DNA Strip Research Articles

Evaluation of the FluoroType Mycobacteria assay for identification of mycobacteria.

This study is the first clinical evaluation report of the novel FluoroType Mycobacteria assay. The assay has the potential to replace the established GenoType NTM product family in identification of culture-enriched mycobacteria. However, our research results suggest that the assay performs suboptimally and may not be feasible for use in all clinical settings.

Current status of Helicobacter pylori resistance to clarithromycin and levofloxacin in Vietnam: Results from molecular analysis of gastric biopsy specimens

ObjectivesThe management of Helicobacter pylori in Vietnam is becoming progressively more difficult due to increasing antibiotic resistance, particularly to clarithromycin (CLR) and levofloxaxin (LVX). In Vietnam, the selection of an H. pylori eradication regimen is predominantly based on empirical evidence. However, molecular analysis aimed at identifying H. pylori antibiotic-resistant genotypes is a promising method in antibiotic susceptibility testing. In this study, we aimed to determine the rates of genotypic H. pylori resistance to CLR and LVX by using DNA strip technology in Vietnam. MethodsWe performed DNA-strip technology-based testing on 112 patients with H. pylori-positive gastroduodenal diseases to detect 23S rRNA and gyrA mutations. ResultsHelicobacter pylori genotypic resistance to CLR and LVX was evident in 81.3% and 53.6% of the patients, respectively, and dual resistance was observed in 48.2%. The 23S rRNA A2142G and A2143G mutations accounted for 1.8% and 79.5% of cases, respectively. The gyrA N87K, D91N, D91G, and D91Y mutations were present in 37.5%, 11.6%, 5.4%, and 5.4% of patients, respectively. All four gyrA mutations were observed in both the naïve and failure patients. We further found an association between the 23S rRNA A2143G mutation and a history of CLR use as well as between the gyrA N87K mutation and a history of LVX use. ConclusionsWe found a very high prevalence of H. pylori resistance to CLR and LVX and dual resistance to these antibiotics in Vietnam. The application of molecular assays is feasible and may improve the management of H. pylori infection in Vietnam.

Identification of animal species by nucleic acid chromatography of hair samples and investigation of its applicability to human biological samples

GeneFields®-Hair is a simple analysis kit that uses nucleic acid chromatography and polymerase chain reaction (PCR) to identify animal species of hair-like food contaminants. In this study, we evaluated GeneFields®-Hair as a simple and rapid method for identifying animal species from hair-like materials collected in forensic science, such as at crime scenes. The use of this kit with other human biological materials (whole blood, head dandruff, nails, saliva, oral mucosa, sebum, and urine) was also investigated. Animal body hair samples were pretreated by grinding in a buffer solution, centrifuged, and the supernatant was used for PCR. Nucleic acid chromatography of the PCR products allowed the identification of the animal species by the presence or absence of coloration on the decision line. For human biological materials, nucleic acid chromatography was performed after the appropriate pretreatment like body hair material. The determination of some animal species was difficult, even if they had a dedicated DNA Strip determination line. Furthermore, animals from the same family but different genera were sometimes detected on the same determination line. All the human biological samples were correctly identified. Smartphone photographs of the coloration of the judgment line were processed using the ImageJ software for quantitative determination.

PCR-dipstick DNA Chromatography for detection of Carbapenemase Producing Carbapenem-Resistant Enterobacteriaceae

