Heat Inactivation Renders Sputum Safe and Preserves
            <i>Mycobacterium tuberculosis</i>
            RNA for Downstream Molecular Tests

Sabiiti, Wilber; Azam, Khalide; Esmeraldo, Ezembro; Bhatt, Nilesh; Rachow, Andrea; Gillespie, Stephen H.

doi:10.1128/jcm.01778-18

Cited by 22 publications

(22 citation statements)

References 32 publications

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“…As shown in table 3, the virus could not be detected in the event of heat treatment at 100°C for 30 mins, and 60 minutes, even if there is no statistical difference between the activated ones at 100°C for 10 mins and the non-inactivated samples. However, our study has shown that the virus loads measured in the heat-inactivated samples were consistent with those in the unheat-inactivated ones, with sufficient amounts of RNA preserved for downstream rRT-PCR testing, which concurs with the conclusions of Wilber Sabiiti et al and Boris Pastori et al [16][17][18] . The RNA preservation may be due to the preservation solution, which contains guanidine isothiocyanate, sodium dodecylsarcosine, and dithiothreitol.…”

Section: Rrt-pcr Results Of Inactivated At 100°csupporting

confidence: 90%

Effect of Heat inactivation on Real-Time Reverse Transcription PCR of the SARS-COV-2 Detection

Liu

2020

Preprint

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Background: Real-time reverse transcription PCR (rRT-PCR) is commonly used to diagnose SARS-CoV-2 infection. Heat inactivation prior to nucleic acid isolation may allow safe testing, while the effects of heat inactivation on SARS-CoV-2 rRT-PCR detection result need to be determined. Methods: 14 positive nasopharyngeal swab specimens were inactivated at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min, and another 2 positive nasopharyngeal swab specimens were also inactivated at 100°C for 10min, 100°C for 30min, 100°C for 60min, after which the samples were isolated and detected by rRT-PCR. Results: All 14 heat treated samples remained positive. The range of threshold cycle (Ct) values observed when detecting ORF1a/b was 27.228-34.011 in heat-treated samples, while 25.281-34.861 in unheated samples, and the range of threshold cycle (Ct) values observed at the time of detecting N was 25.777-33.351 in heat-treated samples, while 24.1615-35.433 in unheated samples, on basis of which it showed no statistical difference otherwise a good correlation of Ct values between the heat-inactivated samples and the untreated samples. However, the 2 samples inactivated at 100°C 30min, 100°C 60min turned into negative. Conclusions: Heat inactivation at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min shall not affect the detection results of Real-Time Reverse Transcription PCR of the SARS-COV2. Furthermore, it is recommended to inactive nasopharyngeal swab specimens 10min at 100°C before RNA extraction in consideration of efficiency and reliable results. Key Words: SARS-CoV-2, Heat Inactivation, rRT-PCR, Comparison

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Section: Rrt-pcr Results Of Inactivated At 100°csupporting

confidence: 90%

Effect of Heat inactivation on Real-Time Reverse Transcription PCR of the SARS-COV-2 Detection

Liu

2020

Preprint

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“…In order to perform the test in a Biosafety Level 2 laboratory, samples were previously inactivated. MTBc inactivation was achieved using a dry-block at 95˚C for 30 minutes, this allowed easier and safer heat-inactivation compared to a boiling water bath, as previously shown by other Authors [16,17].…”

Section: Discussionmentioning

confidence: 72%

Simultaneous detection of Mycobacterium tuberculosis complex and resistance to Rifampicin and Isoniazid by MDR/MTB ELITe MGB® Kit for the diagnosis of tuberculosis

et al. 2020

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The MDR/MTB ELITe MGB ® Kit on the ELITe InGenius ® platform (ELITechGroup SpA, Italy) is the first system for simultaneous detection of the Mycobacterium tuberculosis complex (MTBc) genome and the main mutations responsible for resistance to Isoniazid (inhA, katG) and Rifampicin (rpoB), from decontaminated and heat inactivated samples. In this study we compared the performance of the MDR/MTB ELITe MGB ® Kit (ELITe) with culture in 100 pulmonary and 160 extra-pulmonary samples. The sensitivity and specificity of ELITe compared to culture for pulmonary samples were 98.0% and 98.0% respectively; for extrapulmonary samples the overall sensitivity was 86.3% (80% for urine, 85% for biopsy and gastric aspirate and 95% for cavitary fluid) and specificity was 100%. Genotypic Isoniazid and Rifampicin susceptibility typing was feasible in 96% of sputum MTBc-positive samples and 43% of extra-pulmonary samples; all samples were found to be drug susceptible by phenotypic and ELITe (100% agreement). Detection of mutations in the rpoB, kat G or inhA genes was evaluated on 300 spiked samples (60 per biological matrix) and all resistance profiles were correctly identified by ELITe. Molecular agreement between ELITe and Xpert was 98.0% and 93.3% for pulmonary and extra-pulmonary samples, respectively. In conclusion, our results provide evidence to support the use of MDR/MTB ELITe MGB ® Kit in combination with ELITe InGenius ® for the diagnosis of MTBc and the detection of Rifampicin and Isoniazid resistance-related mutations in both pulmonary and extra-pulmonary samples. This system simplifies the laboratory workflow, shortens report time and is an aid in choosing appropriate therapeutic treatment and patient management.

