TITLE: Absence of Candida africana in Ugandan Pregnant Women: Results from a Pilot Study
AUTHORS: Bwambale Jonani, Ssebuliba Joshua, Atuhairwe Susan, Gerald Mboowa, Mike N Kagawa, Beatrice Achan

[DESCRIPTION]
Candida africana, an emerging variant identified since 2001 as a distinct species or variety within the Candida albicans complex, exhibits a global prevalence of 1.67%. This study outlines the methodology employed to investigate the presence of C. africana vaginitis in Uganda through a pilot study conducted in three hospitals in central Uganda, focusing on pregnant women receiving antenatal care. The protocol details the procedures implemented for the collection of vaginal swabs from pregnant women and the utilization of laboratory techniques including microscopy, fungal culture, and MALDI-ToF analysis. Notably, the prevalence of C.africana vaginitis was zero, establishing a baseline data tp inform future exploration into cryptic yeast species associated with vulvovaginal candidiasis in Uganda

[BEFORE_START]
Data on age, gestation period, clinical signs and symptoms were collected using a Research Ethics Committee approved structured questionnaire from all pregnant women included in the study. This proceeded the informed consent to participate in the study provided by the pregnant woman 

[GUIDELINES]
Inclusion Criteria 
Pregnant women who presented with clinical symptoms of either vaginal/vulva Itching, vaginal discharge, history of previous VVC infections, and those with on-going antifungal treatment for vulvovaginal candidiasis were included in the study
 
Exclusion Criteria
Pregnant women with signs of bleeding per vaginum were excluded from the study. This was due to the increased potential for interference with accuracy of test results, discomfort for the patient, and increased risk of introducing pathogens into the vaginal area

[STEPS]
SECTION: Specimens collection
1.1. Explain the specimen collection procedure to study participant (Pregnant woman) and gain consent. Advise woman to pass urine if needed.
SECTION: Specimens collection
1.2. Place the pregnant woman in a lithotomy position (lying on her back with her legs flexed and spread apart). Ensure privacy is maintained 
SECTION: Specimens collection
1.3. 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
SECTION: Specimens collection
1.4. 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
SECTION: Specimens collection
1.5. Complete microbiology request form, and attach it to the signed consent form from the participant
SECTION: Specimens collection
1.6. Place all the 2 high vaginal swabs from a participant in biohazard Ziploc bag with accompanying forms, and store at 2 - 8oC until they are transported to the Microbiology Laboratory to testing 
SECTION: Specimen Transportation
2. 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
SECTION: Specimen processing
3. Physical inspection of the specimen
SECTION: Specimen processing
3.1. 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 
SECTION: Specimen processing
5. 10% Potassium Hydroxide (10% KOH) wet preparation examination
SECTION: Specimen processing
5.1. Take a clean, glass slide free of grease
SECTION: Specimen processing
5.2. 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
SECTION: Specimen processing
5.3. 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
SECTION: Specimen processing
5.4. Examine the clear sample at low magnification (10x). Examine the whole coverslip from corner to corner in a zigzag pattern
SECTION: Specimen processing
5.5. Turn to a high magnification (40x). Adjust the light entering the condenser as you inspect at high power. 
SECTION: Specimen processing
5.6. Examine if present, type of branching of hyphae, budding, non-budding cells, septation and thickness of hyphae
SECTION: Specimen processing
5.7. 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
SECTION: Specimen processing
5.8. 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
SECTION: Specimen processing
4. Direct gram Stain 
SECTION: Specimen processing
4.1. Using one of the swabs, prepare a thin smear of the sample (discharge) on a clean, dry glass slide
SECTION: Specimen processing
4.2. Fix and dry on a slide heater block at 37oC
SECTION: Specimen processing
4.3. Add crystal violet to cover the smear and wait for 1 minute
SECTION: Specimen processing
4.4. Drain off the crystal violet by rinsing with clean water, being careful not to let the flow of water fall directly on the smear
SECTION: Specimen processing
4.5. Add gram-iodine solution to cover the smear and leave for 1 minute
SECTION: Specimen processing
4.6. Rinse with water in the same manner as in 9.4 above
SECTION: Specimen processing
4.7. Flood the slide with 50% acetone - alcohol for 5 seconds  
SECTION: Specimen processing
4.8. Rinse again with water
SECTION: Specimen processing
4.9. Counter stain with safranin for 1 minute and rinse in water. Dry on slide warmer at 37oC
SECTION: Specimen processing
4.10. 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
SECTION: Specimen processing
4.11. 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).  
SECTION: Specimen processing
4.12. Determine the number of organisms and the N-scores for each morphotype as shown in Table I below
 

