Culture and Molecular Method for Detection of Mycobacterium tuberculosis Complex and Mycobacterium avium subsp. paratuberculosis in Milk and Dairy Products

Messelhäußer, Ute; Kämpf, Peter; Hörmansdorfer, Stefan; Wagner, Bettina; Schalch, B.; Busch, Ulrich; Höller, Christiane; Wallner, Peter; Barth, G.; Rampp, Albert

doi:10.1128/aem.06322-11

Cited by 10 publications

(7 citation statements)

References 13 publications

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“…Culturing these organisms requires complex media and specialist equipment and is therefore costly in terms of time and reagents. In addition, these bacteria typically exhibit a low plating efficiency, where only a proportion of viable cells in a culture will grow into colonies; thus, culture is not always a reliable way to detect low numbers of cells present in a sample (Messelhausser et al , ; Fawzy et al , ). This problem is exacerbated by the need to treat clinical samples with harsh chemicals to inactivate contaminating microbes that will overgrow samples during the long incubation periods, but also reduces the viable population of mycobacteria (Grant et al , ; Medeiros et al , ).…”

Section: Introductionmentioning

confidence: 99%

The development and use of Actiphage^® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

Swift

Meade

Barron

et al. 2019

Microbial Biotechnology

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Summary Here, we describe the development of a method that exploits bacteriophage D29 as a lysis agent for efficient DNA extraction from low numbers of mycobacterial cells. This method (Actiphage®) used in combination with PCR achieved rapid and sensitive (LOD ≤ 10 cell ml−1) detection and identification of viable, pathogenic mycobacteria in blood samples within 6 h. We demonstrate that mycobacteriophage D29 can be used to detect a range of mycobacteria from clinical blood samples including both Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis without the need for culture and confirms our earlier observations that a low‐level bacteraemia is associated with these infections in cattle. In a study of M. bovis‐infected cattle (n = 41), the sensitivity of the Actiphage® method was 95 % (95 % CI; 0.84–0.99) and specificity was 100 % (95% CI; 0.92–1). We further used Actiphage® to demonstrate viable Mycobacterium avium subsp. paratuberculosis is present in the blood of Johne’s infected cattle. This method provides a revolutionary new tool for the study of infections caused by these difficult to grow pathogens.

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Section: Introductionmentioning

confidence: 99%

The development and use of Actiphage^® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

Swift

Meade

Barron

et al. 2019

Microbial Biotechnology

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“…M ycobacterium avium subspecies paratuberculosis (MAP) is the cause of Johne's disease ( JD): granulomatous enteritis in domestic livestock and has been associated with Crohn's disease (CD) in humans (Messelhäusser et al, 2012;Banche et al, 2015). Association of MAP with CD has been reported in the recent past and bacilli were isolated from intestines and blood of the patients (Greenstein, 2003;Feller et al, 2007;Mendoza et al, 2009;Rani et al, 2010;Rosenfeld and Bressler, 2010;Chiodini et al, 2012;Naser et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Bio-Contamination Estimates of Mycobacterium Avium Subspecies Paratuberculosis in Fresh Cottage Cheese (Paneer) Sold in Rural, Semi-Urban and Peri-Urban Regions of South Uttar Pradesh using Multiple Diagnostic Tests

Stephen¹,

Singh²,

Singh³

et al. 2016

Adv. Anim. Vet. Sci.

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Paneer (fresh cottage cheese) samples collected from local dairy shops in rural (Farah), peri-urban and urban (Mathura and Agra) regions of South Uttar Pradesh were screened by six tests combinations to estimate the rate of 'bio-contamination' and 'bio-typing' of MAP (paraTB bacilli). Of 55 paneer samples screened, 54.5, 32.7, 54.5, 74.5, 50.9 and 63.6% were positive by microscopy, IS900 PCR, indigenous ELISA kit, Dot-ELISA, Latex Agglutination test and indirect Fluorescent Antibody tests, respectively. Except 2 (3.6%), all the positive samples were detected by minimum two tests. Of the six test combinations used and compared, microscopy (AFB vs iFAT and IS900 PCR were best combinations for the detection of MAP in fresh cottage cheese (paneer) samples. High rate of bio-contamination of paneer sold by the local dairy shops was due to the use of milk supplies contaminated with high bio-load of MAP. Milk supplies were sourced from domestic livestock belonging to local rural areas, where animal health care facilities and knowledge on bio-contamination of MAP were non-existent. Therefore, in order to produce clean milk, free of MAP bacilli it is absolutely essential to control infection in the domestic livestock.

