Sunday 26 December 2010

Streptococcus anginosus group

Scanning a gram stain of a body fluid, you come across what appears to be a jumble of purple (gram positive) dots within a white blood cell. At first glance the dots do not appear to be arranged in clusters as would be expected if Staphylococci, nor do they appear to be very convincing chains of cells as Streptococci would exhibit. Focusing up and down through the depth of material on the slide, no clear ghost cells bearing sporadic beading of crystal violet (purple-blue) stain as might be expected with mycobacterium is evident. (See previous post on Mycobacterium tuberculosis).

What the heck is this? An organism?…an artifact of staining?

A colleague encountered this situation however, with the specimen site in mind and a scan of the smear quickly suspected Streptococcus anginosus group as the culprit in this infection.

Some background;

In 1956, microbiologist W. D. Miller began to isolate and group a number of oral streptococci which later became known as the Streptococcus milleri group, named in his honour. Being streptococci they are gram positive, catalase negative cocci occurring in chains however the milleri group exhibited considerable variability in hemolysis, Lancefield group antigen and other properties typically employed in streptococcal identification. Recently, with the advent of DNA analysis, it was determined that this group consists of three distinct species designated S. anginosus, S. constellatus and S. intermedius. This rearrangement of the S.milleri group is now known collectively as the Streptococcus anginosus group. The historical name of Strepticoccus milleri group, however, continues to remain popular in Britain and parts of Europe.

The Streptococcus anginosus group of organisms are found as normal flora, particularly in the mouth and gastrointestinal tract. As opportunists they have been implicated in systemic infections at multiple body sites and are particularly associated with abscess formation. As such, if isolated from blood culture sterile fluids or abscesses, it is critically important that these organisms not be dismissed as contaminants.

Specifically, alpha or gamma hemolytic members of the S.anginosus group might erroneously be dismissed as the less pathogenic viridians streptococci. Beta-haemolytic members of the S.anginosus group organisms may also be initially confused with S.pyogenes , S.agalactiae, S.equisimilis and large colony group Group ‘G’ streptococci. It is therefore imperative that Streptococcus anginosus group be considered whenever such colonies are evident on cultures of abscesses and normally sterile sites. A complete identification is warranted and the isolates should never be reported solely by their Lancefield grouping. Also, although many of the commercial identification systems, whether manual or automated have the individual Streptococcus anginosus group species in their databases, studies suggest that the accuracy of specific speciation might be as low as 75%. Again, because of the serious pathogenicity of this group, one must be absolutely certain of the identification in reporting.

Streptococcus anginosus species isolated from Abdominal Fluid (Below)

Clues that an isolate might be one of the S.anginosus group;
  • Alpha, beta or gamma streptococci isolated from normally sterile site such as blood*, tissue & body fluids.
  • Isolated from abscess.
  • Slower growing colonies that may not appear until 36 – 48 hours of incubation.
  • Colonies that may exhibit enhanced growth in CO2 or an anaerobic environment.
  • Small colonies on sheep blood agar, typically less that 0.5 mm diameter.
  • Colonies on agar media give off a characteristic caramel or butterscotch odour.
  • Gram stain (see below)
*isolating S.anginosus from the blood should alert the clinician that there is a focus (abscess) elsewhere in the body serving as the source of the bacteremia.

To remember the species comprising the Streptococcus anginosus group, just think of the acronym of America’s Central Intelligence Agency, the CIA where now the letters can stand for Constellatus, Intermedius & Anginosus.

Jumble of Gram Positive Dots within White Blood Cells in Abdominal Fluid
Are these Streptococci?? Do they offer a clue to specific identification?
(Click On Photo To Enlarge for Better Viewing)

Streptococcus anginosus
Gram Stain of isolated colony taken from Blood Agar Plate of above specimen
This more familiar appearance of Streptococcal cells is what is usually seen even in direct smears of species other than the anginosus group.