Carbapenemase producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are an epidemiologically important emerging public health threat. Early and reliable detection of CP-CRE is important for containment of these pathogens. The aim of this study was to evaluate and observe the sensitivity of polymerase chain reaction (PCR)-dipstick DNA chromatography (GeneFields CPE) for detection of carbapenemase genes (NDM, OXA-48, KPC, IMP) in comparison to conventional PCR. In this cross-sectional study, a total of 145 carbapenem resistant Enterobacteriaceae isolates were subjected to test for carbapenemase by phenotypic mCIM and genotypic- conventional PCR and PCR-dipstick DNA Chromatography method. In PCR dipstick DNA chromatography assay, after multiplex PCR a DNA-DNA hybridization-based detection system is applied where amplified products can be easily interpreted by observing a blue line in DNA strip visually within 15 minutes without gel electrophoresis. All the CP-CRE isolates (100%) were positive for either NDM or OXA-48 by PCR-dipstick DNA Chromatography assay, whereas 142 (97.9%) isolates were detected by conventional PCR. The positivity rate of NDM, OXA-48 and both NDM and OXA-48 among CP-CRE isolates were 73.8%, 51.7% and 25.5% respectively. The sensitivity, specificity of PCR-dipstick DNA Chromatography assay for detection of NDM and OXA-48 were 100.0%, 92.7% and 98.6%, 97.2% respectively. This assay showed excellent agreement with conventional PCR for detection of CP-CRE. PCR-dipstick DNA chromatography may be an excellent and robust diagnostic tool in terms of rapidity and sensitivity for the detection of CP-CRE.

Aspects of Stage-by-Stage Diagnosis of Pulmonary Mycobacteriosis in Real Clinical Practice

The objective: to analyze the effectiveness of etiological verification of pulmonary mycobacteriosis when using various biological materials at different diagnostic stages.Subjects and Methods. In Center for Diagnosis and Rehabilitation of Respiratory Diseases, CTRI, the results of stage-by-stage examinations of 142 patients (from 2018 to 2022) were analyzed. A retrospective, prospective, cohort, open study was conducted. Inclusion criteria: pulmonary mycobacteriosis etiologically verified by comprehensive microbiological examinations (molecular genetic and cultural tests) (with retrospective analysis after all stages of examination). Exclusion criteria: HIV infection. 115 women and 27 men were enrolled in the study, the age ranged from 21 to 86 years, the mean age was 57,9 years.All patients referred to CTRI for examination due to newly detected changes in the lungs (bronchiectasis, focal changes, and cavities). For etiological verification of the diagnosis, microbiological sputum testing was performed (Stage 1 of the examination); when negative results were obtained, bronchoscopy was prescribed and bronchobiopsies were used for microbiological and molecular genetic diagnostics – Stage 2. In case of negative results of Stage 2, the patient was recommended to undergo a diagnostic resection of the lung – Stage 3 of the examination. At all stages, specimens were sent for comprehensive microbiological testing: fluorescent microscopy to detect acid-fact mycobacteria, real-time polymerase chain reaction (PCR-RV) to detect NTBM DNA. Identification of NTBMB was carried out by molecular genetic methods (PCR for the presence of MTB DNA/NTBM (SYNTOL, Russia), and on DNA strips (GenoType Mycobacterium CM\AS Hain Livescience, Germany)). Specimens were cultured on a liquid medium in the ВАСТЕС MGIT960 (BD, USA).The diagnosis of mycobacteriosis was established in accordance with the diagnostic criteria proposed in the guidelines of the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) (2020): detection of NTBM in 2 sputum samples or in 1 sample of bronchobiopsy specimens or in 1 surgical specimens [15].The studied data were entered to the Microsoft Office Excel sheet (Microsoft, USA) for statistical processing; the level of statistical significance of differences was taken as 0.05.Results. It was found that slow-growing NTBM were identified as etiologically significant in 93.0%, of which 78.1% was M. avium, fast-growing NTBM were represented only by M. abscessus, 7% of observations. During X-ray examination, the bronchiectatic form – 52.1% (74/142) was detected more often compared to the abdominal – 30.9% (44/142) and focal forms – 16.9% (24/142) (p<0.001). When verifying mycobacteriosis in patients with a negative sputum test – Stage 2 of the examination – the effectiveness was 91.0% (80/88). Regardless of the radiological form of the disease and dissemination of the disease, bronchoscopy revealed pathology of the tracheobronchial tree in 85.2% of cases, mainly in the form of bronchitis with purulent secretions.