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“…Culture methods remain the “gold standard” for diagnosing tuberculosis (TB), even now in the 21st century [1] . Culture methods can be used to isolate live Mycobacterium tuberculosis , and are more sensitive than acid-fast bacilli (AFB) staining (e.g., Kinyoun/Ziehl-Neelsen staining), which detects both live and dead bacilli, and culture methods can reliably detect mycobacteria present at a concentration of about 100–1000 colony forming units (CFU)/mL of specimen [ 2 , 3 ]. Growing cultures also permit species identification and drug effectiveness testing [4] .…”

Section: Introductionmentioning

confidence: 99%

A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity

Wang

Takeuchi²,

Jain

et al. 2020

EBioMedicine

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Background Nucleic acid amplification tests (NAATs) are widely used to diagnose tuberculosis (TB), but cannot discriminate live bacilli from dead bacilli. Live bacilli can be isolated by culture methods, but this is time-consuming. We developed a de novo TB diagnostic method that detects only live bacilli with high sensitivity within hours. Methods A prospective study was performed in Taiwan from 2017 to 2018. Sputum was collected consecutively from 1102 patients with suspected TB infection. The sputum was pretreated and heated at 46°C for 1 h to induce the secretion of MPT64 protein from live Mycobacterium tuberculosis . MPT64 was detected with our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling. We compared our data with those obtained using a culture test (MGIT), a smear test (Kinyoun staining), and a NAAT (Xpert). Findings The limit of detection for MPT64 in our culture-free ultrasensitive ELISA was 2.0 × 10 −19 moles/assay. When the criterion for a positive response was set as an absorbance value ≥17 mAbs, this value corresponded to ca . 330 CFU/mL in the culture method – almost the same high-detection sensitivity as the culture method. To confirm that MPT64 is secreted from only live bacilli, M. bovis BCG was killed using 8 μg/mL rifampicin and then heated. Following this procedure, our method detected no MPT64. Our rapid ultra-sensitive ELISA-based method required only 5 h to complete. Comparing the results of our method with those of culture tests for 944 specimens revealed a sensitivity of 86.9% (93/107, 95% CI: 79.0–92.7%) and a specificity of 92.0% (770/837, 95% CI: 89.9–93.7%). The performance data were not significantly different (McNemar's test, P = 0.887) from those of the Xpert tests. In addition, at a ≥1+ titer in the smear test, the positive predictive value of our culture-free ultrasensitive ELISA tests was in a good agreement with that of the culture tests. Furthermore, our culture-free ultrasensitive ELISA test had better validity for drug effectiveness examination than Xpert tests because our test detected only live bacilli. Interpretation Our culture-free ultrasensitive ELISA method detects only live TB bacilli with high sensitivity within hours, allowing for rapid diagnosis of TB and monitoring drug efficacy. Funding Matching Planner Program from JST (VP29117939087), the A-STEP Program from JST (AS3015096U), Waseda University grants for Specific Research Projects (2017A-015 and 2019C-123), the Precise Measurement Technology Promotion Foundation to E.I.

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Culture and Molecular Method for Detection of Mycobacterium tuberculosis Complex and Mycobacterium avium subsp. paratuberculosis in Milk and Dairy Products

Cited by 10 publications

References 13 publications

The development and use of Actiphage^® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

The development and use of Actiphage^® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

Bio-Contamination Estimates of Mycobacterium Avium Subspecies Paratuberculosis in Fresh Cottage Cheese (Paneer) Sold in Rural, Semi-Urban and Peri-Urban Regions of South Uttar Pradesh using Multiple Diagnostic Tests

A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity

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Culture and Molecular Method for Detection of Mycobacterium tuberculosis Complex and Mycobacterium avium subsp. paratuberculosis in Milk and Dairy Products

Cited by 10 publications

References 13 publications

The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

Bio-Contamination Estimates of Mycobacterium Avium Subspecies Paratuberculosis in Fresh Cottage Cheese (Paneer) Sold in Rural, Semi-Urban and Peri-Urban Regions of South Uttar Pradesh using Multiple Diagnostic Tests

A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity

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The development and use of Actiphage^® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

The development and use of Actiphage^® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals