DNA Polymerase Chain Reaction Research Articles

Reliability of hemoglobin A2 value as measured by the Premier Resolution system for screening of β-thalassemia carriers.

Accurate quantification of hemoglobin (Hb) A2 is vital for diagnosing β-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE). We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA2 cutoff value for β-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the β- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing. HbA2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias,-0.33; r, 0.991), β-thalassemia (bias,-0.27; r, 0.927), and non-β-thalassemia carriers (bias,-0.36; r, 0.928). An HbA2 cutoff value of≥4.0 % for β-thalassemia screening achieved 100 % sensitivity and 99.6 % specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3 , 99.8, 97.3, 99.8, and 99.7 %, respectively. We also identified a rare β-Hb variant, Hb La Desirade [HBB:c.389C>T], associated with β-thalassemia and co-inherited with a single α-globin gene. The Premier Resolution HPLC is a reliable and accurate method for routine β-thalassemia carrier screening, aligning with existing CE methods.

Early detection of a possible multidrug-resistant Acinetobacter baumannii outbreak in the local hospital setting by using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), oxacillinase gene profiles, and antibiograms.

Detecting the source of a potential outbreak of multidrug resistant (MDR) Acinetobacter baumannii is necessary to be investigated. This study aimed to detect the possibility of A. baumannii outbreak in a hospital setting using a combination of random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), antibiograms, and the presence of oxacillinase genes. The antibiogram of 31 clinical isolates and six environmental isolates of A. baumannii were determined by Vitek® 2 Compact. Oxacillinase genes (OXA-23, -24, -51, and -58) were detected by PCR, and RAPD-PCR was conducted using DAF-4 and ERIC-2 primers. The Similarity Index and dendrogram were generated using GelJ v2.3 software. The antibiograms showed that all MDR A. baumannii isolates has very limited susceptibility to cephalosporins, but mostly susceptible to tigecycline. All isolates were positive for bla OXA-51-like gene, thirty-two of 37 total isolates (86.5%) were positive for bla OXA-23-like gene, and none were positive for bla OXA-24-like and bla OXA-58-like genes. RAPD-PCR showed that the DAF-4 primer on average had more band visualization and lower Similarity Index's variation compared to the ERIC-2. The discriminatory power of DAF-4 was 0.906. There was a significant correlation between the DAF-4 dendrogram pattern with the antibiogram (r=0.494, p<0.001) and the presence of bla OXA-23-like gene (r=0.634, p<0.001) from all ICU A isolates. Six out of fourteen ICU A isolates belonged to the same cluster with >95% Similarity Index, while one clinical isolate having an identical dendrogram and antibiogram pattern with an environmental isolate within this cluster. There is a high probability of MDR A. baumannii outbreak within ICU A detected by multiple analysis of RAPD-PCR, antibiogram and the bla OXA-23-like gene profiles. This combinatorial approach is conceivable to mitigate possible outbreak situations of A. baumannii in the local hospital without sophisticated microbiology laboratory.

The CD19+ B cell as a marker for the febrile children infected with influenza A and Omicron variant.

H3N2 and Omicron are common pathogens of respiratory infections in children. This study aimed to explore dynamic changes of lymphocyte subsets and the diagnostic value of CD19+ B cell in children infected with influenza A and Omicron. One hundred and sixty-five in-patients with H3N2, 175 in-patients with Omicron variant, and 50 age-matched healthy children from Children's Hospital of Soochow University were included in this study. The participants underwent 13 respiratory pathogens by DNA polymerase chain reaction (PCR), sputum culture, severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) DNA PCR, routine blood, and lymphocyte subset assays within 24 h of admission. The neutrophils, neutrophil-to-lymphocyte ratio, and monocyte-to-lymphocyte ratio in the H3N2 and Omicron groups were significantly higher than in the control groups (p < 0.05). However, the lymphocytes and eosinophils in the H3N2 and Omicron groups were lower than the control groups (p < 0.05). The CD3+ T cell, CD3+ CD4+ T cell, CD3+ CD8+ T cell, CD3- CD19+ B cell, and natural killer cell were lower in the H3N2 and Omicron groups than in the control group (p < 0.05). The CD3- CD19+ cell in the Omicron group was higher than that in the H3N2 group but lower than that in the control group (p < 0.05). In addition, CD3- CD19+ cell had good diagnostic value for H3N2 (area under the receiver operating characteristic curve = 0.902, p < 0.05). The children with H3N2 were more likely to have lower lymphocytes than children with Omicron. Additionally, B-cell count had good diagnostic value for H3N2.

