Liquid Culture Method Research Articles

Decomposition of Polythene by Bacteria Isolated from Waste Disposal Sites in Gusau City

The accumulation of polythene can be hazardous and can cause various environmental problems. This work was aimed at studying the biodegradation of polythene by bacteria isolated from waste disposal areas in North West, (Zamfara state) Nigeria. A total of twenty soil samples were collected from four different locations, such as waste disposal areas, hospitals, roadsides, and petrol pumps station. Spread plate method was carried out on Nutrient agar for the growth of bacteria. A total of 13 bacteria were isolated from different areas. The total bacteria count in hospital ranged from 80 × 10-4 - 248 × 10-4 cfu/mL, roadsides ranged from 116 × 10-4 - 208 × 10-4 cfu/mL, petrol pump ranged from 88 × 10-4 - 184 × 10-4 cfu/mL and waste disposal areas ranged from 116 × 10-4 - 264 × 10-4 cfu/mL. The microbial species associated with the polythene materials were identified as Pseudomonas sp, Bacillus sp, Staphylococcus aureus, Providencia stuartii, Clostridium perfringes, Corynebacterium diphtheria, Listeria monocytogen. The bacteria isolates were screened for polyethylene degradation using mineral salt medium containing polyethylene as sole source of carbon and nitrogen for their ability to degrade polyethylene for the period of 2 months. The efficacy of the microbes in the degradation of polythene were analyzed using liquid culture method, among the bacteria Pseudomonas aeruginosa degraded 75% of polythene, Bacillus subtilis 71.4% in a period of 2 months.

A liquid culture method assisted by ATP analysis for accelerating laboratory experiments on ultraviolet disinfection of airborne bacteria

The solid culture method for measuring the efficiency of ultraviolet (UV) disinfection of airborne bacteria is time-consuming, typically taking 12–48 h. To expedite such experiments, this study proposed a liquid culture method assisted by adenosine triphosphate (ATP) analysis, as a liquid culture is faster than a solid culture, and measurement of ATP does not require waiting for visible colonies to form. Escherichia coli (E. coli) was used as the experimental bacterium. This study first compared the log reduction of bacteria in liquid as measured by the proposed method and by the traditional solid culture method. The minimum liquid culture time was determined for different bacterial concentration ranges. Finally, the feasibility of the proposed method was validated by UV disinfection experiments on airborne bacteria. The results indicated that the proposed method measured a similar log reduction to that of the solid culture method in liquid experiments. The minimum liquid culture time for E. coli in 105-106 colony forming units (CFU)/mL was 2 h. The validation experiments demonstrated that the proposed method is capable of measuring the UV disinfection efficiency of airborne bacteria. The proposed method can accelerate laboratory experiments on UV disinfection of airborne bacteria, which in turn can support the effective design and utilization of UV disinfection in real life.

Effect of different microplastics on the mobilization of soil inorganic phosphorus by exomycorrhizal fungi

<p>Microplastics (MPs) can affect the phosphorus (P) cycle in soil by changing soil properties and the function of microbial communities, but their impact on soil P availability remains unclear. To explore the effects of different types of MPs on the mobilization of soil inorganic P by exomycorrhizal fungi, Lactarius delicious (Ld) was used as the test strain, and polystyrene microspheres (PS), polypropylene (PP) and poly(lactic acid) (PLA) were used as test materials. The soil was used as the only P source for the media. The effect of different types of MPs on the mobilization of soil inorganic P by exomycorrhizal fungi was studied using liquid culture method. The results showed that exposure to MPs at low to medium concentrations (10 or 100 mg L-1) activated inorganic P in soil by stimulating the secretion of additional organic acids and hydrogen ions by ectomycorrhizal fungi, improving the available P in soil. The effect of mobilizing inorganic P was more significant in the treatment groups exposed to low and medium concentrations of biodegradable PLA. This may be related to the fact that PLA stimulated the ectomycorrhizal fungi to secrete more organic acids, and the release of a large amount of lactic acid after PLA was biodegraded. However, when the exposure concentration of MPs further increased, the growth of ectomycorrhizal fungal mycelium may be inhibited, the secretion of organic acids and hydrogen ions may be reduced, and the mobilization ability of soil inorganic P may be limited. In addition, it should be noted that high concentrations of non biodegradable MPs (PS and PP) had significant limiting effects on the mobilization of soil inorganic P by exomycorrhizal fungi. Therefore, our study results provide evidence that the presence of MPs may reduce the availability of soil P. Further research should focus on maintaining or improving P availability in plant-microbe systems in MP-polluted soil.</p>