Molecular characterisation of second-line drug resistance among drug resistant tuberculosis patients tested in Uganda: a two and a half-year\u2019s review

BackgroundSecond-line drug resistance (SLD) among tuberculosis (TB) patients is a serious emerging challenge towards global control of the disease. We characterized SLD-resistance conferring-mutations among TB patients with rifampicin and/or isoniazid (RIF and/or INH) drug-resistance tested at the Uganda National TB Reference Laboratory (NTRL) between June 2017 and December 2019.MethodsThis was a descriptive cross-sectional secondary data analysis of 20,508 M. tuberculosis isolates of new and previously treated patients’ resistant to RIF and/or INH. DNA strips with valid results to characterise the SLD resistance using the commercial Line Probe Assay Genotype MTBDRsl Version 2.0 Assay (Hain Life Science, Nehren, Germany) were reviewed. Data were analysed with STATAv15 using cross-tabulation for frequency and proportions of known resistance-conferring mutations to injectable agents (IA) and fluoroquinolones (FQ).ResultsAmong the eligible participants, 12,993/20,508 (63.4%) were male and median (IQR) age 32 (24–43). A total of 576/20,508 (2.8%) of the M. tuberculosis isolates from participants had resistance to RIF and/or INH. These included; 102/576 (17.7%) single drug-resistant and 474/576 (82.3%) multidrug-resistant (MDR) strains. Only 102 patients had test results for FQ of whom 70/102 (68.6%) and 01/102 (0.98%) had resistance-conferring mutations in the gyrA locus and gyrB locus respectively. Among patients with FQ resistance, gyrAD94G 42.6% (30.0–55.9) and gyrA A90V 41.1% (28.6–54.3) mutations were most observed. Only one mutation, E540D was detected in the gyrB locus. A total of 26 patients had resistance-conferring mutations to IA in whom, 20/26 77.0% (56.4–91.0) had A1401G mutation in the rrs gene locus.ConclusionsOur study reveals a high proportion of mutations known to confer high-level fluoroquinolone drug-resistance among patients with rifampicin and/or isoniazid drug resistance. Utilizing routinely generated laboratory data from existing molecular diagnostic methods may aid real-time surveillance of emerging tuberculosis drug-resistance in resource-limited settings.

Patients with non-tuberculous mycobacteriosis in clinical practice

Incidence and prevalence of non-tuberculous mycobacteriosis (NTM) are recorded in many countries, but the rates vary. There are no official statistics on the prevalence and incidence of NTMs in the Russian Federation at the moment. The regulatory and expert clinical database for NTMs has not yet been developed. The aim of thе article is to share the experience of the Federal State Scientific Institution “Central Research Institute of Tuberculosis” (FSSI “CRIT”) in working with patients with NTMB over the past 7 years. Methods. The study included 190 patients with a confirmed diagnosis of NTMB who were treated at the FSSI “CRIT” in 2011 – 2018. Before the diagnosis of NTMB, all patients were followed for a long time by various specialists (pulmonologists, general practitioners) for various chronic respiratory diseases. NTMs were isolated from the diagnostic material by the culture method. The material was inoculated into a liquid medium in an automated BACTEC MGIT 960 system and into Levenshtein – Jensen dense medium until the culture growth was obtained. The HTM species was determined using DNA strips GenoType® Mycobacterium CM by Hain Lifescience (Germany). Results. More than half of the examined patients were registered with a phthisiatrician for various clinical forms of pulmonary tuberculosis (disseminated, fibro-cavernous, focal). In 85.6% of cases, no MBT DNA was found in the sputum and skin tests with recombinant tuberculosis antigen (Diaskintest®) were negative. Nevertheless, the changes in the lungs detected on the chest CT and even a single detection of acid-fast microorganisms (AFB) without typing led to an erroneous diagnosis of tuberculosis. In some patients (6.8%), a combination of pulmonary tuberculosis and NTMB was diagnosed. Bronchiectasis and COPD were the most common chronic nonspecific lung diseases. 66.7% of the patients had centrilobular foci characterized by the involvement of not only the bronchial wall but also the pulmonary vessels. NTMB resistance to a wide range of antibacterial drugs was determined in 118 patients (out of 190). The prolonged use of chemotherapy drugs revealed a wide range of drug intolerance. Side effects were handled in various ways: drug replacement (with consideration to the sensitivity), temporary withdrawal, and drug discontinuation. Conclusion. This work reflects the long-term experience of the FSSI “CRIT” staff with patients with NTMs, which can be used in creating national recommendations on NTMs.

