MYCO-TB: the first IVD kit suitable for the digestion and decontamination of extra-pulmonary specimens to detect Mycobacteria

Extra-pulmonary mycobacterial infections are characterized by a paucibacillary nature and extra-pulmonary samples consist of different matrices; the processing of these samples requires a high level of manual skills and non-standardized procedures. The aim of this study was to compare the performance of MYCO-TB with MycoPrep on extra-pulmonary samples in terms of Mycobacteria detection, culture contamination and suitability for molecular assay. This prospective study was conducted on 201 extra-pulmonary samples from suspected cases of mycobacterial infection. Specimens were divided into two equal aliquots; one was decontaminated with MYCO-TB the other with MycoPrep. The contamination rate of liquid cultures was significantly different: 2.5% (5/201) for MYCO-TB and 7.5% (15/201) for MycoPrep (p = 0.036). At least 1 Mycobacterium tuberculosis complex (MTBc) positive culture was detected in 6 specimens treated with MYCO-TB and 8with MycoPrep, without significant differences in times to positivity (TTP) in liquid culture. No Xpert MTB/RIF Ultra invalid results were obtained with samples decontaminated with MYCO-TB. The MYCO-TB kit had greater activity than MycoPrep in the digestion and decontamination of extra-pulmonary specimens for the detection of Mycobacteria, supporting the use of MYCO-TB in this type of sample. Ready-to use reagents, rapid protocol and single-sample formulation of MYCO-TB reduced the level of manual skills required as well as the risk of sample contamination.


Scientific Reports
| (2021) 11:13706 | https://doi.org/10.1038/s41598-021-93182-z www.nature.com/scientificreports/ In our routine protocol we currently use MycoPrep (Becton Dickinson, BD, USA). The aim of this study was to compare the performance of MYCO-TB with MycoPrep, both based on NALC and NaOH, in terms of Mycobacteria detection and culture contamination in extra-pulmonary samples.

Materials and methods
Samples. This prospective study was conducted at the Microbiology Unit of S. Orsola-Malpighi University Hospital, a referral centre for the diagnosis of mycobacterial infections for the metropolitan area of Bologna (Italy).
The study was approved by the Ethics Committee of Area Vasta Emilia Centro (AVEC), Bologna, Italy (Study protocol n.133/2020/Sper/AOUBo). All the experiments were conducted according to the principles expressed in the Declaration of Helsinki.
For this comparative study, a volume of 10 ml was required for cavitary fluids and gastric aspirates, 50 ml for urine (centrifuged, washed and suspended in 10 ml of Phosphate buffered saline (0.067 M, pH 6.8, PBS), while for the remaining biological samples (swab, biopsy, purulent exudate and lymph-node) the volume was adjusted to 10 ml with PBS. Before decontamination, all samples were analysed by acid-fast microscopic examination using Ziehl-Neelsen staining and divided into two 5 ml aliquots, which were decontaminated with MYCO-TB or MycoPrep in parallel, as described below.

Decontamination with MycoPrep (Becton Dickinson). Each MycoPrep kit contains NaOH solution
and a sealed glass ampule of lyophilized NALC, to preserve its mucolytic activity 4 . 5 ml of NALC-NaOH solution were added to an equivalent volume of sample. After vortexing for 15 s, decontamination continued at room temperature for 23 min.
PBS was added to bring the volume up to 45 ml and mixed by inversion to neutralize the decontamination reaction.
After centrifugation at 3000g for 15 min the supernatant was discarded and the bacterial pellet resuspended with 2 ml of PBS.

Decontamination with MYCO-TB (Copan).
Each MYCO-TB kit consists of 4 tubes with different coloured caps filled with ready-to-use reagents formulated for single sample use: The neutralizing solution and resuspension reagent are both composed of Sodium phosphate, Sodium chloride and Potassium phosphate. 5 ml of specimen was transferred to the reaction tube, and 5 ml of MYCO-TB reagent was added. The tube was vortexed for 30 s and left at room temperature for 5 min.
Neutralizing solution was added to the reaction tube to a volume of 50 ml and mixed by inversion. The reaction tube was centrifuged at 3300g for 5 min and the supernatant discarded. The sediment was resuspended in 2 ml of resuspension solution.