Isolation of Plastic Degrading Fungi from Plastic Dumped Sites

Plastics that are biodegradable can be considered environment friendly, they have an increasing range of potential application and are driven by the growing use of plastics in packaging. In this study, the biodegradation of Low-density Polyethylene films was analyzed about 1 month of incubation in liquid culture method. In this investigation, soil samples were collected from two different plastic dumped sites and the fungus were isolated through spread plate technique from diluted soil samples. Among the six fungal isolates were tested for checking its degradation ability of plastics. This study showed the cultural analysis for its ability to adhere and grow on the LDPE films. The activity enzyme produced by these fungal isolates was responsible for the biodegradation. Isolating plastic-degrading fungi from plastic dumped sites serves several important purposes, Understanding the role of fungi in plastic degradation can shed light on the ecological impact of plastic pollution on ecosystems. It can also help identify potential solutions to mitigate this impact. Isolating fungi from plastic dumped sites contributes to the exploration of microbial biodiversity in these environments. It can lead to the discovery of novel fungal species with unique abilities to degrade plastic.

Identification of Mycobacterium tuberculosis Resistance to Common Antibiotics: An Overview of Current Methods and Techniques.

Multidrug-resistant tuberculosis (MDR-TB) is an essential cause of tuberculosis treatment failure and death of tuberculosis patients. The rapid and reliable profiling of Mycobacterium tuberculosis (MTB) drug resistance in the early stage is a critical research area for public health. Then, most traditional approaches for detecting MTB are time-consuming and costly, leading to the inappropriate therapeutic schedule resting on the ambiguous information of MTB drug resistance, increasing patient economic burden, morbidity, and mortality. Therefore, novel diagnosis methods are frequently required to meet the emerging challenges of MTB drug resistance distinguish. Considering the difficulty in treating MDR-TB, it is urgently required for the development of rapid and accurate methods in the identification of drug resistance profiles of MTB in clinical diagnosis. This review discussed recent advances in MTB drug resistance detection, focusing on developing emerging approaches and their applications in tangled clinical situations. In particular, a brief overview of antibiotic resistance to MTB was present, referred to as intrinsic bacterial resistance, consisting of cell wall barriers and efflux pumping action and acquired resistance caused by genetic mutations. Then, different drug susceptibility test (DST) methods were described, including phenotype DST,genotype DST and novel DST methods. The phenotype DST includes nitrate reductase assay, RocheTM solid ratio method, and liquid culture method and genotype DST includes fluorescent PCR, GeneXpert, PCR reverse dot hybridization, ddPCR, next-generation sequencing and gene chips. Then, novel DST methods were described, including metabolism testing, cell-free DNA probe, CRISPR assay, and spectral analysis technique. The limitations, challenges, and perspectives of different techniques for drug resistance are also discussed. These methods significantly improve the detection sensitivity and accuracy of multidrug-resistant tuberculosis (MRT) and can effectively curb the incidence of drug-resistant tuberculosis and accelerate the process of tuberculosis eradication.

Comparing diagnostic accuracy: Xpert MTB/RIF Ultra vs. Xpert MTB/RIF for diagnosis of pulmonary tuberculosis

