Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Vermamoeba vermiformis as the etiological agent in a patient with suspected non-Acanthamoeba keratitis.

Vermamoeba vermiformis (V. vermiformis) is one of the most common free-living amoeba (FLA) and is frequently found in environments such as natural freshwater areas, surface waters, soil, and biofilms. V. vermiformis has been reported as a pathogen with pathogenic potential for humans and animals. The aim is to report a case of non-Acanthamoeba keratitis in which V. vermiformis was the etiological agent, identified by culture and molecular techniques. Our case was a 48-year-old male patient with a history of trauma to his eye 10 days ago. The patient complained of eye redness and purulent discharge. A slit-lamp examination of the eye revealed a central corneal ulcer with peripheral infiltration extending into the deep stroma. The corneal scraping sample taken from the patient was cultured on a non-nutritious agar plate (NNA). Amoebae were evaluated according to morphological evaluation criteria. It was investigated by PCR method and confirmed by DNA sequence analysis. Although no bacterial or fungal growth was detected in the routine microbiological evaluation of the corneal scraping sample that was cultured, amoeba growth was detected positively in the NNA culture. Meanwhile, Acanthamoeba was detected negative by real-time PCR. However, V. vermiformis was detected positive with the specific PCR assay. It was confirmed by DNA sequence analysis to be considered an etiological pathogenic agent. Thus, topical administration of chlorhexidine gluconate %0.02 (8 × 1) was initiated. Clinical regression was observed 72 h after chlorhexidine initiation, and complete resolution of keratitis with residual scarring was noticed in 5 weeks. In conclusion, corneal infections due to free-living amoebae can occur, especially in poor hygiene. Although Acanthamoeba is the most common keratitis due to amoeba, V. vermiformis is also assumed to associate keratitis in humans. Clinicians should also be aware of other amoebic agents, such as V. vermiformis, in keratitis patients.

Serological and molecular detection of Toxoplasma gondii infection in apparently healthy horses in eastern of Spain.

Toxoplasmosis is one of the most common parasitic zoonoses and represents a significant health risk for humans, especially for immunodeficient patients. The main transmission route is by oral uptake of oocysts and consumption of undercooked meat of infected animals. Different species have been evaluated as possible reservoirs of the parasite, but few studies have been carried out to examine the role of horses in transmission of the disease. Given the proximity of these animals to humans and the widespread consumption of their meat in many countries, including the Mediterranean basin, it is important to determine the prevalence of T. gondii infection in this species. In this study, blood samples from 105 horses were collected and the presence of T. gondii was evaluated by serological and molecular methods. Antibodies against T. gondii of 12 horses (11.43%) were detected by enzyme-linked immunosorbent assay (ELISA), whereas 29 horses (27.62%) showed positive for PCR. Seroprevalence was related to use of the animals, being higher in horses used for dressage than in others. Purebreds had higher seroprevalence than crossbred animals. No differences between breed, sex or age were found. The results of this study confirm the presence of T. gondii infection in horses, highlighting the need to analyse the meat of this species before human consumption and to control of this infection in horses, as they could be an important reservoir of this zoonotic parasite.

Unveiling genetic variants: Tetra-primer ARMS-PCR diagnosis and structural insights into BLAD, BC, and DUMPS in Pakistani cattle herds.

