Can Thin Layer Agar Test Play a Key Role in the Diagnosis of Tuberculosis in Low-Resource Settings?

Background: Fast, reliable, and cost-effective tests are recommended for tuberculosis diagnosis and drug susceptibility testing, especially in resource-limited settings. Objectives: This study aimed to evaluate the performance of thin-layer agar for tuberculosis diagnosis and drug susceptibility testing. Methods: Samples were collected from patients with presumptive tuberculosis and tested using thin-layer agar for tuberculosis and drug susceptibility testing in parallel with Lowenstein Jensen culture method for tuberculosis diagnosis and proportion method for drug susceptibility testing as the gold standard. Receiver operating characteristic curve analysis was performed to calculate the performance parameters. Results: Thin-layer agar method showed sensitivity and specificity values of 96.63% and 62.50%, respectively, for the isolation of Mycobacterium tuberculosis directly from specimens. Drug susceptibility results using thin-layer agar showed sensitivity values for isoniazid, rifampicin), ethambutol and streptomycin were 94.74%, 86.84%, 94.74% and 81.58%, respectively, while the specificity values were 100%, 100%, 86.27% and 100% for isoniazid, rifampicin, ethambutol and streptomycin, respectively. Results were available in a median time of 16 days for thin-layer agar and 25 days for the conventional method. Conclusions: The thin-layer agar method is a relatively rapid, simple, and cost-effective method for the diagnosis and drug susceptibility testing of M. tuberculosis. It may be a useful tool for establishing tuberculosis laboratories in resource-limited settings because it does not require expensive equipment and a high level of training. Our study may help in choosing the appropriate treatment and control of tuberculosis.

Azole resistant Aspergillus fumigatus clinical isolate screening in azole-containing agar plates (EUCAST E.Def 10.1): low impact of plastic trays used and poor performance in cryptic species

Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedurés performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n=91) and cryptic species (n=52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multi-dish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harbouring TR 34 -L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/L. Isolates harbouring G54R and TR 46 -Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole-resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto ; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.

Cell Block Optimization: A Comparative Study of Quality Variables in 4 Different Cell Block Methods

<b><i>Objective:</i></b> The cell block (CB) is an important adjunct to cytological preparations in diagnostic cytopathology. Optimizing cellular material in the CB is essential to the success of ancillary studies such as immunohistochemistry (IHC) and molecular studies (MS). Our aim was to identify which CB method was most suitable in a variety of specimen types and levels of cellularity. <b><i>Study Design:</i></b> We assessed 4 different CB methods, thrombin clot method (TCM), MD Anderson method (MDAM), gelatin foam method (GFM), and agar method (AM), with descriptive observations and ranking of the methods based on quantity of cells and morphological features. <b><i>Results:</i></b> TCM performed best in ranking for both quantity of cells and morphological features, followed by MDAM, GFM, and AM. Lack of adjuvant in the MDAM resulted in some unique morphological advantages which, however, also resulted in inconsistent performance. In low cellularity cases insufficient cells were frequently identified on slides from MDAM and AM CBs. Technique touch time was similar for all methods, with total processing time being shortest for TCM followed by MDAM, GFM, and AM. <b><i>Conclusions:</i></b> TCM was the most robust CB technique, retaining high scores for ranking of quantity and morphology in a variety of specimen cellularities and specimen types.

UJI AKTIVITAS ANTIBAKTERI EKSTRAK DAN FRAKSI ASCIDIAN (Lissoclinum badium) DARI PERAIRAN PULAU MANTEHAGE

ABSTRACTLissoclinum badium is a type of ascidian that contains bioactive compounds. This study aims to determine of presence of antibacterial activity from extracts and fractions of Lissoclinum badium collected from Mantehage Island Manado against Escherichia coli and Staphylococcus aureus bacteria. Samples were extracted by maceration method using 95% ethanol solvent and fractionated using solvents of chloroform, n-hexane and methanol. Antibacterial activity was carried out by the disk diffusion agar method. The results showed that the ethanol extracts an methanol fraction had activity to inhibit the growth of Escherichia coli bacteria with strong category. Meanwhile, against the Staphylococcus aureus the ethanol extracts, chloroform and n-hexane fractions had ability to inhibit the growth of bacteria with weak category.. Keywords: Antibacterial activity, Lissoclinum badium, Escherichia coli, Staphylococcus aureus. ABSTRAKLissoclinum badium merupakan salah satu jenis tunikata yang memiliki kandungan senyawa bioaktif yang dapat digunakan untuk pengobatan berbagai penyakit. Penelitian ini bertujuan untuk mengetahui adanya aktivitas antibakteri dari ekstrak dan fraksi Lissoclinum badium yang diperoleh dari Pulau Mantehage Manado terhadap Escherichia coli dan Staphylococcus aureus. Sampel diekstraksi dengan metode maserasi menggunakan pelarut etanol 95% dan fraksinasi menggunakan pelarut metanol, kloroform, dan n-heksan. Aktivitas antibakteri dilakukan dengan metode difusi agar cakram kertas. Hasil penelitian menunjukkan bahwa ekstrak etanol dan fraksi metanol memiliki aktivitas untuk menghambat bakteri Escherichia coli dan Staphylococcus aureus dengan daya hambat kuat. Sedangkan untuk fraksi kloroform dan fraksi n-heksan memiliki aktivitas untuk menghambat bakteri Staphylococcus aureus saja dengan daya hambat sedang. Kata kunci: Aktivitas antibakteri, Lissoclinum badium, Escherichia coli, Staphylococcus aureus.

