Tuesday, 22 November 2011

Streptococcus pneumoniae

Streptococcus pneumoniae (Bacteria)
Pneumococcal meningitis

Recently a 45 year old gentleman presented himself to the emergency department of our facility with suspected meningitis. The attending ER physician performed a lumbar puncture on the subject to obtain a sample of cerebral spinal fluid (CSF) which was noticeably cloudy. The specimen was sent to the laboratory where chemistry would measure the protein and glucose levels, haematology would perform a cell count and differential of the cells present, and to microbiology where we’d attempt to identify any infecting agent.

A number of different organisms can infect the blood stream (bacteraemia/septicaemia) and make their way to the meninges, the membranes that surround and protect both the brain and spinal cord.

The infection causes an inflammatory response and can produce various symptoms such as;
  • Headache
  • Stiff neck
  • Photophobia (dislike of bright lights)
  • Fever and chills
  • Drowsiness, confusion and possible delirium

Pneumococcal meningitis may not result in a rash which is common in Meningococcal meningitis (Neisseria meningitidis)

Pneumococcus can be found as commensal (normal) flora in up to 40% of the population and is the most common cause of meningitis in adults and in children over the age of two. The organism may reside in the back of the throat and in the nose where it can be passed on to others through close physical contact such as kissing or through coughs and sneezes. If the opportunity presents itself, this opportunist may invade the body to cause serious infections such as bacteraemia or meningitis. Still rather rare pneumococcal meningitis occurs at a rate of about 2 in every 100,000 of the Canadian population (1).

Predisposing factors for acquiring pneumococcal meningitis may include;

  • Diabetes
  • Compromised immune system
  • Pneumococcal pneumonia
  • Alcoholism
  • Head injury
  • Lacking a spleen
  • Other major chronic diseases

Laboratory Testing;
Chemistry - CSF is analyzed for it’s glucose and protein levels. Bacteria consume glucose for energy and a drop in the CSF to serum glucose ratio may provide a clue that a bacterium is the invading agent. Viruses do not utilize glucose for energy but may hijack the invaded cells ‘cellular mechanisms’ to produce protein necessary for its own reproduction an multiplication. Glucose levels that remain in the normal range with an increase of protein level may suggest a viral encephalitis. In our patient the levels were as follows;

In our patient;
  • Glucose = 2.3 mmol/L (2.2 - 3.9 mmol/L Normal Range)
  • Protein = 3.65 g/L (0.12 - 0.60 g/L Normal Range) -High
Haematology - Host cells present in the CSF are enumerated and differentiated which provide further clues to the nature of the illness. When an organism invades the body responds with an increase of white blood cells (WBC) to combat the invader. In other words, and increase in the number of WBC’s above the normal range suggests an infection. The type of WBC produced in response to the invader again provides further clues as to the invader. Neutrophils (=PolyMorphoNucleocytes=PMN) are the primary response to bacterial infections while viral agents may stimulate Lymphocytes.

In our patient;

  • Lymphocytes = 0.2 X 10^9/L (1.00 - 4.00 X 10^9/L Normal Range) -Low
  • Monocytes = 0.7 X 10^9/L (0.00 - 1.20 X 10^9/L Normal Range)
  • Neutrophils (PMN) = 17.2 X 10^9/L (2.00 - 7.50 X10^9/L) -High

Microbiology - In micro, we perform a gram stain on the specimen in an attempt to visualize the invading organism. A cytosine is usually performed which simply concentrates (via centrifugation) an aliquot of the CSF specimen onto a glass microscope slide for staining. In addition to concentrating the possibly small numbers of bacteria, the cytosine process ‘flattens’ the Neutrophils, thereby spreading them out and allowing better viewing of any bacterial cells present, particularly if ingested by the PMN.

The gram stain process not only makes the stained bacteria easier to visualize but may assist in differentiation of the invading bacterial species. Pneumococci (Streptococcus pneumoniae) are gram positive cocci and will appear as dark bluish-purple dots in pairs or short chains. Meningococcal (Neisseria meningitidis) are gram negative diplo-cocci and appear as red ‘coffee bean-like’ cells - in pairs with flattened adjacent sides. (gram negative bacilli may indicate a bacterial pathogen such as Haemophilus species while a gram positive bacillus may suggest Listeria species.)

In the case that presented to our lab, the CSF which is normally as clear as pure water, appeared as a cloudy or milky solution. Increased cellular immune response, the presence of bacterial cells and by-products may account for this altered appearance. Xanthachromia, a yellow colour due to the breakdown of red blood cells that may have entered the CSF was not observed. (This may be present in a Sub-arachnoid haemorrhage.)

The gram stain revealed gram positive cocci in pairs and short chains. Pneumococci often appear ‘lancet’ shaped which the adjacent pair of cells have a flattened common side while the opposite side is somewhat pointed or elongated.

Given the age of the patient and the gram stain result, pneumococcus was immediately suspected as the most likely culprit in this infection. A Quellung capsular swelling reaction was performed on the CSF to confirm our suspicions. I have described the Quellung Reaction elsewhere in this blog, and will only mention here that it is a specific and definitive test for pneumococcus. The Quellung reaction was observed allowing the lab to report a definitive diagnosis of pneumococcal meningitis to the doctor within an hour of the specimen being taken. Immediate treatment with appropriate antibiotics is imperative to prevent potentially catastrophic consequences.

Microbiological Results shown below;

Gram stain of Cytospin preparation of CSF (X1000)
Gram positive cocci in pairs and short chains suggestive of Streptococcus pneumoniae
Cystospin process has concentrated both the bacterial cells as well as the neutrophils (pink cells)
(click on photo to enlarge for better viewing)

Still at 1000X magnification but photo enlarged showing greater detail

Gram stain of Cytospin preparation of CSF showing greater detail. (X1000)
Insert on right may reveal evidence of capsule as clearing around bacterial cell.
(click on photo to enlarge for better viewing)

Quellung reaction in preparation of above CSF. (X1000 -Methyene Blue Counter Stain)
Clearing revealing presence of capsule viable around bacterial cells.
(click on photo to enlarge for better viewing)

Another view - ditto
(click on photo to enlarge for better viewing)

Quellung again at X1000 however I enlarged a portion of the photo to better illustrate the capsule surrounding the pneumococcal cells.
(click on photo to enlarge for better viewing)

Sheep Blood Agar Plate of CSF incubated overnight in 5% CO2 revealing alpha-hemolytic colonies (greening zones around colonies) typical of Streptococcus species. Colonies have a slight depression in the center typical of some Streptococcus pneumoniae strains. Insert shows colonial features in greater detail while purple arrow 'B' shows where colonies were tested for bile solubility (10% Sodium deoxycholate), another characteristic used as a confirmatory test for Streptococcus pneumoniae. Organism was sensitive to Optochin Taxo P Discs* (2) (not shown) adding further evidence of this bacterium being pneumococcus.
Phenotypic /biochemical identification was performed with the Phoenix Identification system (2) which also provided an antibiotic sensitivity summary. The patient would have been treated emperically when meningitis was first suspected or when the gram stain result (presumptive pneumococcal meningitis) result was sent STAT to to the attending ER physician.

(1) Meningitis Research Foundation of Canada
(2) BD™ Becton-Dickenson Diagnostics, Franklin Lakes, N.J. USA

Saturday, 5 November 2011

Neoscytalidium dimidiatum

Neoscytalidium dimidiatum (formerly Scytalidium dimidiatum)

-Arthric synamorph of Nattrassia mangiferae
-Species, until recently known under the picnidial synamorph name of Hendersonula toruloidea occurs on a wide variety of tropical fruit trees, sometimes causing branch rot.

Macroscopic; Rapid growth quickly filling the plate, mature within three days. Effuse, hairy or wooly colonies which are white to greyish (or dark grey to blackish brown) with a cream colour to an ochraceous-yellow reverse. Melanin deficient mutants may occur.
Growth inhibited by cycloheximide therefore will not grow on selective media such as that used for dermatophyte isolation.

Microscopic; Branched and septate hyphae with no conidiophores. Arthroconidia (3-6 X 5-15 µm) develop and are 1-2 cells in length, flattened on the ends and may be rectangular, square, as well as oval to roundish becoming barrel shaped. The wide hyphae (6 -10 µm), and arthroconidia are brown (melanin) while the narrower side branches of the hyphae tend to produce pale arthroconidia. Melanin free variants (previously known as Scytadidium hyalinum) do occur, producing hyphae and arthroconidia that are invariably colourless but are identical to Neoscytalidium dimidiatum in every other way.

A picnidial form very occasionally develops in very old cultures and is currently considered to be Fusicoccum dimidiatum (formerly thought to be Nattrassia mangiferae. Picnidia, when present are large (100 - 300 µm). Picnidial conidia are hyaline when young with age but as they age may develop 1- 5 septa with a dark brown central region. Picnidia may be induced by prolonged growth on sterilized lemon or banana skins.

Pathogenicity; Known to cause nail and skin infections with rare reports of deep-seated infections such as brain infections, sinusitis, lymphadenitis and endophthalmitis with higher incidence of fungemia in immunocompromised patients. Infection is primarily found in patients from tropical to sub-tropical regions.