Staphylococcus aureus enterotoxin coding genes identified from patients with atopic dermatitis in Iraq by molecular analysis

The present study determined the frequency of Staphylococcus aureus enterotoxins (B, C, Luk-pv) of atopic dermatitis isolated from (AD) cases in Laboratories of Baghdad. 54 Staphylococcus aureus isolates were subjected to primary identification tests using various methods (cultural characteristics, gram staining, biochemical tests, and vitek2 system). This study used antibiotic disc diffusion in fifty-four S. aureus isolates. Multidrug resistance (MDR) against different antimicrobial agents applied to polymerase chain reaction to amplify different genes coding for Staphylococcal enterotoxins, including 3 types (seb, sec and luk-pv), To ensure that the sequences of these genes match NCBI, DNA sequencing was performed for isolate No. (3). As a result of this study, 25 isolates had a multidrug resistance (MDR) percent (46.2%) against different antimicrobial agents—the results of DNA extraction and polymerase chain reaction directed to amplify the specific enterotoxin coding genes. This study showed that the (seb) gene is present in isolates of staph aureus bacteria isolated from patients with atopic dermatitis 12 /25 at a percentage (48 %). Furthermore, the absence of the sec gene in all Staphylococcus aureus isolates isolated from patients with atopic dermatitis—the results of the detection of the luk-pv 23\25 (92%) gene encoded for lukucidin.Polymerase chain reaction using different primers successfully identified Staphylococcus aureus enterotoxins (B, C)and luk-pv, luk-pv gene, which was the most frequent. Keywords: Staphylococcus aureus enterotoxin genes, polymerase chain reaction, Staphylococcus aureus, atopic dermatitis

Molecular variability of Fusarium oxysporum f. sp. lini isolates by RAPD analysis

Seven isolates of Fusarium oxysporum f. sp. lini collected from different linseed growing areas in India were analyzed by random amplified polymorphic DNA-polymerase chain reaction. Two isolates (FOL 1 and FOL 2) found non-pathogenic to linseed crop. Twenty random decamer primers were used for amplification. All the primers revealed scorable polymorphism among the isolates and a total of 156 bands were recorded. Amplification patterns of non-pathogenic Fusarium isolates were entirely distinct from those of pathogenic isolates. Seventeen primers generated isolate specific bands which would be highly useful in the identification and characterization of Fusarium isolates. RAPD markers showed substantial genetic diversity among the different isolates with maximum of 0.89 between FOL1 and FOL6. UPGMA cluster analysis was used to generate a dendogram showing relationship between them. Isolates were clustered in to three groups corresponding to their genetic similarity and physiological reactions. RAPD markers can be quick and reliable alternative for differentiating isolates of Fusarium oxysporum f. sp. liniin to different groups.

Epidemiological study of antimicrobial-resistant bacteria in healthy free-ranging bantengs (Bos javanicus) and domestic cattle.

