Amplification Of Specific Genes Research Articles

Molecular Detection of Anaplasma marginale in Capybara (Hydrochoerus hydrochaeris) from Corrientes, Argentina.

Monitoring wildlife health is essential for understanding global disease patterns, particularly as vector-borne infections extend the geographic ranges and thereby hosts due to environmental shifts. Anaplasma marginale, primarily impacting cattle, has economic implications and has been found in diverse hosts, yet its presence in capybaras (Hydrochoerus hydrochaeris), influential in tick-borne pathogen spread, lacks comprehensive understanding. From 2015 to 2022, 14 capybaras were surveyed across two different areas of northeastern Argentina. In 1 of 14 (7%) capybaras, the presence of A. marginale was confirmed through the amplification of specific genes, msp5 and msp1β. In addition, A. marginale DNA was detected in the capybara's blood sample through quantitative PCR, with a cycle threshold value of 30.81 (800 copies per reaction). Amplification of a fragment of the msp1α gene revealed PCR products of three different sizes, suggesting the presence of at least three coinfecting A. marginale variants in the capybara host. This study suggests that capybaras are wild hosts for A. marginale in the Ibera Wetlands in Argentina, potentially influencing the infection dynamics of both domestic and wild species. This finding highlights the necessity for thorough studies on the role of capybaras in disease dynamics, crucial for understanding wildlife health and the spread of disease.

Identification and characterisation of emerging fish pathogens Aeromonas veronii and Aeromonas hydrophila isolated from naturally infected Channa punctata.

Naturally infected Channa punctata exhibiting bacterial septicemic syndrome including ulcerations along with mortality records were collected from a fish farm in Assam during winter season (early November 2020 to early January 2021). The moribund fishes were subjected for bacterial isolation followed by identification of the bacteria. Two dominant emerging bacterial pathogens were identified as Aeromonas veronii (isolate ZooGURD-01) and Aeromonas hydrophila (isolate ZooGURD-05) by standard biochemical characterization and 16S rRNA and rpo B gene amplification. Re-infection experiments of both the bacterial isolates in healthy disease-free C. punctata showed similar symptoms to that of natural infection thus confirming their virulence. The LD50 calculated during challenge test for both the isolates ZooGURD-01 and ZooGURD-05 found to be pathogenic at 2.6 × 104 and 1.6 × 104CFU/fish respectively. Further PCR amplification of specific virulent genes (aerolysin, hemolysin and enterotoxin) confirmed pathogenicity for both isolates. Histopathological examinations of liver and kidney in re-infection experiments showed prominent changes supporting bacterial septicaemia. Antibiotic sensitivity pattern showed that the isolates ZooGURD-01 and ZooGURD-05 were sensitive to 22 and 19 out of 25 antimicrobials respectively. The present study was the first report on the mortality of farmed C. punctata associated with natural infection caused by A. veronii and A. hydrophila with no record of pathogenicity of A. veronii in C. punctata.

Ozone as a disinfectant in laboratory surfaces against the SARS-cov-2 coronavirus

Introduction: The treatment of surfaces with ozone has become important due to the ease of the SARS-CoV-2 virus to reach places where it is not normally disinfected with chemical treatments. Ozone can be supplied from two sources: ozone generators and electrostatic air purifiers, both of which leave no residues that damage the environment. Ozone is highly effective against bacteria, fungi, mold, and virus inactivation. The objective of this work was to investigate the disinfection of surfaces naturally contaminated with SARS-CoV-2 and bacteria by using ozone plasma. Material and methods: We examined the disinfection capacity of ozone plasma against the SARS-CoV-2 and bacteria, through a study of natural contamination in situ. Amplification of specific genes by real-time reverse transcription-polymerase chain reaction of SARS-CoV-2 and microbiological culture of bacteria was performed before and after the disinfection process. Results: SARS-CoV-2 was not detected in all assays; bacteria were not cultivable after disinfection with ozone plasma. Conclusion: Disinfection with ozone plasma technology can be an alternative for their use in a shortage situation of others disinfects. Implications for the use of disinfection technologies of surfaces lab’s and the safety of laboratory personnel are discussed.