08/01/11: Added this photo of Streptococcus anginosus in a liver asperate (above)
Gram positive cocci in pairs and chains-the textbook description of Streptococcus.

Studies have suggested that there may exist a correlation between S.anginosus group’s specific species and site of infection or clinical manifestation. There appears to be a marked association between S.intermedius and infections of the central nervous system. S.anginosus is the species most frequently associated with the gastrointestinal and genitourinary tracts and S. constellatus was most frequently identified from specimens from the respiratory system.

A quick reference chart of some of the characteristics
the Streptococcus anginosus group may exhibit.

Related Papers;

Streptococcus milleri group: Renewed Interest In An Elusive Pathogen

Streptococcus anginosus (Streptococcus milleri): The Unrecognized Pathogen

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Sunday 21 November 2010

Schistosoma mansoni & haematobium



Primary human species are;
  • Schistosoma mansoni
  • Schistosoma haematobium
  • Schistosoma japonicum
  • (S.mekongi & S.intercalatum encountered less frequently)
Schistosomes infect about 200 million people worldwide, particularly in endemic regions such as Asia, Africa and South America. They have plagued man throughout history with evidence of infection (ELISA) in Egyptian mummies from the predynastic period (3100 B.C.) as well as Schistosome eggs detected in mummified kidneys (1250 B.C. to 1000 B.C.)

Schistosomes are often referred to as 'blood' trematodes as they differ from other trematodes because they infect humans by penetrating intact skin to gain entry to the circulatory system rather than infection through ingestion. In other words, Schistosomes infect humans through direct larval (cercariae) penetration rather than the ingestion of metacercariae.
Schistosomes are also unique among the flukes in that there are both a male and female organisms.
Schistosoma eggs also lack an operculum which characterizes other fluke eggs.

Their life cycle is as follows;
  • Eggs in feces or urine are passed into water
  • Larvae are liberated and penetrate the intermediate host snail where they further develop.
  • Cercariae emerge from the snail while in the water
  • Cercariae penetrate the skin of humans in contact with the water
  • Larval migration begins through the circulatory system where they may enter alveoli to produce hemoptysis. Organisms mature in the liver before entering specific veins specific to the infecting species. (S.haematobium in veins of the bladder, S.japonicum in veins of small intestine & S.mansoni in veins of the large intestine)
  • Eggs are passed to continue the cycle.
Pathogenicity of Schistosomes depends on the host's immune response to the various stages of infection, previous host exposure and the worm burden.
Symptoms include cercarial dermatitis, acute schistosomiasis (Katayama fever) and related tissue egg deposition. Acute schistosomiasis begins when the adult female begins laying eggs.

In the circualtory system it is believed the organisms either becomes covered with host soluble blood group antigens, lipoproteins, or develops antigens similar to the host's so that it excapes the host's immune response. For this reason, adult worms in the veins evoke little immune response.

Symptoms may vary in intensity but can include malaise, fever, abdominal tenderness or hepatic pain.

Infection with S.mansoni or S. japonicum may cause diarrhea. S. haematobium causes hematuria.

Schistosomiasis should be considered with any patient from endemic areas who has had exposure to untreated water and presents with symptoms previously mentioned. Diagnosis is confirmed with the identification of Schistosome eggs recovered the patient.

All eggs are embryonated when passed
All eggs are easily differentiated by their appearance
  • S.mansoni eggs are large (110-170 µm), oval and have a lateral, 'rose thorn' spine.
  • S.haematobium eggs are large, oval (110-170 µm) and have a terminal (end) spine.
  • S.japonicum eggs are smaller (55-90 µm), round and have a 'crooked finger' spine.
S.haematobium eggs can be recovered from centrifuged urine specimens
S.mansoni & S.japonicum from fecal specimens although on occasion both may be recovered from urine as well.

Praziquantel is the drug of choice in treating schistosomiasis. O & P examinations should be conducted periodically for up to a year post treatement to ensure erradication.