Antimicrobial-resistant microorganisms (ARMs) have been increasing among wild animals. Interactions occurring at the interface between wildlife, humans, and livestock can lead to the transmission of ARMs. Thus, the prevalence of ARMs in wild and domestic animals should be determined to address and prevent this issue. This study aimed to determine the resistance patterns of cefotaxime (CTX)-resistant Escherichia coli and identify the presence of extended-spectrum beta-lactamase (ESBL) genes in ESBL-producing E. coli among a population of wild banteng (Bos javanicus) and domestic cattle kept on farms located close to the Lam Pao non-hunting area, Kalasin province, Thailand. Forty-five fecal samples were taken from wild bantengs inhabiting the Lam Pao non-hunting area in Thailand, alongside 15 samples from domestic cattle. Bacterial culture, triple sugar iron, and motile indole lysine tests were conducted to identify E. coli. A polymerase chain reaction (PCR) was conducted for specific confirmation. MacConkey agar supplemented with 2 μg/mL of CTX was used to identify CTX-resistant E. coli, which would be used to identify ESBL production based on a double-disk synergy test. Extended-spectrum beta-lactamase-producing samples were subjected to disk diffusion tests to determine resistant patterns, and the sizes of PCR bands and DNA sequencing were used to differentiate ESBL gene types. All samples tested positive for E. coli. Forty-five isolates from 15 banteng samples and three isolates from one domestic cattle sample displayed CTX-resistant and ESBL-producing traits. The banteng and domestic cattle populations exhibited nine and three distinct resistant patterns, respectively. The PCR results indicated that the banteng isolates harbored the following genes: Cefotaxime-M1 (n = 38), CTX-M9 (n = 5), and the SHV group (n = 2). All three isolates from the domestic cattle sample contained the CTX-M1 gene. Classification of ESBL genes based on the DNA sequences of the banteng isolates showed the characteristics of CTX-M15 (n = 20), CTX-M55 (n = 6), CTX-M14 (n = 5), and CTX-M79 (n = 1). The three domestic cattle isolates exhibited the characteristics of CTX-M15, CTX-M55, and CTX-M79. Despite no previous antibiotic applications, approximately one-third of the banteng samples displayed CTX resistance, indicating ARM contamination within the ecosystem. The similarity in ESBL genes between the banteng and domestic cattle populations suggests potential gene transmissions between these animal groups. However, the initial source of ARMs remains unclear, as the banteng population exhibited more ESBL genes than the domestic cattle, suggesting the possibility of multiple ARM sources. These findings raise concerns because the banteng population inhabits an area that is an important source of freshwater and nourishes the entire north-east region of Thailand and other South-east Asian countries, including Laos, Cambodia, and Southern Vietnam.

Metal-DNA interactions: Exploring the impact of metal ions on key stages of forensic DNA analysis.

Forensic DNA analysis continues to be hampered by the complex interactions between metals and DNA. Metal ions may cause direct DNA damage, inhibit DNA extraction and polymerase chain reaction (PCR) amplification or both. This study evaluated the impact of metal ions on DNA extraction, quantitation, and short tandem repeat profiling using cell-free and cellular (saliva) DNA. Of the 11 metals assessed, brass exhibited the strongest PCR inhibitory effects, for both custom and Quantifiler Trio quantitation assays. Metal ion inhibition varied across the two quantitative PCR assays and the amount of DNA template used. The Quantifiler Trio internal PCR control (IPC) only revealed evidence of PCR inhibition at higher metal ion concentrations, limiting the applicability of IPC as an indicator of the presence of metal inhibitor in a sample. Notably, ferrous ions were found to significantly decrease the extraction efficiency of the DNA-IQ DNA extraction system. The amount of DNA degradation and inhibition in saliva samples caused by metal ions increased with a dilution of the sample, suggesting that the saliva matrix provides protection from metal ion effects.

Evaluation of the Impact of Some Single-Nucleotide Gene Polymorphisms on the Development of Adenomatous Polyps of the Colon in Patients with Irritable Bowel Syndrome.

