Development of a combined RLEP/16S rRNA (RT) qPCR assay for the detection of viable M. leprae from nasal swab samples

Background Leprosy continues to be a health problem in endemic areas. More than 200,000 new cases of leprosy per year suggest that transmission of the disease is still ongoing, presumably as airborne infection through nasal droplets. Late diagnosis supports continued transmission and increases the individual risk for functional disabilities. Laboratory tools are considered beneficial to facilitate early detection and clinical assessment of cases. The aim of this study was to validate molecular tools allowing detection, quantification and assessment of viability of M. leprae from nasal swab samples which are easy to obtain without the need of any invasive procedures. Methods Validation of two real-time PCRs detecting M. leprae DNA (RLEP qPCR) and RNA (16S rRNA RT qPCR) was conducted on “must not detect”/“must detect” samples and 160 pre-treatment nasal swab samples from 20 clinically diagnosed multibacillary (MB) leprosy patients from Togo. Results Both assays were 100% M. leprae specific and showed analytical sensitivities of three templates each. Out of 20 clinically diagnosed MB leprosy patients, 15 (75.0%) had a positive RLEP qPCR result from nasal swab samples. The 16S rRNA RT qPCR detected viable bacilli in nasal swab samples of ten out of these 15 RLEP positive patients (66.7%). Conclusion The combined RLEP/16S rRNA (RT) qPCR assay provides a sensitive and specific tool to determine the bacterial load and viability of M. leprae from nasal swab samples and is applicable for early diagnosis, monitoring treatment response and investigating the role of nasal carriage of M. leprae in human-to-human transmission through aerosol infection. Electronic supplementary material The online version of this article (10.1186/s12879-019-4349-9) contains supplementary material, which is available to authorized users.


DNA Extraction
For DNA extraction procedures the Gentra Puregene method (Qiagen, Hilden, Germany) is used with minor modifications of the manufacturer's protocol as described below.

▲Ambient Temperature (AT) is defined as 14-25°C!
To avoid the contamination risk all reagents must be aliquoted in single-use-volumes and stored as described below: • CLS buffer is used as transport-and storage medium for diagnostic PCR samples. It is aliquoted by 700 µl and stored at AT until expiry date.
• PPS-solution and DNA Hyd. are aliquoted according to the amount of samples subjected to DNA extraction and stored at AT until expiry date.
• Isopropanol is aliquoted to 700 µl and stored at AT in the dark until expiry date.
• Ethanol (70%) is stored at AT in the dark until expiry date and filled from the bottle to a 50 ml falcon tube before use (if ethanol needs to be diluted from 100% to 70%: 35ml of 100% Ethanol is added to 15 ml distilled water directly before use). Any remaining ethanol in the 50 ml falcon tube is discarded directly after use.

Performance of DNA Extraction
Using the Qiagen Puregene method, gDNA can be extracted from nasal swab samples.
The extracted DNA is suitable for further downstream applications (e.g. PCR, LAMP, sequencing etc.).

Prearrangements
Keep clinical samples in CLS always in an upright position!
The samples must all be labelled with patient/sample ID and/or laboratory numbers to ensure the correct allocation of patients and samples.
Upon arrival of samples at the laboratory, specimens are heat-inactivated by incubation at 95°C in the thermomixer for 15 minutes. If the extraction procedure is not carried out on the same day, store samples in the fridge at 4°C for up to 1 month or freeze samples at -20°C for long-term storage. ▲Specimens are not infectious anymore but nevertheless should be treated as "possibly infectious" agents and handled with care! Do not remove the swab stick prior to step 6! Before starting the extraction procedure, prepare one empty reaction tube as negative extraction control: Add 700 µl CLS buffer and label the tube as "ExCo" and the date of extraction. The extraction control will be treated the same way as the clinical samples. 3. The Proteinase K is inactivated at 95°C for 10 minutes in the thermomixer.

Protein Precipitation
4. Place samples in an ice bath for 5 minutes.

Add 230 µl Protein Precipitation Solution (PPS)
6. Vortex vigorously at high speed for 20 seconds to mix PPS uniformly with cell lysate (in CLS). 7. Place the samples in an ice bath for 5 minutes.
8. Centrifuge at 13.000 rounds per minute (rpm) for 5 minutes. The precipitated proteins will form a tight pellet. Remove swab stick carefully and discard.
If the protein pellet is not tight, repeat step 6, followed by a repeated incubation on ice for 5 minutes, then repeat step 8.
During centrifugation prepare the respective number of new 2 ml reaction tubes containing 700 µl Isopropopanol (see next paragraph, "DNA Precipitation", step 9).

9.
Pour the supernatant containing DNA into a new 2 ml reaction tube containing 700µl 100% Isopropanol (leaving behind the precipitated protein pellet).

10.
Mix by inverting tubes gently for 10 times.

12.
Pour off the supernatant. Add 700 µl 70% Ethanol and invert tubes to wash DNA pellets.

13.
Centrifuge at 13.000 rpm for 5 minutes. Carefully pour off the ethanol.
▲The pellet may be loose so pour slowly and watch the pellet. 14.
Invert and drain tubes on a clean absorbent paper towel (each tube at a different spot) and allow tubes to air dry at AT or in the thermomixer at 65°C until tubes are completely dry (20 min.).
The ethanol must be completely evaporated as this may inhibit PCR.

16.
Rehydrate the DNA by carefully pipetting up-and down for 20 times.

17.
Incubate eluted DNA in the thermomixer for 30 minutes at 65°C.

18.
Optional: Measure quality and quantity of extracted whole-genome DNA in a photometer.

Storage of DNA Extracts
DNA extracts may be stored at 4°C until further processing for up to 1 week. For long-term storage DNA extracts are stored at -20°C.
Avoid repeated freezing and thawing of the samples as this may lead to degradation of DNA.

