Use Of Polymerase Chain Reaction Research Articles

Isolation and identification of enteric bacteria from different water sources at Mymensingh city, Bangladesh and their antibiogram study

The goal of the current study was to separate and characterize the enteric bacteria found in water samples taken from various Mymensingh municipal sources and places. A total of 40 water samples were examined. Through the use of polymerase chain reaction (PCR), biochemical testing, staining, and culturing enteric bacteria were isolated and identified. The antibiotic resistance phenotype was examined using the disk diffusion assay. Out of 40 isolates that tested positive for bacterial growth, 3 (7.5%) were identified as Salmonella spp., 6 (15%) as Shigella spp., 14 (35%) as E. coli, and 3 (7.5%) as Vibrio spp. Molecular detection of Salmonella spp. were confirmed by PCR-based detection of the bcfC gene, Shigella species by the invC gene, E. coli by the 16S rRNA gene, and Vibrio spp. by the groEL gene. Furthermore, PCR-confirmed isolates were tested for antibiotic resistance to 12 routinely used antibiotics. All Salmonella isolates were 100% resistant to Amoxicillin but 100% sensitive to Azithromycin. However, varied sensitivity was seen against Cefepime (80%), Gentamycin (80%), Levofloxacin (75%), Cotrimoxazole (74%), and Amikacin (70%). The isolates also showed resistance to Ceftazidime (70%), as well as Ceftriaxone (33%). Shigella spp. isolates showed increased resistance to Cefepime (83%) and Ceftazidime (67%). Additionally, resistance to Amoxicillin (17%) and Colistin (10%) was shown to be lower. In addition, Azithromycin, Levofloxacin, Cotrimoxazole, Ceftriaxone, Amikacin, and Gentamicin showed nearly no resistance or significant susceptibility E. coli isolates showed stronger resistance patterns to amoxicillin (100%), and colistin (93%). Cefepime (72%), Cotrimoxazole (72%), and Azithromycin (72%). The lower levels of resistance to ceftriaxone, amikacin, ceftazidime, levofloxacin, and gentamicin were also seen. The higher resistance patterns were shown by the isolated Vibrio spp. against Ampicillin (100%), Cefepime (81%), Cefixime (72%), Amoxicillin (70%), Erythromycin (67%), and Ceftazidime (67%). On the other hand, isolates with high and moderate sensitivity were found to be gentamicin, levofloxacin, doxycycline, cotrimoxazole, and amikacin. The study’s conclusions point to recent and ongoing contamination of the water sources, putting residents who depend on the water for drinking at risk for gastrointestinal illnesses.

Easy extraction of Ganoderma boninense liquid sample using portable on‐chip device

Detecting Ganoderma boninense in Indonesia is crucial for effectively controlling and mitigating the spread of basal stem disease in oil palm fields. While there is ongoing development of tolerant plants, no such plant has been successfully created yet. Consequently, researchers are actively studying detection methods for Ganoderma boninense. One established and highly accurate approach is the use of polymerase chain reaction (PCR) techniques for molecular detection. However, this method requires time‐consuming sample preparation, which can pose challenges in plantation settings. To address this problem, a portable lab‐on‐chip device has been introduced. This technology enables easy and automatic DNA retrieval from liquid samples by absorbing lysed DNA using magnetic beads. An efficient mechanism for manipulating the magnetic bead within the semiconductor has been successfully implemented. The extraction process typically takes around 15 minutes using a modified methodology on the chip device approach. The chip facilitates the retrieval of two samples with a capacity of 120 µL for each sample. The PCR method was utilized to validate the equivalence of the lab‐on‐chip device extraction to the standard extraction method. This represents a promising alternative for expedited and simplified detection of Ganoderma boninense in field conditions.

Genetic Insights: Examining CNTNAP2 rs7794745 Gene Polymorphism and Its Impact on ABLLS Assessment in Middle Euphrates Children in Iraq with Autism Spectrum Disorder

Objective: examine the effect of CNTNAP2 gene polymorphism on behavioral therapy response in Iraqi children with autism spectrum disorder using ABLLS-r.
 Methods: In this study, 150 samples for autism spectrum disorder were obtained from AL-SIBTEIN ACADEMY, however owing to many challenges, the final number of samples was 50 patients. Polymerase Chain Reaction-Restriction refers to a technique that combines the use of polymerase chain reaction (PCR) with restriction enzymes to amplify and analyze certain DNA sequences. Fragment Length Polymorphism (PCR-RFLP) was used to genotype the CNTNAP2 gene at rs7794745. All 25 ABLLS-R repertoires and all four primary components had their dependent parameters determined by aggregating and converting the ABLLS data to a percentage. This provided us with the proportion of finished assignments from each repertoire.
 Results: After correlating the genotype data (AA/AT/TT) with the behavioral therapy response as indicated by ABLLS-R, we only discovered a statistically significant difference between the frequency of AA/TT genotypes in the rs7794745 variant in patients and the social interaction domain. Conversely, the remaining 24 subdomains exhibited no discernible correlation.
 Conclusion: This research topic could provide significant information on targeting and categorizing which skill areas need to be developed based on genetic models' influence.

