January 2016 – Lablogatory

Microbiology Case Study: 45 Year Old Woman with Subarachnoid Hemorrhage

Case History:

A 45 year-old woman was hospitalized for a bilateral subarachnoid hemorrhage with right-sided intraventricular hemorrhage secondary to a basilar artery aneurysm. On hospital day 2, after endovascular coiling of her aneurysm, she developed seizure-like activity. She was found to have new bilateral thalamic and brainstem infarcts with increased hydrocephalus and had an external ventricular drain placed.

She showed some minimal neurological improvements over the next 2 weeks until hospital day 17 when her mental status acutely declined and she developed a fever, leukocytosis, and meningeal signs. 8cc of cloudy, yellow fluid was collected via lumbar puncture. Analysis of the fluid showed pleocytosis (cell count of 54K) with a neutrophil predominance and markedly elevated protein.

Laboratory Identification:

Initial review of the spinal fluid gram stain showed many polymorphonuclear leukocytes and what appeared to be paired gram negative cocci suspicious for Neisseria meningitidis. However, many organisms lacked the more characteristic “kidney-bean” shape. Further inspection of the gram stain revealed many gram negative organisms which more closely resembled bacilli. Given the patient’s history and clinical course, it was determined the pathological agent was most likely a gram negative coccobacillus. On culture, the bacteria formed smooth, round, opaque colonies on Blood and Chocolate agar and was lactose non-fermenting on MacConkey agar. The bacterial colonies were also oxidase negative.

Mass spectrometry identified the organism as Acinetobacter radioresistens.

Rare gram positive appearing organisms of similar shape were also located on the gram stain. This demonstrates that Acinetobacter is known to occasionally retain the crystal violet stain leading to cases of initial misidentification.

Discussion:

Acinetobacter radioresistens is one of about 30 species of bacteria included in the Acinetobacter genus. Acinetobacter is characterized as a gram negative, aerobic coccobacillus which is non-motile, non-fermentative, and oxidase-negative. It grows well on standard aerobic media and typically forms smooth, round, mucoid colonies at 37°C. Acinetobacter is a water organism which preferentially colonizes aquatic, humid, and tropical environments; perhaps accounting for the increased incidence of Acinetobacter infections between the months of July and October.

While there have been reported cases of community-acquired Acinetobacter pneumonia in Southeast Asia and Australia, in most areas of the world Acinetobacter is known primarily as an agent of nosocomial infections. Studies show that an estimated 33% of healthcare workers are colonized with Acinetobacter and that it is one of the most prevalent bacteria isolated from the white coats of medical students. Despite its ubiquity in hospitals, Acinetobacter infections are relatively rare. Many patients may be colonized with it, but Acinetobacter only usually causes disease in immunocompromised and/or critically-ill patients with long hospitalizations. At particular risk are ventilated patients supported with multiple lines, drains, and catheters. Acinetobacter is reported as the pathological agent in a small percent of ventilator-associated pneumonias, central line-associated bloodstream infections, catheter-associated urinary tract infections, and surgical site infections. It is also recognized as a cause of nosocomial meningitis in neurosurgical patients with external ventricular drains, especially those with a history of intracranial hemorrhage and recent prior antibiotic therapy.

Acinetobacter infections are of particular concern because several species demonstrate resistance to many antimicrobials. Acinetobacter baumanni, the species responsible for the majority of Acinetobacter infections, has demonstrated resistance to 1^st-3^rd generation cephalosporins, macrolides, penicillins, and aminoglycosides. Because these infections are robust and difficult to treat, patients with Acinetobacter infections have a 25-75% mortality risk depending on the site of their infection and their baseline cardiopulmonary and immune status. Currently, carbapenems are considered the gold standard treatment.

-Elaine Amoresano, MD, is a 1^st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Zika Virus

If you’re an infectious disease geek and you’ve been following the news, you know that the Zika virus is causing a pandemic in Central and South America and is linked to cases of microcephaly in those regions. It’s gotten bad enough fast enough that the CDC has issued travel warnings for pregnant women and women of child-bearing age.

