Gene Polymerase Chain Reaction Research Articles

A pilot study: Examining cytoskeleton gene expression profiles in Pakistani children with autism spectrum disorder.

Finding effective pharmacological interventions to address the complex array of neurodevelopmental disorders is currently an urgent imperative within the scientific community as these conditions present significant challenges for patients and their families, often impacting cognitive, emotional, and social development. In this study, we aimed to explore non-invasive method to diagnose autism spectrum disorders (ASD) within Pakistan children population and to identify clinical drugs for its treatment. The current report outlines a comprehensive bidirectional investigation showcasing the successful utilization of saliva samples to quantify the expression patterns of profilins (PFN1, 2, and 3); and ERM (ezrin, radixin, and moesin) proteins; and additionally moesin pseudogene 1 and moesin pseudogene 1 antisense (MSNP1AS). Subsequently, these expression profiles were employed to forecast interactions between drugs and genes in children diagnosed with ASD. This study sought to delve into the intricate gene expression profiles using qualitative polymerase chain reaction of profilin isoforms (PFN1, 2, and 3) and ERM genes extracted from saliva samples obtained from children diagnosed with ASD. Through this analysis, we aimed to elucidate potential molecular mechanisms underlying ASD pathogenesis, shedding light on novel biomarkers and therapeutic targets for this complex neurological condition. (n = 22). Subsequently, we implemented a diagnostic model utilizing sparse partial least squares discriminant analysis (sPLS-DA) to predict drugs against our genes of interest. Furthermore, connectivity maps were developed to illustrate the predicted associations of 24 drugs with the genes expression. Our study results showed varied expression profile of cytoskeleton linked genes. Similarly, sPLS-DA model precisely predicted drug to genes response. Sixteen of the examined drugs had significant positive correlations with the expression of the targeted genes whereas eight of the predicted drugs had shown negative correlations. Here we report the role of cytoskeleton linked genes (PFN and ERM) in co-relation to ASD. Furthermore, variable yet significant quantitative expression of these genes successfully predicted drug-gene interactions as shown with the help of connectivity maps that can be used to support the clinical use of these drugs to treat individuals with ASD in future studies.

Direct 16S/ITS rRNA Gene PCR Followed by Sanger Sequencing for Detection of Mycetoma Causative Agents in Dakar, Senegal: A Pilot Study Among Patients with Mycetoma Attending Aristide Le Dantec University Hospital.

Mycetoma can be caused either by fungi or aerobic Actinomycetes. A precise identification of the causal agents is critical for the therapeutic outcome. Thus, this study aimed to identify the pathogens of mycetoma using 16S/ITS rRNA gene polymerase chain reaction (PCR) followed by Sanger sequencing directly on grains. In sum, 32 samples including 15 black grains, 12 red grains, and five white/yellow grains collected from patients with mycetoma at the Aristide Le Dantec University Hospital in Dakar, Senegal, between October 2014 and September 2020 were submitted to PCR/sequencing. For black grain eumycetoma, the ITS rRNA region was targeted. Similarly, the 16S rRNA gene was targeted for red grain actinomycetoma. These two regions were targeted in parallel for white/yellow grains, which could be of either bacterial or fungal origin. The age of the patients ranged from 14 to 72years with a mean age of 36 ± 14years. Thirteen (86%) of the 15 samples with black grains, were successfully sequenced with only one established eumycetoma pathogen, Madurella mycetomatis identified in 11 (73%). Cladosporium sphaerospermum was identified in one sample. For the 16S rRNA sequencing of red grains, a 58.3% (7/12) success rate was obtained with Actinomadura pelletieri identified in six samples. Among the five samples sequenced twice, the 16S rRNA allowed us to identify the causative agent in 2 cases, A. madurae in one, and A. geliboluensis in the other. The ITS rRNA identified 3 fungi, of which none was a mycetoma agent. Overall, direct 16S/ITS rRNA sequencing of the grains for detecting and identifying mycetoma pathogens was successful in 59.4% of cases. Fungi, led by M. mycetomatis, were the predominant pathogens identified. Two probable new mycetoma agents, C. sphaerospermum, and A. geliboluensis were identified and both deserve to be confirmed in further studies.