Pediatric intracranial empyema complicating otogenic and sinogenic infection

ObjectiveTo describe and compare clinical and microbiological features, surgical and medical management, and outcomes of children with otogenic and sinogenic intracranial empyema (IE) in an institution with an established multidisciplinary protocol. To use the study findings to inform and update the institutional algorithm. MethodsRetrospective analysis was carried out on the electronic healthcare records of all children with oto-sinogenic IE admitted in a 5-year period. ResultsA total of 76 patients were identified and treated according to an institutional protocol. Two distinct groups were identified: intracranial empyema related to otogenic infection (OI-IE, n = 36) or sinogenic infection (SI-IE, n = 40). SI-IE was seen in older children and had a significantly higher morbidity. Sub-dural IE was seen in a minority (n = 16) and only in SI-IE and required urgent collaborative ENT-neurosurgery. Extra-dural IE occurred more frequently and was seen in both SI-IE and OI-IE. No death and overall low morbidity were observed. Particularities found in SI-IE and OI-IE groups (as thrombosis, microbiology, antibiotic treatment, duration and outcome) permitted the delineation of these groups in our updated algorithm. ConclusionThe presence of a collaborative multidisciplinary protocol permits the step-wise co-ordination of care for these complex patients in our institution. All patients received prompt imaging, urgent surgical intervention, and antibiotic treatment. Microbiological identification was possible for each patient and antibiotic rationalization was permitted through use of Polymerase chain reaction (PCR) testing in cases of sterile cultures. Of note, intracranial empyema related to sinogenic infection is shown to have significantly more severe clinical presentation, a higher morbidity, and a longer duration of antibiotic therapy than that related to otogenic infection. Study findings allowed for the update and clarification of the institutional protocol, which now clearly demarcates the clinical presentation, biological evidence, radiology, surgical and medical treatments in children with oto-sinogenic IE.

A Paradigm Shift in Non-Small-Cell Lung Cancer (NSCLC) Diagnostics: From Single Gene Tests to Comprehensive Genomic Profiling.

Lung cancer imposes a burden on the health care system worldwide affecting 2 million people and causing 1.8 million deaths in 2021.More than 85% of all lung cancer cases are reported under Non-small-cell lung cancer (NSCLC). It is critical to discover gene alterations to treat non-small cell lung cancer successfully. The CAP/IASLC/AMP recommendations supported use of polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) EGFR (epidermal growth factor receptor) mutations and ALK (Anaplastic lymphoma kinase) rearrangements, respectively. A study presented in the annual meeting of the American Society of Clinical Oncology (ASCO) in Chicago emphasized the need for comprehensive genomic profiling (CGP) before single gene tests (SGTs) since it demonstrated that SGT can result in the depletion of precious biopsy samples. As a result, the efficacy of thorough genetic Profiling (CGP) is reduced, preventing patients from receiving valuable genetic information about their tumors.

Using a combination of phospholipid fatty acids profiles and DNA-based sequencing analyses to detect shifts in the biofloc microbial community in different carbon sources and carbon/nitrogen ratios.

A 35-day study investigated the impact of carbon sources and carbon/nitrogen (C/N) ratios on the microbial community of biofloc. For this purpose, we utilized a combination of phospho-lipid fatty acids (PLFAs) profiles and DNA-based sequencing methods to investigate changes in the microbial community composition and structure. The experiment involved three carbon sources including Dextrin (DEX), corn starch (CS) and wheat bran (WB) at two C/N ratios (19 and 30). The results indicated that WB and CS were found to decrease nitrogen metabolite concentration while increasing total suspended solids and bacterial density compared to DEX. The treatments exhibited variations in microbial communities and the use of polymerase chain reaction/ denaturing gradient gel electrophoresis analysis revealed distinct dominant bacterial species linked to carbon sources and C/N ratios. Furthermore, the highest levels of bacteria and protozoa PLFAs biomarkers were observed in the C/N30 ratio and WB treatment while the ratio for poly-β-hydroxybutyrate/PLFAs and fungi biomarkers displayed a decrease. Also, by incorporating the results of PLFAs profile and conducting a principal component analysis, the treatments were categorized into distinct groups based on both the carbon source and C/N ratios. Overall, both methods yield consistent results. PLFAs offered additional insights into the microbial composition beyond bacterial structure while DNA-based analysis provided finer taxonomic resolution.