If you’d like to get up-to-date on this outbreak, check out Maryn McKenna’s blog on National Geographic. Also, the CDC has great information for clinicians and laboratory professionals.

Microbiology Case Study: Immunocompromised 65 Year Old Man

Case History:

A 65 year old man presents to the emergency room with acute onset back pain. Of note, the man was diagnosed with Burkitt’s lymphoma two months prior and had recently received a course of chemotherapy. During the workup for his back pain, a chest CT is obtained and reveals a 2 cm pulmonary nodule in the left upper lobe with a surrounding “groundglass halo” highly suspicious for invasive fungal involvement. A fine needle aspiration (FNA) of the nodule is performed and tissue is sent for histopathologic examination as well as bacterial and fungal culture.

Chest CT showing a 2 cm nodule in the left upper lobe

Silver stain of the FNA specimen showing ribbon-like fungal elements with rare septations

Scotch-tape preparation reveals rarely septate hyphae with internodal rhizoids and pyriform sporangia supported by funnel-shaped apophyses.

Laboratory Identification:

One rapidly growing white colony was identified that became grey over time. The colony was a “lid-lifter” that began pushing at the lid after only a few days. Microscopically, the organisms had broad hyphae with single and branching sporangiophores. At the ends of the sporangiophores there were pyriform, or pear-shaped, sporangia sitting atop funnel-shaped apophyses. Rhizoids were found to be internodal, or arising from the hyphae between the sporangiophores. Based on this morphology, the fungal organisms were identified as Lichtheimia corymbifera complex (formally Absidia corymbifera).

Discussion:

Lichtheimia corymbifera is an organism within the phylum Zygomycota and is one of two pathogenic species in the genus Lichtheimia. This organism is known as an uncommon cause of Zygomycosis and is only implicated in approximately 5% of cases. As in most cases of Zygomycosis, exemplified in our patient, Lichtheimia corymbifera most often affects immunocompromised patients. It is ubiquitous in the environment and is associated with decaying plant matter and soil. Disease is caused by inhalation of spores.

Important points for laboratory identification:

Lichtheimia

Growth at 35-37°C (capable of growth up to 50°C)
Inhibited by media containing cycloheximide
Internodal rhizoids
Pyriform sporangia
Apophysis

Compared to other common Zygomycetes:

Mucor

No rhizoids
Round sporangia
No apophysis

Rhizopus

Nodal rhizoids (directly opposite of the sporangiophores)
Round sporangia
No apophysis

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

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

The Value of Electives (Both Internal and External) During Pathology Residency

It’s been a while since my last blog. I haven’t had as much time and energy as I would like this past year. For now, I’ll just say…appreciate all that your chief residents do because much more time and effort lies beneath the surface than everyone is able to see.

But the topic for this blog is the value of electives during pathology residency. Our programs vary with respect to electives in terms of number and ability to take them externally or not. My previous program had five electives that could be taken internally or externally. However, external electives did not receive salary support and I didn’t take any electives although I could’ve during my two years in Chicago. Since we had a decent number of electives, many residents scheduled them internally during fourth year to have lighter months to study for boards although a handful did utilize them during earlier years for external electives.

My current program has two and we do receive salary support with external electives. For my first, I had an extra month of hematopathology internally because I wanted to see another perspective on one of my chosen subspecialties. Internal electives are good to spend more focused time in an area of subspecialty interest that you may have for fellowship. It also allows for the opportunity to develop deeper relationships with the faculty who will most likely be writing your letters of recommendation for that fellowship. They may also provide you with the opportunity to become more involved in research and/or publications (eg – book chapters, case reports, research articles) with a mentor and these are all helpful in enhancing your fellowship and future job applications and build up your CV.