Выявление патогенов картофеля с использованием различных методов диагностики

Abstract. Potato diseases have far reaching implications for the agricultural industry as they not only reduce productivity and quality of potato crop but also negatively affect the livelihood of farmers. The purpose of the studies detection and identification of potato phytopathogens by visual assessment, real time polymerase chain reaction (PCR) and molecular genetic screening for phytopathogen resistance genes. Methods. Visual evaluation of each potato variety for major fungal, viral and bacterial diseases was carried out during the period of full sprouting, at plant height of 15–20 cm and in the phase of full flowering. The percentage of plants affected by diseases in relation to the total number of inspected plants was calculated. Diagnosis of resistance of potato samples to diseases was carried out on the basis of PCR. DNA markers of phytopathogen resistance genes YES3-3A, RYSC3, Ry186, TG 689, Gro1-4-1, Gpa2-2, PVX were used for molecular screening of potato. Infection of samples by PCR-RV (real-time) was determined using reagent kits of Syntol LLC Potato Virus X and Potato Virus Y, Potato spindle tuber viroid-PB, Clavibacter michiganensis subsp. sepedonicus-PB, Dickeya spp-PB. Results. According to the results of visual evaluation, the absence of symptoms of wrinkle mosaic in 20% of hybrids, mottle mosaic in 40%, mosaic leaf curl in 96 %, leaf curl in 72 hybrids, black leg in 100%, phytophthorosis in 88% of hybrids, alternaria in 84 %, rhizoctoniosis in 96 % of hybrids were observed. According to the results of PCR-RV analysis in 21 samples detected virus Y. The scientific novelty consists in the diagnosis of potato viruses using traditional visual assessment with the use of molecular markers that allow to identify correlation relationship between trhis methods.

Molecular characterization of DNAH6 and ATPase6 (Mitochondrial DNA) genes in asthenozoospermia patients in the northern region of India

BackgroundMale infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. The present study was undertaken to assess the association of DNAH6 and ATPase6 genes in asthenozoospermia patients in the northern region of India.MethodsA total of 60 semen samples were collected for the study, of which 30 were from the case group and 30 were from the control group. The semen samples for the case group (asthenozoospermia) and control groups were collected from IVF and Reproductive Biology Centre, Maulana Azad Medical College, New Delhi. Sperm count and motility were classified as per World Health Organization (WHO 2021) protocol. A total genomic DNA was extracted as per the stranded TRIZOL method with little modification.ResultsIn-vitro molecular characterizations of DNAH6 and ATPase6 genes in both groups were checked by Polymerase Chain Reaction (PCR). The 675 bp and 375 bp amplicons were amplified using PCR for ATPase6 and DNAH6 genes. Our study results showed a significant (P ≤ 0.05) null deletion of DNAH6 and ATPase6 genes in asthenozoospermia patients as compared to the control. We found the significant null deletion of DNAH6 in case 45.0%, and the control group was 11.7%. However, in the case of APTase6, it was 26.7% and 10.0%, respectively.ConclusionsOur study concluded that the presence of DHAH6 and ATPase6 genes had a significant impact on male infertility.

Microbiological culture versus 16S/18S rRNA gene PCR-sanger sequencing for infectious keratitis: a three-arm, diagnostic cross-sectional study.

To compare the diagnostic performance of microbiological culture and 16S/18S rRNA gene polymerase chain reaction (PCR)-Sanger sequencing for infectious keratitis (IK) and to analyse the effect of clinical disease severity on test performance and inter-test concordance. This was a three-arm, diagnostic cross-sectional study. We included all eligible patients who presented with presumed bacterial/fungal keratitis to the Queen's Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous culture (either direct or indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. The bacterial/fungal genus and species identified on culture were confirmed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Relevant clinical data were also collected to analyze for any potential clinico-microbiological correlation. Main outcome measures included the diagnostic yield, test accuracy (including sensitivity and specificity), and inter-test agreement [including percent agreement and Cohen's kappa (k)]. A total of 81 patients (86 episodes of IK) were included in this study. All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p = 0.13). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4-95.3%), followed by indirect culture (85.4%; 95% CI, 71.6-93.5%) and PCR (73.5%; 95% CI, 59.0-84.6%), with 100% specificity noted in all tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement = 81.8-86.2%, Cohen's k = 0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with greater infection severity. This study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower).