Role of Nanotechnology in Forensic Medicine to Solve Medico-legal Case Work in Pakistan - A Review Study

Background: Nanotechnology is relatively a new emerging field of Forensic Science and is being used to solve many medico-legal cases all over the world. Some of its use is the most prevalent in biomedical science, material science, biological indicators, food science, Trace Evidence, real-time crime scenes, and terrorist attacks as a nanomaterial to the medico-legal investigations. It is now utilized in the medico-legal casework in Pakistan in Forensic Medicine. Aim: To analyze and compare the forensic value of nanotechnology and methods used to solve the medico-legal investigations relating to nanomaterials in Forensic medicine in Pakistan, Pakistan. Method: This is a review study focusing on the currently available data in research articles all over the world and in Pakistan about the use of nano-material in medico-legal casework for slaving medico-legal investigations in Pakistan. Extensive research is being conducted on the use of nanomaterials. Various analysis methods for nanomaterials, both old and new, have been gathered from previous research and compared. The statistical significance of their use as forensic evidence in criminal investigations and medico-legal cases has been analyzed in the field of Forensic Medicine. Results: The field of nanotechnology plays a crucial role in forensic investigations. This is supported by numerous publications both globally and in Pakistan. Nanomaterials have been instrumental in solving many forensic cases by leveraging advanced technology and high-tech tools. The Pakistan Forensic Science Agency in Lahore (PFSA) has effectively utilized nanomaterials in DNA analysis and various serological tests. Notably, nanomaterials have greatly aided in identifying crime scene evidence through the use of Polymerase Chain Reactions (PCR). Practical Implication: New nanotechnologies are being utilized in DNA fingerprinting to solve medico-legal cases in forensic investigations. Advancements include the use of capillary electrophoresis, nano-gold, nano-silver, and nanoparticles of titanium dioxide (TiO2) combined with SEM, TEM, and FTIR(56). Currently, there are ongoing developments to create new nano-probes, nano-devices, and nano-chips to combat the threat of terrorism in Pakistan. Conclusion: Nanotechnology is being very extensively used in crime-scene investigations to solve medico-legal cases in Forensic Medicine, especially in various ways and techniques in fingerprinting, DNA analysis, explosive detection, drug detection, and time since death estimation. Examination of body fluids, document analysis, explosive residual inspection, and nano-biosensors are benefited by nanotechnology. It is concluded by the review research on the use of nanotechnology that it is a more accurate, fast, reliable, secure system and an Armour for medico-legal investigations in Forensic Medicine. Keywords: Nanotechnology , DNA analysis, Forensic value, Forensic Medicine ,Nanoparticles

The association of rs4646994 ACE and rs1799983 eNOS gene polymorphisms with metabolic effects of dapagliflozin in patients with diabetic nephropathy and essential hypertension