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“…(i) RNA extraction. Extraction of RNA was performed as previously described (20,22). Briefly, 100 l of an extraction control (Vitalbacteria; SOI Group, UK) was added to each tube prior to RNA extraction.…”

Section: Confirmation Of M Tuberculosis In Liquid Culturementioning

confidence: 99%

“…Sequence-specific primers and TaqMan probes dually labeled for M. tuberculosis 16S rRNA and for the extraction control (EC) target were procured from MWG Eurofins, Germany. Master mix preparation, PCR test conditions, and ampli- fication were set and performed as previously demonstrated (19,20,22). The sensitivity and specificity of the primers and probes for MBLA were previously tested against nontuberculosis mycobacteria, including a wide range of respiratory pathogens, and none of them was found to be amplified (19).…”

Section: Confirmation Of M Tuberculosis In Liquid Culturementioning

confidence: 99%

Molecular Bacterial Load Assay Concurs with Culture on NaOH-Induced Loss of Mycobacterium tuberculosis Viability

et al. 2019

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Effective methods to detect viable Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB), are urgently needed. To date, cultivation of M. tuberculosis is the gold standard, which depends on initial sample processing with N-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH), chemicals that compromise M. tuberculosis viability and, consequently, the performance of downstream tests. We applied culture and the novel molecular bacterial load assay (MBLA) to measure the loss of M. tuberculosis viability following NALC-NaOH treatment of M. tuberculosis H37Rv pure culture and clinical sputum samples from pulmonary TB patients. Compared to the bacterial loads of untreated controls, NALC-NaOH treatment of M. tuberculosis reduced the MBLA-detectable bacillary load (estimated number of CFU [eCFU] per milliliter) by 0.66 ± 0.21 log10 at 23°C (P = 0.018) and 0.72 ± 0.08 log10 at 30°C (P = 0.013). Likewise, NALC-NaOH treatment reduced the viable count on solid culture by 0.84 ± 0.02 log10 CFU/ml at 23°C (P < 0.001) and 0.85 ± 0.01 log10 CFU/ml at 30°C (P < 0.001), respectively. The reduction in the viable count was reflected by a corresponding increase in the time to positivity of the mycobacterial growth indicator tube (MGIT) liquid culture: 1.2 days at 23°C (P < 0.001) and 1.1 days at 30°C (P < 0.001). This NaOH-induced M. tuberculosis viability loss was replicated in clinical sputum samples, with the bacterial load dropping by 0.65 ± 0.17 log10 from 5.36 ± 0.24 log10 eCFU/ml to 4.71 ± 0.16 log10 eCFU/ml for untreated and treated sputa, respectively. Applying the model of Bowness et al. (R. Bowness, M. J. Boeree, R. Aarnoutse, R. Dawson, et al., J Antimicrob Chemother 70:448–455, 2015, https://doi.org/10.1093/jac/dku415) revealed that the treated MGIT time to culture positivity of 142 ± 7.02 h was equivalent to 4.86 ± 0.28 log10 CFU, consistent with the MBLA-measured bacterial load. Our study confirms the contribution of NALC-NaOH treatment to the loss of viable bacterial counts. Tests that obviate the need for decontamination may offer an alternative option for the accurate detection of viable M. tuberculosis and treatment response monitoring.

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Heat Inactivation Renders Sputum Safe and Preserves Mycobacterium tuberculosis RNA for Downstream Molecular Tests

Cited by 22 publications

References 32 publications

Effect of Heat inactivation on Real-Time Reverse Transcription PCR of the SARS-COV-2 Detection

Effect of Heat inactivation on Real-Time Reverse Transcription PCR of the SARS-COV-2 Detection

Simultaneous detection of Mycobacterium tuberculosis complex and resistance to Rifampicin and Isoniazid by MDR/MTB ELITe MGB® Kit for the diagnosis of tuberculosis

Molecular Bacterial Load Assay Concurs with Culture on NaOH-Induced Loss of Mycobacterium tuberculosis Viability

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