Prevalence, virulence genes, and antimicrobial profiles of Escherichia coli O157:H7 isolated from healthy cattle in Tunisia.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is associated with intestinal infection in humans and is considered an important cause of food-borne diseases. The aim of the study was to assess the incidence of E. coli O157:H7 in fecal samples of healthy cattle collected in slaughterhouses (n = 160) and from five farms (n = 100). E. coli isolates were detected on MacConkey agar. A total of 236 E. coli isolates were recovered from fecal samples of healthy cattle. We used sorbitol MacConkey medium to detect non-sorbitol fermenting colonies. These bacteria were examined for the presence of O157:H7 antigen by latex agglutination. The isolation of E. coli O157:H7 has been confirmed with PCR amplification of rfbEO157 and fliCH7 specific genes for serogroup O157 and with multiplex PCR of stx1, stx2, eaeA, and ehxA. All isolates were examined for their susceptibility to 21 antibiotics by the disc diffusion method. Of the 236 E. coli isolates, 4.2% (10/236) were positive for STEC O157:H7. Shiga toxin gene (stx2) and ehxA were present in 70% of isolates, stx1 and eae were confirmed in 60% of the isolates. Other virulence factors screened (fimH, sfa/focDE, cdt3, traT, iutA, and hlyA) were present among the 10 isolates. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole/trimethoprim. All isolates belong to the phylo-group E. This is the first study of the incidence of E. coli O157:H7 in cattle in Tunisia. Our finding proves the existence of STEC O157:H7 in healthy animals producing food for human consumption which could be a source of food-borne disease.

Molecular Identification of Babesia spp. and Anaplasma marginale in Water Buffaloes in Veracruz and Tabasco, Mexico: A Retrospective Study.

Two hundred and thirty-three blood samples of water buffalo were collected on four farms in Veracruz state and Tabasco state, Mexico, to detect and confirm the identities of Babesia and Anaplasma spp. sequences. Nested PCR assays were used for the amplification of specific genes encoding B. bovis rhoptry-associated protein (RAP-1), B. bigemina SpeI-AvaI restriction fragment, and Anaplasma marginale major surface protein 5 (MSP5). Using DNA sequencing and BLASTn analysis for DNA homology hemoparasite identification, the identities of the hemoparasites were established by comparing the nucleotide sequences obtained in this study with those available in the GenBank database at the National Center for Biotechnology Information (NCBI). Water buffalo infection with at least one of the hemoparasites under study was detected in 45% (105/233) of the blood samples, while a mixed infection with B. bovis and B. bigemina was detected in 6.4% (15/233) of samples. For this cross-sectional study, mixed infections with the three hemoparasites were not detected. BLASTn analysis revealed that the nucleotide sequences of the water buffalo isolates shared sequence identity values ranging from 88 to 100% with previously published gene sequences of B. bovis, B. bigemina, and A. marginale. The current results confirm that water buffalo, as cattle, are also carriers of hemoparasite infections that are tick-transmitted, and suggest that they probably have an important role in the epidemiology of bovine babesiosis in Mexico.

Portable Electrochemical DNA Sensors Based on Gene Amplification Reactions to Screen and Identify Pathogen and SNPs.

In this paper, we introduce portable sensors based on genetic measurements that can be used in the field for the diagnosis of infectious diseases and disease risk based on SNPs (single nucleotide polymorphisms). In particular, the sensors are based on electrochemical measurements that can be performed with printed electrodes and small measuring devices. Indicator molecules that can bind to nucleic acid molecules in various ways are already known, and some of these molecules have electrochemical activity. First, we investigated the change in their electrochemical responses in a solution system. As a result, we searched for nucleic acid-binding molecules whose current value changes in the presence of DNA. In addition, when we measured the change in the current value, associated with the amplification of specific genes, such as PCR (polymerase chain reaction) and LAMP (loop-mediated isothermal amplification), we found that the current value decreased with the number of amplifications, indicating that specific genes can be monitored electrochemically. Based on this principle, we showed that pathogenic microorganisms and viruses, such as Salmonella, O157 E. coli, hepatitis B virus, periodontal disease bacteria, antibiotic-resistant bacteria and influenza virus, were able to be measured. The method was also applied to the diagnosis of SNPs, such as ApoE (apolipoprotein E), which is a risk factor for Alzheimer’s disease. Rapid PCR was available with a microfluidic device, and a simple method was also presented with the isothermal amplification of LAMP.