BACKGROUND: Bovine leukocyte adhesion deficiency (BLAD), bovine citrullinemia (BC), and deficiency of Uridine monophosphate synthetase (DUMPS) are the common autosomal recessive disorders affecting the global dairy industry. BLAD leads to poor wound healing and recurrent infections. In BC, ammonia builds up leading to neurological disorders and death. DUMPS results in developmental abnormalities. METHODOLOGY: In this study, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) based diagnostic tests were optimized for BLAD, BC, and DUMPS. A total of 250 animals (58 indigenous and 192 Holstein Friesian (HF)) were screened from all across Pakistan. In addition to validation of ARMS-PCR results through Sanger sequencing, the protein modeling provided structural insights of the disease-associated reported SNPs. Pathway analysis illustrated gene functions under normal and mutated conditions. Furthermore, haplotype and phylogenetic analysis of ASS1 (Argininosuccinate synthetase) gene were performed on study samples and NCBI retrieved sequences. RESULTS: The study's focus was to screen the herds for prevalence of carriers of genetic disorders, as they are the main source of disease dissemination. One animal was found carrier for BC, whereas no carriers were found for BLAD and DUMPS. The protein models corroborated the reported amino acid change in BLAD, and protein truncation in both BC and DUMPS proteins. SNPs found in NCBI retrieved sequences were either silent or missense and had no effect on protein structure. DNA network presented graphical illustration of haplotype interactions and phylogenetic analysis conferred evolutionary landscape of ASS1 gene. The combination of these approaches produced an in-depth genetic picture of BC in Pakistani cattle. CONCLUSION: The development of diagnostic tests and identification of the heterozygous BC sample underscores the significance of constant monitoring to avoid the unwanted dissemination of mutant alleles among Pakistani cattle, thereby promoting the general well-being and sustainability of the dairy sector.

Molecular survey of Toxoplasma gondii B1 gene in pigs from various localities in Korea.

Toxoplasma gondii, a common protozoan parasite, poses significant public health risks due to its potential to cause toxoplasmosis in humans and can be contracted from pigs, which are considered its critical intermediate host. The aim of this study is to evaluate the prevalence of T. gondii in slaughtered pigs for human consumption, emphasizing the zoonotic implications and the need for improved biosecurity and monitoring practices in pig farming. A total of 1,526 pig samples (1,051 whole blood samples and 384 lung tissue samples from the local slaughterhouse and 91 aborted fetus samples from local farms) were collected throughout the whole country of Korea in 2020. Among them, 6 (0.4%) were found to be infected with T. gondii by nested PCR. When compared by sample type, the prevalence of T. gondii was significantly higher in the aborted fetus samples (2.2%, 2/91) than in the blood (0.3%, 3/1,051) and lung tissue samples (0.3%, 1/384). The B1 gene sequence of T. gondii was similar (97.9-99.8%) to that of the other T. gondii isolates. This study represents the first molecular genotyping survey of T. gondii in the lung tissue of fattening pigs and aborted fetuses in Korea. Our findings indicated the importance of adopting preventive measures including the implementation of rigorous farm hygiene protocols and the promotion of public awareness about the risks of consuming undercooked pork. By addressing the gaps in current control strategies and encouraging the One Health approach, this study contributes to the development of more effective strategies to mitigate the transmission of T. gondii from pigs to humans, ultimately safeguarding public health.

Evaluation of phage-based decontamination in respiratory intensive care unit environments using ddPCR and 16S rRNA targeted sequencing techniques.

Background: Klebsiella pneumoniae is a major cause of hospital-acquired infections (HAIs), primarily spread through environmental contamination in hospitals. The effectiveness of current chemical disinfectants is waning due to emerging resistance, which poses environmental hazards and fosters new resistance in pathogens. Developing environmentally friendly and effective disinfectants against multidrug-resistant organisms is increasingly important. Methods: This study developed a bacteriophage cocktail targeting two common carbapenem-resistant Klebsiella pneumoniae (CRKP) strains, ST11 KL47 and ST11 KL64. The cocktail was used as an adjunctive disinfectant in a hospital's respiratory intensive care unit (RICU) via ultrasonic nebulization. Digital PCR was used to quantify CRKP levels post-intervention. The microbial community composition was analyzed via 16S rRNA sequencing to assess the intervention's impact on overall diversity. Results: The phage cocktail significantly reduced CRKP levels within the first 24 hours post-treatment. While a slight increase in pathogen levels was observed after 24 hours, they remained significantly lower than those treated with conventional disinfectants. 16S rRNA sequencing showed a decrease in the target pathogens' relative abundance, while overall species diversity remained stable, confirming that phages selectively target CRKP without disrupting ecological balance. Discussion: The findings highlight the efficacy and safety of phage-based biocleaners as a sustainable alternative to conventional disinfectants. Phages selectively reduce multidrug-resistant pathogens while preserving microbial diversity, making them a promising tool for infection control.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

A Cost-Effective Approach for Single-Stranded DNA Amplification Using Primer-Blocked Asymmetric PCR.