 
 0 = no morphotype, 1+ = < one morphotype, 2+ = 1–4morphotypes, 3+ = 5-30 morphotypes, and 4+ = > 30 morphotypes
 
SECTION: Specimen processing
4.13. 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
SECTION: Culture
6.     Sabouraud Dextrose Agar (SDA) supplemented with Chloramphenicol
SECTION: Culture
6.1. 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
SECTION: Culture
6.2. Incubate the plates at 25–30°C for 24hours 
SECTION: Culture
6.3. Note the growth, color, elevation, size, margins, texture, of the colonies. Report No fungal growth after incubation for 10 days 
SECTION: Culture
7. ChromAgar Candida Media 
SECTION: Culture
7.1. Obtain an isolated colony from the SDA and inculcate it on a subdivided ChromAgar plate. Incubate for 24hrs at 35 - 37oC
SECTION: Culture
7.2. Note the color of the colony and identify the yeast species based on colony color, according to the manufacture's guidelines  
SECTION: Culture
8. Germ Tube Test
SECTION: Culture
8.1. Suspend a very small inoculum of the yeast cells obtained from an isolated colony in 0.5mL of Human serum. Incubate at 35 - 37oC for no longer than 3 hours 
SECTION: Culture
8.2. 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. 
SECTION: Culture
8.3. 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
SECTION: Culture
9. Corn Meal Agar Morphology 
SECTION: Culture
9.1. Obtain an isolated colony from the primary culture plate (SDA)
SECTION: Culture
9.2. 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 
SECTION: Culture
9.3. Incubate the corn meal agar plate at 30oC for 72hours
SECTION: Culture
9.4. After 72hours, examine the areas under the coverslip for the presence of blastoconidia, arthroconidia,  pseudohyphae, hyphae, or chlamydospores 
SECTION: Culture
9.5. Record the presence or absence of chlamydospores, and any other features seen 
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10. Extraction procedure
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.1. Perform extraction procedure in duplicate
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.2. Label two 1.5 mL microcentrifuge tubes for each sample, including QC strains 
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.3. Add 500 µL of 100% Ethanol to each.
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.4. Add approximately 50 µL of 0.1 mm diameter silica beads to each tube, using the scaling on the microcentrifuge tube
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.5. 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
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.6. 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. 
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.7. Suspend the collected material in the 1.5 mL tube. Centrifuge for 3 minutes at 14,000g
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.8. Remove the Ethanol supernatant
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.9. Re-suspend the pellet in 20µL of 70% formic acid. Vortex briefly
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.10. Add 20µL of acetonitrile. Vortex briefly and Centrifuge for 3 minutes at 14,000g
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.11. 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
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.12. 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 
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.13. 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.
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.14. Air dry for two minutes.
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.15. 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.
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.16. 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.
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
10.17. Interprete the resulting spectra using Bruker database
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
11. Performance specification 
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
11.1. Bruker allows for any score above 2.0 to be acceptable for a species identification
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
12. Interpreting results
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
12.1. 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)
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
12.2. If the control strains generate scores < 2 or incorrect identification, the results should not be interpreted, and the extraction/submission process must be repeated.
SECTION: Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry
12.3. If no species identification is made, repeat extraction/submission process using a new subculture
SECTION: Specimens collection
1. High Vaginal Swab