Objective — to establish possible associations of rs4646994 ACE and rs1799983 eNOS gene polymorphisms with metabolic effects of dapagliflozin in patients (pts) with diabetic nephropathy (DN) and essential hypertension (EH).
 Materials and methods. Clinical investigation and examinations involved 171 pts, from them 85 (49.7%) females and 86 (50.3%) males with DN of I—IV stages and EH of II—III stages, aged 31 to 82 years old (an average age was 59.38±1.58 years). They were also genotyped for rs4646994 (I/D) ACE and for rs1799983 (G894T) eNOS gene polymorphisms with the use of polymerase chain reaction. Among the patients with I/D polymorphism of ACE gene there were 24 (14.0%) pts with genotype II, 32 (18.7%) pts with genotype ID and 26 (15.2%) subjects with genotype DD. Among the participants with G894T eNOS gene polymorphism GG genotype was revealed in 29 (16.9%) pts, GT genotype was found in 33 (19.3%) cases and 27 (15.8%) examined persons had TT genotype. Standard parameters of lipid profile were measured in all included pts with a use of an immune enzyme method. Glucose oxidase and immune enzyme methods were used to define serum glucose and insulin levels; level of glycosylated hemoglobin (HbA1C) was detected by fluid chromatography method. Insulin resistance (IR) indices such as HOMA‑IR, triglyceride‑glucose index (TGGI), METS‑IR (metabolic score for insulin resistance) index were calculated by known formulas. Cardiometabolic risk (CMR) of the pts was estimated by ­METS‑IR index. Body mass index (BMI), body fat percentage (BFP), total fat mass (TFM) and fat mass index (FMI) in all the pts were calculated by standard known formulas. Serum uric acid concentration was measured with phosphovolframic method. Dapagliflozin 10 mg/daily was added for 24 weeks to the basic standard antidiabetic therapy including gliclazide or glimepiride and metformin combined with lipid lowering therapy containing atorvastatin. The standard therapy was not changed during the whole period of observation.
 Results. No association has been revealed between I/D ACE and G894T eNOS gene polymorphisms in pts with DN and EH, with a noted improvement of lipid blood spectrum due to administration of dapagliflozin in a dose of 10 mg/daily for 24 weeks. It has been established that allele D was associated with a reduction of dapagliflozin efficacy in the correction of carbohydrate metabolism disorders in pts with DN and EH. At presence of allele D (ID and DD) in genotype, the levels of glycemia, insulinemia, HbA1C, IR indices such as HOMA‑IR, TGGI, METS‑IR decreased less effective than in the case of allele D absence (genotype II). In pts with DN and EH who had G894T eNOS gene polymorphism, dapagliflozin 10 mg/daily improved muscle sensitivity to insulin and decreased CMR depended on genotype in the following order: GT >TT >GG, whereas the intensity of glycemia, insulinemia, HbA1C and HOMA‑IR index reduction was found to be associated with genotype as follows: GG >GT >TT. Allele I compared with allele D of I/D ACE gene polymorphism in pts with DN and EH was revealed to be associated with more significant reduction of BFP, TFM and FMI due to dapagliflozin administration. In pts with DN and EH who had G894T eNOS gene polymorphism dapagliflozin 10 mg/daily reduced a body mass due to its influence on fat accumulation but not on muscle tissue both in phenotype of visceral obesity (genotype GG) and in phenotype of sarcopenic obesity (genotype GT). In the realization of hypouricemic effects of dapagliflozin in patients with DN and EH, both genetic factors (D and T alleles of I/D ACE and G894T eNOS gene polymorphisms correspondingly), and metabolic factors (initial levels of HbA1C and UA together with anthropometric parameters characterized visceral obesity phenotype such as BMI, TFM and FMI) were involved.
 Conclusions. In pts with DN and EH, the association of I/D ACE (rs4646994) and G894T eNOS (rs1799983) gene polymorphisms with metabolic effects of dapagliflozin 10 mg/daily has been established. A presence of allele D of I/D ACE and allele T of G894T eNOS gene polymorphisms in genotype was concluded to be associated with less significant metabolic effects of dapagliflozin compared with an absence of these alleles in genotype.

Source tracking of fecal contamination in Asian green mussels (Perna viridis) harvested in Manila Bay, Philippines by molecular detection and genotyping of Cryptosporidium spp.

Manila Bay, a multipurpose body of water located around Metro Manila, Philippines, is progressively deteriorating because of massive pollution. Reports have shown that the bay and its aquatic resources (i.e., seafood) are contaminated with fecal matter and enteric pathogens, posing a threat to public health and industry. This problem raises the need for a microbial source tracking methodology as a part of the rehabilitation efforts in the bay. Bivalve mollusks cultivated in water can serve as sentinel species to detect fecal pollution and can complement water monitoring. With the use of polymerase chain reaction and DNA sequence analysis, this study detected Cryptosporidium spp. in Asian green mussels (Perna viridis) cultivated and harvested in Manila Bay and sold in Bulungan Seafood Market, Parañaque, Philippines, from 2019 to 2021 with an overall occurrence of 8.77% (n = 57). The analysis of the 18S rDNA segment revealed three genotypes from Cryptosporidium-positive samples, namely, Cryptosporidium sp. rat genotype IV (60%), C. galli (20%), and C. meleagridis (20%). These findings suggest fecal pollution in bivalve cultivation sites coming from sewage, nonpoint, and agricultural sources. The presence of C. meleagridis, the third most common cause of human cryptosporidiosis, in mussels poses a threat to human health. Thus, there is a need to establish routine detection and source tracking of Cryptosporidium spp. in Manila Bay and to educate seafood consumers on food safety.

The association of rs1799983 eNOS gene polymorphism with dyslipidemia and efficacy of atorvastatin in correction of lipid exchange disorders in patients with diabetic nephropathy and essential hypertension

