2016 – Page 14 – Lablogatory

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.