield. Therefore, culture of the vegetation may be the only way to make a diagnosis.

In the microbiology laboratory, Curvularia spp. will grow on routine fungal media as a darkly pigmented mold in a relatively short time. On lactophenol cotton blue prep, Curvularia spp. produce large conidia that usually contain 4 cells that are divided by transverse septations. The conidia take on a curved appearance due to swelling of the subterminal cell, which is often the largest and most deeply pigmented. If identification is necessary beyond the genus level, panfungal PCR assays followed by sequencing of ribosomal genes may be useful in providing a species level diagnosis from fresh or paraffin embedded tissue.

For localized infections, surgical treatment alone may be adequate in some cases.  In infections that are extensive or if there is dissemination, treatment with newer triazoles, such as posaconazole or voriconazole, have shown a broad spectrum of activity against dematiceous molds. Amphotericin B is also another effective option. While susceptibility testing can be performed on clinically significant Curvularia spp. infections, interpretative breakpoints have not been defined and clinical correlation is lacking.

In the case of our patient, she remained on a ventilator following surgery and with the identification of mold on histology, she was started on micafungin. She was switched to amphotericin B after the mold was classified as Curvularia spp. Her condition did not improve despite therapy and she died 3 weeks after surgery.

 

-Azniv Azar, MD, is a fourth year anatomical and clinical pathology resident at the University of Mississippi Medical Center.

Stempak

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the director of the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement, and resident education.