Tuesday 28 September 2010

Trichomonas vaginalis

(Parasite - Flagellate)

Trichomonas vaginalis is a human parasite which has worldwide distribution and is most commonly isolated from the female genital tract. In the male, Trichomonas vaginalis has been isolated from the prostate so it may be necessary to treat both sexual partners in order to prevent re-infection.

Trichomonas infection in the female may be suspected if a thin frothy white discharge is observed. Diagnosis is best made using a wet preparation from a freshly collected swab. The organism is sensitive to both temperature and drying so it should be sent to the laboratory as quickly as possible without refrigeration. On receipt, a drop of sterile saline is placed on a glass microscope slide and with the swabs contents expelled, it is cover-slipped and examined under low magnification (100-250X).

Trichomonas is roughly pear shaped and is between 7 to 23 µm long by about 5 to 15 µm in size. It normally has 1 posterior and 4 anterior flagella which provides a rather rapid and jerky motility which draws one’s attention when examining fresh preparations. Even in specimens that have been somewhat delayed in transit, an undulating membrane running along a portion of the cell, may be seen beating, An axostyle is also evident running the length of the cell. Other structures may not be evident on an unstained preparation. Trichomonas is only found as a trophozoite as it has no cyst stage.

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Two Trichomonas vaginalis cells (center) seen in a wet prep of a vaginal swab

Trichomonas is site specific and when found in the human genital tract it is diagnostic for Trichomonas vaginalis. Care must be taken not to contaminate the swab with fecal material as the non-pathogenic Trichomonas hominis can be found in stools. Trichomonas tenax may be found as a commensal in the oral cavity.

Gram Stain of Trichomonas in vaginal swab
(note size comparison to wbc's)

Although it is possible for an experienced technologist to see Trichomonas vaginalis in a gram stain, it is not the preparation of choice. A Hematoxylin stain as employed for fecal material examination would stain Trichomonas however the simple wet prep remains both cheaper and quicker. Other tests such as monoclonal antibody, enzyme immunoassay and latex agglutination have been developed. Serological tests have not proven to be effective .
Treatment with Metronidazole (Flagyl) is usually effective although resistant strains have been described.

Short video of motile Trichomonas vaginalis cells in wet prep. Flagellar movement and undulating membrane motion occasionally evident. Will replace with a better video when I get a more active specimen.
(Click on Lower Right Hand of Control Bar to view Full screen)

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Monday 27 September 2010

Mycobacterium tuberculosis in Gram Stain

Mycobacterium tuberculosis
(In Gram Stain Of Peritoneal Dialysis Fluid)

So, you find yourself the lone technologist manning the micro lab during the evening shift. A dialysis fluid arrives for routine analysis and a gram stain is performed on a cytospin(1) of the sample. Under the microscope no bacteria are initially evident during repeated scans - yet something catches the technologist’s eye. It may have been the odd string of purple-blue dots or perhaps thin strands of clearing where the surrounding material uniformly retains the safranin gram counterstain.

An astute colleague recently encountered that very scenario and immediately knew something was amiss. She immediately suspected the presence of either Nocardia, or due the site, more likely Mycobacterium species. A fluorescent acid-fast(2) Auromine-O stain was performed to confirm her suspicious of presence Mycobacterium species.

The patient was a 72 year old Oriental gentleman with chronic renal failure receiving peritoneal dialysis. The specimen was sent to the provincial health laboratories the following morning where further analysis by AMTD(3) confirmed the identity as Mycobacterium tuberculosis (TB).
The diagnosis came as a total surprise to the doctor in charge.

Below are some photographs I took of this rather interesting specimen. Additional cytospins were made with more material deposited on the slide resulting in more Mycobacteria per field. The initial gram stain was even more challenging than what is seen in the gram stains that follow.

Gram Stain of Peritoneal Dialysis Fluid ;
(All photos 1000X Magnification)

Can you spot the purple-blue dots in the lower right quadrant of the photograph above?
(click on photo to enlarge)

If one examines the cell wall of a Mycobacterium under an electron microscope, it resembles the gram positive cell wall structure. Mycobacteria, however, have a high content of mycolic acid associated with the cell wall which resists staining by the traditional gram stain method. For this reason the gram stain is not routinely used to visualize Mycobacteria. Alternative stains are employed such as the Ziehl-Neelsen, or Rhodomine-Auromine stains. The Mycobacteria are stained using these stains and the high mycolic acid content resists decolourization using a mild acid-alcohol solution.

'Ghost Cells' A clue to the presence of Mycobacteria in a gram-stained specimen
(Click on photo to enlarge for better viewing)

One clue found in a gram stain that may suggest the presence of Mycobacteria are the ghost like cells. These appear as a clear (unstained) line in the shape and size of a bacillus and are due to the mycolic acids resisting retention of the gram stain. As the bacterial cells are not stained they appear as a clear line or 'ghost cell' surrounded by material retaining the counterstain. Look carefully at the photo above (click on it to enlarge for better viewing) and look for the clear ghost cells in the left. Focusing the microscope up and down may bring areas retaining the gram stain into better focus thereby revealing the parts of the cell wall staining purple-blue as seen on the right. (arrows point identical areas to where the Mycobacteria are visible under varying focus) [contrast this appearance with the 'dots' that Streptococcus anginosus may exhibit in direct specimen gram stains]

Gram stain retained as purple-blue dots spaced between clear areas of high mycolic acid content that resists the stain. Easy to overlook by the untrained eye.