Wastewater surveillance for antibiotic resistance genes during the late 2020 SARS-CoV-2 peak in two different populations

ABSTRACT The United States Centers for Disease Control and Prevention reported a rise in resistant infections after the coronavirus disease 2019 (COVID-19) pandemic started. How and if the pandemic contributed to antibiotic resistance in the larger population is not well understood. Wastewater treatment plants are good locations for environmental surveillance because they can sample entire populations. This study aimed to validate methods used for COVID-19 wastewater surveillance for bacterial targets and to understand how rising COVID-19 cases from October 2020 to February 2021 in Portugal (PT) and King County, Washington contributed to antibiotic resistance genes in wastewater. Primary influent wastewater was collected from two treatment plants in King County and five treatment plants in PT, and hospital effluent was collected from three hospitals in PT. Genomic extracts were tested with the quantitative polymerase chain reaction for antibiotic resistance genes conferring resistance against antibiotics under threat. Random-effect models were fit for log-transformed gene abundances to assess temporal trends. All samples collected tested positive for multiple resistance genes. During the sampling period, mecA statistically significantly increased in King County and PT. No statistical evidence exists of correlation between samples collected in the same Portuguese metro area.

Agroclimatic zone-wise prevalence of biofilm forming methicillin resistant staphylococcus aureus in West Bengal

The present study envisages the prevalence of biofilm producing, methicillin resistant Staphylococcus aureus, isolated from cattle milk of different agro-climatic zones of an eastern state of India, West Bengal. The milk samples (n=168) were collected from three predominant agro- climatic zones of South Bengal, viz. new alluvial, coastal saline and red laterite. A total of 146 Staphylococcus aureus isolates were recovered, confirmed by biochemical tests and polymerase chain reaction (PCR). Through PCR of mecA gene, it was found that 45 strains were methicillin resistant, encompassing 28.57% new alluvial zone, 30.19% red laterite region and 29.16% coastal saline region. Determination of biofilm formation was done, both phenotypically by microtiter plate method and genotypically by PCR of icaA and icaD genes, specific for biofilm formation in Staphylococcus aureus. Phenotypic study revealed that 14 isolates were strong biofilm producers, 79 were moderate and the 47 were weak biofilm producers. PCR studies also showed that a total of 30 isolates were icaA positive and 25 isolates were icaD positive. Although no significant difference was found in the occurrence of icaA positive isolates, the possession of icaD by the Staphylococcus aureus isolates varied significantly according to different agro-climatic zones.

Identification of algD and oprL genes in Pseudomonas aeruginosa isolated from multiple environments and different local sources in Iraq