The number of cases of irritable bowel syndrome is growing worldwide, in which adenomatous polyps can develop as a result of microinflammation of the colonic epithelium. Our study was aimed at the identification of the possible effect of single-nucleotide polymorphisms on the risk of the development of irritable bowel syndrome-related colonic adenomatous polyps. The study involved 187 irritable bowel syndrome patients. The single-nucleotide polymorphisms were investigated by the polymerase chain reaction method and DNA was extracted with the phenol-chloroform: interleukin-1β gene-31C/T (rs1143627), -511C/T (rs16944); interleukin-6 gene-174G/C (rs1800795); interleukin-10 gene-592C/A (rs1800872), -819T/C (rs1800871), -1082A/G (rs1800896); Toll-like receptor-2 gene Arg753Gln (rs5743708); Toll-like receptor-4 gene Thr399ile (rs4986791), Asp299Gly (rs4986790); and metalloproteinase-9 gene-8202A/G (rs11697325). The study of polymorphic loci was checked for compliance with the Hardy- Weinberg equilibrium using Fisher's exact test along with the analyses of the frequency of alleles and the genotypes. The association of diseases with G allele Toll-like receptor-2 gene Arg753Gln (rs5743708) was revealed in irritable bowel syndrome patients with adenomatous polyps of the colon (P < .0006) and AG single-nucleotide polymorphisms s of Toll-like receptor-2 gene (χ2 = 12.78, P < .002); A allele had a protective effect. The AG genotype metalloproteinase-9 gene-8202A/G (rs11697325) polymorphism in irritable bowel syndrome patients with adenomatous polyps of the colon had a protective effect (P < .05). AA genotype interleukin-10 gene-1082A/G (rs1800896) polymorphism in the irritable bowel syndrome patient (χ2 = 33.97, 4.0E-8) can be considered as the risk for adenomatous polyps of the colon in irritable bowel syndrome. G allele Toll-like receptor-2 gene Arg753Gln (rs5743708) and AA genotype interleukin-10 gene-1082A/G (rs1800896) polymorphisms can be the marker of the emergence of adenomatous polyps of the colon concomitant with irritable bowel syndrome.

Candidate germline biomarkers of lenalidomide efficacy in mantle cell lymphoma: the Fondazione Italiana Linfomi MCL0208 trial.

In the Fondazione Italiana Linfomi MCL0208 phase 3 trial, lenalidomide maintenance (LEN) after autologous stem cell transplantation (ASCT) in mantle cell lymphoma (MCL) improved progression-free survival (PFS) vs observation (OBS). The host pharmacogenetic background was analyzed to decipher whether single-nucleotide polymorphisms (SNPs) of genes encoding transmembrane transporters, metabolic enzymes, or cell-surface receptors might predict drug efficacy. Genotypes were obtained via real-time polymerase chain reaction of the peripheral blood germ line DNA. Polymorphisms of ABCB1 and VEGF were found in 69% and 79% of 278 patients, respectively, and predicted favorable PFS vs homozygous wild-type (WT) in the LEN arm was 3-year PFS of 85% vs 70% (P< .05) and 85% vs 60% (P< .01), respectively. Patients carrying both ABCB1 and VEGF WT had the poorest 3-year PFS (46%) and overall survival (76%); in fact, in these patients, LEN did not improve PFS vs OBS (3-year PFS, 44% vs 60%; P= .62). Moreover, the CRBN polymorphism (n= 28) was associated with lenalidomide dose reduction or discontinuation. Finally, ABCB1, NCF4, and GSTP1 polymorphisms predicted lower hematological toxicity during induction, whereas ABCB1 and CRBN polymorphisms predicted lower risk of grade ≥3 infections. This study demonstrates that specific SNPs represent candidate predictive biomarkers of immunochemotherapy toxicity and LEN efficacy after ASCT in MCL.

Monkeypox: A Histopathological and Transmission Electron Microscopy Study.