IntroductionWith the development of Xpert MTB/RIF (Xpert) and its latest version Xpert MTB/RIF Ultra (Ultra), diagnosis of tuberculosis has improved dramatically. Only few have compared the Ultra to Xpert, especially in varied geographic locations. The ability of Ultra to detect tuberculosis in pulmonary samples was thus compared in the current study to the Xpert for the quick identification of MTBC and resistance to rifampicin (RR) in sputum samples. MethodologyAdults and adolescents (>10 years) with suspicion of pulmonary tuberculosis were enrolled for the study. All the samples were subjected to smear microscopy, liquid culture and molecular methods Xpert MTB/RIF as well as Xpert Ultra in parallel. ResultsA total of 143 sputum samples were cultured in liquid media, with 52 of them proved to be culture positive. Using culture as the “reference standard,” the Xpert Ultra detected MTBC in 45 samples, with sensitivity and specificity of 86.5% (74.2%–94.4%) and 89.9% (81.0%–95.5%), respectively. While the sensitivity and specificity, of Xpert was found to be 76.9% (63.2%–87.5%) and 93.7% (85.8%–97.9%) respectively. Xpert Ultra also detected 78.8% smear negative culture positive samples in comparison to Xpert MTB/RIF which detected 63.6% smear negative samples. ConclusionXpert Ultra can offer a valid TB diagnosis in a fraction of the time it takes to perform a culture and has higher sensitivity than Xpert MTB/RIF. This is especially true for paucibacillary samples with negative sputum smear.

National cross-sectional cluster survey of tuberculosis prevalence in Timor-Leste: a study protocol

IntroductionTimor-Leste has one of the world’s highest estimated tuberculosis (TB) incidences, yet the data which informs this estimate is limited and the true burden of TB disease is not known....

Cultivation Strategies of Kenaf (Hibiscus cannabinus L.) as a Future Approach in Malaysian Agriculture Industry

In recent years, kenaf has gained significant global attention as a more cost-effective, adaptable, and manageable alternative to other fibre crops. India and China, with nearly 70% of the global kenaf production, have emerged as the leading producers of kenaf plants. While kenaf was traditionally valued for its paper production, it has evolved into a multipurpose crop with diverse industrial applications over the past two decades. Conventional soil-based cultivation methods for kenaf require up to six months for plant maturity. However, in vitro propagation techniques offer a promising alternative that enables faster growth and reduced labour costs. In vitro propagation can be achieved using solid and liquid media, with limited research available on the pure liquid culture method for kenaf. This review aims to introduce and compare the production of kenaf using solid and liquid media, with a specific focus on the emerging country of Malaysia, which seeks to harness the potential of kenaf cultivation for the 15th Sustainable Development Goal, “life on land”, and its contribution to the economy.

A Comparative Analysis of Microscopy, Culture, and the Xpert Mycobacterium tuberculosis/Rifampicin Assay in Diagnosing Pulmonary Tuberculosis in Human Immunodeficiency-Positive Individuals.

Individuals with human immunodeficiency virus (HIV) infection have an increased likelihood of developing tuberculosis (TB). The primary objective of this study was to compare the diagnostic accuracy of microscopy, culture, and the XpertMycobacterium tuberculosis/Rifampicin (MTB/RIF) assay in the diagnosis of pulmonary TBin sputum samples of HIV-infected patients. The secondary objectives were to evaluate the sensitivity and specificity of these three methods along with a comparison of diagnostic approaches for detecting drug-resistant strains. This prospective, laboratory-based study was done in the Microbiology Department of IGIMS, Patna. The study included sputum samples of 102 individuals who were HIV-positive and exhibited symptoms indicative of tuberculosis. Out of 102 individuals suspected of having tuberculosis, 18 tested positive for M. tuberculosis. Male individuals between the ages of 31 and 40 were more affected by both HIV and tuberculosis, and in most of these cases, their CD4 cell count was below 200 cells/µl. Among the 102 sputum samples collected, 18% (18 samples) were found to be positive using the Mycobacterium Growth Indicator Tube (MGIT) 960 liquid culture method. Two samples were contaminated, and 14.7% (15 cases) tested positive using the cartridge-based nucleic acid amplification test (CBNAAT) method. Additionally, 3.92% (four samples) were positive using the ZN staining method. The study found that Xpert MTB/RIF outperformed other methods in identifying resistance to RIF, showed better agreement with gene sequencing results for RIF resistance, and had higher accuracy in detecting tuberculosis cases, including both smear-positive and smear-negative cases.

Electrophoretic deposition of gentamicin and chitosan into titanium nanotubes to target periprosthetic joint infection.