Modern on-site tool for monitoring contamination of halal meat with products from five non-halal animals using multiplex polymerase chain reaction coupled with DNA strip

Meat adulteration is a persistent problem in the food industry in many countries. This unfair practice can potentially not only affect the economy of a country but also the confidence and health of its citizens. Additionally, it can aggravate allergies or damage religious beliefs (e.g., Muslims). The aim of this study was to develop a simple and rapid technique for the on-site inspection of several targets using multiplex polymerase chain reaction (PCR) coupled with DNA strip for detecting contamination with products from five non-halal animals (monkeys, dogs, rats, pigs, and cats) in foods certified to be halal. Species-specific primers were retrieved from the mitochondrial genes of each non-halal animal and modified with a tag sequence and biotin. The selected primers showed high specificity for each non-halal animal. The PCR products obtained after amplification were detected using DNA strip. The limit of detection ranged from 0.01 ppm to 1 ppm (dogs, 1 ppm; cats, 0.1 ppm; pigs, 0.01 ppm; monkeys, 0.01 ppm; and rats, 0.01 ppm). Beef meatballs spiked with non-halal meat in different ratios were prepared to validate the developed technique, which presented 100% accuracy in detection. In addition, the developed technique was applied to verify 115 samples of meat products commercially available in Thailand. Sixteen samples were contaminated with pig DNA, but contamination with the DNA of other non-halal animals was not found. Therefore, this technique has high potential for on-site monitoring of non-halal animal contaminants in halal products in Thailand.

НОВІ ФЕНО-ГЕНОТИПІЧНІ ТЕХНОЛОГІЇ В ДІАГНОСТИЦІ ТУБЕРКУЛЬОЗУ

NEW PHENO-GENOTYPIC TECHNOLOGIES IN DIAGNOSIS OF TUBERCULOSIS O. A. Zhurilo, A. I. Barbova, L. M. Sladkova Abstract Widespread drug-resistant tuberculosis is a global problem and can have a negative impact on the global tuberculosis situation. Rapid diagnosis of the disease and early initiation of effective treatment based on the selection of personalized chemotherapy regimens are the basis for preventing the spread of tuberculosis. Microbiological methods which can justify the etiology of the process and determine the drug susceptibiliity of the pathogen are of particular importance for the diagnosis of tuberculosis. The review is focused on current methods of microbiological diagnosis of tuberculosis, including classical microbiological (diagnostic microscopy; solid and liquid media cultures) and modern molecular genetic tests (DNA strips; GeneXpert; multiplex PCR and other). The article presents data on modern methods of bacteriological and molecular genetic diagnosis of tuberculosis, their effectiveness and role in verifying the diagnosis of tuberculosis. The effectiveness of their integrated use to improve the quality of research and rapid detection of patients with M. tuberculosis has been proven. The place of the described methods in the diagnostic algorithm of tuberculosis laboratories is estimated. Further prospects for the diagnosis of tuberculosis associated with the use of new technologies are considered. The information on the current state of microbiological diagnostics of tuberculosis is generalized, the importance of development and introduction of the latest technologies in the diagnostic process is emphasized. Key words: tuberculosis, mycobacteria tuberculosis, drug resistance, methods of bacteriological diagnostics, molecular genetic technologies, PCR, sequencing.