Antimicrobial Susceptibility Profiles among Pseudomonas aeruginosa Isolated from Professional SCUBA Divers with Otitis Externa, Swimming Pools and the Ocean at a Diving Operation in South Africa.

SCUBA divers are predisposed to otitis externa caused by Pseudomonas aeruginosa, which is becoming increasingly multi-drug resistant (MDR). The present work assessed the antibiotic resistance profiles of P. aeruginosa obtained from SCUBA divers and their environment in Sodwana Bay, South Africa. Bacterial isolates from a total of 137 random water and ear swab samples were identified using biochemical and molecular methods. P. aeruginosa strains were further evaluated for antibiotic susceptibility using the Kirby–Bauer assay. Double disk synergy test (DDST) to confirm metallo-β-lactamase (MBL) production and PCR amplification of specific antibiotic resistance genes was performed. All (100%) 22 P. aeruginosa isolates recovered were resistant to 6 of the β-lactams tested including imipenem but exhibited susceptibility to trimethoprim–sulfamethoxazole. MBL production was observed in 77% of isolates while the most prevalent extended-spectrum β-lactamase (ESBL) genes present included blaAmpC (86.9%) followed by blaTEM (82.6%). Sulfonamide resistance was largely encoded by sul1 (63.6%) and sul2 (77.3%) genes with a high abundance of class 1 integrons (77.3%) of which 18.2% carried both Intl1 and Intl2. P. aeruginosa found in Sodwana Bay exhibits multi-drug resistance (MDRce) to several pharmaceutically important drugs with the potential to transfer antibiotic resistance to other bacteria if the judicious use of antibiotics for their treatment is not practiced.

Rapid and Sensitive Pathogen Detection by DNA Amplification Using Janus Particle-Enabled Rotational Diffusometry

Rapid and sensitive detection of infectious bacteria is in all-time high demand to prevent the further spread of the infection and allow early medical intervention. In this study, we use rotational diffusometry (RD), a natural phenomenon characterized by Janus particles, to detect pathogens like Escherichia coli by performing amplification of specific genes. This biosensing method is used to measure the change in viscosity of the fluid in the presence and absence of DNA in the solution by capturing images of modified microbeads at 10 Hz by a CCD camera followed by cross-correlation algorithm analysis. Using rotational diffusometry, we have achieved E. coli detection with 50 pg/μL DNA with a measurement time of 30 s and a sample volume of 2 μL. This sensitivity was achieved with 30 thermal cycles for three different amplicons, viz., 84, 147, and 246 bp. Meanwhile, in the case of 10 and 20 thermal cycles, the detection sensitivity was achieved with 0.1 and 1 ng/μL DNA concentrations for a 246 bp amplicon. Compared with conventional PCR, this technique appears to improve the detection time, thereby reaching a turnaround time of less than 60 min. Other studies showed a successful identification of DNA amplification up to 10 thermal cycles with different sizes of amplicons. The effect of DNA concentration, amplicon size, and the number of thermal cycles on the detection of E. coli was examined in detail and represented in the form of three maps. These maps show the clear difference and the advantages of RD method in comparison with conventional PCR. This unconventional and rapid biosensing method can be used further for downstream application of nucleic acid amplification-based pathogen detection and early disease control.