Schistosoma mansoni egg in concentrate (X400)
(Click on photos to enlarge for better viewing)


Schistosoma mansoni wallpaper (1024 X 768)
(note lateral 'rose-thorn' spine on egg)

I once encountered S.haematobium in the urine of a young Egyptian child however it was prior to my attempts at documenting interesting specimens in photographs. I have never personally seen a S.japonicum.

Update; I recently took some photos of S.haematobium from a preparation obtained from our Pathology department. Unfortunately I don't have much information on this patient's history nor the stain used. However, it does make for a pretty photo!

Schistosoma haematobium
(Note terminal spine)
(Click on photo to enlarge for better viewing)

Schistosoma haematobium (X400)
(Terminal spine at bottom barely visible)
New -August 2012:  Obtained from our pathology department - bladder biopsy with Schistosoma haematobium embedded in tissue.  Patient was of middle eastern heritage with relatively recent travel to that region but I have no further information.  Schistosoma egg is in a deteriorated state in these tissue section photos, leaving not much more than the outline with little internal structure remaining.

 Deteriorated S.haematobium embedded within bladder tissue (X500 Nikon: Giemsa?)

Two deteriorated S.haematobium eggs embedded in bladder tissue.  (X500 Nikon: Giemsa?)

New -Added November 08, 2013
Fresh urine sample from patient from Africa.  Total urine sample was centrifuged to concentrate and re-suspended sediment in a smaller volume.  Wet preparation was examined under the light microscope.

Schistosoma haematobium -unstained concentrate
(400+10X, DMD-108)

Schistosoma haematobium -unstained concentrate (note terminal spine)
(400X, DMD-108)

Schistosoma haematobium -unstained concentrate
Still fresh, the parasite was still alive with continuous movement and cytoplasmic streaming within the organism.  (400X, DMD-108)

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Sunday 14 November 2010

Bacterial Vaginosis

A number of nasties can infect the female genital tract. The Previous post dealt with the parasite Trichomonas vaginalis. Other common infections are Chalamydia infections, yeast infections and bacterial vaginosis. While Neisseria gonorrhoea can be isolated from the vagina, a cervical swab is a superior specimen for detecting gonnococcus (G.C.) I'll do a post on Neisseria gonnorhoea infection at a later date.

Bacterial vaginosis is characterized by a thin malodorous vaginal discharge due to an alteration of bacterial flora caused by a shift in the acidity (pH) of the vagina. It's referred to as a 'vaginosis' rather than an 'vaginitis' because 'itis' implies an inflamation which would be accompanied by a proliferation of white blood cells (wbc's). Here, in a bacteria vaginosis, the bacteria flourish without a significant increase in wbc's.