In vitro amplification of single-stranded oligonucleotide libraries presents a significant challenge due to the potential for excessive byproduct formation. This phenomenon largely affects the quality of the ssDNAs created using the most commonly used methods, e.g., asymmetric PCR, biotin-streptavidin separation, or lambda exonuclease digestion of dsDNA. Here, we describe an improved protocol that combines primer-blocked asymmetric PCR (PBA-PCR) with emulsion PCR and a cost-effective downstream process that altogether alleviates byproduct formation without distorting the sequence space of the ssDNA library. In PBA-PCR, the reaction mixture is complemented with a 3'-phosphate-blocked limiting primer that decreases mispriming, thus reducing polymerization of DNA byproducts. The downstream process includes mixing of the PBA-PCR product with excess reverse complement of the 3'-phosphate-blocked limiting primer and removal of dsDNA strands via biotin-streptavidin separation, yielding purified ssDNAs. In conclusion, we have devised a universally applicable approach for simple and cost-effective production of ssDNA libraries and unique ssDNA sequences with on-demand labeling. Our protocol could be beneficial for a variety of uses, such as generating aptamer libraries for SELEX, creating unique molecular identifiers for a wide range of sequencing applications, providing donor DNA for CRISPR-Cas9 systems, developing scaffold nanostructures, and enabling DNA-based data storage. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Amplification of ssDNA libraries using PBA-PCR Alternate Protocol 1: Amplification of ssDNA libraries using emulsion PBA-PCR with a simplified extraction of PBA-PCR products Basic Protocol 2: Purification of PBA-PCR products to remove dsDNA and conversion of 3'-blocked primer to double-stranded complexes Alternate Protocol 2: Purification of PBA-PCR products to remove both dsDNA and blocking primers from the reaction mixture Support Protocol: Analysis of PBA-PCR products by gel electrophoresis.

Efficient cloning of linear DNA inserts (ECOLI) into plasmids using site-directed mutagenesis.

This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

Detection of Pseudomonas aeruginosa pus wound isolate using a polymerase chain reaction targeting 16S rRNA and gyrB genes: A case from Indonesia.

Infectious wounds on the skin surface are easily colonized by bacteria from pyogenic group that manifest as inflammation, such as Pseudomonas aeruginosa. P. aeruginosa is a Gram-negative bacterium and an opportunistic pathogen known for causing invasive state in critically ill and immunocompromised patients. The aim of this study was to detect the 16S rRNA and gyrB genes in P. aeruginosa using polymerase chain reaction (PCR) method. The sample in this study was pus isolate from a 5-year-old boy with leg wounds. The bacteria were isolated on brain heart infusion broth (BHIB) media and identified with molecular identification. Sequencing and BLAST analysis were carried out to determine the similarity of gene identity by comparing sample sequence with other isolate sequences on the Gene Bank. The results of molecular identification showed amplification DNA band of around 934 base pairs (bp) for 16S rRNA and 225 bp for gyrB gene. The BLAST program demonstrated that the sample had 99.89% similarity with P. aeruginosa strain XC4 (accession code ON795960.1) for the 16S rRNA gene. Meanwhile, the gyrB gene exhibited 99.10% similarity with the P. aeruginosa strain PSA-1.2 (accession code KP172300.1).

An overview of Buruli ulcer in Australia.