The aim of this study was to investigate the spread of the genes algD and oprL in P. aeruginosa within the genetic structure of environmental sources of isolates and various clinical studies, and to evaluate a successful alternative for diagnosing Pseudomonas aeruginosa (P. aeruginosa) among various sources. In this study, 100 samples were collected from various clinical and environmental sources. Twenty-eight isolates of P. aeruginosa bacteria were isolated. Nine pathological samples were taken from wounds, burns, blood, and ear infections in three governorates: Salah al-Din, Baghdad, and Kirkuk. Five isolates of P. aeruginosa bacteria were collected from contaminated water from the Salah Al-Din and Baghdad governorates. The isolates were diagnosed phenotypically through the colony's shape, colour, odour and interaction with Gram stain. Additionally, the VITEK 2 system was used to confirm the diagnosis of the isolates. The results show that all the isolates belonged to the bacteria P. aeruginosa. The results of the molecular diagnosis using a polymerase chain reaction (PCR) technique for three genes with specialised primers (16SrRNA, algD, and oprL) show that most of the bacterial isolates from different sources belong to the type P. aeruginosa because they contain the specific gene 16SrRNA except for five isolates (M5, M7, M8, M9 and W1). Four of these belong to isolates with a pathogenic source and one was isolated from water. The results of the PCR for the algD gene show that all isolates contain the gene except for four diseased samples. Samples M5, M7, M8, and M9 were without the gene, while all isolates had the oprL gene. A sequencing analysis of the DNA was then performed for the isolates that were not associated with the specialised primers of the above genes to ensure their genera (M5, M7, M8, and M9), in addition to a sample of 15 isolated from soil (S6) that was associated with all special primers for the same genes for the purpose of comparison. The results show that isolates M5, M7, M8 and M9 were diseased isolates belonging to Escherichia coli, while the 15 isolated from soil (S6) were diagnosed as P. aeruginosa.

Evaluation of Viral Subgenomic RNAs and Antigen Presence in SARS-CoV-2 PCR Positive Cases

Polymerase chain reaction (PCR) and antigen test (AgT) are frequently used in the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Routine PCR tests that detect the virus genome cannot determine whether the virus is infectious or not. However, detection of subgenomic RNA (sgRNA) produced during the replication period may indicate active viral infection. Active virus detection can offer various health and economic benefits from isolation time to treatment. Antigen tests are also considered as indicators of infectiousness since they can detect viruses above a certain load amount. The aim of this study was to use two different subgenomic RNAs and antigen test instead of genomic RNA to examine the relationship with each other and the clinic in terms of infectiousness. Evaluating the antigen test together with subgenomic RNA as an indicator of infectiousness may show the importance of this test. SARS-CoV-2 PCR positive 109 naso/oropharyngeal swab samples stored at -80 °C were included in the study. In order to confirm the PCR positivity of these samples, E gene PCR was performed and AgT, and E and N sgRNA quantitative real-time reverse transcription-PCR (RT-qPCR) detection was performed. Of the 109 SARSCoV-2 PCR positive samples, 83 (76.14%) had antigen test positivity, 88 (80.73%) had E gene sgRNA, 96 (88.07%) had N gene sgRNA and 97 (89%) had at least one sgRNA positivity.The antigen test was found positive in 77.3% of the samples in which at least one sgRNA was detected and in 66.7% of the negative samples and this difference was not statistically significant (p= 0.475). The difference between E sgRNA and AgT positivity was significant (p= 0.023). N sgRNA was positive in 98.9% of E sgRNA positive samples and 42.9% of the negative samples and this difference was statistically significant (p= 0.0001). The AgT positivity rate was found to be 98.15% (53/54) for cycle threshold (Ct) value ≤ 25, 57.14% (12/21) for Ct 25-30, and 52.94% (18/34) for Ct ≥ 30. The difference in antigen test positivity between E gRNA Ct value ≤ 25 and > 25, ≤ 29 and > 29, < 30 and ≥ 30 was statistically significant (p= 0.0001). Antigen test positivity appears to be associated with viral load and infectivity, as expected. In our study, it has been shown that sgRNAs and AgT which are indicators of infectiousness can be detected at least 10 days after the symptom period. Using these two tests together could detect infective individuals with higher accuracy and shorten the duration of hospital stay and isolation.