Xpert MTB/RIF Ultra. Samples strongly suspected of MTBc infection were analysed by Xpert MTB/RIF
Ultra (Ultra, Cepheid, USA). For each decontamination system 500 μl of sample were mixed with Sample Reagent (containing NaOH and isopropanol) in a 1:3 ratio for 15 min at room temperature, poured into a singleuse disposable cartridge and subsequently loaded onto the GeneXpert module according to the manufacturer's instructions 5 . The system automatically interpreted all results from fluorescent signals into the following categories: invalid, if PCR inhibitors were detected with amplification failure, negative or positive. Positive results were divided into 5 categories depending on bacterial load: the relationship between the rpoB Ct value and input CFU allows samples to be classified as "high, " "medium, "low, " and "very low" and to define strains as susceptible or resistant to Rifampicin depending on the detection of mutations in the rpoB gene, while the "trace" category identifies the paucibacillary samples which are IS6110/IS1081 positive but rpoB negative, with indeterminate Rifampicin susceptibility. The contamination rate of solid media was similar for both systems: 8.5% (17/201) and 6.0% (12/201) on LJ; 3.5% (7/201) and 4.0% (8/201) on LJ PACT for MYCO-TB and MycoPrep respectively. Table 1 shows the agreement between MYCO-TB and MycoPrep methods in terms of culture contamination: overall agreement was 97.79% with moderate concordance (k = 0.489).

Discussion
Culture is considered the gold standard for microbiological diagnosis of mycobacterial infections, but contaminating organisms can limit diagnostic performance, therefore the type of decontamination system could affect Mycobacteria detection 6 . This is the first study evaluating the performance of MYCO-TB kit, developed by Copan, for sample digestion and decontamination to detect Mycobacteria in extra-pulmonary samples. www.nature.com/scientificreports/ Only two studies have been performed on MYCO-TB, both limited to respiratory samples. De Geyter and colleagues, from University Hospital of Brussels, evaluated the performance of MYCO-TB in comparison to Zephiran method on 387 pulmonary specimens 7 . The contamination rates on solid media were 22% with both the Zephiran and MYCO-TB method while in liquid media it was calculated only for the MYCO-TB Kit (4%) as Zephiran method is incompatible with the MGIT system.
Our group compared MYCO-TB to MycoPrep on 162 respiratory samples 8 reporting the same overall contamination rate in liquid and solid media (1.8%). Furthermore, we showed that extended time of decontamination (5 and 10 min) with MYCO-TB did not affect MTBc detection in terms of TTP in liquid culture and Ultra performance.
In this study we have shown that the MYCO-TB kit has greater activity than MycoPrep digesting and decontaminating extra-pulmonary specimens with an overall contamination rate of 1.5% and 2.5%, respectively. In particular, the decontamination activity of MYCO-TB was significantly higher than MycoPrep in liquid culture, where 10 MycoPrep treated samples (5 urine and 5 purulent samples) did not successfully complete the incubation required, due to highly contaminated biological matrices. We exclude that this difference could be caused by the people conducting the work or a difference in working environments as the two dedicated laboratory technicians performed the experiments simultaneously in the dedicated laboratory of biosafety level 3. The stronger decontamination activity of MYCO-TB kit could have potential downstream clinical benefits for patients with TB or NTM abscesses, where contaminating organisms present in highly purulent samples might prevent mycobacterial detection.
On However, 2 out of 8 MTBc-positive cultures, a cavitary fluid (sample number 155) and a gastric aspirate (sample number 163), detected after MycoPrep treatment, were not detected after decontamination with MYCO-TB. We can speculate that this could be due to the very low Mycobacteria load in these samples, proven by no growth on solid media, trace and negative Ultra results and a high TTP in MycoPrep-treated samples (23.04 and 36.58 days, respectively). A limit of this prospective study is the small sample size of MTBc-positive samples detected, which prevented us from accurately calculating sensitivity and specificity. However, the low number of MTBc-positive culture detected reflects the epidemiology of extra-pulmonary TB in our setting.
In conclusion, the ready-to-use reagents, formulation for single-sample and the rapid protocol of MYCO-TB (5 vs. 23 min with MycoPrep) made it possible to optimize manual skills, avoid errors and reduce the risk of contamination during sample processing for Mycobacteria detection.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.