Periprosthetic joint infection (PJI) occurs in 1%-2% of primary total hip and knee arthroplasties; the rate can reach 20% in individuals at risk. Due to the low local bioavailability of systemic antibiotics and possible off-target effects, localized drug delivery systems are of great importance. Our aim was the electrophoretic deposition (EPD) of gentamicin and chitosan in Titanium (Ti) nanotubes to establish a local, prolonged antibiotic delivery. Nanotubes were created on Ti wire with a two-step anodization process. For drug deposition, EPD and the air-dry methods were compared. For a prolonged drug release, gentamicin and crosslinked chitosan were deposited in a two-step EPD process. Drug release was quantified by fractional volume sampling. The Ti wires were tested against Staphylococcus aureus by agar dilution and liquid culture methods. MC3T3-E1 osteoblastic cell viability was determined with trypan blue. Nanotubes were characterized by a 100 nm diameter and 7 μm length. EPD allowed a higher amount of gentamicin deposited than the air-dry method. Drug deposition was controllable by adjusting the voltage and duration of the EPD process. The crosslinked chitosan layer allowed diffusion-driven release kinetics for up to 3 days. Gentamicin-loaded Ti wires significantly inhibited bacterial growth and resulted in a larger inhibition zone compared to unloaded wires. Twenty-four hours of incubation with loaded wires did not have a significant effect on osteoblast viability. Gentamicin-loaded Ti nanotubes represent a promising approach for PJI prevention, as well as a valuable preclinical tool for the investigation of localized drug delivery systems created on Ti surface.

Drug Resistance Patterns in Tubercular Meningitis patients from North India (S25.007)

<h3>Objective:</h3> We aim to study the extensiveness of drug resistance in patients of Tubercular Meningitis (TBM) in our population. <h3>Background:</h3> Drug-resistant TBM is a potentially underrecognized entity, and delay in the institution of appropriate therapy may pave the way for development of further drug resistance by facilitating selective growth of resistant bacilli. Due to the paucibacillary nature of CNS-TB, it is difficult to isolate mycobacteria from the CSF. Thus, there is limited data regarding drug susceptibility in TBM. <h3>Design/Methods:</h3> All suspected TBM patients from across North India presenting to the emergency and those hospitalized in our institute were screened and recruited if they fulfilled the inclusion and exclusion criteria. CSF samples were processed by putting up smear for Ziehl-Neelsen staining and inoculation into the LJ culture media (solid culture) and MGIT 960 (liquid culture). Drug susceptibility patterns of MTB isolates from CSF samples were assessed for first and second-line drugs by phenotypic (MGIT 960) liquid culture method. <h3>Results:</h3> Out of 309 CSF samples processed, 11.65% (36/309) were culture positive. MDR-TB was detected in 16.66% (6/36) of isolates. However, the prevalence of Pre-XDR-TB and XDR-TB among isolates was 5.55% (2/36) and 2.77% (1/36), respectively. Fluoroquinolone (FQ) resistance was observed in 16.66% (6/36) isolates. Of the 6 FQ-resistant isolates, 2/6 were Pre-XDR and 4/6 were mono-FQ resistant. Resistance to second-line injectables was observed in 8.33% (3/36) isolates. Mono-resistance to isoniazid was observed in 8.33% (3/36) of isolates. <h3>Conclusions:</h3> The prevalence of MDR in culture positive TBM patients was observed to be high (16.66%) in our study. Fluoroquinolone resistance was observed both in MDR-TB and sensitive TBM. Rapid detection of drug resistance is essential in a clinically relevant time frame for the timely initiation of appropriate treatment and better patient outcomes. <b>Disclosure:</b> Mr. Modi has nothing to disclose. Kusum Sharma has nothing to disclose. Dr. SINGLA has nothing to disclose. Ms. Sood has nothing to disclose.