An osteoimmunomodulatory Ca2+/Zn2+-doped scaffold promotes M2 macrophage polarization via the src-mediated chemoking signaling pathway to enhance osteoinduction

Bone defects disrupt physiological functions of various cells including immune cells, osteoblasts, and osteoclasts. Restoring the coordinative effects of cells between osteogenesis and the immune microenvironment is essential and challenging for bone regeneration. Here, we developed an osteoimmunomodulatory Ca2+/Zn2+ doped polycaprolactone electrospinning scaffold to enhance osteoinduction by modulating the polarization of macrophages. To endow nucleation sites for Ca2+ deposition, epitaxial crystallization was conducted to yield the conformal nano-lamellae on the scaffold surface. The Zn2+ was then embedded into the mineralized scaffold via an ion exchange method. Such an approach remarkably improved the deposition amount of Ca2+ and Zn2+, showing a highly sustained iron release. This hybrid scaffold was capable to regulate the M2-type polarization of macrophages, forming an immune-osteogenic microenvironment. As a result, the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) was enhanced via activating the Src-mediated chemoking signaling pathway as verified by gene sequencing, western blotting, and polymerase chain reaction results. In vivo evaluation further demonstrated that the hybrid scaffold remarkably promoted the bone regeneration in a critical-sized rat cranial defect. Compared to the control group, the bone volume/total volume (BV/TV) of the hybrid scaffold group was increased by 2.6 times. This work provides a feasible strategy for fabricating bone repair substitutes that regulate the bone immune microenvironment and coordinate multicellular functions.

Coexistence of β-lactamase genes and biofilm forming potential among carbapenem-resistant Acinetobacter baumannii in Lahore, Pakistan.

Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced β-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM-1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE.

Detection fimH Gene in The Urine of UTI Patients at The Puskesmas Banyuanyar, as a Marker for The Presence of Escherichia coli

Urinary Tract Infection (UTI) is a bacterial infection characterized by a urine culture showing bacteriuria with bacterial counts exceeding 100,000 per milliliter of urine. Polymerase Chain Reaction (PCR) is a method that utilizes in-vitro enzymatic reactions to amplify specific DNA targets. This process synthesizes new DNA strands complementary to the target sequences using enzymes and oligonucleotide primers within a thermocycler. The fimH gene is among the most important of the UPEC strains and is associated with severe urinary tract infections. The aim of this study was to identify the presence of the fimH gene in urine samples from UTI patients at the Puskesmas Banyuanyar in Surakarta using Polymerase Chain Reaction (PCR). The research employed the Quota Sampling Technique, collecting a total of five urine samples from individuals diagnosed with urinary tract infections. The results of the control isolates using pure Escherichia coli isolates showed that the fimH gene PCR amplified control with annealing temperature of 52°C. The analysis of clinical isolates of Escherichia coli from the urine of urinary tract infection patients revealed that the fimH gene was PCR amplified in all five samples.

Impact of the Reverse 2-Step Algorithm for Clostridioides difficile Testing in the Microbiology Laboratory on Hospitalized Patients.

Multistep laboratory testing is recommended for the diagnosis of Clostridioides difficile infection (CDI). The aim of this study was to present the impact of multistep CDI diagnostic testing in an academic hospital system and evaluate the toxin B gene polymerase chain reaction (PCR) cycle threshold (Ct) values of PCR-positive tests. In October 2022, our system began reflex testing all PCR-positive stool samples with the C. DIFF QUIK CHEK COMPLETE (Techlab), an enzyme immunoassay-based test with results for the glutamate dehydrogenase antigen (GDH) and C difficile toxin A/B. Hospital-onset (HO) CDI and CDI antibiotic use before and after testing were tracked. Ct values were obtained from the Infectious Diseases Diagnostic Laboratory. Receiver operating curve analysis was used to examine the sensitivity and specificity for identifying GDH+/toxin+ and GDH-/toxin- at various Ct thresholds. The HO-CDI rate decreased from 0.352 cases per 1000 patient-days to 0.115 cases per 1000 patient-days post-reflex testing (P < .005). Anti-CDI antibiotics use decreased, but the decrease was not commensurate with CDI rates following reflex testing. PCR+/GDH+/toxin+ samples had a lower mean Ct value than PCR+/GDH-/toxin- samples (23.3 vs 33.5, P < .0001). A Ct value of 28.65 could distinguish between those 2 groups. Fifty-four percent of PCR+/GDH+/toxin- samples had a Ct value below that cut-off, suggesting the possibility of CDI with a negative toxin test. Reflex testing for a laboratory diagnosis of CDI results in rapid, systemwide decreases in the rate of HO-CDI. Additional research is needed to distinguish CDI from C difficile colonization in patients with discordant testing.