Currently, I’m on an external elective at the institution where I’ll do both my hematopathology and molecular genetic fellowships. I’m laying the groundwork for molecular hematopathology research now that hopefully results in data analysis over the ensuing months to culminate in an abstract submission for the American Society of Hematology (ASH) which has a deadline only a month after I start fellowship. I also want to use this time away to get to know people at my future program better, prepare for my eventual move here, and study for boards. Hopefully, I’ll also get a sense of the daily work flow as I am also attending signouts and intra- and interdepartmental conferences so that I can manage my time as efficiently as I can from day 1 of fellowship. I really like the culture and people here, but that’s subject matter for a future blog. I also am enjoying the benefits of attending inter-program activities as TMC is the largest medical center in the world with active interaction and collaboration between member hospitals. Not so much in my case since I obtained both my consecutive fellowships last year as a PGY-3, but for many, the value of an early external elective is that it can be seen it as an “audition” rotation to obtain a desired fellowship. You may even be able to ask for an interview before you finish (which saves you time and money). I also have some friends who were offered fellowship spots at the end of their elective rotation because they impressed the fellowship director. Obtaining fellowship positions is pretty competitive and there tends to be fewer spots than there are for residency. And in many cases, positions are not even available if an internal candidate is chosen early (even during their PGY-1) so anything to augment your fellowship application is a plus.

Now that I’ve mentioned external electives, I’d like to give some advice on setting up an external elective. First, start as EARLY as possible! Even a year or more before isn’t too early to ask about getting the ball rolling. Start preparing and updating your CV from your PGY-1 as you’ll need this for both external elective and fellowship applications. Scan and keep a PDF of all your vaccinations and work-related health requirements (eg – PPD/Quantiferon results, flu vaccine, hepatitis B testing, MMR and hepatitis B antibody titers, and N-95 fit testing) as well because its likely you’ll also have to include this in your external elective application.

I initiated the elective rotation request about a half year prior to the actual rotation. And even then, that was not early enough and everything came down to the wire. It’s far more complicated than when we applied for elective rotations as a medical student and takes much more time. Since we are now physicians, you are more than likely required to have at least a medical permit in that state to rotate and this process can take a while. Also the back-and-forth between program coordinators and the respective GME departments can appear glacial at times, and in my case, was the main cause of delay. I hit several delays at obtaining paperwork (especially between Christmas and New Year’s when many staff were off at both programs, my medical school, and the Texas Medical Board where I needed paperwork from). It can be time-consuming to have to make multiple phone calls, and often, the process cannot be completed until its antecedent step has been approved. I know residents who have had to postpone external rotations because paperwork between GME departments (eg – PLAs or malpractice agreements) were not in place in time. Maintaining constant and open communication between all parties involved is a must and sometimes easier said than done the more people that are involved.

In addition to obtaining the state medical permit (which generally requires an application fee; in my case, $135), there may be other requirements that are also time-consuming and financially expensive. You may be required to do pre-employment-type health screening (in my case, a $36 urine drug screen) at your own cost as you are not a true employee. I also had to become ACLS certified (at $120, despite being BLS certified and a former American Red Cross CPR instructor). But since I’m going to be a fellow here, I can get it reimbursed through my GME funds and would have to do it later anyways so I might as well do it now. But if you are not doing an elective at your future fellowship institution, it’s good to find out what items may incur cost in your application for your elective since you are not likely to get reimbursed and so you can decide whether those expenses are acceptable. One way to defray costs is to apply for grants such as the ASCP subspecialty grant which is administered to six residents twice a year (Jan/Aug). In fact, the next deadline is this Friday, Jan 15^th! You can find more information on how to apply at http://www.ascp.org/Residents/Resident-Grants.

Chung

-Betty Chung, DO, MPH, MA is a fourth year resident physician at Rutgers – Robert Wood Johnson University Hospital in New Brunswick, NJ.

“Flustration”

Contrary to popular belief, “irregardless” is not a word. The correct word is “regardless.” One can be frustrated or one can be flustered, but it is incorrect to say one is “flustrated.”When these common grammatical errors are made, it can be very irksome for some Type A Personality laboratory professionals. For those in the field of lab safety, however, this can be a lesson for learning how to improve the lab safety culture.