The global outbreak of human monkeypox virus (hMPXV1) in 2022 highlighted the usefulness of dermatological manifestations for its diagnosis. Infection by the human monkeypox virus thus necessitated inclusion in the diagnostic repertoire of dermatopathology. To assess the histopathological and microscopical findings of cutaneous lesions related to hMPXV infection, we analyzed skin biopsies from patients with positive MPXV DNA polymerase chain reaction presenting with a typical course of hMPXV1 infection. The most prominent histopathological findings were ascribable to a pustular stage in which epidermal necrosis with areas of non-viable keratinocytes and a "shadow cell" appearance were evident; in some cases, the deep portion of the hair follicle and the acrosyringial epithelium were affected. The main cytopathic modifications included ballooning keratinocytes, followed by Guarnieri bodies and a ground glass appearance of the keratinocytes' nuclei, together with a dense mixed inflammatory cell infiltrate with prominent neutrophil exocytosis. Transmission electron microscopy analysis demonstrated viral particle aggregates in the cytoplasm of keratinocytes, without any involvement of the nucleus. Interestingly, we also found the presence of viral particles in infected mesenchymal cells, although to a lesser extent than in epithelial cells. Through this study, we contributed to expanding the histological and microscopic knowledge of the human mpox virus, a key step to understanding current and potential future trends of the disease, as well as of other Orthopoxvirus infections.

Design of a Test to Identify Mutagenic Effects of Hair Dye using Proteus mirabilis

The purpose of this study was to determine the possible mutagenicity of hair dye using the auxotroph’s methionine Proteus Mirabilis as a test. The Ames test was employed to identify reverse mutations in auxotroph’s P. Mirabilis treated with hair dyes. The results demonstrated a significant increase (P< 0.05) in reverse colonies of auxotroph’s P. mirabilis treated with 250 and 500 mg/mL of hair dyes in comparison to the negative control. Reverse mutations in the metE gene of the auxotroph’s P. Mirabilis were identified using DNA sequencing and polymerase chain reaction. The results confirmed the presence of the metE gene at a percentage of (100%) in all isolates treated with hair dyes at 250 and 500 mg/mL. Using BLAST software, the metE gene’s sequence was compared to the gene sequence of a standard isolate. Point mutations in the DNA of the metE gene altered protein translation, according to a genetic study of metE gene isolates treated with hair dyes. Finally, point mutations in the metE genes were identified using amino acid translation analysis of auxotroph’s P. mirabilis isolates treated with hair dyes. Point mutations in the metE gene cause many amino acids to be converted to methionine, which enables auxotroph’s P. mirabilis to grow without the need for methionine from an external source. The study found that the test for identifying mutagens in hair dyes was simple to use and cheap. It has also shown high efficacy in altering the amino acid and nitrogenous base sequences in the metE genes, thus their effect on methionine synthesis.

Partial Thyroid Hormone-Binding Globulin Deficiency: A Case Report and Literature Review.

Thyroxine binding globulin (TBG) deficiency is a rare thyroid disease, mostly caused by genetic mutations and acquired by X-linked recessive inheritance. The clinical features of children with TBG deficiency and their family members were summarised and the Serpina7 gene mutation was analysed, providing a reference for the differentiation of TBG deficiency. Thyroid function was detected in TBG deficient patients, and genetic analysis was performed using polymerase chain reaction (PCR) and direct DNA sequencing to detect the characteristics of TBG mutants. Using "thyroxine binding globulin, gene and mutation" as keywords, PubMed (biomedical literature database), Web of Science and other databases were searched for relevant studies to collect and summarise relevant information. The TBG (14.7 μg/mL), 70% triiodothyronine (T3) (<0.3 nmol/L), total T3 (Tr3) (<0.05 ng/mL) and thyroxine (T4) (14.72 nmol/L) values were lower than normal, while the thyrotropin (TSH) (2.33 uIU/mL), free T3 (FT3) (1.62 pmol/L), and free T4 (FT4) (11.39 pmol/L) values were normal. These values indicate a TBG partially deficient phenotype. Using PCR amplification and direct sequencing of the target gene, a missense mutation in exon 4 of the Serpina7 gene was found in the patient and the father, and the nucleic acid variant was C.909 (exon 4) g > T; the patient was heterozygous and the father was hemizygous. The literature search retrieved a total of 45 studies, most of which were related to mutations in the Serpina7 gene. The mutation locations included exons, introns, enhancers and promoters, with exons the predominant location. A total of 49 variants of the Serpina7 gene were identified. Serpina7 C.909G (P.L303F) is a mutation acquired from the father by X-linked recessive inheritance. The main clinical features of TBG deficiency patients are low serum T4, T3 and TBG levels, normal TSH, FT3 and FT4 levels, and no clinical manifestations.