Physiological response of ectomycorrhizal fungi (Lactarius delicious) to microplastics stress

&lt;p&gt;The pollution and toxicological effects of microplastics in soil have gradually attracted widespread attention, but studies on the ecological effects of microplastics on soil microorganisms and their mechanisms are still very scarce. To explore the physiological response of ectomycorrhizal fungi (Lactarius delicious, Ld) to microplastic stress, Ld was used as the test strain, and 80 nm and 4 μm monodisperse polystyrene microspheres (PS-MPs) were selected. The solid plate method and the liquid culture method were used to study the effect of PS-MPs with two particle sizes on the growth, nutrient content, reactive oxygen species (ROS) production, organic acid secretion and other indicators of the strain Ld. The results showed that with increasing PS-MPs concentration, the biomass of strain Ld decreased gradually. When the strain Ld was exposed to high concentrations of PS-MPs (200-300 mg L-1), PS-MPs with particle size of 4 μm showed stronger inhibition of biomass than PS-MPs with particle size of 80 nm. After exposure to the PS-MPs environment of two sizes, the concentration of PS-MPs increased (0-300 mg L-1), and the contents of phosphorus (P), potassium (K), H2O2 and superoxide anion radical (O2·-) in Ld mycelia increased significantly (p &amp;lt; 0.05). This showed that the antioxidant substances in Ld could not remove excess ROS, but could reduce the stress of microplastics by increasing the absorption of P and K from the environment, which was beneficial to its own metabolic activities. In addition, the strain Ld could secrete oxalic acid, acetic acid and succinic acid into the culture medium, which the amount of oxalic acid secreted was the highest. We speculated that strain Ld responded to the stress of microplastics by secreting a large amount of organic acids. Therefore, the mechanism of the influence of microplastics on ectomycorrhizal fungi may involve oxidative stress, and the mycelia responded to the toxicity of microplastics by secreting more organic acids and increasing the absorption of P and K. The results of this study will provide a scientific basis for the study of the acute toxicity of microplastics to ectomycorrhizal fungi.&lt;/p&gt;&#x0D;

WITHDRAWN: Analysis of the application value of molecular diagnostic technology of mycobacterium tuberculosis

To evaluate the application value of molecular diagnostic technology for tuberculosis. Firstly, the sensitivity and specificity were compared between sputum smear and isothermal amplification (IA, Di’ao Biological Technology Co., Ltd, Guangzhou, China) with solid culture as the standard method. Secondly, the detection rate was compared between liquid culture and molecular diagnostic methods, including IA and Xpert MTB/RIF. Thirdly, the minimum detection limit of IA and Xpert MTB/RIF was compared. Finally, IA was used from 2019 to February 2020, and Xpert MTB/RIF was used from March 2020, the contribution of molecular diagnosis to the etiological diagnosis of tuberculosis in Lishui District in Nanjing was compared in 2019 and 2020. The sensitivity of IA and sputum smear was 100% and 54.55%, respectively ( P < 0.05), and the specificity was 97.66% and 98.44%, respectively( P >0.05). IA was more consistent with solid culture( Kappa =0.924) than smear( Kappa =0.624). The detection rates of IA, Xpert MTB/RIF and liquid culture were 75.00%, 77.50% and 77.50%, respectively ( P > 0.05). The minimum detection limit of Xpert MTB/RIF was lower than that of IA. In 2019 and 2020, molecular diagnosis accounted for 31.58% and 39.28% of positive etiology, respectively, and the incidence of positive molecular pathogens was 1.25/100,000 and 2.24/100,000, respectively. Molecular detection technology improves the detection rate of tuberculosis etiology. Each TB designated detection hospital can select molecular detection methods according to local conditions, which will help control the spread of tuberculosis.

Biodegradation and Bioremediation of Polymer by Microbial Assisted Novel Process

The degradation of polystyrene (PS) and polyurethane (PU), both are foams, is a budding challenge due to increasing white pollution. The present investigation has focused on the microbial assisted biodegradation. Various indigenous microorganisms were isolated from sewage soil. PS – PU foam and bioplastic was used to screen the soil bacteria with biodegradation potential. The screened bacteria were subjected to biodegradation assay such as soil burial and liquid culture in the presence of PS – PU foams and bioplastic in a growth medium. Two microorganism were isolated-- Pseudomonas aeruginosa and Aspergillus niger. The degradation rate based on weight loss is conducted by in vitro assay for 10 and 35 days. The maximum degraded foam was analyzed through Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy and PS – PU foam and bioplastic act as a control without microorganisms. Results of the present study showed that the maximum degradation and weight loss in the polyurethane foam occurred in the soil burial method by Pseudomonas aeruginosa compared with Aspergillus niger. The soil burial method is more significant than liquid culture method. There is no weight reduction in the polystyrene foam in both organism and methods. For the degradation of synthetic polymers the isolated Pseudomonas aeruginosa and Aspergillus niger were encapsulated for future studies.