BACKGROUND: Buruli ulcer (BU) is caused by Mycobacterium ulcerans, an environmental pathogen that causes severe skin and soft-tissue necrosis. In Australia, cases of BU are acquired in endemic regions, which include Victoria and Far North Queensland, but those who have visited these regions can present to health practitioners anywhere. OBJECTIVE: This article provides Australian general practitioners with an overview of BU, including its epidemiology, transmission, clinical features, diagnosis and management. DISCUSSION: BU can manifest as an ulcer or as a non-ulcerated skin lesion, such as a plaque, nodule or oedema. Diagnosis can be achieved with a dedicated Mycobacterium ulcerans polymerase chain reaction (PCR) test performed on a wound swab. Swabs on non-ulcerated disease have a high false negative rate, and a PCR test should be performed on a tissue biopsy to confirm disease. Most cases are managed with prolonged antibiotic therapy - commonly a combination of oral rifampicin and clarithromycin or fluroquinolone (moxifloxacin or ciprofloxacin) - and wound dressings.

Characterization of Helicobacter pylori iceA and babA2 virulence genes in dyspeptic patients at a teaching hospital in Ghana.

Introduction: Helicobacter pylori (H. pylori) infection is endemic in Africa. It is a major aetiological factor in the development of peptic ulcer disease and distal gastric cancers. Existing data shows that clinical outcomes are dependent on the virulence of the infecting strain, host´s susceptibility, and environmental factors. In Ghana, a previous study showed that the majority of symptomatic individuals harboured cagA and vacA virulent strains. The main objective of this study was to characterize and assess the significance of other virulence factors, specifically iceA and babA2 in Ghana. Methods: H. pylori iceA and babA2 genes were investigated in dyspeptic patients at the Korle Bu Teaching Hospital (KBTH), Accra, Ghana. The study employed a cross-sectional design consecutively recruiting patients with upper gastrointestinal symptoms for endoscopy. Nucleic acid was extracted from gastric biopsies using a commercial kit (QIAGEN DNeasy tissue kit). H. pylori babA2 and iceA genes were amplified using extracted deoxyribonucleic acid (DNA) and primers by polymerase chain reaction (PCR). Results: majority, (71.1%), of the study participants, were H. pylori positive when tested with urease-campylobacter-like organism (CLO). In total, 46 H. pylori urease CLO-positive samples were randomly analyzed by PCR for iceA, of which, 12 (26%) and 7 (15%) were found to have iceA1 and iceA2 respectively. Of the CLO-positive samples, 9 were randomly analysed for babA2 by PCR. Three samples were babA2 positive and 6 were babA2 negative. Conclusion: in Ghana, although H. pylori is endemic, iceA prevalence is rather low and probably exerts a limited effect on bacterial virulence. Further evaluation would be required, not only to determine association with other virulence factors but more importantly, inter-relationships with wider host and environmental factors that impact on disease pathogenesis.

Evaluating the efficacy of three Y-STRs commercial kits in degraded skeletal remains.

Y chromosome short tandem repeats (Y-STRs) typing is a useful tool in scenarios such as mass graves analysis or disaster victim identification and has become a routine analysis in many laboratories. Not many comparisons have been performed with the currently available commercial kits, much less with degraded skeletal remains. This research aims to evaluate the performance of three commercial Y-STR kits: Yfiler™ Plus, PowerPlex® Y23, and Investigator® Argus Y-28 in 63 degraded skeletal remains from mass graves. PowerPlex® Y23 yields more reportable markers and twice the RFU on average, while Yfiler™ Plus and Investigator® Argus Y-28 exhibited a similar behaviour. Additionally, Argus Y-28, which has not been tested with this kind of samples in literature before, showed a good performance. Finally, a predictive model was attempted to be developed from quantification and autosomal STR data. However, no acceptable model could be obtained. Nevertheless, good Y-STR typing results may be expected if at least 50 pg DNA input is used or 13 autosomal markers were previously obtained.

Molecular Identification of Mycobacterium leprae in the Leprosy Patients.