Impact of electrolyzed water treatment on bacterial communities in food washing processes

Electrolyzed water is expected to have a sterilizing effect on bacteria and viruses, which makes it useful for hygiene management, such as cleaning dishes or medical equipment. Electrolyzed water treatment may affect the cellular structure of microbes, leading to DNA leakage or degradation. Here, we conducted a next-generation sequencing (NGS) analysis of bacterial diversity in washing solution to elucidate the impact of cleaning food surfaces with electrolyzed water. Modified primers were used to evaluate the differences between the existing primers used to obtain NGS sequences and through statistical analysis. The NGS reads obtained using the modified primers tended to reveal more Enterobacteriaceae sequences, and fewer mitochondrial sequences than did those obtained with conventional primers. An UniFrac distance analysis and Nonmetric multidimensional scaling (NMDS) revealed that the results obtained with the modified primer set made the differences between samples more distinct. Real-time polymerase chain reaction (PCR) using the template DNA obtained from an electrolyzed water or sodium hypochlorite solution wash revealed that some of the DNA could not be amplified using PCR of the 16S rRNA genes suggesting that the DNA fragmentation had occurred due to the electrolyzed water treatment. Our results revealed that the bacterial species removed from the food surfaces varied depending on the washing treatment. Electrolyzed water treatment may be advantageous for removing Enterobacteriaceae from leafy vegetables.

Tuberculosis of the thyroid gland: A rare case report

Abstract Tuberculosis (TB) of the thyroid gland is exceedingly rare, even in regions like India, where tuberculosis is prevalent, with an estimated incidence of 0.1–0.4%. While tuberculosis can affect various organs, its involvement in the thyroid, pancreas, striated, and cardiac muscle is uncommon. Diagnosis poses a challenge due to the lack of distinctive clinical features—microscopic examination revealing epithelioid granulomas, sometimes with caseous necrosis and Langhans-type giant cells, aids in diagnosis. Confirmation typically involves detecting acid-fast bacilli through staining techniques like Ziehl Neelsen (ZN), molecular diagnostic methods such as TB Gene Expert or polymerase chain reaction for Mycobacterium tuberculosis (TB PCR), and histopathological examination of excised specimens. Recently, we encountered a case involving a 21-year-old male presenting with a 2-month history of diffuse neck swelling. Clinical examination suggested thyroid swelling, confirmed by ultrasound, showing a cystic lesion with necrotic areas, suggestive of thyroiditis, accompanied by bilateral cervical lymphadenopathy. Fine Needle Aspiration Cytology revealed necrotizing granulomatous thyroiditis, prompting further investigation for tuberculosis. TB Gene Expert and ZN staining performed on fine needle aspiration cytology (FNAC) material confirmed tuberculosis, leading to anti-tuberculous treatment, which resulted in a positive response. In presenting this case, we aim to highlight the rarity of tuberculosis affecting the thyroid gland and emphasize the importance of considering minimally invasive diagnostic methods, such as FNAC and molecular techniques, before resorting to surgical interventions. This case underscores the significance of clinicians’ awareness regarding unusual tuberculosis presentations and the availability of advanced diagnostic tools to ensure timely and appropriate management.

A study to assess the prevalence and risk factors for Clostridioides difficile infection in patients with inflammatory bowel disease in a tertiary care hospital in Northern India