A study on microbial deterioration of low-density polyethylene

Plastics have been widely used as a packing material in the form of low-density polyethylene (LDPE) they are very versatile in nature, cost efficient and facile. However, they are not decomposable and resistant to microbial attack which is a major cause of persistent and long-term environmental pollution. In this study, the isolation of polythene degrading microorganisms was done from plastic wastes dumped site. The effect of the pre-treatment on the biodegradation of polythene was analyzed during one month of incubation period in liquid (shaker) culture method. To check the efficiency of biodegradation, weight method was performed. The bacterial species found associated with the degrading materials were found to be as Gram-positive cocci and Gram-negative rods; and these were identified as Staphylococcus simiae and Proteus vulgaris with the help of an online advanced bacterial identification software. Several biochemical assays were performed to determine the effective biodegradation of polyethylene bags. This work reveals that Proteus vulgaris possess greater potential to degrade polythene as compared to Staphylococcus simiae. Proteus vulgaris showed better degradation efficiency in case of both pre-treated and untreated samples, proving to be a potent microbe in degrading polythene.

Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise l-cysteine, l-lysine and l-arginine. It was found that l-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but l-lysine or l-arginine addition did not enhance growth. Brucella broth supplemented with l-cysteine (0.4 mM), l-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Effect of Glucose, Agar Supplementation and Bacterial Cell Density on the in vitro Liquid Culture of Steinernema jeffreyense

Entomopathogenic nematodes (EPNs) of the family Steinernematidae are effective biological control agents against important pest insects. The in vitro liquid culture method of mass production is used to produce EPNs of high quantity, quality and with reduced cost by upscaling. The first step in liquid mass production is the use of shake flasks to obtain monoxenic infective juvenile (IJ) inoculum for optimisation purposes, followed by upscaling to a desktop fermenter. This study was undertaken to assess the impact of the addition of agar and glucose to the culture medium, as well as assessing the impact of bacterial cell density inoculum on IJ recovery and yield. Steinernema jeffreyense was cultured in 250 ml Erlenmeyer flasks, with the mutualistic bacterium Xenorhabdus khoisanae. In this study the impact of four different agar and glucose concentrations showed negligible impact on nematode recovery and yield. Different initial bacterial inoculum densities inoculated to the culture medium showed a low inoculum density of 2 % (6 × 106 cells) of the bacteria culture to be the optimal inoculum concentration. A bacterial growth curve for X. khoisanae in tryptic soy broth, showed 40–44 h to be the optimum time for introduction of the initial nematode inoculum into the complex medium.

The value of gene chip detection of bronchoalveolar lavage fluid in the diagnosis of nontuberculous mycobacterial lung disease.

Nontuberculous mycobacteria (NTM) are mycobacteria other than mycobacterium tuberculosis complex (MTBC) and mycobacterium leprae. NTM can cause infection in many human tissues and organs and is most commonly seen in the lungs. Clinically, the symptoms and signs of nontuberculous mycobacteria lung disease (NMLD) are very similar to those of tuberculosis (TB). Because most NTMs are resistant to conventional anti-TB drugs, the rapid diagnosis of NMLD is the key to treatment. This study aimed to use gene chip technology to examine bronchoalveolar lavage fluid (BALF) from NMLD patients to explore the value of this technique for the rapid diagnosis of NMLD in BALF. A retrospective analysis of 308 patients with NMLD treated at Fuzhou Pulmonary Hospital from January 2018 to June 2020 was performed. BALF was collected from the patients. Gene chip detection (Capital Bio Corporation, Chengdu, China) and BACTEC MGIT960 (Becton, Dickinson and Company, MD, USA) liquid culture were performed to compare the NTM positive detection rates between the two methods. The NTM strain isolated from liquid culture were identified by rDNA sequencing and the results of identification were compared with those of gene chip detection using BALF specimens. A total of 221 cases of NTM were detected in 308 BALF specimens by the gene chip method; the positive rate was 71.75% (221/308). A total of 218 cases of NTM were detected by the liquid culture method, and the positive rate was 70.78% (218/308). There was no significant difference in the positive rate of NTM detected in BALF specimens between the two methods (χ2=0.138 P=0.804>0.05); 187 cases were detected with both sequencing and gene chip detection, and the coincidence rate of strain identification with the two methods reached 96.79% (181/187). Sequencing of 218 strains of NTM was carried out; eight species were identified, and the top four species were M. intracellulare (131/218, 60.09%), M. avium (48/218, 22.02%), M. abscessus (27/218, 12.38%), and M. kansasii (5/218, 2.29%). Gene chip technology can rapidly detect NTM in BALF and accurately identify bacterial species. It has important clinical value in the early diagnosis and treatment of NMLD.