Very often grammatical errors are made because people simply do not remember what is correct, or the correct use of the word was never explained to them. If we hear something that is wrong, we may make a judgement about the person who said it—they are not well-educated, they are lazy, etc. We tell ourselves a story, and often, unfortunately, it is inaccurate.

You walk into the laboratory and you see the new tech Judy working at the bench with no gloves. Last week you spoke to Judy about the same issue, and she told you she would wear them from now on. In your frustration you believe Judy to be obdurate and someone who willfully violates lab safety practices. Because these things are in your mind, the conversation you are about to have with Judy will not go well.

Consider the following options:

Judy ran out of gloves and doesn’t know where to get more.
Judy went to get more gloves in the store room, but there is a combination lock on the door and she doesn’t know what it is.
Judy has developed a skin reaction to the gloves and is embarrassed to bring it up.
Judy just received a phone call that her mother is very ill and she is quite upset.
Judy saw the supervisor working without gloves and assumed you spoke to her last week because it’s your job to look out for safety.

These are just some of the possible influences on Judy’s decision not to wear gloves. To have good conversations about safety, your job is to determine the real issue without telling yourself stories first. Maybe everyone in the lab says “irregardless.” Maybe no one ever told Judy the correct word to use.

If you want to make a difference in your lab safety culture, think about the sources of influence on staff behavior. Ask about the reasons for the behavior, and work patiently to educate people about the consequences of unsafe actions. Use these tactics to reduce the amount of “flustration” you may feel when working to promote safety every day.

Scungio 1

-Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

Microbiology Case Study: 4 Year Old Girl with Diarrhea

A 4-year-old girl with no past medical history had been feeling unwell for one day following a barbecue she had attended a few days prior. Her symptoms worsened to include colicky abdominal pain and bloody diarrhea, with as many as eight bowel movements per day. This persisted for the following two days; thereafter, she presented to the hospital also complaining of fever, nausea, and vomiting. She was found to be dehydrated and pale on exam, and was admitted for intravenous rehydration. Fecal leukocyte testing and stool cultures were sent. A Gram stain of the pathogen isolated from stool culture is shown in Figure 1.

camp1 — Figure 1. Gram stain showing Gram-negative, curved rods

An infectious etiology is highly suspected given this patient’s presentation, leading to work-up with fecal leukocytes and stool cultures. The presence of fecal leukocytes, which was positive in this patient, is a strong indicator of inflammatory diarrhea. Bacterial stool culture allows for detection of Salmonella, Shigella, Campylobacter, E. coli O157:H7, Yersinia, Aeromonas, and Plesiomonas.

Many different culture mediums are used to isolate bacterial gastrointestinal pathogens. In addition to the routine 5% sheep blood agar and MacConkey agar, a case of infectious diarrhea requires further workup to rule out the above mentioned pathogens. Sorbitol-MacConkey agar is a variant of traditional MacConkey agar, and is used to detect E. Coli O157:H7, which differs from other E. coli strains by its inability to ferment sorbitol, thus forming colorless colonies on this media. Xylose lysine deoxycholate (XLD) and hektoen enteric (HE) agars are utilized for the selection and differentiation between Salmonella and Shigella. A sweep of bacteria growing on the blood agar plate and subsequent oxidase testing is used for detection of Aeromonas and Plesiomonas, which are oxidase positive organisms unlike normal fecal flora which is oxidase negative. Cefsulodin-irgasan-novobiocin (CIN) agar is used for the selection and differentiation of Yersinia, which utilizes inhibitory substances (cefsulodin, irgasan, novobiocin, bile salts, and crystal violet) to prevent the growth of most bacteria. The agar also contains a pH indicator that turns red or pink when mannitol is fermented; with Yersinia having a characteristic ‘bull’s eye’ colonies with red centers and clear edges. CIN is incubated at room temperature for 48 hours. Finally, Campy CVA agar is a selective media for Campylobacter containing antimicrobial agents cefoperazone, vancomycin, and amphotericin B (CVA) which inhibit normal fecal flora. This media is incubated at 42°C under microaerophilic conditions, which support the growth of Campylobacter jejuni and C. coli.