Objectives: The prevalence of Clostridioides difficile infection (CDI) is on rise among patients with inflammatory bowel disease (IBD). This study sought to describe the prevalence and risk factors of CDI in patients with IBD as compared to non-IBD controls. Materials and Methods: This was a prospective study conducted at a Department of Microbiology in collaboration with a Department of Gastroenterology. The patients with IBD and controls without IBD presenting with diarrhea were included in the study. The screening test for C. difficile infection was done by glutamate dehydrogenase (GDH) assay and toxin detection by enzyme-linked immunoassay (ELISA). Anaerobic culture for C. difficile was done on a selective cycloserine cefoxitin fructose agar and polymerase chain reaction (PCR) was done for Toxin A (TcdA) and Toxin B (TcdB) gene detection. C. difficile infection was confirmed if GDH and toxin ELISA or PCR were positive. Statistical Analysis: Data were analyzed with the Statistical Package for the Social Sciences version 20.0.The numerical variables were presented by means and standard deviations. Comparison of continuous variables was done using Student’s t-test. Categorical variables were analyzed by Chi square test. P&lt;0.05 was considered to be statistically significant. Results: A total of 160 cases and 112 age- and gender-matched control were included in IBD group and nonIBD group, respectively. Only one culture was positive, 12 and six were positive for GDH ELISA and TcdA and TcdB ELISA, respectively, and 7 were positive by PCR for toxin genes. The factors found significantly associated with CDI were proton-pump inhibitors use (P = 0.001), levofloxacin (P =0.001), and azathioprine (P =0.042). Using PCR as a reference method for C. difficile toxin detection, the sensitivity, and specificity of GDH ELISA and ELISA for toxins were 100%, 96.8% and 85.7%, and 100%, respectively. Conclusions: The prevalence of CDI among patients with IBD has been found to be low, that is (only 4.4%) in this study population.

Molecular Detection of Toxoplasma gondii Oocytes in Soil Samples from Guilan Province, Northern Iran.

Soil is an appropriate substrate for the storage and transmission of oocytes of Toxoplasma gondii. Ingestion of soil contaminated with T. gondii oocysts is a major transmission route of human and animal toxoplasmosis. The present study was carried out to investigate soil contamination with T. gondii oocysts in urban and rural areas of Guilan Province, northern Iran. Overall, 208 soil samples were collected from 16 cities and villages in Guilan Province, northern Iran from Oct 2020 to Nov 2021. Soil samples were investigated using modified sucrose flotation technique. Real-time polymerase chain reaction was used to detect presence of T. gondii DNAs in the samples. Positive samples were further analyzed using nested polymerase chain reaction for GRA6 gene. Moreover, six selected positive samples were used for amplifying and sequencing of the GRA6 gene. Overall, 31 samples were positive for T. gondii with frequency of 14.9% and ranging from 10.9% in rural areas to 16.3% in urban areas. Statistical analysis showed significant differences between the seasons (P=0.003). The phylogenetic analysis illustrated that our six sequences were similar and closely related to Type I strain of T. gondii. Results showed relatively high levels (14.9%) of T. gondii oocytes in soil samples of Guilan Province, northern Iran, which provided essential data for the effective prevention and control of toxoplasmosis in the region.

Human buccal epithelial cells as a model system for molecular analysis of DNA, RNA and protein

We report use of human buccal epithelial cells as an easy model system for isolation and molecular analysis of genomic DNA, RNA, and protein to study any gene of interest by Polymerase Chain Reaction (PCR), RNA expression by Reverse Transcription-PCR (RT-PCR), protein-profiling, and expression by western blot as well as DNA-methylation by Msp I/Hpa II-restriction digestion. We used simple methods to isolate genomic DNA, RNA and protein from human buccal cells collected by oral swab and cultured them in-vitro. The microscopic observation of haematoxylin and eosin (EA-50) stained cells, genomic PCR of house-keeping genes (GAPDH and β-actin), RT-PCR of GAPDH and β-actin mRNAs and whole cell protein-profiling by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) were carried out. Expression of β-actin protein in supernatant and pellet fractions of the cells was determined by western blot analysis. MTT-assay was carried out to assess the cell viability and cell growth. Green Fluorescence Protein (GFP)-DNA was expressed in these cells by transient transfection. DNA-methylation in the genome was analyzed by Msp I/ Hpa II restriction digestion and agarose gel electrophoresis. Thus these methods can be used for molecular analysis of DNA, RNA and protein from the human buccal epithelial cells for studying and monitoring health, disease, population genetics/genomics and epidemiology under different conditions.