A cytospin of dialysis fluid containing a larger amount of material resulting in more bacterial cells per microscopic field. Short rows of dots are visible, however they are unlike the chain of cocci as a Streptococcus would appear.

Mycolic acids also assist Mycobacterium's ability to survive. They are similarly responsible in resisting the uptake of antibiotics used to eradicate the organism. The also resist being engulfed and killed by macrophages - a cellular defense mechanism of the body.

(1) Cytospin - a fluid specimen is added to a miniature funnel that is clamped to a microscope slide above a blotting spacer. The assembly is placed in a special centrifuge which then, under force deposits the liquid, under force onto the microscope slide. Solid material such as cells and bacteria are concentrated and somewhat flattened onto the glass slide and excess fluid is wicked away by the blotting material. This microscope slide is then stained by the desired method and examined under the microscope. This maximizes detection of any bacteria if present in the specimen.

(2) Acid-Fast/Auromine-O -Auromine-O, Auromine-Rhodomine, and Ziehl-Neelsen stains are known as 'Acid-Fast' stains. The two former stains are fluorescent stains which glow a bright yellow to apple-green under a particular fluorescent wavelength as seen under a fluorescent microscope. The later (Z-N) is examined under a traditional light microscope and Mycobacteria will appear bright red against a green (malachite green) or blue (methylene blue) counterstained background. Mycobacteria resist being decolourized by a mild acid-alcohol solution and as they retain these stains, the cells are said to be 'acid-fast'.

(3) AMTD - An acronym for Amplified Mycobacterium tuberculosis Direct test. This is a DNA probe test that looks for a particular and unique sequence of nucleic acids within the microbes genome that only occurs in Mycobacterium tuberculosis. Finding its presence confirms the organism is Mycobacterium tuberculosis and not a 'MOT' (Mycobacterium Other than Tuberculosis). Other species of Mycobacteria exist and not all have the same devastating consequences assiciated with TB. AMTD is a very rapid test for identifying TB in a sample compared to the weeks to months required to isolate and identify the bacilli by conventional culture techniques.

Our fluorescent microscope does not permit the attachment of the camera at this time so I'm currently unable to take photographs of the bacilli fluorescing in the acid-fast stain employed by our laboratory.

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Monday 6 September 2010

Cryptococcus neoformans

Cryptococcus neoformans

Worldwide distribution often found in soil contaminated with bird excrement, in particular, pigeon droppings have frequently been implicated.

Cryptococcus neoformans is a rapidly growing typically round yeast (5-10µm) capable of producing polysaccharide capsules which often surround the cell. A 1% peptone solution might aid encourage capsule production. Some species &/or strains of Cryptococcus will not produce capsules in-vitro. Capsule production is best demonstrated using the India-ink (or Nigrosen) negative staining technique. Colonies producing capsules on culture are often evident by their glistening wet or mucoid appearance whereas colonies which fail to produce capsules or have diminished ability to form capsules typically produce dull, creamy, butyrous colonies. Cryptococcus neoformans can be sub-typed further based on serological response to capsular antigens.

Click on Photo to Enlarge
Urease Test Positive: (urea split to release ammonia which raises the pH of the phenol red indicator in the media turning it from a straw colour to a bright pink.)
C.neoformans and other Cryptococci inhibited by Cycloheximide.
C.neoformans can be differentiated from other Cryptococcus species using the Caffeic acid test (a substance used as a substrate to demonstrate phenoloxidase activity. If phenoloxidase is present, it breaks down caffeic acid to melanin with resulting brown-black colour production.) Note: phenoloxidase is inhibited by the presence of glucose so culture the yeast on a glucose-free media such as Cornmeal-Tween 80 agar.
C.neoformans grows well at 25oC as well as 37oC. Some other Cryptococcus species will not grow at 37oC.
Cryptococcus neoformans & most other Cryptococcus species do not produce pseudohyphae.

Clinical Manifistations;
Cryptococcus infections can be found with increasing frequency amongst HIV patients and others who are immunocomprimised however Johns Hopkins Medical center has noted an interesting pattern regarding the serotypes of C.neoformans.
C.neoformans v. neoformans is the most common, usually afflicting immunocompromised hosts while C.neoformans v. gattii is most common in immunocompetent hosts.

Cryptococcus neoformans infection is primarily acquired through inhalation and may invade;
  • Respiratory system - (Sputum, Broncheal Wash, Lung Biopsy)
  • Central Nervous System (CNS) - (Lumbar Puncture)

Also implicated in;
  • Skin infections
  • Bone infections
  • Other sites (disseminated)

Treatment; (dependent on site of infection)
Amphotericin B

Prognosis varies.

The photos on this post were taken from speimens obtained from an 80 year old woman of Indian heritage who presented with respiratory distress. Unfortunately she succumbed to her infection.

Gram stain of sputum specimen 1000X showing cell & size variations
(Inset: Top -Cell showing Capsule, Bottom -Budding cell and clear capsule)

India Ink preparation taken from SAB isolate showing numerous Cryptococcal cells surrounded by clear capsule (negative staining)
(Inset: Enlarged photo of Cryptococcal cell & budding daughter cell surrounded by clear capsule)

Rather unremarkable round Cryptococcal cells taken from Cornmeal Agar Plate

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