Post-transcriptional screen of cancer amplified genes identifies ERBB2/Her2 signaling as AU-rich mRNA stability-promoting pathway

Amplification of specific cancer genes leads to their over-expression contributing to tumor growth, spread, and drug resistance. Little is known about the ability of these amplified oncogenes to augment the expression of cancer genes through post-transcriptional control. The AU-rich elements (ARE)-mediated mRNA decay is compromised for many key cancer genes leading to their increased abundance and effects. Here, we performed a post-transcriptional screen for frequently amplified cancer genes demonstrating that ERBB2/Her2 overexpression was able to augment the post-transcriptional effects. The ERBB1/2 inhibitor, lapatinib, led to the reversal of the aberrant ARE-mediated process in ERBB2-amplified breast cancer cells. The intersection of overexpressed genes associated with ERBB2 amplification in TCGA datasets with ARE database (ARED) identified ERBB2-associated gene cluster. Many of these genes were over-expressed in the ERBB2-positive SKBR3 cells compared to MCF10A normal-like cells, and were under-expressed due to ERBB2 siRNA treatment. Lapatinib accelerated the ARE-mRNA decay for several ERBB2-regulated genes. The ERBB2 inhibitor decreased both the abundance and stability of the phosphorylated inactive form of the mRNA decay-promoting protein, tristetraprolin (ZFP36/TTP). The ERBB2 siRNA was also able to reduce the phosphorylated ZFP36/TTP form. In contrast, ectopic expression of ERBB2 in MCF10A or HEK293 cells led to increased abundance of the phosphorylated ZFP36/TTP. The effect of ERBB2 on TTP phosphorylation appeared to be mediated via the MAPK-MK2 pathway. Screening for the impact of other amplified cancer genes in HEK293 cells also demonstrated that EGFR, AKT2, CCND1, CCNE1, SKP2, and FGFR3 caused both increased abundance of phosphorylated ZFP36/TTP and ARE-post-transcriptional reporter activity. Thus, specific amplified oncogenes dysregulate post-transcriptional ARE-mediated effects, and targeting the ARE-mediated pathway itself may provide alternative therapeutic approaches.

P-146 Clinical usefulness of next generation sequencing by liquid biopsy for advanced gastric cancer

Recently, most clinical trials are designed on the basis of protein expression or gene amplification of specific genes. Although, only ERBB2 amplification showed targetable biomarker in advanced gastric cancer (AGC). This study aimed to evaluate the profile of gene alterations and protein expression in AGC by using detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA).

Disinfection of N95 masks artificially contaminated with SARS-CoV-2 and ESKAPE bacteria using hydrogen peroxide plasma: Impact on the reutilization of disposable devices

IntroductionOne of the serious consequences of the SARS-CoV-2 pandemic is the shortage of protective equipment for health personnel. N95 masks are considered one of the essential protective equipment in the management of patients with COVID-19. The shortage of N95 masks implies potential health risks for health personnel and significant economic losses for the health institution. The objective of this work was to investigate the disinfection of N95 masks artificially contaminated with SARS-CoV-2 and ESKAPE bacteria by using hydrogen peroxide plasma.Material and methodsWe examined the disinfection capacity of hydrogen peroxide plasma against the SARS-CoV-2 and 2 members of the ESKAPE bacteria (Acinetobacter baumannii and Staphylococcus aureus) through a study of artificial contamination in situ of N95 masks. Amplification of specific genes by real-time reverse transcription polymerase chain reaction of SARS-CoV-2 and microbiological culture of ESKAPE bacteria was performed before and after the disinfection process.ResultsSARS-CoV-2 was not detected in all assays using 5 different concentrations of the virus, and A baumannii and S aureus were not cultivable with inoculums of 102 to 106 CFU after disinfection tests of N95 masks with hydrogen peroxide plasma.ConclusionDisinfection of N95 masks by using the hydrogen peroxide plasma technology can be an alternative for their reuse in a shortage situation. Implications for the use of disinfection technologies of N95 masks and the safety of health personnel are discussed.