BACKGROUND: Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is a major port of entry and exit. Molecular probes have shown certain potential for the detection and identification of M. leprae in patients. The aim of this study was to identify M. leprae in nasal swab specimens using polymerase chain reaction (PCR)-based assays followed by gene sequencing methods. This observational study examines 64 anterior nasal swab samples taken from pretreatment leprosy patients, on-treatment and completed leprosy treatment in Bulukumba, South Sulawesi, Indonesia. METHODS: samples were analyzed by molecular detection methods according to the standard methods at the Clinical Microbiology Laboratory of Hasanuddin University. Descriptive statistics were utilized to summarize patient demographics and outcomes. RESULTS: This study uses PCR to detect the M. leprae deoxyribonucleic acid (DNA) from nasal swab specimens. Data were collected from 64 patients with a percentage of male patients 51.54%. Based on the age category, the group 45-46 years was the most frequent (39.05%). PCR detection proline-rich antigen gene of a 531 bp DNA fragment from M. leprae, was positive in eight patients, and they were multibacillary. Furthermore, PCR was positive in 5 (31.25%) of 16 new leprosy patients, 2 (8.69%) of 23 on-treatment patients, and 1 (4%) of 25 treatment completed patients. Based on the results of the phylogenetic tree and analysis of 8 positive results detected by M. leprae from leprosy patients, almost all samples have a level of similarity, except for sample Ua7. CONCLUSIONS: M. leprae cannot grow in vitro, so molecular diagnostic tools were used to confirm the disease. This study predominantly of males with the age above 45 years of age being the most common. Eight M. leprae were positive from nasal swab leprosy patients. The sequencing findings provide insight into the genetic diversity of the genus M. leprae, so it is necessary to consider the detection of whole-genome sequence.

Identification of Nontuberculous Mycobacterium Species by Polymerase Chain Reaction - Restriction Enzyme Analysis (PCR-REA) of rpoB gene in Clinical Isolates.

BACKGROUND: Nontuberculous mycobacteria (NTM) infections are an emerging global health concern with increasing incidence. Conventional identification methods for NTM species in clinical settings are prone to errors. This study evaluates a newer method, polymerase chain reaction-restriction enzyme analysis (PCR-REA) of the rpoB gene, for NTM species identification. The study identified NTM species in clinical samples using conventional biochemical techniques and compared the results with PCR-REA of the rpoB gene. This cross-sectional study was conducted at a tertiary health-care center in North India over 18 months, analyzing both pulmonary and extrapulmonary samples. METHODS: Two hundred and forty-seven NTM isolates were identified using phenotypic and biochemical methods. The same isolates were subjected to rpoB gene amplification by PCR followed by REA using Msp I and Hae III enzymes. RESULTS: Conventional methods identified 12 different NTM species (153 slow-growing and 94 rapid-growing), whereas PCR-REA identified 16 species (140 slow-growing, 107 rapid-growing). The Mycobacterium avium intracellulare complex was the most common species isolated. PCR-REA demonstrated higher resolution in species identification, particularly in differentiating within species complexes. CONCLUSIONS: PCR-REA of the rpoB gene proves to be a simple, rapid, and more discriminative tool for NTM species identification compared to conventional methods. This technique could significantly improve the diagnosis and management of emerging NTM infections in clinical settings.

Diagnostic Accuracy of Polymerase Chain Reaction Against Giemsa Staining on Tissue Biopsy for Cutaneous Leishmaniasis.

OBJECTIVE: To evaluate the diagnostic accuracy of a commercial real-time polymerase chain reaction (PCR) kit targeting 18S rRNA against Giemsa-stained tissue slides in patients clinically suspected of cutaneous leishmaniasis (CL). STUDY DESIGN: Cross-sectional analytical study. Place and Duration of the Study: Department of Microbiology, Armed Forces Institute of Pathology / National University of Medical Sciences, Rawalpindi, Pakistan, from July to December 2022. METHODOLOGY: Samples of skin tissue in 98 patients suspected of CL were evaluated. These samples were subjected to Giemsa-staining for microscopy and real-time PCR. Sensitivity, specificity, and accuracy of the PCR were calculated keeping Giemsa-stained tissue slide microscopy as gold standard. RESULTS: Out of the 98 tissue samples, 37 were found positive for leishmaniasis on PCR while 13 were found Leishmania positive on microscopy of Giemsa-stained slides. The sensitivity, specificity, and accuracy of the PCR for the detection of Leishmania species were 100%, 71.8%, and 91.8%, respectively with 100% negative predictive value. CONCLUSION: This study demonstrates that the commercial PCR is a reliable diagnostic test for the diagnosis of CL. The ease, rapidity, and reliability of the PCR make it a dependable tool in diagnostic repertoire of CL. KEY WORDS: Giemsa stain, Leishmania spp., Polymerase chain reaction, Viasure.

