Introduction. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.Gap statement. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.Aim. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.Methodology. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of M. abscessus subsp. massiliense (MAM).Results. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between M. abscessus subsp. abscessus/subsp. bolletii (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology.Conclusions. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients' sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.
- MeSH
- atypické mykobakteriální infekce * diagnóza farmakoterapie MeSH
- cystická fibróza * komplikace mikrobiologie MeSH
- DNA terapeutické užití MeSH
- intracelulární infekce bakterií Mycobacterium avium * epidemiologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- Mycobacterium abscessus * genetika MeSH
- Mycobacterium avium komplex genetika MeSH
- netuberkulózní mykobakterie MeSH
- sputum mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.
- Publikační typ
- časopisecké články MeSH
Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.
- MeSH
- DNA bakterií klasifikace genetika MeSH
- feces chemie mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce normy MeSH
- lyofilizace MeSH
- Mycobacterium avium subsp. paratuberculosis klasifikace genetika izolace a purifikace MeSH
- nemoci skotu diagnóza MeSH
- paratuberkulóza diagnóza mikrobiologie MeSH
- referenční standardy MeSH
- senzitivita a specificita MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An important prerequisite for the effective control, timely diagnosis, and successful treatment of mycobacterial infections in both humans and animals is a rapid, specific, and sensitive detection technique. Culture is still considered the gold standard in the detection of viable mycobacteria; however, mycobacteria are extremely fastidious and slow-growing microorganisms, and therefore cultivation requires a very long incubation period to obtain results. Polymerase Chain Reaction (PCR) methods are also frequently used in the diagnosis of mycobacterial infections, providing faster and more accurate results, but are unable to distinguish between a viable and non-viable microorganism, which results in an inability to determine the success of tuberculosis patient treatment or to differentiate between an active and passive infection of animals. One suitable technique that overcomes these shortcomings mentioned is the phage amplification assay (PA). PA specifically detects viable mycobacteria present in a sample within 48 h using a lytic bacteriophage isolated from the environment. Nowadays, an alternative approach to PA, a commercial kit called Actiphage™, is also employed, providing the result within 6-8 h. In this approach, the bacteriophage is used to lyse mycobacterial cells present in the sample, and the released DNA is subsequently detected by PCR. The objective of this review is to summarize information based on the PA used for detection of mycobacteria significant in both human and veterinary medicine from various kinds of matrices.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Infection with Toxoplasma gondii has usually been connected with consumption of improperly treated meat. However, contaminated water and products of plant origin have emerged as new sources of infection in the last few years. Here, 292 vegetable samples-carrot, cucumber and lettuce-obtained from nine farms in the Czech Republic were examined using triplex real time PCR targeting two specific T. gondii sequences. Irrigation water and water used for washing of vegetables were also included. Overall, a positivity rate of 9.6% was found in vegetables. The concentration varied between 1.31 × 100 and 9.00 × 102 oocysts/g of sample. A significant difference was found between the positivity of vegetables collected directly from fields and that of vegetables collected from farm storage rooms (4.4-8.6% vs 10-24.1%, respectively). All samples of irrigation water and water used to rinse vegetables were negative. Genotyping based on restriction fragment length polymorphism (RFLP) analysis using seven markers revealed the exclusive presence of genotype II.
- MeSH
- bezpečnost potravin MeSH
- farmy * MeSH
- genotyp MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- potravinářská parazitologie * MeSH
- Toxoplasma * klasifikace genetika izolace a purifikace MeSH
- zelenina parazitologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.
- MeSH
- chov zvířat MeSH
- divoká zvířata mikrobiologie MeSH
- hlášení nemocí normy MeSH
- incidence MeSH
- Mycobacterium avium subsp. paratuberculosis izolace a purifikace MeSH
- paratuberkulóza ekonomika epidemiologie prevence a kontrola MeSH
- přežvýkavci mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The objective of this study was to analyze the changes in the microbiota of milk products during fermentation and storage. Two kinds of Yoghurt, one Kefir, and one Acidophilus milk were observed during the fermentation process and storage using 16S rDNA amplicon sequencing. Cow's, goat's, raw and pasteurized milk were also examined. The most represented organisms in all manufactured products were shown to be those of the phylum Firmicutes. In some products, Proteobacteria, Bacteroidetes and Actinobacteria were also present in high amounts.
- MeSH
- Bacteria klasifikace genetika MeSH
- DNA bakterií chemie genetika MeSH
- fermentace MeSH
- fylogeneze MeSH
- kozy MeSH
- kysané mléčné výrobky mikrobiologie MeSH
- mléko MeSH
- ribozomální DNA chemie genetika MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- skladování potravin MeSH
- skot MeSH
- společenstvo * MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mycobacterium marinum patří mezi pomalu rostoucí, netuberkulózní (environmentální, atypická) mykobakterie se zoonotickým potenciálem. Vyskytuje se ve vodním prostředí a vyvolává onemocnění u ryb a jiných vodních živočichů označované jako mykobakteriózy. U lidí vyvolává primárně kožní onemocnění, které bývá nejčastěji lokalizováno na horních končetinách. Onemocnění se objevuje nejčastěji v souvislosti s akvarijním prostředím, a proto také bývá označováno jako „fish tank granuloma“. Léčba je, jako v případě všech mykobakteriálních onemocnění, komplikovaná a zdlouhavá. Pro definitivní určení patogenního agens by vždy mělo být provedeno vyšetření biologického materiálu laboratoří specializujících se na diagnostiku mykobakteriálních onemocnění. Zásadním bodem pro diagnostiku je správné odebrání vzorku a také posouzení anamnézy pacienta. Základními laboratorními metodami jsou kultivace a mikroskopie. Pro zařazení do druhu jsou nejčastěji využívány moderní biologické metody, jako hmotnostní spektrofotometrie (MALDI), polymerázová řetězová reakce (PCR), hybridizační sondy nebo sekvenování.
Mycobacterium marinum is a slowly growing non-tuberculous (environmental, atypical) mycobacterium with zoonotic potential. It occurs in the aquatic environment and causes diseases in fish and other aquatic animals known as mycobacterioses. In humans, it primarily causes skin infections, which are most commonly located in the upper limbs. The disease commonly appears in connection with the aquarium environment and is thus referred to as fish tank granuloma. As with all mycobacterial diseases, treatment is complicated and lengthy. For a definitive determination of the pathogen, biological materials should always be examined in a laboratory specializing in diagnosing mycobacteria. Critical for the right diagnosis is proper sample collection and assessment of the patient’s history. To detect mycobacteria, culture and microscopy are generally used. Species are identified using modern biological methods such as mass spectrometry (MALDI), polymerase chain reaction, hybridization probes or sequencing.
- MeSH
- diagnostické techniky a postupy MeSH
- klinické laboratorní techniky metody MeSH
- lidé MeSH
- Mycobacterium marinum izolace a purifikace patogenita růst a vývoj MeSH
- mykobakteriózy * diagnóza etiologie mikrobiologie MeSH
- nemoci zvířat diagnóza etiologie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