Neoscytalidium dimidiatum on SAB agar after 3 days incubation.
(click on photo to enlarge for better viewing)

Neoscytalidium dimidiatum on SAB agar Reverse & Surface after 5 days growth.
Surface shows impression where hyphae were removed in celluloid tape examination.
(click on photo to enlarge for better viewing)

Hyphae 40X LPCB

Broad Septate Branching Hyphae (6 -10 µm)
Note narrower, un-pigmented side branches.
(click on photo to enlarge for better viewing)

Dark (melanin), Broad, Septate, Branching Hyphae (6 -10 µm)
Arthroconidia present. (X400)
(click on photo to enlarge for better viewing)

Broad septate hyphae and contiguous arthroconidia (LPCB X 400)
(click on photo to enlarge for better viewing)

(click on photo to enlarge for better viewing)

Pigmented arthroconidia as contiguous cells.
(click on photo to enlarge for better viewing)

Arthroconidia (LPCB X600)
(click on photo to enlarge for better viewing)

Broad septate hyphae and two celled conidia.
Unstained preparation from Corn Meal Agar (magnification not noted)
(click on photo to enlarge for better viewing)

Friday, 4 November 2011

Slide Culture Technique

Preparation of a slide culture can be useful in the study and identification of an unknown fungal isolate. While the 'sticky tape' preparation is quick and easy to perform, it can be disruptive, with salient features altered or destroyed. The slide culture when performed properly is more gentle allowing for the examination of intact structures & features as they naturally developed. The technique is quite easy to perform and I have outlined the basic steps in the photos that follow.

(1) Start by getting a plate of fungal media, Saboraud-Dextrose as shown above, and some means of cutting the agar. Pictured here is a sterile scalpel however they can be relatively pricey for such a small job. I prefer to use a microscope cover slip as the edge is thin, sharp and straight. A 20 X 20 mm cover slip can be used however the 20 X 40 cover slip allows for batter grip. Simply plunge or drag the edge of a cover slip into the agar surface, cutting out small blocks of agar somewhat about 1/2 to 3/4 of an inch square. as shown below.
(some people have used the open end of a sterile test tube as a punch to cut out a round plug of agar for the same purpose)

(2) Above you can see how I sliced the agar into squares that would be slightly smaller than a 20 X 20 mm glass microscope cover slip, using a glass cover slip as a knife. Cut as many squares as you require for the current and near future projects.

(3) A row of squares cut into the agar is seen above. Cut as many as you need for your project and the remaining plate can be placed into a plastic bag and refrigerated for future projects. (note the expiry date of the agar)

(4) Remove an agar square that you have cut into the plate using the same cutting tool (scalpel or cover slip) that you used to previously.

(5) Place the agar block onto a clean glass microscope slide. You can place one per slide or two per slide as I sometimes do. You can also place two different agars on the same slide. (eg. SAB and Corn Meal)

(6) The slide can then be placed in a clean petrie dish which will prevent contamination and preserve moisture during incubation. I like to raise the slide off the bottom of the petrie dish. Moisture in the between the plastic surface of the dish and the glass slide itself might create enough surface tension between the two making removal of the glass slide difficult without disrupting the delicate growth. You can use plastic or wooden stir sticks for this purpose. Some people sacrifice an uninoculated media plate such as blood agar and place the slide directly onto the agar surface. The agar acts as a source moisture during incubation however the surface tension may make remove of the slide somewhat more difficult.

(7) Using a sterile instrument (loop, needle, etc), transfer some of the fungus (spores, conidia etc) from the specimen being cultured to each of the four sides of the agar block. Just a quick touch of the isolate to the four edges as shown below.

(8) Transfer the fungus to the agar block's sides. It isn't necessary to smear the entire side but rather just touch the center of the agar block.

(9) After inoculation, place a clean cover slip on the surface of the agar block. (Remember to do this as it is easy to forget!!).

(10) A few drops of sterile water can be added to the petrie dish as an additional source of moisture which may be beneficial to slow growing fungi which may dry out with prolonged incubation. In general this is not necessary as the dish will be partially sealed up.

(11) The plate is now partially sealed with Parafilm™ or a bit of cellulose tape. If fully sealed the plate may fog up and moisture condense on specimen. The tape partially seals the plate, still allowing it to breath. The sealing of the plate also minimizes contamination of both the culture and the environment.

The slide culture is now ready for incubation. Incubate the slide at an appropriate temperature (room temperature to 30C for most fungi) and for an appropriate length of time. Fast growing fungi can overgrow the agar block very quickly so monitor the growth visually daily. One may wish to make several side cultures of the same fungus and examine one per day to observe how the structures develop.

To examine the slide culture, remove the slide from the petrie dish and then gently remove the cover slip from the agar block using plastic forceps or gloved fingers. The fungus should have adhered to some extent to the glass cover slip. Place a drop of LPCB* onto a clean microscope slide and then place the cover slip from the slide culture (growth down) onto the LPCB. The slide is now ready for examination under the light microscope. Don't throw the agar block away as if you remove it you will find that the fungus also adhered to the microscope slide. Just add LPCB and cover slip the growth. You get two cultures from one block - top and bottom.

*LPCB: Lacto Phenol Cotton Blue stain.