Absence of Candida africana in Ugandan Pregnant Women: Results from a Pilot Study

High Vaginal Swab

Explain the specimen collection procedure to study participant (Pregnant woman) and gain consent. Advise woman to pass urine if needed.

Place the pregnant woman in a lithotomy position (lying on her back with her legs flexed and spread apart). Ensure privacy is maintained

Put on sterile gloves, and gently separates the vaginal lips (labia) to access the vaginal canal. Inserts a sterile cotton swab into the vaginal canal until some slight resistance is felt.  Rotated the swab 8 times in one direction to collect a sample from the vaginal walls

Remove the swab carefully to avoid contamination and place it in its plastic casing. Collect two swabs from each participant. Label each HVS tube with the participants unique identification number, the time and date of collection

Complete microbiology request form, and attach it to the signed consent form from the participant

Place all the 2 high vaginal swabs from a participant in biohazard Ziploc bag with accompanying forms, and store at 2 - 8^oC until they are transported to the Microbiology Laboratory to testing

Vaginal swabs in Ziploc bags were placed in a portable cooler box along with the questionnaire, request form, consent form, and transported to the analysis laboratory within 8 hours of collection

Physical inspection of the specimen

The sample swabs were first carefully examined to enable selection of the correct part of the sample likely to contain the fungus. Caseous or bloody areas were selected. Attention was paid to the color and thick consistency of the vaginal secretion

Direct gram Stain

Using one of the swabs, prepare a thin smear of the sample (discharge) on a clean, dry glass slide

Examine at low power (10-40x), then under oil immersion (100x) for the presence of gram organisms. Yeast cells are gram positive. Note the size, shape and arrangement of the fungal elements

Perform a Nugent scoring by by evaluating each Gram-stained smear for large gram-positive rods ( Lactobacilli morphotype), small Gram-variable rods ( Gardnerella vaginalis morphotypes), small gram-negative rods (Bacteroides species morphotypes), and curved gram-negative rods ( Mobiluncus spp. morphotypes).

Determine the number of organisms and the N-scores for each morphotype as shown in Table I below

A	B	C	D
Score	Lactobacillus(Large gram-positive rods)	Gardnerella and Bacteroides	Curved gram-negative rods
0	4+	0	0
1	3+	1+	1+ or 2+
2	2+	2+	3+ or 4+
3	1+	3+
4	0	4+

0 = no morphotype, 1+ = < one morphotype, 2+ = 1–4morphotypes, 3+ = 5-30 morphotypes, and 4+ = > 30 morphotypes

A total Nugent score ≤ 3 indicated that the smear was negative for BV, 4-6 indicated altered vaginal flora, 4–6 with polymorphonuclear cells indicated altered vaginal flora with inflammatory conditions, and ≥7 indicated that the smear results were consistent with BV

Fix and dry on a slide heater block at 37^oC

Add crystal violet to cover the smear and wait for 1 minute

Drain off the crystal violet by rinsing with clean water, being careful not to let the flow of water fall directly on the smear

Add gram-iodine solution to cover the smear and leave for 1 minute

Rinse with water in the same manner as in 9.4 above

Flood the slide with 50% acetone - alcohol for 5 seconds

Rinse again with water

Counter stain with safranin for 1 minute and rinse in water. Dry on slide warmer at 37^oC

10% Potassium Hydroxide (10% KOH) wet preparation examination

Take a clean, glass slide free of grease

Using a Pasteur pipette, place a large drop of 10% KOH solution on the slide. Gently mix the swab with a drop of 10% KOH

Cover the sample with a coverslip. Leave the sample on damp cotton wool for 2 to 5 minutes to allow the clearing process to take place

Examine the clear sample at low magnification (10x). Examine the whole coverslip from corner to corner in a zigzag pattern

Turn to a high magnification (40x). Adjust the light entering the condenser as you inspect at high power.

