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SAMPLE COLLECTION AND PROCESSING(Mycology) BY-DR. POORNIMA SEN |
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Introduction Successful laboratory diagnosis of mycoses is dependent not only mycology expertise of clinical laboratory but also upon quality of specimens provided for analysis.
Inappropriately collected, stored and processed samples may lead to misdiagnosis and unnecessary delay in therapy. |
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DOs & DON’Ts From active lesions, old lesions should be avoided
Sufficient amount of specimen
All aseptic precautions should be followed
Prefer to collect before instituting the therapy
Use sterile containers and devices, label them appropriately
All clinical specimens should be considered as potential biohazards. |
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Types of samples Skin scrapings
Hairs and nails
Respiratory tract specimens (sputum, BAL)
Blood
CSF
Urine
Pus
Tissue
Ocular specimen
Other sterile body fluids( peritonial, pericardial, synovial fluid and vitreous humor)
Stool
Mouth and vaginal scrapings |
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Skin scrapings Clean the area with 70% isopropanol, collect scrapings with flat edge of sterile blade
Painful nicking and bleeding should be avoided
Margins of the lesion- best place to scrap
Edges of loose skin at margin of lesion can be trimmed off
Roof of vesicle can also be gently shaved off
Once visible material is on the slide-place in petri dish KOH smear and culture
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Hair and nails Dull lusture less hair should be pulled out and placed in clean & dry container
Hair clippings should not be accepted
Nails should be cleaned with 70% alcohal , excess keratin should be scraped off and portion of the infected nail should be clipped.
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Respiratory tract specimens Sputum is best specimen for chronic pneumonia
Collection should be in disposable, wide mouth, screw capped container of around 100 ml capacity
Avoid spilling over rim of container, wipe off with tissue if any spill material is there
Sputum can be induced by hypertonic saline if patient does not have any productive cough
Appropriate smear is wet mount or calcoflour white
If patient has difficulty in coughing, physiotherapy is given to induce the secretions
BAL can be taken directly from bronchoscope through aspiration
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Blood PROCEDURE OF SAMPLE COLLECTION
Operator’s hands should be clean and dry and gloves should be worn
Disinfect venepuncture site by applying 70% alcohal in water with 1% iodine for 1 min and allow to dry
Take out the sample(10 ml) with syringe and avoid touching and recontaminating the punctured site while taking out blood
Change the needle avoiding touching the shaft and transfer in to blood culture bottles. |
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While incubating these samples-
Temp.- 25 ° to 30° c
Blood culture bottles- Adequate aeration must be ensured, residual vacuum should be removed
Duration- for most isolates of yeasts- 1week
for dimorphic fungi -4 to 6 weeks
with lysis centrifugation method-3 weeks
Histoplasma and Cryptococus –Lysis centrifugation method is preferred.
Malassezia furfur- Media enriched with appropriate fatty acid should be used |
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CSF Collected by lumbar puncture
Best site for LP- L3 & L4
After cleaning (with alcohal iodine solution) and anaesthetizing the skin around 3 to 5 ml of fluid should be collected
CSF for fungal culture-centrifuged when volume > 2ml
Supernatant-chemical or serological test
Sediment-for culture and india ink
Filteration of CSF can be used instead of centrifugation
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Urine Mid-stream urine (MSU) should be collected in a wide mouth container
Catheter specimen of urine is now a days avoided
In infants and children collections poses a problem, urine can be aspirated through supra pubic stab
Once collected a specimen it should be transported to lab with out delay (delay of >1-2 hr should be avoided)
Around 50-200 ml of o centrifuged urine is sufficient for isolating organism |
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A wet mount of urine may occasionally show yeast cells compatible with Blastomyces or Cryptococcus
The salt content of urine makes India ink clump so if Cryptococcus is suspected ,little sterile water should be added to the sediment of urine
In infection with Blastomyces or Cryptococcus patient may have prostatic infection so it indicates utility of collecting urine after prostatic massage
Candida species are commonly isolated from urine and species identification is not routinely recommended.
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Pus and tissue Pus should be collected from infected site using sterile syringe
Outer surface of site should be thoroughly cleaned
Swab should not be collected from a closed lesion which is surgically opened
Care should be taken as pus from lesions sometimes may contain granules
For tissue small pieces may be minced with scissors and large pieces may be ground in sterile mortar pestle in biosafety hood
Can be used for wet mount and calcoflour white preparations |
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Mouth and vaginal scrapings When thrush is suspected lesion should be scrapped gently with a wooden spatula and transfer the material on a clean glass slide
For vaginal smears cervical cytology spatula is used
Trichomonas-seen on wet mount
Culture is usually not required for Candida
Oral or vaginal lesion of Histoplasmosis or Paracoccidioidomycoses can also be diagnosed by using Geimsa stain for former and calcoflour white for latter |
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Ocular specimens Corneal scrapings are ideally used for direct examination and culture media
Avoid repeated scrapings on cornea
Vitreous humor sample should be centrifuged to increase the sensitivity, Candida and Aspergillus are the most common fungal pathogens isolated .
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DIRECT MICROSCOPY METHODS |
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Incubation conditions Temp. 25 °-30 °c
Provide a humid environment to avoid drying of plates during 4-6 weeks of incubation.
Place containers of water with in the incubator
Conversion of dimorphic fungi in to yeast form 35-37 °c
Higher temprature may be needed to test temp. tolerance |
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