Fun With Microbiology (What's Buggin' You?): November 2010

Trematodes=flatworms=flukes

Phylum=Platyhelmithes
Family=Schistosomatidae

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.

Morphology;
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.

Treatment;
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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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)

Ditto

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).