Objective — to search an association of rs1799983 endothelial nitric oxide synthase (eNOS) gene polymorphism with dyslipidemia (DLP) and the efficacy of atorvastatin used in correction of lipid exchange disorders in patients with diabetic nephropathy (DN) and essential hypertension (EH).
 Materials and methods. The study had been conducted in the clinical Department of Arterial Hypertension and Kidney Disease at L. T. Mala National Therapy Institute of NAMS of Ukraine (Kharkiv). Clinical examinations involved 89 patients (pts) (43(48.3%) females and 46(51.7%) males) with DN of II—IV stages and EH of II—III stages aged 34 to 78 years old (an average age is 57.11±1.89 years old). They were also genotyped for rs1799983 eNOS gene polymorphism (G894T) with a use of polymerase chain reaction. GG genotype was revealed in 29(32.6%) of pts, GT genotype was found in 33(37.1%) of cases and 27(30.3%) participants of the study had TT genotype. With the use of immune enzyme method, serum levels of total cholesterol (TC), high‑density lipoprotein cholesterol (HDL‑C) and triglycerides (TG) were measured in all included pts. Very low density lipoprotein cholesterol (VLDL‑C), low‑density lipoprotein cholesterol (LDL‑C), non‑HDL‑C and coefficient of atherogenicity were calculated by known formulas. Atorvastatin was prescribed to all the pts included in study for correction of lipid exchange disorders in daily doses of 10—20 mg (an average daily dose was 16.7±0.91 mg) for 24 weeks.
 Results. Rs1799983 eNOS gene polymorphism in pts with DN and EH was found to be associated with lipid parameters characterized a phenotype of diabetic DLP with its peculiar disorders in the systems of reverse cholesterol transport and lipoprotein lipolysis of TG‑containing VLDL. In this case a dominant G allele was associated with less significant lipid disorders than a recessive T allele. A homozygous state with allele G (genotype GG) in pts with DN and EH was associated with abnormalities in the system of TG‑containing VLDL lipolysis and an appearance of T allele in genotype (genotypes GT and TT) made an additional negative contribution to the state of lipid exchange and transport. In pts with genotype GT abnormalities in reverse cholesterol transport added to disorders in the system of TG‑containing VLDL lipolysis and in pts with genotype TT there was a combination of disorders in the systems of direct, reverse cholesterol transport and lipoprotein lipolysis. The study of hypolipidemic effects of atorvastatin depended on genotype of rs1799983 eNOS gene polymorphism showed that in GG genotype atorvastatin demonstrated mainly a hypocholesterolemic action while in genotypes contained T allele (GT and TT) we observed a reduction of hypocholesterolemic effect against the background of significant hypotriglyceridemic action and elevation of HDL‑C.
 Conclusions. In pts with DN and EH rs1799983 eNOS gene polymorphism was associated with diabetic DLP. A recessive allele T was associated with significant lipid disorders compared with a dominant G allele. A significant hypocholesterolemic effect of atorvastatin was observed in case of a presence of G allele in pts genotype while a presence of T allele in pts genotype (GT and TT) was associated with a reduction of atorvastatin cholesterol lowering action.

Biosensor for PCR amplicons by combining split trehalase and DNA-binding proteins: A proof of concept study

The use of polymerase chain reaction (PCR) technology in low-cost settings has gained significant attention due to its ability to amplify and detect specific bacterial pathogen genes, aiding in the diagnosis of infectious diseases. PCR amplicons can be visualized by conventional endpoint agarose gel electrophoresis and fluorochrome-enabled real-time PCR. However, this is not practical in on-field tests due to cumbersome instrumentation, labor-intensive reaction preparation, and long time-to-results. Many studies have combined microfluidic devices or electrochemical dyes with PCR technology to enhance in-field operability. However, the high cost of manufacturing high-precision microfluidic chips and the dependence on non-portable readout equipment limit their further development. In this paper, we present a proof-of-principle study of a novel method combining split enzyme technology and DNA-binding proteins for the convenient and efficient detection of amplified genetic material from bacterial pathogens. The amplicon binding split trehalase assay (ABSTA) relies on incorporating specific recognition sequences of DNA-binding protein SpoIIID in tandem within one of the PCR primers. Applied by a Gram-type specific PCR assay, ABSTA was capable of discriminating Staphylococcus devriesei and Escherichia coli in <90 min after colony PCR amplicons bound split trehalase fragments-fused SpoIIID and triggered split enzyme complementation. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. The glucose produced by restored enzymatic activity was detectable by glucometer. With limited requirements for reaction preparation and the compatibility of ABSTA with commercially available handheld glucometers, this test platform has substantial potential to be implemented into a future point-of-care (POC) diagnostic tool for detecting pathogen specific genes with further improvement.

Single-Point Nail Sampling to Diagnose Onychomycosis Caused by Non-Dermatophyte Molds: Utility of Polymerase Chain Reaction (PCR) and Histopathology

The three most commonly used methods for diagnosing non-dermatophyte mold (NDM) onychomycosis are culture, polymerase chain reaction (PCR), and histopathology. Toenail samples from 512 patients (1 sample/patient) with suspected onychomycosis were examined using all three diagnostic tests. A statistically significant association was found between PCR and histopathology results, as well as between fungal culture and histopathology results. All PCR-positive and culture-positive dermatophyte samples were confirmed by histopathology. However, 15/116 (12.9%) of culture-positive NDM samples had negative histopathology results, while all PCR-positive NDM samples were confirmed by histopathology. The overall rate of dermatophyte detection was higher using PCR compared to culture (38.9% vs. 11.7%); the lower rate of NDM detection by PCR (11.7% vs. 38.9%) could be attributed to the restriction of the assay design to seven pre-selected targets. When repeat sampling in the clinic is not possible, a combination of NDM detection by PCR and positive histopathology of hyphae may be a proxy for NDM infection, particularly where the NDM occurs without a concomitant dermatophyte. There was a high degree of correlation between negative PCR and negative histopathology. A negative PCR result with negative histopathology findings may be a reliable proxy for the diagnosis of non-fungal dystrophy.