A reconfigurable sandwich structure switchable DNA-based metamaterial

In this paper, a tunable DNA-based metamaterial is designed and simulated in 170–340 THz range. This metamaterial can be transformed from an ON mode with a low resistance state of the DNA strip to its OFF mode with a high resistance state. Three Structures with containing different combinations metal layers are designed and simulated. Structure 1 with Ag/DNA/Ag and Au/DNA/Au strategies achieves field enhancement factors (FEF) 2.18 and 2.07, respectively. Structure 2 (Au/DNA/Dirac, Dirac/DNA/Au, Ag/DNA/Dirac, or Dirac/DNA/Ag) achieves the FEF values 14.11, 10.70, 13.75, or 9.62, respectively, while the FEF value of Structure 3 with Dirac/DNA/Dirac reaches 59.8. The FEF value of Structure 3 can be modulated from 59.8 to 91.96 as Fermi energy increasing from 0 to 60 meV. Moreover, the FEF value is also enhanced through increasing the magnetic field strength. The Structure 3 exhibits convertibility and sustainable modulation lines between two opposing patterns. The proposed structure reveals a switchable feature based on the resistance characteristics of DNA strips, which can be revealed as an ON/OFF switch sensor. Moreover, the switching performance of Structures 3 and 2 is significantly higher than Structure 1. Therefore, Structures 3 and 2 can be set to be an optical memristor or optical gate.

Biocatalytic cascades operating on macromolecular scaffolds and in confined environments

Biocatalytic cascades guide complex, efficient and selective intracellular transformations. These unique features originate from the spatial organization of the biocatalysts in confined cellular environments that allow the directional channelling of reaction intermediates across the cells. Here we address efforts directed towards the development of synthetic cell analogues and supramolecular ensembles acting as nano/microenvironments for operating biocatalytic cascades. Multienzyme systems are integrated within metal–organic frameworks, polymersomes, lipid-stabilized microdroplets and hydrogel microparticles acting as cell-like containments. Also, multienzyme systems are spatially positioned on one-dimensional DNA wires, two-dimensional DNA strips or origami tiles, and three-dimensional DNA origami bundles or cages, and specific protein–protein interactions or peptide–protein complexes provide versatile scaffolds for engineering enzyme assemblies. Biocatalytic cascades operating on these scaffolds or in confined nano/microenvironments reveal substantially enhanced reaction yields compared with the analogous diffusional mixtures of the biocomponents. Mechanistic pathways accounting for the enhanced biocatalytic activities and future challenges in developing and applying biocatalytic cascades are presented. Spatial organization of biocatalytic cascades can improve their performance. In this Review Article, Itamar Willner and colleagues discuss technologies to artificially confine and localize enzyme cascades, the origin of observed rate enhancements and potential applications of such designed systems.

New technologies for the diagnosis of drug-resistant tuberculosis

Widespread drug-resistant tuberculosis is a global challenge that has a potential to have a negative impact on the global TB situation. Rapid TB diagnosis and early initiation of effective treatment based on the individualized chemotherapy regimens underpin efforts in prevention of further spread of tuberculosis. Microbiological methods of TB diagnosis that provide evidence of the TB process etiology and identify drug susceptibility of the pathogen play a very important role. The review covers the current and emerging methods of the microbiological TB diagnosis including conventional microbiological (microscopy of diagnostic material, cultural studies on solid media and culture fluids) and modern molecular genetic tests (DNA strips, biological microchips, multiplex PCR, GeneXpert and diagnostic systems of the new generation and point-of-care tests) and demonstrates the role of the described methods in the diagnostic TB laboratory algorithm. The example of the Microbiology Department of the Central TB Research Institute illustrates the effectiveness of modern developments in the diagnosis of drug-resistant tuberculosis and their impact on improving treatment success in the clinical departments of the CTRI. The review presents further prospects of drug-resistant TB diagnosis in relation to the new technologies such as Next Generation Sequencing. The conclusion summarizes information on the current state of microbiological diagnosis of drug-resistant tuberculosis and emphasizes the importance of developing and introducing new technologies into the diagnostic process.