Our patient’s culture grew gray, non-hemolytic colonies on Campy CVA agar (Figure 2). The organism was identified as Campylobacter jejuni by MALDI-TOF MS (matrix-assisted laser desorption/ionization, time of flight mass spectrometry).

camp2 — Figure 2. Bacterial colonies growing on Campy CVA agar

Campylobacter are gram-negative, microaerophilic, curved or spiral rods in the family Campylobacteriaceae. They are widely distributed in animals and infection is most often transmitted by contaminated foods, particularly undercooked chicken. The species most commonly associated with human infections are C. jejuni and C. coli, with C. jejuni accounting for the large majority. Infection with C. jejuni has been linked with subsequent development of Guillain-Barre syndrome two to three weeks following the initial illness. Our patient improved following two days of IV fluids and antibiotics with no subsequent follow up after discharge.

References:
Manual of Clinical Microbiology, 11th edition

-Said Albahra, MD, 1^st year Anatomic and Clinical Pathology resident at the University of Texas Southwestern Medical Center.

-Erin McElvania TeKippe, Ph.D., D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.

Chromosome Structure, Staining, and Naming

Chromosome Structure

In a post back in September, I quickly summarized the abnormalities that can occur with chromosomes as a whole (such as deletions, insertions, transversions, etc). There is so much more to learn (more than I could possibly put into one blog post), because the way chromosomes behave, depends on their structure and DNA sequence. For instance, genes with the same DNA sequence will behave differently depending on where they are located on a chromosome as well as the effect of the surrounding DNA sequence.

So how exactly is the immense length of DNA compacted into a chromosome? Let’s take a DNA sequence and see just how it makes up a chromosome. A single molecule of DNA spools around histone protein cores forming bead like structures called nucleosomes. Between each nucleosome is a sequence of DNA termed “linker DNA.” The amino acids associated with histones are lysine and arginine. The super coiled form is compacted and can be visualized as a karyotype in laboratory testing.

name1 — Image courtesy of http://ghr.nlm.nih.gov/handbook/basics/chromosome

The centromere is the connection point of the duplicated chromosome, while telomeres are the endpoints. The short arm of the chromosome is termed “p” and the long arm of the chromosome is termed “q.” If we take these two chromosome arms into consideration, there are three types of chromosome morphology:

Metacentric – Chromosome arms are equal in length
Sub-centric – One arm is longer than the other
Acro-centric / Telocentric – One arm is extremely small or even missing

Chromosomal Staining Methods

As I mentioned above the complete set of chromosomes for an individual can be visualized via a karyotype. I’ve listed a few of the ways this can be accomplished:

G-Banding – Chromosomes are stained with giesma stains. The appearance differs based on the treatment of chromosomes prior to staining.
Q-Banding – Chromosomes are stained with fluorescent dyes, quinacrine or quinacrine mustard. Q-Band staining is similar to G-banding in that the fluorescent regions represent the AT-rich regions of the chromosome.
R-Banding – Results from heat treatment in a phosphate buffer followed by staining with Giesma dyes.
C-Banding – Centromere staining that results from alkali treatment.

TYPE OF BANDING	STAINING SUMMARY
G-Banding	· Geisma stain · AT-rich regions stain darker than GC-rich regions
Q-Banding	· Quinacrine fluorescent dye stains AT-rich regions
R-Banding	· Banding pattern is opposite G-banding
C-Banding	· Stains heterochromatic regions close to the centromeres · Usually stains the entire long arm of the Y chromosome

So how do you exactly identify chromosomal location based on banding patterns?