Comparative Analysis of FCGR Gene Polymorphism in Pulmonary Sarcoidosis and Tuberculosis.

The clinical outcome of sarcoidosis (SA) is very similar to tuberculosis (TB); however, they are treated differently and should not be confused. In search for their biomarkers, we have previously revealed changes in the phagocytic activity of monocytes in sarcoidosis and tuberculosis. On these monocytes we found a higher expression of receptors for the Fc fragment of immunoglobulin G (FcγR) in SA and TB patients vs. healthy controls. FcγRs are responsible for the binding of immune complexes (ICs) to initiate an (auto)immune response and for ICs clearance. Surprisingly, our SA patients had a high blood level of ICs, despite the abundant presence of FcγRs. It pointed to FcγR disfunction, presumably caused by the polymorphism of their (FCGR) genes. Therefore, we present here an analysis of the occurrence of FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B variants in Caucasian SA and TB patients, and healthy individuals with the use of polymerase chain reaction (PCR) and real-time PCR. The presented data point to a possibility of supporting the differential diagnosis of SA and TB by analyzing FCGR2C, FCGR3A and FCGR3B polymorphism, while for severe stages of SA also by studying FCGR2A variants. Additionally, the genotyping of FCGR2A and FCGR3B might serve as a marker of SA progression.

P498 Pneumocystis Jirovecii pneumonia: Clinical profile and diagnostic modalities

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesIn developing countries like India, the diagnosis of Pneumocystis jirovecii infection is often made either by conventional staining or clinically. This study was planned to know the utility of polymerase chain reaction (PCR) in diagnosing Pneumocystis jirovecii pneumonia (PJP), to compare the PCR results with that of the staining technique, and also to correlate the results with the radiological condition of patients from a tertiary care center in India.MethodA retrospective study included 3500 adult in-patients with symptoms of lower respiratory tract infection. Sputum and bronchoalveolar lavage samples were proceeded for both staining and PCR for multicopy mtLSU gene and singlecopy DHPS fas gene of P. jirovecii.ResultsThe present study was carried out in the Department of Microbiology, Max, Hospital, Saket, New Delhi, India. Between the years 2019 and 2022, the patients with clinically suspected pneumocystis pneumonia who attended outpatient or admitted to the Departments of Medicine, Gastroenterology, and Neurology, etc. of our hospital were retrospectively analyzed. The details of these patients were analyzed as per a well-structured proforma that included the detailed clinical history and laboratory data from the hospital records. A total of 3500 patients were included in the present study. Of these 3500 patients, 1.42% (50/3500) patients of pneumocystis pneumonia were confirmed, based on positivity by at least any two of the above-mentioned techniques. The age of the patients ranged from 29 to 80 years (mean 32 years). Among these 50 patients, 34 (68%) were males and 16 (32%) were females and the male-to-female ratio was 2:1. A total of 10 patients had human immunodeficiency virus (HIV) infection, while the other 25 were HIV negative. The immune status of five patients was unknown. Fever and dyspnea (n = 44, 88%), non-productive cough and abnormal auscultation sound (n = 30, 60%), and also chills and weight loss (n = 10, 20%) were the documented clinical symptoms of PJP. Radiological diagnosis of Pneumocystis pneumonia was noticed in 34 cases. Foamy alveolar casts were present in 19 cases. Silver methenamine stain enhanced the rounded, helmet, or cleft forms of sporozoites. Inflammatory infiltrate was mainly polymorphonuclear. All the cases were confirmed as P. jiroveci by PCR amplification.ConclusionClinicians often face the diagnostic dilemmas presented in the study. Individual modalities of the diagnosis are available, but all have drawbacks with varied sensitivity and specificity. Combining the available clinical, radiological, and microbiological modalities to reach early diagnosis can go a long way to avoid misdiagnosis and unnecessary delays in treatment.

Shiga toxin-producing Escherichia coli clonal complex 32, including serotype O145:H28, in the UK and Ireland.