Low-cost molecular methods to characterise gastrointestinal nematode co-infections of goats in Africa

BackgroundVeterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings.MethodsGoats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and ‘nemabiome’ internal transcribed spacer 2 (ITS-2) amplicon sequencing.ResultsBoth DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species.ConclusionsThese data represent the first ‘nemabiome’ sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition.Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas.Graphical

Highly purified DNA-containing cell envelopes from fungi for direct use in PCR

Efficient DNA sample preparation from fungi with the rigid cell walls is still critical for successful polymerase chain reaction (PCR), one of the basic platforms in molecular diagnostics of fungi, especially in medical mycology. Common methods that involve different chaotropes to yield DNA samples have found a limited application for fungi. Here we describe a novel procedure for efficient production of permeable fungal cell envelopes with DNA inside as suitable templates for PCR. This procedure is facile, relies on boiling of fungal cells in aqueous solutions of selected chaotropic agents and additives and enables to remove RNA and proteins from PCR template samples. The use of chaotropic solutions containing 7 M urea, 1% sodium dodecyl sulfate (SDS), up to100 mM ammonia and/or 25 mM sodium citrate was the best option to yield highly purified DNA-containing cell envelopes from all fungal strains under study, including clinical Candida and Cryptococcusisolates. After treatment with the selected chaotropic mixtures, the fungal cell walls had undergone loosening and were no longer a barrier to release DNA in PCR as evident from electron microscopy examinations and successful target gene amplifications. Overall, the developed simple, fast, and low-cost approach to produce PCR-suitable templates in the form of DNA encased by permeable cell walls can find application in molecular diagnostics.

Outbreak of Burkholderia multivorans among patients at two acute-care hospitals in California, August 2021–July 2022

Background:Burkholderia multivorans are gram-negative bacteria typically found in water and soil. B. multivorans outbreaks among patients without cystic fibrosis have been associated with exposure to contaminated medical devices or nonsterile aqueous products. Acquisition can also occur from exposure to environmental reservoirs like sinks or other hospital water sources. We describe an outbreak of B. multivorans among hospitalized patients without cystic fibrosis at 2 hospitals within the same healthcare system in California (hospitals A and B) between August 2021 and July 2022. Methods: We defined confirmed case patients as patients without cystic fibrosis hospitalized at hospital A or hospital B between January 2020 to July 2022 with B. multivorans isolated from any body site matching the outbreak strain. We reviewed medical records to describe case patients and to identify common exposures. We evaluated infection control practices and interviewed staff to detect exposures to nonsterile water. Select samples from water, ice, drains, and sink splash zone surfaces were collected and cultured for B. multivorans in March 2022 and July 2022 from both hospitals. Common aqueous products used among case patients were tested for B. multivorans. Genetic relatedness between clinical and environmental samples was determined using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic polymerase chain reaction (Rep-PCR). Results: We identified 23 confirmed case patients; 20 (87%) of these were identified at an intensive care unit (ICU) in hospital A. B. multivorans was isolated from respiratory sources in 18 cases (78%). We observed medication preparation items, gloves, and patient care items stored within sink splash zones in ICU medication preparation rooms and patient rooms. Nonsterile water and ice were used for bed baths, swallow evaluations, and ice packs. B. multivorans was cultured from ice and water dispensed from an 11-year-old ice machine in the ICU at hospital A in March 2022 but no other water sources. Additional testing in July 2022 yielded B. multivorans from ice and a drain pan from a new ice machine in the same ICU location at hospital A. All products were negative. Clinical and environmental isolates were the same strain by RAPD and Rep-PCR. Conclusions: The use of nonsterile water and ice from a contaminated ice machine contributed to this outbreak. Water-related fixtures can serve as reservoirs for Burkholderia, posing infection risk to hospitalized and immunocompromised patients. During outbreaks of water-related organisms, such as B. multivorans , nonsterile water and ice use should be investigated as potential sources of transmission and other options should be considered, especially for critically ill patients.Disclosures: None

Molecular prevalence and factors associated with Ehrlichia canis infection in dogs from the North Pantanal wetland, Brazil.