Preparation of PANTA transport medium ▲Work under sterile conditions in a laminar Flow!
1) Distil 850 ml water and filter through sterile bottle top filter.
2) Filter glycerol through sterile bottle top filter (this will take a while due to the consistency). ▲Store 500 µl PANTA transport medium in 2 ml screw cap tubes at 4-8°C for max. 6 months!

Preparation of RNAlater transport medium
RNAlater RNA Stabilization Reagent may form a precipitate during storage below room temperature (15-25°C). In this case, re-dissolve the precipitate by heating to 37°C with agitation before use.
▲Work under sterile conditions in a Laminar Flow! 1) Add 500 µl RNAlater reagent using RNAse free filter tips with a 1000µl pipette to each 2ml RNAse free screw-cap tube under sterile conditions! 2) Store RNAlater transport tubes at ambient temperature (AT) for up to 6 months (or between 4-8°C for long term storage up to 12 months).

Stabilization of swab samples in PANTA/RNAlater for RNA/DNA extraction ▲The swab samples may be transported at AT for up to seven days! Apply the following conservation steps directly upon arrival of clinical samples in the laboratory! Work in a biosafety cabinet class II!
All samples in PANTA and RNAlater, respectively are supposed to be directly subjected to DNA/RNA extraction.
2) Incubate specimens for 5 minutes (min). at AT, vortex each sample every minute for 10 sec.
3) Inactivate samples at 95°C for 5 min., incubate on ice for 5 min. 4) Pellet material by centrifugation at 5000g at AT for 5 min., remove swab stick carefully and discard supernatant, keep pellets on ice and continue DNA/RNA extraction with pellet or freeze pellets at -20°C for up to 3 months (or -80°C for up to 12 months) for DNA/RNA extraction at a later point in time.

DNA Extraction
For DNA extraction procedures the "My Budget DNA extraction kit mini" (Bio-Budget, Krefeld, Germany) is used with minor modifications of the manufacturer's protocol as described below.

Storage and preparation of reagents
To decrease the risk of contaminations, all reagents should be aliquoted in single-use-volumes and stored as described below. Avoid repeated thawing and freezing of Proteinase K!

▲ Ambient temperature (AT) is defined as 14-25°C!
• The lysis buffer TLS is used as transport-and storage medium for diagnostic PCR samples. TLS is aliquoted by 400 µl in 2 ml screw-cap tubes and stored at AT for up to 12 months. • Buffers TBS, HS, MS and EB are stored at AT.
• Ethanol (96%) is stored at room temperature in the dark and filled from the bottle to a 50 ml falcon tube before use.

Before using the Bio Budget DNA extraction kit for the first time:
-Add 15 ml Ethanol 96% to Buffer HS -Add 35 ml Ethanol 96% to Buffer MS -Dissolve lyophilized Proteinase K in 1.5 ml DEPC treated water, aliquote by 250 µl in 2 ml screw-cap tubes (one aliquot for 10 samples) and store at -20°C for up to 12 months.

▲ All centrifugation steps are conducted at AT!
The extracted DNA is suitable for further downstream applications (e.g. PCR, LAMP, sequencing etc.)

Prearrangements
Keep clinical samples in TLS always in an upright position! The samples must all be labelled with patient/sample IDs and/or laboratory numbers to ensure the correct allocation of patients and samples.
Upon arrival of samples at the laboratory, specimens are heat-inactivated by incubation at 95°C in the thermoshaker for 15 minutes (min). If the extraction procedure is not carried out on the same day, store samples in the fridge at 4°C for up to 1 month or freeze samples at -20°C for long-term storage.

▲Specimens are not infectious anymore but nevertheless should be treated as "possibly infectious" agents and handled with care! Do not remove the swab stick prior to step 6!
Before starting the extraction procedure, prepare one empty reaction tube as negative extraction control: Add 400 µl TLS buffer and label the tube as "ExCo" and the date of extraction. The extraction control will be treated the same way as the clinical samples.
Thaw one aliquot of Proteinase K per 10 samples (including the negative extraction control!).

DNA extraction from swab samples and slit skin smears
1. Add 25 µl Proteinase K to each sample and vortex vigorously for 5 seconds (sec).
3. Prepare 1.5 ml reaction tubes (two for each sample) and label them for steps 7 and 16.
4. Put spin columns in 2.0 ml collection tubes (one for each sample) and label spin columns.   IV) Generate 10 mg/ml Lysozyme solution by adding 10 mg Lysozyme per 1 ml DEPC treated/RNAse free water, label aliquots with "L" and the date of preparation; store at -20°C for up to 6 months.

Material, reagents and instruments
General preparation prior to each extraction:

▲ Work under a fume hood wearing nitrile gloves! ß-Mercaptoethanol is a competent RNAse inhibitor preventing RNA degradation during lysis of mycobacterial cell walls and compartments! Inhalation and contact with human skin is toxic! ▲ Turn off Air-Con
A) Add 3.5µl x n (n = no. of samples processed) ß-Mercaptoethanol (ß-ME) to 350µl x n Buffer RLT Plus. Buffer RLT Plus containing ß-ME is stable at AT for up to one month.

-a very competent RNAse inhibitor -samples (or previously frozen pellets) must remain on ice!
1) Thaw frozen pellets on ice.

RNA extraction and purification:
▲ All centrifugations are conducted at ambient temperature (AT)! 10) Add 350 µl Ethanol 70% to the flow through in RNA collection tubes from step 9, pipet 5 times slowly up and down to mix.