Introduction. Shiga toxin-producing Escherichia coli (STEC) O157:H7 has been the most clinically significant STEC serotype in the UK for over four decades. Over the last 10 years we have observed a decrease in STEC O157:H7 and an increase in non-O157 STEC serotypes, such as O145:H28.Gap Statement. Little is known about the microbiology and epidemiology of STEC belonging to CC32 (including O145:H28) in the UK. The aim of this study was to integrate genomic data with patient information to gain a better understanding of the virulence, disease severity, epidemic risk assessment and population structure of this clinically significant clonal complex.Methodology. Isolates of E. coli belonging to CC32 (n=309) in the archives of public health agencies in the UK and Ireland were whole-genome-sequenced, virulence-profiled and integrated with enhanced surveillance questionnaire (ESQ) data, including exposures and disease severity.Results. Overall, diagnoses of STEC belonging to CC32 (290/309, 94 %) in the UK have increased every year since 2014. Most cases were female (61 %), and the highest proportion of cases belonged to the 0-4 age group (53/211,25 %). The frequency of symptoms of diarrhoea (92 %), abdominal pain (84 %), blood in stool (71 %) and nausea (51 %) was similar to that reported in cases of STEC O157:H7, although cases of STEC CC32 were more frequently admitted to hospital (STEC CC32 48 % vs O157:H7 34 %) and/or developed haemolytic uraemic syndrome (HUS) (STEC CC32 9 % vs O157:H7 4 %).The majority of STEC isolates (268/290, 92 %) had the stx2a/eae virulence gene combination, most commonly associated with progression to STEC HUS. There was evidence of person-to-person transmission and small, temporally related, geographically dispersed outbreaks, characteristic of foodborne outbreaks linked to nationally distributed products.Conclusion. We recommend more widespread use of polymerase chain reaction (PCR) for the detection of all STEC serogroups, the development of consistent strategies for the follow-up testing of PCR-positive faecal specimens, the implementation of more comprehensive and standardized collection of epidemiological data, and routine sharing of sequencing data between public health agencies worldwide.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines.

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Utility of Polymerase Chain Reaction in Nasopharyngeal Swabs for Identifying Respiratory Bacteria Causing Community-Acquired Pneumonia.

ABSTRACTTimely identification of a pathogen in lower respiratory tract infections (LRTI) can support appropriate antibiotics use. The difficulty of obtaining lower respiratory tract (LRT) samples limits the utility of point-of-care syndromic molecular assays. We assessed the performance of the FilmArray Pneumonia plus panel (FilmArray PP) in nasopharyngeal (NP) swab for detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Patients in the study included retrospectively consenting adults who attended the emergency department of Lausanne University Hospital between February 2019 and August 2020 for a community-acquired LRTI, with available NP swab and a high-quality LRT sample. These samples were tested with the FilmArray PP (cutoff of ≥104 copies/mL). Positive (PPA) and negative percent agreement (NPA) of FilmArray PP in NP swab were calculated, using (i) FilmArray PP in LRT sample and (ii) standard microbiological tests as reference standards. To assess the performance of a lower detection cutoff, NP samples were also tested with an in-house PCR (cutoff of ≥10 copies/mL) for S. pneumoniae and H. influenzae. Overall, 118 patients were included. FilmArray PP in LRT sample and standard microbiology tests detected S. pneumoniae in 19/118 and 12/118, H. influenzae in 44/118 and 19/118, and M. catarrhalis in 14/118 and 0/118, respectively. Using LRT FilmArray PP as reference, PPA and NPA of FilmArray PP on NP were 58% and 100% for S. pneumoniae, 61% and 100% for H. influenzae, and 57% and 99% for M. catarrhalis. Using standard diagnostic tests as reference, PPA and NPA were 58% and 96% for S. pneumoniae, 74% and 87% for H. influenzae, and indefinite and 92% for M. catarrhalis. Using a lower cutoff on NP (≥102 copies/mL), PPA was 68% for S. pneumoniae and 77% for H. influenzae with LRT FilmArray PP as reference. FilmArray PP in NP swabs has a limited PPA for identifying the most common etiologies of community-acquired LRTI irrespective of the reference standard, preventing its use for withholding antibiotics. The PCR detection cutoff does not explain the low PPA. The excellent NPA suggests the use of NP PCR results for rapidly targeted antimicrobial therapy.IMPORTANCE Timely identification of a pathogen in patients with lower respiratory tract infections is of paramount importance to avoid inappropriate antibiotic prescription. We aimed to evaluate the performance of a rapid syndromic molecular assay in nasopharyngeal swabs for identifying the most common bacterial causes of lower respiratory tract infections in adults (Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis). Our data show that nasopharyngeal molecular assay has a good concordance with lower respiratory tract sample when positive but not when negative. A positive result is therefore concordant with a lower respiratory tract infection and can be used to target antibiotics. Nevertheless, a negative result does not have a good concordance, so it cannot be used to withhold antibiotics. Our findings illustrate the potential utility of these easily collected samples for the management of patients with lower respiratory tract infections.