Canine monocytic ehrlichiosis is a vector-borne disease caused by the obligatory intracellular bacterium Ehrlichia canis, which is distributed across tropical and subtropical regions worldwide. Its prevalence within dog populations is high in municipalities located across the Pantanal biome, but it remains unknown in Barão de Melgaço, Mato Grosso, Brazil. This study aimed to determine the molecular prevalence and factors associated with E. canis infection in dogs domiciled in Barão de Melgaço. A cross-sectional study was carried out to investigate the prevalence of E. canis infection in 369 dogs from urban and rural areas in Barão de Melgaço, North Pantanal wetland, Brazil. Initially, the dogs were examined, and, through a questionnaire, the risk factors were investigated. Blood samples were subjected to DNA extraction and PCR was performed to estimate the prevalence of E. canis infection. The molecular prevalence of E. canis infection in dogs was 42.5% and none of the studied variables were significantly associated with polymerase chain reaction (PCR) positivity (p > 0.05). The high molecular prevalence demonstrates an increased transmission of the agent across the city. This also indicates that attention needs to be paid to E. canis infection and control measures should be introduced to prevent its transmission. The demographic and clinical risk factors commonly associated with E. canis infection in this study were not associated with PCR positivity.

Deteksi Gen SLC30A8 pada Sampel Darah Penderita Diabetes Melitus Tipe 2 Dengan Metode Polymerase Chain Reaction

Background: The SLC30A8 gene is a zinc efflux transporter associated with insulin secretion expressed by the pancreas. The SLC30A8 gene causes abnormalities in insulin synthesis, maturation &amp; secretion as well as abnormalities in the body's effectiveness in responding to glucose metabolism. Polymerase Chain Reaction (PCR) is a DNA amplification method that can be used to detect the presence of the SLC30A8 gene in type 2 diabetes mellitus patients. Methods: This study used a descriptive research method conducted at the Molecular Biology Laboratory of Sekolah Tinggi Ilmu Kesehatan Nasional. The sampling technique in this study used a total sampling technique on 25 type 2 diabetes mellitus patients who were members of the Prolanis Diabetes Mellitus Puskesmas Bulu Sukoharjo, Central Java. The detection of the SLC30A8 gene went through several stages, namely DNA isolation, qualitative testing of DNA isolates, quantitative testing of DNA isolates and PCR Results: The results showed the presence of the SLC30A8 gene in all blood samples from type 2 diabetes mellitus patients using the PCR method. The primer used with forward primer 5'-GGACGAAAGAGTTCCCATAGCG-3' and reverse primer 5'- ATAGCAGCATGTTTTGAAGGTGGC-3' attaches specifically to chromosome 8 with a product length of 429 bp. Conclusion: All blood samples of type 2 diabetes mellitus patients detected the SLC30A8 gene

PCR-dipstick DNA Chromatography for detection of Carbapenemase Producing Carbapenem-Resistant Enterobacteriaceae

Carbapenemase producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are an epidemiologically important emerging public health threat. Early and reliable detection of CP-CRE is important for containment of these pathogens. The aim of this study was to evaluate and observe the sensitivity of polymerase chain reaction (PCR)-dipstick DNA chromatography (GeneFields CPE) for detection of carbapenemase genes (NDM, OXA-48, KPC, IMP) in comparison to conventional PCR. In this cross-sectional study, a total of 145 carbapenem resistant Enterobacteriaceae isolates were subjected to test for carbapenemase by phenotypic mCIM and genotypic- conventional PCR and PCR-dipstick DNA Chromatography method. In PCR dipstick DNA chromatography assay, after multiplex PCR a DNA-DNA hybridization-based detection system is applied where amplified products can be easily interpreted by observing a blue line in DNA strip visually within 15 minutes without gel electrophoresis. All the CP-CRE isolates (100%) were positive for either NDM or OXA-48 by PCR-dipstick DNA Chromatography assay, whereas 142 (97.9%) isolates were detected by conventional PCR. The positivity rate of NDM, OXA-48 and both NDM and OXA-48 among CP-CRE isolates were 73.8%, 51.7% and 25.5% respectively. The sensitivity, specificity of PCR-dipstick DNA Chromatography assay for detection of NDM and OXA-48 were 100.0%, 92.7% and 98.6%, 97.2% respectively. This assay showed excellent agreement with conventional PCR for detection of CP-CRE. PCR-dipstick DNA chromatography may be an excellent and robust diagnostic tool in terms of rapidity and sensitivity for the detection of CP-CRE.

