Protocol for detection of Leishmania braziliensis, skin commensals and host pro-inflammatory gene expression from lesion swabs in human cases of cutaneous leishmaniasis
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Supplies

Sample collection

swabs

For each patient, two sterile swabs (provided by Biomeme) are held together and used to swab the inner border and ulcer of the lesion (encircled 20x). A second pair of swabs is used to sample from normal skin on the contralateral side of the same patient. Swabs are then processed as follows:

  1. Immerse each pair of swabs into separate eptubes containing 1ml BLB/tube from Biomeme M1 RNA kit, twirl in media for approx. 1 min, discard swabs.
  2. Attach standard lab 1cc syringe to Biomeme filter tip. Pump lysate up/down 10x, expel volume
  3. Immediately move to BPW protein wash (1ml), pump up/down 3x, expel volume
  4. Immediately move to BWB wash buffer (1ml), pump up/down 1x, expel volume
  5. Immediately move to BDW drying wash (1ml), pump up/down 1x, expel volume and excess BDW
  6. Discard syringe but keep filter tip
  7. Attach 20cc and pump up and down with air 10x to dry the column.
  8. remove and discard the syringe. Connect new Biomeme ‘slip-tip’ 1 cc syringe to the column and draw up 150 ul of Biomeme Elution Buffer (BEB). Pump up/down 3x with BEB.
  9. Expel the elution buffer into a final collection tube. Typical elution volume is ~120ul, and contains both RNA and DNA. We use appox. 15ul of this for any of the assays listed below, which means a single eluate is enough for at least 6 QPCR assays.

fine needle aspirate (FNA)

After the physician has applied local anesthesia to the site of the lesion, a FNA is collected and dispensed into 1ml of BLB and processed in the same way as swabs (detailed above)

biopsy

1/3 to 1/2 of a 4-5mm punch biopsy is placed into a 5ml screw-cap sample collection tube containing 1/2-inch ball bearing (from homogenization sample collection kit) and 2ml of BLB buffer (from M1 sample prep kit). Samples are then processed as follows:

  • with cap tightened, vigorously shake 5ml screw-cap by hand for 30sec to disrupt tissue in BLB
  • let sit at room temp until all other patients and samples have been processed
  • vigorously shake for another 30sec when ready to resume processing
  • allow particulate to settle to bottom of tube
  • use sample tip from M1 sample prep kit to draw up 1ml. Avoid drawing up particulates.
  • move to an empty eptube and process extract as indicated above for swabs.

Inflammation panel

The primers and probes below are for the detection of host transcripts present in cellular material on recovered from swabs, FNAs, or biopsies.

name Sequence (5’ -> 3’)
hIL1B fwd CAA AGG CGG CCA GGA TAT AA
hIL1B rev CTA GGG ATT GAG TCC ACA TTC AG
hIL1B probe /56-FAM/AGA GCT GTA /ZEN/ CCC AGA GAG TCC TGT/3IABkFQ/
hGZMB fwd GTA CCA TTG AGT TGT GCG TG
hGZMB rev CAT GCC ATT GTT TCG TCC ATA G
hGZMB probe /56-FAM/CCT CCA GAG /ZEN/ TCC CCC TTA AAG GAA GT/3IABkFQ/
h18S (+ control) fwd provided by Biomeme
h18S (+ control) rev provided by Biomeme
h18S (+ control) probe provided by Biomeme

Microbial panel

The primers and probes below are used for detection of Staphylococcus aureus (Saur), Streptococcus pyogenes (Spyo) and Leishmania braziliensis (Lbra)

  • Leishmania braziliensis primer/probes are from Weirather et al., 2011
  • Streptococcus pyogenes primer/probes were adapted from CDC here
  • Staphylococcus aureus primers and probes were provided by Biomeme Inc.
name Sequence (5’ -> 3’)
Saur fwd provided by Biomeme
Saur rev provided by Biomeme
Saur probe provided by Biomeme
Spyo fwd GCA CTC GCT ACT ATT TCT TAC CTC AA
Spyo rev GTC ACA ATG TCT TGG AAA CCA GTA AT
Spyo probe /56-FAM/CCG CAA CTC /ZEN/ ATC AAG GAT TTC TGT TAC CA/3IABkFQ/
Lbra fwd TGC TAT AAA ATC GTA CCA CCC GAC A
Lbra rev AAA TGG CAT ACA GAA ACC CCG TTC
Lbra probe /56-FAM/GCC TCT GGG /ZEN/ TAG GGG CGT TCT GCA A/3IABkFQ/
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