Comparative study of Legiolert with ISO 11731-1998 standard method-conclusions from a Public Health Laboratory

BackgroundLegionella pneumophila (L. pneumophila) is responsible for 96% of Legionnaires' disease (LD) and 10% of all worldwide pneumonia cases. Legiolert™, a liquid culture method for most probable number (MPN) enumeration of L. pneumophila, was developed by IDEXX Laboratories. The method detects all serogroups of L. pneumophila in potable and non-potable water samples. ObjectiveThe goal of this study is to establish that Legiolert is a suitable alternative method to meet testing requirements in Spain for the enumeration of Legionella in water samples. MethodologyThe laboratory analyzed 118 environmental water samples from the Barcelona region (56 potable and 62 non-potable) in parallel by the Standard method for detection and enumeration of Legionella (ISO 11731:1998) and by Legiolert. Comparison of the recovery of the alternative method (Legiolert) and the Standard was made using ISO 17994:2014 and McNemar's binomial test statistical methods. Results44 samples were positive for Legionella (36 potable and 8 non-potable). Legiolert and the Standard method detected a similar percentage of positive samples, with Legiolert being slightly higher (31 vs 30%) and detecting higher concentrations of Legionella within the samples. ISO 17994:2014 analysis of the potable water samples found Legiolert was more sensitive than the Standard at detecting Legionella, even when complete Legionella species (L. spp.) results were considered for both methods. The two methods also demonstrated equivalent detection of L. spp. according to the McNemar's test. The comparison is significantly more in favor of Legiolert when only L. pneumophila results are considered. Each confirmation run with material extracted from positive Legiolert wells contained L. pneumophila, giving the method a specificity of 100%. Although statistical results for non-potable waters are not included because of the low number of samples, the two methods trended towards equivalence. ConclusionsRelative to the Standard method, Legiolert has a greater sensitivity and selectivity, and appears to have higher recovery for L. pneumophila, and equivalent recovery when L. spp. is included in the comparison. Legiolert also has high specificity. The procedural advantages of Legiolert allow laboratories to save on resources, costs, and time and consequently to test more frequently. In conclusion, the study finds IDEXX Legiolert a suitable alternative to ISO 11731:1998.

Evaluation of Legiolert™ for the Detection of Legionella pneumophila and Comparison with Spread-Plate Culture and qPCR Methods.

Legionella pneumophila, the organism responsible for Legionnaires' disease, a potentially lethal pneumonia, is an opportunistic bacterium spread via inhalation of contaminated, aerosolized water. The detection and control of L. pneumophila is crucial to reduce the risk it poses to human health. L. pneumophila is generally detected and quantified by the plating method, ISO 11731:2017 and by qPCR. ISO 11731 is based on the filtration of the water sample through a membrane, which is placed on selective agar medium, and after colony growth, presumptive Legionella are then confirmed by subculturing, serology, or PCR. Quantitative Polymerase Chain Reaction (qPCR) is based on the amplification of a DNA sequence specific to L. pneumophila, usually within the mip gene. The objective of this study was to compare these methods to a new, liquid culture method based on the Most Probable Number (MPN) technique, Legiolert™/Quanti-Tray® with data obtained with ISO 11731 and a viability quantitative qPCR (v-qPCR), for quantification of L. pneumophila in potable and non-potable waters. Data showed that the Legiolert method revealed concentrations of L. pneumophila greater than ISO 11731 and generally similar results to those of v-qPCR. The Legiolert method was highly specific and easy to use, representing a significant advancement in the quantification of L. pneumophila from potable and non-potable waters.