In studying disease and mutation, we follow a specific type of nomenclature to designate the regions that are of interest to us. Let’s take for instance something like the 22q11.2 deletion. What do all of these numbers and letters mean? To quickly summarize, 22q11.2 deletion syndrome occurs from the deletion of a small piece of chromosome 22 at a location: q11.2

22q11.2 DELETION
22	· Chromosome 22
q	· Long arm of chromosome (q)
1	· Region 1
1	· Band 1
2	· Sub band 2

So now, when we add in karyotope information you might see something like the following:

46, XY, del(8)(q21)

When you break it down, it states the patient is male (XY) and has a deletion in the long arm (q) of chromosome 8 at region 2, band 1

Translocation nomenclature can get a little more confusing:

46, XX, t(3;12)(p12.1;p11)

This designates a female has a translocation between the short arms (p) of chromosomes 3 and 12 and region 1, band 2, sub band 1; and region 1 band 1 respectively.

An example of Down syndrome: 47, XX + 21 (Female has an extra chromosome 21)

An example of Klinefelter Syndrome: 47, XXY (Male with extra X chromosome)

What do “FISH” have to do with Molecular Biology?

FISH, an acronym for Fluorescent In-Situ Hybridization, is a method used to detect and visualize protein, RNA, and DNA structures in the cell. FISH analysis is a relatively fast method that provides great resolution as it incorporates fluorescent probes labeled for detection of specific regions, deletions, and translocations. The images below show the difference between FISH and Karyotype images.

name2 — http://www.mun.ca/biology/scarr/FISH_chromosomes_300dpi.jpg

name3 — http://biochem.co/wp-content/uploads/2008/08/chromosomes-hmale.jpg

Robertsonian Translocations (ROB)

Robertsonian translocations are of importance because they involve translocating most of one entire chromosome to the centromere of another chromosome. They can be balanced or unbalanced. A balanced translocation usually results in no health difficulties because there isn’t a gain or loss of genetic material. However, due to the duplication or deletion of genetic material in an unbalanced translocation, syndromes and other malformations are likely to occur. The chromosome pairs common for Robertsonian translocations include translocations between 13 and 14, 14 and 21, and 14 and 15.

During a Robertsonian Translocation two chromosomes (typically acrocentric in formation) will break apart at their centromeres. The long arms will fuse to form a single chromosome and the short arms will also join to form a product. Typically the product created by the short arms contains nonessential genes and is eventually lost through cell division. Most people with ROB have only 45 chromosomes in each cell containing all of the essential genetic material and appear normal.

An example of a balanced Robertsonian Translocation would be when the long arms of chromosomes 14 and 21 fuse together. Phenotypically, the heterozygous carrier would appear normal because there are two copies of the major chromosome arms, resulting in two copies of the essential genes. However, children of the carrier could inherit an unbalanced translocation that causes Trisomy 21 (Down Syndrome).

Test your Knowledge!

Which two amino acids are associated with histones?
What type of chromosome morphology is shown:
If you wanted to stain chromosomes to see varying regions that were AT-rich, which type of stain would you use?
Describe the following karyotype results: 46, XX, t(1;14)(p21.3; p17.6)

Answers

Lysine and Arginine
Sub-centric
G-Banding (although Q-Banding also will produce darker regions that are AT-rich)
Female patient, with a translocation between the p and q arms of chromosomes 1 and 14, and region 2, band 1, sub band 3; and region 1 band 7, sub band 6 respectively.

References:

Buckingham, L. (2012). Molecular Diagnostics: Fundamentals, Methods and Clinical Applications (2^nd ed.). Philadelphia: F.A. Davis Company.

Coleman, W.B, Tsonagalis, G.J. (2005). Molecular Diagnostics: For the Clinical Laboratorian. New York: Springer-Verlang

Searle, B. Rarechromo.org. The Rare Chromosome Disorder Support Group. 1996. Web. 19 Dec. 2015.

L Noll Image_small

-LeAnne Noll, BS, MB(ASCP)^CM is a molecular technologist at Children’s Hospital of Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.