Escherichia coli and their potential transmission of carbapenem and colistin-resistant genes in camels

BackgroundCamels harbouring multidrug-resistant Gram-negative bacteria are capable of transmitting various microorganisms to humans. This study aimed to determine the distribution of anti-microbial resistance among Escherichia coli (E. coli) isolated from the feces of apparently healthy camels in Egyptian abattoirs. Additionally, we sought to characterize Shiga toxin-producing E. coli (STEC) strains, assess their virulence potential, and investigate the possibility of camels spreading carbapenem- and colistin-resistant E. coli.Methods121 fecal swaps were collected from camels in different abattoirs in Egypt. Isolation and identification of E. coli were performed using conventional culture techniques and biochemical identification. All isolates obtained from the examined samples underwent genotyping through polymerase chain reaction (PCR) of the Shiga toxin-encoding genes (Stx1 and Stx2), the carbapenemase-encoding genes (blaKPC, blaOXA−48, blaNDM, and blaVIM), and the mcr genes for mcr-1 to mcr-5.ResultBacteriological examination revealed 75 E. coli isolates. PCR results revealed that one strain (1.3%) tested positive for Stx1, and five (6.6%) were positive for Stx2. Among the total 75 strains of E. coli, the overall prevalence of carbapenemase-producing E. coli was 27, with 7 carrying blaOXA48, 14 carrying blaNDM, and 6 carrying blaVIM. Notably, no strains were positive for blaKPC but a high prevalence rate of mcr genes were detected. mcr-1, mcr-2, mcr-3, and mcr-4 genes were detected among 3, 2, 21, and 3 strains, respectively.ConclusionThe results indicate that camels in Egypt may be a primary source of anti-microbial resistance (AMR) E. coli, which could potentially be transmitted directly to humans or through the food chain.

Study of bacterial population dynamics in seed culture developed for ammonia reduction from synthetic wastewater.

The waterbodies have been polluted by various natural and anthropogenic activities. The aquatic waste includes ammonia as one of the most toxic pollutants. Several biological treatment systems involving anoxic and semi anoxic bacteria have been proposed for reducing nitrogen loads from wastewater and increasing the efficiency and cost effectiveness. These bacteria play a vital role in the processes involved in the nitrogen cycle in nature. However, the enrichment, sustainability and identification of bacterial communities for wastewater treatment is an important aspect. Most of the chemolithotrophs are unculturable hence their identification and measurement of abundance remains a challenging task. In this study the different bacteria involved in total nitrogen removal from the wastewater are enriched for 700days under anoxic condition. The synthetic wastewater containing 0.382g/L of ammonium chloride was used. Molecular identification of the bacteria involved in various steps of the nitrogen cycle was carried out based on amplification of functional genes and 16S rRNA gene Polymerase chain reaction followed by DNA sequencing. Change in the abundance of chemolithotrophs was studied using qPCR. The mutual growth of various nitrifiers along with anaerobic bacteria were identified by molecular characterisation of DNA at various time intervals with the different genes involved in the nitrogen cycle. Nitrosomonas species like Nitrosomonas europaea were identified throughout the batch scale studies possessing the genes associated with ammonia oxidizing bacteria and nitrite oxidizing bacteria which act as a complete ammonia oxidizer. The uncultured species of Nitrospira and anammox bacteria were also observed which predicts the coexistence of the anammox and comammox bacteria in a batch scale study. The coexistence of the semi anoxic and anoxic bacteria helped in the growth of these bacteria for a longer duration of time. The nitrite produced by the comammox during nitrification can be utilized by anammox as an electron carrier. The other species of denitrifiers like Pseudomonas denitrificans and Aminobacter aminovorans were also observed. It is concluded that the enrichment of semi anoxic and anoxic bacteria was faster with the increase in growth of the bacteria involved in nitrification, comammox, anammox and partial denitrification process. The bacterial growth is enhanced and the efficiency is increased which can be further used in the development of small pilot scale bioreactor for total nitrogen removal.