A several markers provide clues in the diagnosis of bacterial vaginosis.
  • Potassium hydroxide - a drop added to sample of the discharge produces a fishy odour due to amines released.
  • pH measurement - loss of normal acidity (generally considered >4.5) may suggest a vaginosis diagnosis, however it is not very definitive therefore not usually done in the lab.
  • Observation and bacterial enumeration of genera present in a vaginal gram stain. Vaginosis is characterized by a decrease by the lactic acid producing lactobacilli (hence the name) and an increase in the specific organisms, Gardnerella vaginalis, Mobiluncus species, and anaerobic organisms such as Bacterioides & Prevotella species. The relative proportion and or presence of these organisms can suggest bacterial vaginosis.
  • Clue Cells - the presence of epithelial cells stippled with gram variable bacilli is suggestive of bacterial vaginosis. In Scanning the gram stain, the presence of these epithelial cells coated with gram variable bacilli appear purplish in comparison to the regular pinkish-red epithelial cells present. Why these bacteria tend to adhere is not fully understood.
Specific description of bacterial forms present;
  • Probably the most noticeable bacterial form present in bacterial vaginosis is referred to as 'gram variable' bacilli. These are the Gardnerella vaginalis bacteria whose individual cells can stain both purple and red with the gram stain. That is, some entire cells may be either gram positive (purple-blue) or gram negative (pinkish-red) or both. They are relatively small or short cells. The irregular gram staining properties historically have contributed to placing the organisms in different genera. Previously called both Corynebacterium vaginale (a gram positive genera), and Haemophius vaginalis, (a gram negative genera), before being given it's own genera of Gardnerella. It is a somewhat fastidious organism.
  • Mobiluncus species - various species of the genus may be present in bacterial vaginosis and are observed, if present, as gram negative curved bacilli.
  • Anaerobic bacteria such as Bacteroides and Prevotella also are disproportionate and can be seen as smaller straight gram negative bacilli
The bacteria mentioned above may be present in the normal vagina in small amounts. When whatever causes the conditions to change, these bacteria can overgrow the normal flora and therby cause vaginosis.
  • Lactobacillus species are regular gram positive bacilli (purple-blue rods) which can vary in shape depending on the species and environment. The lactic acid they produce within the healthy vagina keep it slightly acidic which restricts the growth of other species of bacteria.
A guide for enumerating the proportion of the various bacteria forms present in a vaginal smear has been created and is known as the Nugent Score.
By scoring the various bacterial forms and noting the presence of Clue Cells, one can standardize the criteria for determining if the patient does indeed have bacterial vaginosis.

While bacterial vaginosis is a genital, or perhaps a sexual disease, it is not considered a sexually transmitted disease. Sexual contact is not responsible for it's presence nor is it spread through sexual contact. It arises from changes within host allowing for the disproportionate proliferation of native bacterial species.

Now for some photos of what is described above;
(Click on any photo to enlarge for better viewing)

Normal vaginal fora
Gram stain showing a couple of epithelial cells and numerous gram positive lactobacilli (purple rods). Note too that there are few if any white blood cells.
(100X, Gram Stain, Nikon)


Normal Vaginal Flora
All epithelial cells appear similar with no 'Clue Cells' which would appear bluish due to all the
Gardnerella vaginalis organisms adhering to the cells.
(100X, Gram Stain, Nikon)

Gram of bacterial vaginosis
Gram stain (1000X enlarged) showing epithelial cell coated with gram variable bacilli (Clue Cell).
Note gram variable bacilli (Gardnerella vaginalis) , gram negative curved bacilli (Mobiluncus sp.) & regular gram negative bacilli. Straight gram positive lactobacilli as seen in the previous photo are absent.
(1000X, Gram Stain, Nikon)

08/10/11: As an afterthought I added this photo which may better illustrate what is referred to as a 'Clue Cell' in vaginal swab gram stains. Here two epithelial cells are seen however the one on the left is 'coated' with gram variable bacilli (Gardnerella vaginalis) making the cell appear purplish. On lower power scanning, these are usually seen with some frequency in a patient experiencing bacterial vaginosis.
(1000X, Gram Stain, Nikon)

A couple enlarged (1000X) gram stains of gram variable bacilli.
Small, short cells are gram variable -gram negative (pink), gram positive (blue) or both at once.

Gram Stain (1000X) of Mobiluncus species (curtisii)
(Click on photo to enlarge for better viewing)

Mobiluncus species contribute to Bacterial vaginosis. They appear as gram negative (pink) curved bacilli - (look like pink parentheses)
Present are also two large pink epithelial cells

Added February 8th, 2016:
I see from the number of hits that this blog topic receives that it is quite popular.  I thought I might add a few more photos to better illustrate the condition.  

The next series of photos illustrate the condition of bacterial vaginosis where normal vaginal flora is greatly diminished and replaced primarily by Gardnerella vaginalis but possibly also Mobiluncus curtisii and other organisms such as the anaerobic gram negative Bacteroides or Prevotella species.