Abstract 1586: ADAM32 is amplified in a wide range of malignancy

Abstract Cytogenetic analysis using karyotyping and fluorescence in-situ hybridization (FISH) has been the method of choice for identifying chromosomal abnormalities in cancer and other diseases. FISH is a powerful tool enabling detection the amplification or deletion of specific genes of interest using specific fluorescent labelled DNA probes. It has high sensitivity and specificity and enables detection of chromosomal abnormalities present in many genetic disorders. Unfortunately, FISH is labor intensive, gives information only regarding chromosomal changes specifically designated by the nucleotide sequence of the probes. Recently, DNA microarray techniques have allowed the simultaneous detection of vast number of chromosomal abnormalities including deletions, amplifications and translocations. These assays are suitable for large scale screening to discovery previously unknown associations of chromosomal lesions with many types of cancer. In present studies, we performed a retrospective analyses on anonymized data from the Constitutional v3 Array from the Texas Tech University Cytogenetics Lab (Children’s Oncology Group reference lab) on 198 sequential patients to determine the frequency of chromosomal aberrations and their disease associations. The patient population included children and adults. The majority of patients had malignancy, but some had pancytopenia or immune deficiency. We found chromosomal rearrangements, amplifications or deletions at 794 loci. The most commonly affected loci were 8p11.21-8p11.22, 15q11.1-15q11.2, 14q11.2, 14q32.33, 9p24 and 9p13. The 8p11.2 locus was amplified in 81 cases and deleted in 17 cases, an amplification/deletion ratio greater than any other locus. At this locus, the KAT6A, ADAM32 and TACC1 genes are known to associate with cancer, while no associations of GPAT4 and TM2D2 have been reported. The ADAM32 gene amplification, previously identified only in breast cancer, was also found in hepatocellular carcinoma, T-cell Lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, Wilm’s Tumor, neuroblastoma, rhabdomyosarcoma, fibromatosis, chondroblastoma, enchondroma, and giant cell tumor of the bone. An oncogenic role of ADAM32 should be investigated in a broad spectrum of pediatric and adult malignancy. Citation Format: Aditya Rajan, Vijay Tonk, Kishore Bhende, Sharda Singh, Sahil Tonk, Sanjay Awasthi. ADAM32 is amplified in a wide range of malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1586.

Abstract P5-12-03: Genome copy number entropy as predictor of response for neoadjuvant therapy in early breast cancer

Abstract Background Copy Number Alterations (CNAs) represent changes in the copy number of genomic segments of somatic cells due to chromosomal instability. CNAs include gene amplifications or deletions and can be involved in tumorigenesis. We analyzed CNAs data in pre- and post-treatment (ttm) tumors from patients (pts) with early breast cancer (BC) in the neoadjuvant trials GEICAM/2006-03 and GEICAM/2006-14, with the aim to identify CNAs in particular genomic regions (genetic entropy) associated with treatment response. Methods GEICAM/2006-03 (NCT00432172) HER2-negative pts were selectively treated according to clinical subtypes: triple negative (TN) pts were treated with standard anthracycline/taxane -based chemotherapy (AT-CT) +/- carboplatin, while luminal patients were randomized to AT-CT vs. hormonotherapy; GEICAM/2006-14 (NCT00841828) HER2+ pts received AT-CT plus anti-HER2 therapy.Shallow-whole genome Illumina sequencing DNA data from 204 paraffin-embedded tumors (100 pre- and 104 post-ttm) were segmented to obtain CNAs and recurrent altered genomic regions were defined. We used Wilcoxon test to analyze the frequency of altered regions and logistic regression analyses to explore their association with tumor response, in terms of pathological complete response (pCR) in breast and axilla. Validation of altered genes associated with therapy response was performed in the microarray gene expression-based Hatzis dataset (GSE25066) from pts receiving neoadjuvant AT-CT (1). Results A total of 672 regions covering the whole genome were identified upon analysis of CNAs data. Regions were categorized according to their alteration status as amplified, normal and lost. Comparative analysis of alterations revealed 11 regions significantly different (p<0.05) in pre- vs post-ttm tumors. Logistic regression analysis showed that in pre-ttm tumors specific alterations of 8 regions localized in 3 different genomic loci (11q12, 16q22 and 21q22) were significantly associated with pCR (p<0.05). Independent analyses of CNAs data with “CGH regions” and “GISTIC2.0” tools confirmed the special relevance of 2 of these 8 regions (#653 and #654), amplified in the locus 21q22.12. This locus contains 20 genes whose expression was tested in Hatzis dataset (1) (GSE25066): the analysis showed that overexpression of 5 of these 20 genes (CHAF1B, CBR1, CBR3, RCAN1 and SLC5A3) turned out to be significantly higher in the cohort of pts who reached pCR, in agreement with our findings. Some of these genes have already been described as proliferation markers (CHAF1B) or involved in treatment response (CBR1) in BC. Other genes related to BC in this genomic region are the transcription factor RUNX1 and the Lysine Methyltransferase SETD4. Conclusions According to our results, neoadjuvant therapy can modulate genomic aberrations landscape in BC. Our data suggest that amplification of specific genes in the genomic locus (21q22.12) is involved in the neoadjuvant therapy response in early BC. (1): Hatzis et al., JAMA 2011, 305(18) 1873-81 Citation Format: Alba E, Rueda OM, Lluch A, Albanell J, Chin S-F, Chacón JI, Calvo L, De la Haba-Rodriguez J, Bermejo B, Ribelles N, Sánchez-Rovira P, Plazaola A, Barnadas A, Cirauqul B, Ramos M, Arcusa A, Carrasco E, Herranz J, Chiesa M, Caballero R, Santonja A, Rojo F, Caldas C. Genome copy number entropy as predictor of response for neoadjuvant therapy in early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-03.