Utility of polymerase chain reaction for assessment of onychomycosis during topical therapy.

Increased detection of fungi including non-dermatophyte molds (NDMs) using polymerase chain reaction (PCR) methods is well-established. However, the use of PCR to evaluate ongoing onychomycosis treatment outcome has not been investigated. Nail samples from 28 patients receiving topical efinaconazole were evaluated by both KOH/culture and PCR methods across the study period. Detection of microorganisms by PCR was compared to the culture at baseline and end of study at month 24 (M24). Fungal detection by both methods was evaluated with respect to clinical cure observed as 100% visual clearance of the target toenail. By culture, all 28 subjects were dermatophyte-positive at pre-treatment; only 4/28 also exhibited an NDM microorganism. According to PCR, 24/28 subjects were dermatophyte-positive pre-treatment, with 25/28 also exhibiting NDMs. At M24, 18/28 (64.3%) participants had negative KOH/culture results, in contrast to 4/28 (14.3%) negative PCR results. PCR showed higher rates of NDM detection than the culture at baseline as well as M24. Calculations to compare the diagnostic utility of KOH/culture versus PCR found that positive tests with both methods reliably indicate the presence of onychomycosis, but negative PCR correlated better with onychomycosis cure than did KOH/culture. PCR confirmed a high presence of NDMs pre-treatment, and continued presence of NDMs to M24, with unknown significance requiring further investigation. Though both KOH/culture and PCR have diagnostic limitations, PCR showed better overall utility than culture in predicting onychomycosis topical treatment outcome and should be more strongly considered for evaluation of topical therapies.

Association between dopamine transporter gene (DAT1/SLC6A3) variants and infertility in the Turkish females

Objective Worldwide, approximately 10–15% of couples is affected by infertility and infertility is associated with disturbances in female and/or male reproductive systems. The aim of the current study is to investigate the relationship between DAT1 (SLC6A3) VNTR polymorphism with female infertility. Methods Genomic DNA extractions were performed in 98 fertile and 90 infertile females, 3' untranslated region (3’ UTR) variable number tandem repeat (VNTR) polymorphism of DAT1/SLC6A3 was determined by the use of Polymerase Chain Reaction (PCR) method. Results It has been demonstrated that there was no statistically significant difference between female infertile and fertile groups in the terms of DAT1 genotypes (p > .05). Moreover, there was no significant difference regarding the frequencies of 9R and 10R alleles in infertile and fertile groups (p > .05). Conclusion This is the first study for investigating the relationship between DAT1/SLC6A3 gene polymorphism and infertility in females. Our study contributes to the growing awareness of the relationship between dopaminergic system and female infertility despite no significant differences were reported between infertile females and corresponding fertile subjects in DAT1/SLC6A3 gene.

Isolation of Methicillin Resistant (MR) Staphylococci from Chicken Meat Samples

The aim of this study was to determine the presence of methicillin resistant staphylococci (MRS) in retail raw chicken meat samples sold in Hatay. The antimicrobial susceptibility of the isolates was tested for 13 different antimicrobials by disc diffusion method and investigated for resistance genes encoding methicillin (mecA), tetracycline (tetM, tetK), penicillin (blaZ), macrolide (ermA, ermC), lincosamide (lnuA) and aminoglycoside [aac(6′)/aph(2′′), aph(3′)‐IIIa, ant(4′)‐Ia] resistance via the use of polymerase chain reaction (PCR). In addition, the presence of staphylococcal enterotoxin (SE) genes was also searched by PCR. Out of 50 collected chicken meat samples, 11 (22%) MRS was isolated and the following species were determined: S. sciuri (72.7%, 8/11) and S. epidermidis (27.3%, 3/11). While all isolates were resistant to oxacillin, penicillin and ampicillin, various rates of resistance were observed for tetracycline (8, 72.7%), clindamycin (3, 27.3%), trimethoprim‐sulfamethoxazole (2, 18.2%), erythromycin (2, 18.2%) and rifampicin (1, 9.1%). All MRS harbored mecA gene together with blaZ. The tetM gene responsible for ribosomal protection was detected in all phenotypically tetracycline resistant isolates. ermC gene in erythromycin resistant isolates and lnuA gene in clindamycin resistant isolates were detected. None of the isolates was found to be positive for SE genes. The results of this study indicated that contamination of retail raw chicken meat samples with MRS poses a risk to public health due to transmission of these bacteria to humans. Additionally, this study also highlights the importance of monitoring antimicrobial resistant bacteria in animal originated foods.