Bacterial vaginosis - usually appears as rather 'granular' (my personal description) as the entire field is usually stippled with the tiny gram-variable Gardnerella vaginalis.  Compare this appearance to the normal vaginal flora which follows shortly below.
(1000X, Gram Stain, DMD-108)

Bacterial vaginosis - as above, a very 'granular' appearance throughout the field with epithelial cells and epithelial cells stippled with Gardnerella vaginals (Clue Cell).
(1000X, Gram Stain, DMD-108)

Bacterial vaginosis - distinctive granular appearance.  Entire microscope field is often filled with these small gram-variable Gardnerella vaginalis usually appearing primarily purple-blue from the crystal violet in the gram stain.  The Nugent score which assists in making a definitive decision on whether the flora present indicate bacterial vaginosis is really not necessary when the specimen looks as above. 
(1000X, Gram Stain, DMD-108)

Bacterial vaginosis - epithelial cells with no distinctive clue cells in this photo, yet the field is filled with small gram-variable Gardnerella vaginalis.
(1000X, Gram Stain, DMD-108)

Bacterial vaginosis - one epithelial cell stippled (covered) with the small gram-variable Gardnerella vaginalis (clue cell) and one epithelial cell showing less coverage, equal to numbers you see throughout the field.
(1000X, Gram Stain, DMD-108)

Bacterial vaginosis - one last photo of bacterial vaginosis, primarily with the gram-variable Gardnerella vaginalis.
(1000X, Gram Stain, DMD-108)

Mobiluncus species - appear as small curved gram negative bacilli.  They may be present in variable quantities in bacterial vaginosis.
(100X, Gram Stain, DMD-108)

Mobiluncus species - another view showing the rather small, curved gram-negative bacilli which may accompany Gardnerella vaginalis in bacterial vaginosis.
(1000X, Gram Stain, DMD-108)

Gram stains of normal vaginal fora and its consituents follow;

Lactobacilli - Lactobacillus species are the predominant flora found in the vagina and appear as gram postivie (purple-blue) rods.  Lactic acid produced by these bacteria lowers the pH of the vagina and normal creates an environment which is not favorable to the growth of many other bacteria including Gardnerella vaginalis.  When the natural occurring commensal flora changes, invading organisms can capitalize and displace and invade.
(100X, Gram Stain, DMD-108)

Lactobacillus species - another view as above.  These should constituent the primary flora of the human vagina.
(1000X, Gram Stain, DMD-108)

Appearance of normal vaginal flora - Here we see an single epithelial cell in the center of the photo and numerous larger gram positive bacilli (rods).  The appearance of this photo is distinctly different from the previous bacterial vaginosis examples.  The overall appearance is not so 'granular' as the tiny Garnerella vaginalis is not present, or at least present in any appreciable quantities.  While white blood cells may be seen, they are not present in large quantities (pus) which differentiates 'vaginitis' from 'vaginosis'.
(1000X, Gram Stain, DMD-108)

Appearance of normal vaginal flora
(1000X, Gram Stain, DMD-108)

Appearance of normal vaginal flora - lactobacilli, epithelial cells and occasional white blood cells.
(1000X, Gram Stain, DMD-108)

 Appearance of normal vaginal flora - lactobacilli accumulated around an epithelial cell, not to be confused with 'clue cells'.
(1000X, Gram Stain, DMD-108)

Appearance of normal vaginal flora -numerous epithelial cells with lactobacilli distributed throughout the microscopic field.  No WBC's seen in this photo.
(1000X, Gram Stain, DMD-108)

Appearance of normal vaginal flora - numerous epithelial cells, occasional wbc's and the comparably (to G.vaginalis) larger gram positive lactobacilli throughout the field.
(400X, Gram Stain, DMD-108)

 Bacterial vaginosis (left) and typical normal vaginal gram stain (right)
For direct comparison
(Both 1000X, Gram Stain)
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Just for comparison: Vaginal Gram Stain of Yeast Infection (1000X)
Note: epithelial cells with yeast cells (purple) and yeast pseudohyphae (long purple thread-like structure).

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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