Examine if present, type of branching of hyphae, budding, non-budding cells, septation and thickness of hyphae

Look for and evaluate the types and numbers of the yeast cells (round, elongated, ovoid, single and multiple budding). True pseudohyphea crisscross epidermal cells in a random fashion and the strands are usually of a uniform diameter

quantifying fungal elements in a wet preparation examination using the following scale: 1+ (Scanty): Few fungal elements observed; rare hyphae or spores. 2+ (Moderate): Moderate number of fungal structures; hyphae and spores present. 3+ (Abundant): Numerous fungal elements; densely distributed hyphae and spores. 4+ (Very Abundant): Extremely high fungal load; overwhelming presence of hyphae and spore

Sabouraud Dextrose Agar (SDA) supplemented with Chloramphenicol

Streak the sample onto the surface of the medium by gently rolling the swab over a small area of the surface at the edge, and then streak from that area with a loop. Include a positive control plate inoculated with control strains of C.albicans ATCC 10231 in the batch

Incubate the plates at 25–30°C for 24hours

Note the growth, color, elevation, size, margins, texture, of the colonies. Report No fungal growth after incubation for 10 days

ChromAgar Candida Media

Obtain an isolated colony from the SDA and inculcate it on a subdivided ChromAgar plate. Incubate for 24hrs at 35 - 37^oC

Note the color of the colony and identify the yeast species based on colony color, according to the manufacture's guidelines

Germ Tube Test

Suspend a very small inoculum of the yeast cells obtained from an isolated colony in 0.5mL of Human serum. Incubate at 35 - 37^oC for no longer than 3 hours

After incubation, remove a drop the suspension and place it on a microscope slide. Cover with a cover slip and examine under low power magnification for the presence of germ tubes.

A germ tube is defined as an appendage that is half the width and 3 - 4 times the length of the yeast cell from which it arises, without a constriction at the point of origin of the germ tube from the cell

Corn Meal Agar Morphology

Obtain an isolated colony from the primary culture plate (SDA)

Inoculate a plate of corn meal agar containing 1% Tween 80 and trypan blue by making two lines at right angle to each other. Put a cover slip at the "x" intersection of the lines

Incubate the corn meal agar plate at 30^oC for 72hours

After 72hours, examine the areas under the coverslip for the presence of blastoconidia, arthroconidia, pseudohyphae, hyphae, or chlamydospores

Record the presence or absence of chlamydospores, and any other features seen

Extraction procedure

Perform extraction procedure in duplicate

Add 20µL of acetonitrile. Vortex briefly and Centrifuge for 3 minutes at 14,000g

Add 1 μL of the supernatant, taking care not to disturb the pellet, and spot, in duplicate, to a 96-spot reusable stainless steel target plate

Add 1 μL of the Bacterial test standard (BTS) suspension to two spots on the 96-spot reusable stainless steel target plate. Simply allow to air dry

As soon as spots are dry, immediately overlay with 1 μL of HCCA matrix (HCCA = α-Cyano-4-hydroxycinnamic acid). Do not delay the addition of HCCA matrix as it will decrease the quality of your run.

Air dry for two minutes.

As soon as spots are dry, subject them to MALDI-TOF analysis. NOTE: Target must be completely dry before it is inserted into MALDI BioTyper.

Be sure that the location of each specimen on the 96-spot reusable stainless steel target plate is recorded on a sample key spreadsheet. Verify the spreadsheet numbers to make sure they match the original sample numbers.

Interprete the resulting spectra using Bruker database

Label two 1.5 mL microcentrifuge tubes for each sample, including QC strains

Add 500 µL of 100% Ethanol to each.

Add approximately 50 µL of 0.1 mm diameter silica beads to each tube, using the scaling on the microcentrifuge tube

Wet a sterile swab using the ethanol from the suspension (from step 15.3) or sterile deionized water. Press swab against the side of the tube to remove excess fluid

Collect approximately a circle of 1-2 cm diameter of mold from the agar plate using the swab, selecting spores (conidia) and hyphae if possible. Be careful not to pick any agar when picking up the colonies.

Suspend the collected material in the 1.5 mL tube. Centrifuge for 3 minutes at 14,000g

Remove the Ethanol supernatant

Re-suspend the pellet in 20µL of 70% formic acid. Vortex briefly

Performance specification

Bruker allows for any score above 2.0 to be acceptable for a species identification

Interpreting results

At least one of the duplicates for each control strain must generate a score of ≥ 2 with a correct identification (no conflicting results between the duplicates)

If the control strains generate scores < 2 or incorrect identification, the results should not be interpreted, and the extraction/submission process must be repeated.

If no species identification is made, repeat extraction/submission process using a new subculture