Rapid detection and diagnosis of herpetic keratitis using quantitative microfluidic polymerase chain reaction system for herpes simplex and varicella-zoster virus DNA: a case series

BackgroundA microfluidic real-time polymerase chain reaction (PCR) system can rapidly detect the viral DNA in specimens. Detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA in tears is a useful diagnostic tool for herpes simplex virus keratitis (HSK) and herpes zoster ophthalmicus (HZO).MethodsIn total, 20 patients were included in this cross-sectional study. Among them, 8 patients with infectious epithelial HSK and 12 patients with HZO were included in HSK and HZO groups, respectively. In addition, 8 patients with non-herpetic keratitis and 4 healthy individuals without keratitis were included in the control group. Numbers of HSV and VZV DNA copies in tears of all patients and individuals were evaluated using a microfluidic real-time PCR system. Regarding HSV/VZV DNA test, tear specimens were collected by filter paper method using Schirmer’s test paper, and subsequently, DNA was extracted from the filter paper using an automated nucleic acid extractor. Afterward, quantitative PCR was performed using a microfluidic real-time PCR system.ResultsFrom tear collection to real-time PCR result determination, the HSV/VZV DNA test took approximately 40 min. In the HSK group, the sensitivity and specificity of the HSV DNA tests were 100% each. The median value (range) of number of HSV DNA copies for affected eyes was 3.4 × 105 copies/μL (under a lower detection limit of 7.6). In the HZO group, the sensitivity and specificity of the VZV DNA tests were 100% each. The median value (range) of number of VZV DNA copies for affected eyes was 5.3 × 105 copies/μL (under a lower detection limit of 5.6 × 10–2).ConclusionIn conclusion, quantitative PCR for HSV and VZV DNA in tears using a microfluidic real-time PCR system is useful for diagnosing and monitoring HSK and HZO.

The first molecular confirmation of Culex pipiens complex as potential natural vectors of Dirofilaria immitis in Myanmar.

Dirofilariosis, known as one of the most widespread vector-borne zoonotic diseases, is caused by several different species of the nematodes of the genus Dirofilaria, which can be transmitted by Culex, Anopheles and Aedes mosquito vectors. In order to identify key vector mosquitoes of filarial parasites in Myanmar, mosquitoes were collected during three different seasons (summer, rainy and winter) in three townships in Nay Pyi Taw area, Myanmar. DNA extraction and polymerase chain reaction (PCR) analyses were conducted for 185 mosquito pools, with each pool containing 1-10 mosquitoes. Dirofilaria immitis was detected in 20 pools of Culex pipiens complex mosquitoes. The minimum infection rate of mosquitoes was found to be 16.33. The small subunit ribosomal RNA (12S rDNA) gene targeted PCR revealed that the sequences obtained were completely identical to the sequences of D. immitis derived from dogs in China, Brazil and France. The sequences obtained from mitochondrial cytochrome oxidase subunit I (COI) gene PCR exhibited 100% identity with the sequences of D. immitis derived from dogs in Bangladesh, Iran, Japan and Thailand, as well as humans in Iran and Thailand, and mosquitoes in Germany and Hungary. The findings of this study demonstrated that the mosquito species of Cx. pipiens complex are potential mosquito vectors for dirofilariosis in Myanmar.