Microbiology Case Study: 58 Year Old Man with Fatigue and Chills

Case History:

Two weeks after returning from a camping vacation in Cape Cod, a 58 year old man presented to the emergency room with six days of fatigue, fever, chills, arthralgia, myalgia, mild right upper quadrant pain, and a frontal headache. Clinical workup revealed worsening leukopenia, thrombocytopenia, and elevated transaminases when compared to preliminary testing done by the patient’s primary care provider at the onset of his symptoms. His preliminary workup was also negative for Lyme antibody, EBV and CMV IgM, and viral hepatitis markers. At no point did the patient notice a skin rash or a tick anywhere on his person.

Differential Diagnosis:

Lyme Disease
Anaplasmosis
Ehrlichiosis
Babesiosis
Rocky Mountain Spotted Fever
Viral Meningitis
Bacterial Meningitis
Leptospirosis

Blood smear showing granulocyte with intracytoplasmic morulae.

Laboratory Identification:

Anaplasma phagocytophilium was initially identified by PCR. Retrospectively, the blood smears originally examined for Babesia by both hematology and parasitology were reviewed. Both slides showed multiple granulocytes with intracytoplasmic morulae.

Discussion

Anaplasma phagocytophilium is the bacterium responsible for the tick-borne disease known as human granulocytic anaplasmosis. Anaplasma is transmitted to humans primarily through the bite of an infected Ixodes scapularis, the same species of tick which transmits Borrelia burgdorferi (Lyme disease) and Babesia spp. (human babesiosis). Anaplasmosis, Lyme disease, and babesiosis therefore share roughly the same geographical distribution in the United States with northeastern and upper midwestern states reporting the most cases.

Anaplasmosis most commonly presents about 1-2 weeks after a tick bite with the sudden onset of a variety of non-specific symptoms including fever, chills, headache, malaise, myalgia, nausea, and abdominal pain. Anaplasmosis, unlike other tick-borne diseases, rarely causes a rash. Routine blood tests may show thrombocytopenia, leukopenia, or elevated liver enzymes in some patients. Severe clinical presentations, more common in immunosuppressed patients, may include difficulty breathing, hemorrhage, renal failure or neurological problems. Anaplasmosis is estimated to be fatal in less than 1% of cases.

A routine blood smear is the quickest method for establishing an early presumptive diagnosis. Microscopic examination of the smear may reveal microcolonies of Anaplasma known as morulae within the cytoplasm of infected granulocytes. Ehrlichia, in contrast, will preferentially target and form morulae within monocytes. Because not all patients with anaplasmosis have visible morulae, this test is diagnostically insensitive and should be followed by further testing.

Confirmatory serologic testing for anaplasmosis includes an indirect immunofluorescence assay using an Anaplasma phagocytophilum antigen. For the highest sensitivity, this test should be performed on paired serum samples collected at least 2 weeks apart with the first sample taken as early in the disease as possible. A positive test will demonstrate a four-fold rise in antibody titers. Although it is a very sensitive detection method when run with paired samples, the lengthy testing time is less than ideal for patients requiring hospitalization for their disease.

A PCR assay on a sample of whole blood, although only available at a few reference laboratories, is the most efficient and accurate way to detect Anaplasma during the acute phase of the illness. The sample used for PCR testing should be taken before the initiation of antibiotic therapy as it causes the sensitivity of this test to rapidly decline.

Doxycycline is the first line treatment for adults and children of all ages with anaplasmosis as recommended by both the CDC and the AAP Committee on Infectious Diseases. Patients should be treated for at least 3 days after the fever subsides. Standard duration of treatment is 7 to 14 days. Therapy should be initiated immediately when there is a high clinical suspicion of anaplasmosis. A physician should never wait for the results of confirmatory testing to begin treatment. Most patients see improvement within 24-48 hours of treatment and non-response to doxycycline may indicate a different disease process.

Anaplasmosis, like many other tick-borne diseases, is a nationally reportable disease. All cases should be reported to local and state health departments as well as the CDC.

-Elaine Amoresano, MD, is a 1^st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.