Detection of Sarcocystis spp. and Shiga toxin-producing Escherichia coli in Japanese sika deer meat using a loop-mediated isothermal amplification-lateral flow strip.

Game meat potentially harbors a number of parasitic and bacterial pathogens that cause foodborne disease. It is thus important to monitor the prevalence of such pathogens in game meats before retail and consumption to ensure consumer safety. In particular, Sarcocystis spp. and Shiga toxin-producing Escherichia coli (STEC) have been reported to be causative agents of food poisoning associated with deer meat consumption. To examine the prevalence of these microbiological agents on-site at a slaughterhouse, the rapid, simple and sensitive detection method known as the “DNA strip” has been developed, a novel tool combining loop-mediated isothermal amplification and a lateral flow strip. This assay has achieved higher sensitivity and faster than conventional PCR and is suitable for on-site inspection.

Radiation sensitivity of water- borne multi drug resistant <i>Escherichia coli</i>

The spread of antibiotic-resistant microorganisms in the environment is recognized widely as an important public health issue, with concerns about future ability to treat infectious diseases. The main risk to public health is that the resistance genes are transferred from environmental bacteria to human pathogens. Safe water is one of the most important needs in public health in the twenty first century. Radiation sensitivity (D10) is defined as the radiation dose (kGy) required to reduce the number of that microorganism by 10-fold. The aim of this paper is to determine the association between multiple antibiotic resistance and radiation sensitivity (D10).Four hundred and sixty four (464) water samples were collected for assessment. E. coli isolation and identification were done using API 20E, and a PCR based DNA STRIP technology that allows simultaneous detection of virulence genes and confirmation of E. coli isolates. Antibiotic susceptibility testing was also conducted using the Kirby-bauer method. Radiation sensitivity was done using a cobalt 60 source. Sixty-three percent (63%) of the multidrug resistant E. coli strains recorded a multiple antibiotic resistance (MAR) index value of >0.2. The mean radiation sensitivity (D10) of the E. coli isolates is 0.33±0.11 kGy. The study confirmed a high prevalence of multiple antibiotic resistances in E. coli isolates. Lastly, there is no association between multiple antibiotic resistant indexes and radiation sensitivity (D10) of antibiotic resistant E. coli. Keywords : Antibiotics, Escherichia coli, water-borne, multi drug resistant radiation, Sensitivity.

Single-Laboratory Validation of Rapid and Easy DNA Strip for Porcine DNA Detection in Beef Meatballs.

Detection of meat from an animal species is required to avoid misleading food labels to consumers. Recently, we developed an easy-to-use molecular detection method by combining isothermal amplification and a DNA strip, referred to as DNA Strip. Here, we report our single-laboratory validation of DNA Strip to detect porcine DNA in beef meatballs. Our results showed that DNA Strip could specifically amplify the target of porcine DNA, with detection limit to 0.01% admixture of pork in beef meatballs. DNA Strip method was also robust because the use of heat block and laboratory water bath showed no significant differences and were comparable to the reference instrument. DNA Strip can detect porcine DNA within ca 1 h, including DNA extraction, DNA amplification, and detection. These results suggest that DNA Strip is applicable because it is easy to use and capable of detecting pork in beef meatballs with a greater detection limit.