Biofilm Detection on Catheter Associated Uropathogenic Bacteriuria among Fistula Patients Attending National Obstetric Fistula Centre Ningi, Bauchi State, Nigeria

Background: Biofilm production caused by bacteria plays a vital role in catheter associated urinary tract infection (UTI) or bacteriuria being responsible for persistence and recurrent infection. Biofilms forming bacteria are difficult to eradicate due to antimicrobial resistance to the commonly used antibiotic. Biofilms are currently estimated to be responsible for over 65% of nosocomial infections and 80% of microbial infections. This study aimed to perform biofilm detection on uropathogenic bacterial isolates among fistula patients attending National Obstetric Fistula centre Ningi and investigate the antimicrobial susceptibility pattern. Methods: A total of 217 strains of significant bacteriuria were isolated from vesico vaginal fistula (VVF) patients. A cross sectional study was conducted at the hospital. The urine samples were collected and cultured on CLED and blood agar media while confirmation was done using their biochemical reaction. The detection of biofilms formation on the isolates was performed using tube adherence and Congo red agar method. Antimicrobial susceptibility testing was carried out by disc diffusion method on Muller Hinton agar. Results: Out of 217 significant bacteriuria isolated, 38 strains produced biofilms;28 strains tested positive on tube adherence method while 15 strains were positive on Congo red agar method. Bacteria that produced biofim showed multiple drug resistance compared to the platonic bacterial cells. All the biofilm producers showed 100% resistant to septrin, ampiclox, gentamycin and amoxicillin. There was no significant value between tube adherence and Congo red agar method with P value > 0.05. Conclusion: Biofilm detection should form part of routine testing while antimicrobial susceptibility testing is paramount on better choice of antibiotic therapy for proper management to reduce economic lost, treatment failure and drug resistance.

Adaptation of Congo Red Agar Method and Microtiter Plate Assay to Study Biofilm Formation in Streptomyces

Streptomyces has many advantages for exploration in biotechnological applications because of their ability to elaborate a multitude of bioactive molecules and secondary metabolites. Despite the importance of this genus in biotechnology, biofilm formation in Streptomyces is under-investigated. The objective of this research is to adapt two assays for the assessment of biofilm formation in Streptomyces. In the present investigation, we assess and follow biofilm formation in eight Streptomyces strains using quantitative and qualitative methods. The quantitative study based on a staining of the retained biomass in the microtiter plate with crystal violet “5%” and destaining using ethanol/acetone mixture, the concentration of crystal violet in the alcoholic solution reflect the intensity of the attached biofilm. On the other hand, the qualitative one consists of using modified freeman’s method a modified congo red agar method based on the color of colonies. Quantification of biomass by crystal violet staining method confirmed that Streptomyces bellus A43 and Streptomyces bellus A61 are biofilm-forming and this ability increase with the period of incubation. Our results showed that sixStreptomyces strains arenon-slime producing/non-biofilm forming. Two Streptomyces strains are slime producing/biofilm forming; this character vanishes at five days. Further research on genes responsible for biofilm formation in Streptomyces is highly recommended for better understanding of the phenomenon.

Characteristics of bacteriophages of the Staphylococcus aureus variant bovis

Bacteriophages may be an alternative method of treatment for antibiotic-resistant bacteria, including mastitis in cows. Our study describes the initial isolation and bacteriological activity of bacteriophages, circulating on dairy farms, the against S. aureus var. bovis. Samples of cow’s milk secretions with signs of mastitis and sewage water were used as the study material. The isolation and production of pure bacteriophage lines were performed according to the double agar method. The method of studying a single cycle of phage reproduction was used to determine the duration of the latency period. Determination of the spectrum of the lytic activity of bacteriophages against the clinical isolates of the microorganisms was carried out by the drop method. As a result of the research, four phages, specific for S. aureus var. bovis were isolated: Phage SAvB07, Phage SAvB08, Phage SAvB12 and Phage SAvB14. The negative colonies of the isolated phages were 1–2 mm in size, rounded with clear edges, with varying degrees of transparency. The latency period of Phage SAvB14 was 35 min, with the number of active virions increasing by 8 orders. In the study on growth curves of other bacteriophages, taken in the experiment, the latency period was more than 35 min, and their titre increased by only two orders. Phage SAvB07, Phage SAvB08 and Phage SAvB12 were able to lyse the bacterial strains of S. aureus var. bovis in 25–45.6% of the cases (low lytic activity), whereas Phage SAvB14 lysed 94.1% of S. aureus strains were isolated from the cows. Studies have shown that among the bacteriophages we have studied, Phage SAvB14 with a short latency period has the best lytic action on the culture S. aureus var. bovis. The resulting bacteriophage strain can be used to create a bacteriophage-based drug for the treatment of mastitis in cows.