Molecular and in silico analyses for detection of Shiga toxin-producing Escherichia coli (STEC) and highly pathogenic enterohemorrhagic Escherichia coli (EHEC) using genetic markers located on plasmid, O Island 57 and O Island 71.

BACKGROUND: Due to the diversity of Shiga toxin-producing Escherichia coli (STEC) isolates, detecting highly pathogenic strains in foodstuffs is challenging. Currently, reference protocols for STEC rely on the molecular detection of eae and the stx1 and/or stx2 genes, followed by the detection of serogroup-specific wzx or wzy genes related to the top 7 serogroups. However, these screening methods do not distinguish between samples in which a STEC possessing both determinants are present and those containing two or more organisms, each containing one of these genes. This study aimed to evaluate ecf1, Z2098, Z2099, and nleA genes as single markers and their combinations (ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA) as genetic markers to detect potentially pathogenic STEC by the polymerase chain reaction (PCR) in 96 animal samples, as well as in 52 whole genome sequences of human samples via in silico PCR analyses. RESULTS: In animal isolates, Z2098 and Z2098/Z2099 showed a strong association with the detected top 7 isolates, with 100% and 69.2% of them testing positive, respectively. In human isolates, Z2099 was detected in 95% of the top 7 HUS isolates, while Z2098/Z2099 and ecf1/Z2099 were detected in 87.5% of the top 7 HUS isolates. CONCLUSIONS: Overall, using a single gene marker, Z2098, Z2099, and ecf1 are sensitive targets for screening the top 7 STEC isolates, and the combination of Z2098/Z2099 offers a more targeted initial screening method to detect the top 7 STEC isolates. Detecting non-top 7 STEC in both animal and human samples proved challenging due to inconsistent characteristics associated with the genetic markers studied.

Molecular identification of Histoplasma capsulatum in patients with disseminated histoplasmosis and acquired immunodeficiency syndrome.

Histoplasmosis is caused by the fungus Histoplasma capsulatum and is often fatal for individuals with acquired immunodeficiency syndrome (AIDS). Delayed diagnosis is a major factor in worsening coinfection, as it can be mistaken for other diseases. Thus, rapid identification of Histoplasma in immunocompromised patients is essential. Molecular techniques, particularly polymerase chain reaction (PCR), were used in this study to identify H. capsulatum in patients coinfected with histoplasmosis and AIDS. Blood samples from 14 individuals with AIDS and disseminated histoplasmosis were collected and analyzed. The PCR method successfully amplified the fungal region in whole blood samples, while PCR-RFLP analysis confirmed a consistent profile in the samples. Genetic sequencing further confirmed the fungal species. Compared to clinical tests such as fungal culture and urinary antigen detection, molecular analysis proved faster, more sensitive, and cost-effective. These molecular markers can potentially be incorporated into routine diagnostics in the future. Further studies are needed to expand and enhance this diagnostic approach, particularly in patients with nonprogressive clinical forms of histoplasmosis.

Identification of hub genes and potential networks by centrality network analysis of PCR amplified Fusarium oxysporum f. sp. lycopersici EF1α gene.