Genetic mutations at rifampicin resistance-determining region of RpoB gene in conferring resistance to isolated multidrug-resistant tuberculosis strains

Introduction: Drug resistant tuberculosis (TB) among the new and retreatment cases was seen to be 5%. Multidrug resistant TB to at least isoniazid and Rifampicin is an alarming threat to the community and the scenario is even worse for retreatment cases showing rising trend over the years. A systematic analysis of the nature of the resistance conferred may contribute in understanding the common as well as rare genetic alterations that ultimately led to the genotypic resistance. The study thus involves a probe into the most common and rare patterns of genetic mutation that have led to the rifampicin resistance among the retreatment cases. The common and rare codons along with their change in amino acid sequences involved in conferring the resistance were looked for. Methods: The detection of the patterns genetic mutation is done by the Genotype MTBDR plus V2 kits (Hains Life Sciences) based on DNA strip technology (line probe assay). Results: The analysis has shown that only around 14.93% cases reflected exclusive mutations at around 531-533 region of the RpoB gene. Presence of both the wild and mutant gene segments simultaneously (WildType [WT] and MUT3) accounted to a striking percentage of 60.3% showing a heterogenous generation of susceptible and resistant strains at the same time. This pattern analysis showing maximum of heterogenous strains may be suggestive of capturing a generation under the process of conversion from WT to mutant type. About 95% reproducibility in liquid culture system further established the fact that most of the strains might be a cause for the rising attrition toward the first line of drugs. Conclusion: This pattern analysis showing maximum of heterogenous strains may be suggestive of capturing a generation under the process of conversion. The retrospective analysis showed how heterogenous population prevailed in the retreatment groups.

Mitigating PCR /Amplicon Contamination in a High Risk High Burden Mycobacterial Reference Laboratory in a Resource Limited Setting

Background: Nucleic acid amplification techniques have become important machineries in the diagnosis of several diseases in clinical laboratories. PCR contamination/Amplicon Contamination leading to false positivity remains a major concern in these laboratories. Prevention of these contaminations in establishing these Molecular Biology Laboratories has been very crucial over the years. Though closed system PCRs has substantial reduction in the PCR contamination rates the conventional probe based hybridization methods continues to show occurrence of contamination for various reasons. The Study involved checking the crucial parameters as well as the probable candidates of causing the contamination at a high burden setting. Bringing out the most effective interventions in controlling PCR contaminations for future endeavors stood as a priority. The study explored the efficacies of different sets of interventions contributed in the process of reducing the contaminants.Materials and Methods: The detection of the contaminating PCR products or amplicons or contaminating organism is done by the Genotype MTBDR plus V2 kits (Hains Life Sciences) based on DNA strip technologyResults: The pre and post cleaning as well as cleaning of the working surfaces was able to bring down the mean contamination percentage by 36.5%. The combined effect of the cleaning of the work surfaces, the automated pipetting devices and the AC machines along with it filters were able bring down the mean contamination percentage to 53.5% reducing the rate contamination nearly to between 94.6% (mean percentage contamination was 56.5% at the control run).

Mitigating polymerase chain reaction/amplicon contamination in a high-risk high-burden mycobacterial reference laboratory in a resource-limited setting.

Nucleic acid amplification techniques have become important machineries in the diagnosis of several diseases in clinical laboratories. Polymerase chain reaction (PCR) contamination/amplicon contamination leading to false positivity remains a major concern in these laboratories. Prevention of these contaminations in establishing these molecular biology laboratories has been very crucial over the years. Although closed system PCRs have substantial reduction in the PCR contamination rates, the conventional probe-based hybridization methods continue to show occurrence of contamination for various reasons. The study involved checking the crucial parameters as well as the probable candidates of causing the contamination at a high-burden setting. Bringing out the most effective interventions in controlling the PCR contaminations for future endeavors stood as priority. The study explored the efficacies of different sets of interventions contributed to the process of reducing the contaminants. The detection of the contaminating PCR products or amplicons or contaminating organism is done by the genotype MTBDR plus V2 kits (Hains Life Sciences) based on DNA strip technology. The pre- and post-cleaning as well as cleaning of the working surfaces was able to bring down the mean contamination percentage by 36.5%. The combined effect of cleaning the work surfaces, the automated pipetting devices, and the AC machines was able to bring down the mean contamination percentage to 53.5% reducing the contamination rate nearly to 94.6%. Regularly cleaning the work surfaces, the automated pipetting devices, the PCR machine, and the AC machines along with it filters and exposure of UV rays significantly lowers down the mean contamination percentage.