Detection of Biofilm Formation by Different Bacterial Isolates of Contact Lens

Background: Biofilms are groups of microorganisms that collect to each other and with different surfaces by adherence mechanisms. These are formed of cells and extracellular matrix manufactured by these cells. There may be a great problem in some situations e.g. on medical implants and resistance against antibiotics. Objective: The objective of this study is to determine biofilm forming power of bacteria isolated from the conjunctiva, contact lens and the lens storage case by both phenotypic and genotypic detection methods. Methodology: Samples were taken from (36) persons in the period from January 2020 to June 2020 at Ophthalmology Department, Tanta University Hospitals, all the samples were transported to the Medical Microbiology & Immunology Department, Tanta University where bacterial strains were isolated. The biofilm formation phenotypic detection was performed by both tube method and Congo red agar method. The biofilm-forming genes of coagulase negative Staphylococcus (CoNS) and Staphylococcus aureus (ica A) and that of P. aeruginosa (psl A), were detected by PCR. Results: The (216) samples (swabs & discarded lenses) gave rise to a total number of (247) bacterial isolates. By using tube method; (52.3%) were moderately positive, (31.5%) strongly positive and (16.2%) negative for biofilm formation while after using the Congo red agar method; (35.3%) were moderately positive, (38.4%) strongly positive and (26.3%) negative for biofilm formation. Regarding the Staphylococcus aureus isolates, two (50%) of these were containing (icaA) gene. Regarding the (21) CoNS isolates, three (14.3%) contained (icaA) gene. Although all of the Pseudomonas isolates didn't contain pslA (1119 bp) gene, these were positive for biofilm production by phenotypic methods. Conclusion: The majority of the isolates had the capacity to form biofilms. Both tube and Congo red agar methods showed clear significant correlation and detected a high number of biofilm-producing strains. The absence of genes responsible for biofilm formation did not exclude the phenotypic biofilm production by these bacteria which is a common state.

Soft Liner with antimicrobial activity

Objective: To develop a material for denture relining and assess the microbiological and mechanical properties. The proposed liner was obtained through the incorporation of nanostructured silver vanadate (AgVO3) at 0, 1, 2.5, 5, and 10% polyethyl methacrylate (PEMA) containing plasticizer. Methods: Antimicrobial efficacy was evaluated by the Kirby Bauer agar method against Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans and Staphyloccocus aureus (n = 5); and mechanical properties were also assessed through roughness, such as Shore A hardness and tensile test. The results were analyzed by ANOVA and Tukey’s Multiple Comparison test (α = 0.05). Results: The material with AgVO3 at concentrations of 1% and 2.5% showed antimicrobial activity for E. faecalis, and 5% and 10% groups were effective for E. faecalis, P. aeruginosa and C. albicans. In the 5% group, hardness remained unchanged (p < 0.001). None of the tested concentrations significantly changed the roughness and the tensile strength (P > 0.05). Conclusion: Obtaining the material with antimicrobial potential promoted efficacy against E. faecalis, P. aeruginosa and C. albicans, kept the roughness property unchanged, did not change the adhesion property of the material to polymethyl methacrylate, and it maintained the hardness values compatible with resilient denture liners.

Antioxidant and Antimicrobial Screening of Endophytic Fungi Culture Filtrate from Purwoceng (Pimpinella alpina Molk) Leaf

This is a preliminary study to determine the bioactivity potential of purwoceng leaf endophytic fungal metabolites. Endophytic fungi were isolated from purwoceng leaf and their secondary metabolite from culture filtrate were subjected to identify the antimicrobial, antioxidant, and phytochemical screening. The antioxidant activity was screened by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH). The antimicrobial activity was screened using a good agar method toward Salmonella typhi, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, dan Candida albicans. This study obtained five distinctive endophytic fungi isolates named A, B, C, D, and E. The endophytic fungal culture filtrate of C has the most extensive antimicrobial activity with phytochemical screening showing alkaloids, saponins, and terpenoids. The antioxidant potential of all culture filtrates seemed low because the DPPH amount was interfered with by pigment compounds. Culture filtrate of fungi A showed the highest antioxidant activity and contained phenolic and alkaloid compounds.