BACKGROUND: Fusarium wilt is a devastating soil-borne fungal disease of tomato across the world. Conventional method of disease prevention including usage of common pesticides and methods like soil solarisation are usually ineffective in the treatment of this disease. Therefore, there is an urgent need to identify virulence related genes in the pathogen which can be targeted for fungicide development. RESULTS: Pathogenicity testing and phylogenetic classification of the pathogen used in this study confirmed it as Fusarium oxysporum f. sp. lycopersici (Fol) strain. A recent discovery indicates that EF1α, a protein with conserved structural similarity across several fungal genera, has a role in the pathogenicity of Magnaporthe oryzae, the rice blast fungus. Therefore, in this study we have done structural and functional classification of EF1α to understand its role in pathogenicity of Fol. The protein model of Fol EF1α was created using the template crystal structure of the yeast elongation factor complex EEF1A:EEF1BA which showed maximum similarity with the target protein. Using the STRING online database, the interactive information among the hub genes of EF1α was identified and the protein-protein interaction network was recognized using the Cytoscape software. On combining the results of functional analysis, MCODE, CytoNCA and CytoHubba 4 hub genes including Fol EF1α were selected for further investigation. The three interactors of Fol EF1α showed maximum similarity with homologous proteins found in Neurospora crassa complexed with the known fungicide, cycloheximide. Through the sequence similarity and PDB database analysis, homologs of Fol EF1α were found: EEF1A:EEF1BA in complex with GDPNP in yeast and EF1α in complex with GDP in Sulfolobus solfataricus. The STITCH database analysis suggested that EF1α and its other interacting partners interact with guanosine diphosphate (GDPNP) and guanosine triphosphate (GTP). CONCLUSIONS: Our study offers a framework for recognition of several hub genes network in Fusarium wilt that can be used as novel targets for fungicide development. The involvement of EF1α in nucleocytoplasmic transport pathway suggests that it plays role in GTP binding and thus apart from its use as a biomarker, it may be further exploited as an effective target for fungicide development. Since, the three other proteins that were found to be tightly associated Fol EF1α have shown maximum similarity with homologous proteins of Neurospora crassa that form complex with fungicide- Cycloheximide. Therefore, we suggest that cycloheximide can also be used against Fusarium wilt disease in tomato. The active site cavity of Fol EF1α can also be determined for computational screening of fungicides using the homologous proteins observed in yeast and Sulfolobus solfataricus. On this basis, we also suggest that the other closely associated genes that have been identified through STITCH analysis, they can also be targeted for fungicide development.

Neonatal and short-term outcome after late vertical transmission in congenital CMV-infected fetuses following primary first-trimester maternal seroconversion.

OBJECTIVE: To document the course of neonatal and short-term outcomes in pregnancies after first trimester CMV (cytomegalovirus) seroconversion and negative amniotic fluid (AF) CMV PCR. METHODS: We included 375 patients with a first-trimester CMV seroconversion and amniocentesis at ≥21 weeks. Termination of pregnancy (TOP) was offered in case antenatally severe CMV-related fetopathy was documented either by ultrasound or by MRI. AF CMV PCR-negative fetuses underwent a PCR CMV on neonatal urine (NU). Perinatal and short-term infant outcomes were investigated by a questionnaire, sent to parents. RESULTS: AF CMV PCR was positive in 118/375 cases (31.4%). TOP was performed in 46/118 (38.9%) and fetal demise occurred twice. Questionnaires were sent to 327 patients with an overall response rate of 77%. Three groups were considered: Group 1: the early infected group (AF CMV PCR positive; N=62), group 2: the late infected group (AF CMV PCR negative, NU CMV PCR positive; N=7) and group 3: the control group (AF+NU CMV PCR negative; N=160). Compared with group 3, group 1 was more frequently symptomatic at birth (6.2% vs 19.4%; p=0.006). In short-term follow-up, hearing impairment (23.5%; p<0.001), mild motor deficit - defined as abnormal early motor development or the need for physiotherapy in later life (21.6%; p=0.005) - and subnormal vision (15.7%; p=0.02) were significantly more frequent. Compared with group 3, group 2 showed more often jaundice (57.1%; p=0.04) and petechiae (28.6%; p=0.04) at birth, but other short-term symptoms were lacking. CONCLUSION: Although neonates may screen positive on urine for CMV after an AF CMV negative PCR, they show rarely and only mild sequelae in early life.