Antibiotic resistance of Helicobacter pylori in Mongolia.

The resistance of Helicobacter pylori to recently available antibiotic treatment regimens has been recognized as a growing problem. Therefore, the aim of this study was to determine the prevalence of antibiotic resistance among H. pylori strains isolated from Mongolians. All gastric biopsy specimens were obtained during upper gastrointestinal endoscopy from patients referred for the exploration of dyspepsia. The urease positive samples by rapid urease test were cultured according to standard microbiological procedures and H. pylori were grown under microaerophilic conditions on selective Pylori agar. H. pylori antibiotic sensitivity was examined using E-test. In addition, the mutations of the corresponding gene were studied by GenoType HelicoDR DNA strip testing. Three hundred twenty patients, 216 female and 104 male in the ages range of 18 to 83 years were included in this study. Rapid urease test yielded positive results for 65.9% (211/320). Among them, we have successfully obtained 72% H. pylori isolates. The antibiotic resistance rates were 35.5% for clarithromycin, 68.4% metronidazole, 23.0% amoxicillin, 25.0% tetracycline, 28.2% erythromycin and 14.5% nitrofuranton. Resistance for 2 drugs was 34.5% and that of 3 drugs was observed in 14.5% of isolates. The most prevalent mutation was A2147G followed by A2146G and D91Y. The prevalence of H. pylori infection increased among Mongolian population and the prevalence of resistance of H. pylori is very high to metronidazole, and moderate to clarithromycin. The data on antimicrobial susceptibilities provided by the present study is may assist the clinicians on the effectiveness of treatment regimens.

Molecular Detection of Mycobacterium Tuberculosis Complex from Sputum of Clinically Suspected Tuberculosis Cases

Background: Tuberculosis (TB) is a communicable disease and it ranks second of all infectious agents due to co-infection with HIV . The causative agent of tuberculosis is a group of mycobacteria known as Mycobacterium tuberculosis complex. Mycobacterium tuberculosis complex consist of M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti. In PCR study, Most commonly sensitivity is higher in smear positive samples (95-100%) rather than smear negative specimens (46-63%). OBJECTIVE: To detection of Mycobacterium tuberculosis complex by Line Probe Assay. Methods: The study was done from non-interventional approaching study of 50 suspected patients of tuberculosis had visited the TB chest/ DOTS centre. Sputum sample collected early morning in a wide- mouth container from the patients having history of cough more than 2 weeks. Methodology used Z-N staining and for detection of MTB complex was done using MTBDR plus assay, multiplex PCR DNA strip assay (Hain Lifescience, Nehren, Gernamy) which is commercially available. M. tuberculosis secreted an important protein is MPT64, a 24-kDa protein. The major culture filtrate protein (24-kDa) is MPT64 encoded by the RD2 region genes and has been exposed to be an exact antigen that differentiates the M. tuberculosis complex from the mycobacteria other than tuberculosis (MOTT) Species. Results: In 50 samples, out of which 10 (20%) were smear positive & 40(80%) were smear negative. Out of 10 smear positive, 9 (90%) were MTB (Mycobacterium tuberculosis) & 01 (10%) was NTM (Non-tuberculous Mycobacteria) by PCR method. Out of 40 smear negative, 30 (75%) were positive by PCR method. Out of 30, 28(93%) were MTB & 02(7%) were NTM. Rests of the 10(25%) samples were found negative for M. tuberculosis complex. Conclusions: This study proved that PCR is a specific and sensitive method in comparison of sputum microscopy after staining with Z-N technique and it helpful the clinicians ability to diagnose and treat the patients on time. This will ensure early treat to patients and prevent further transmission of disease.