Metagenome complexity and template length are the main causes of bias in PCR-based bacteria community analysis.

Multitemplate PCR is used widely for the study of microbial community diversity. Although such studies have established the abundance of different groups within many natural ecosystems, these reports are limited by uncertainties such as bias and artifacts in the PCR. Bias which is introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. In this study, factors leading to the bias of the multitemplate PCR in bacterial communities were examined and optimized. Comparisons between PCR cycle parameters, DNA polymerases, PCR primer degeneracy, and 16S rRNA gene fragments GC content, revealed that annealing temperatures and DNA structure are predominant factors contributing to the observed bias. Pre-digestion of metagenomic DNA with the restriction enzyme Sau3A I and decreased annealing temperature reduced the bias significantly. The application of these optimized conditions to the ten-species model community in a soil sample verified the validity of these treatments.

Usefulness of multiplex molecular diagnostics in diagnosis and management of critical infections in Picu- A single center retrospective analysis

Background : Sepsis forms a major cause of morbidity and mortality in children.Currently, the gold standard for diagnosis for sepsis is culture. However, they suffer from low yield, delay in providing actionable results and inability to grow fastidious organisms and fungus quickly enough to guide therapy in time. Newer molecular methods like multiplex polymerase chain reaction(PCR) systems have been recommended by Infectious Disease Society of America (IDSA) to improve the yield as well as time to intervention in these life threatening emergencies. Syndrome Evaluation System (SES) is one such test which has shown benefits in neonatal sepsis. Methods : In this retrospective case series, we analysed 57 paediatric cases admitted to our PICU during October 2015 to February 2017. The clinical presentations were sepsis, systemic infections, meningitis and febrile neutropenia. SES is a multiplex PCR based molecular diagnostics involving DNA extraction, multiplex amplification of virulence specific genes followed by sequence specific hybridization. Blood,Cerebrospinal fluid( CSF) and other focal specific samples like Bronchoalveolar Lavage(BAL), etc were tested by SES as well as routine cultures. Decision to change antimicrobial therapy was based on culture and sensitivity results, as well as SES. Outcomes were recorded. Results : 57 cases (28 males, 29 females, mean age 5.72±4.34 years) were analysed. SES and corresponding Blood cultures were done for 26 patients, of which 4 had positive blood cultures, yielding a detection rate of 15.4% vs 26.9% 7 by SES (7/26) .24 CSF and 7 other body fluid samples underwent both cultures and SES. All these cultures were negative. SES was positive in 5 (20.8%)CSF and 7 (100%) other body fluids. Overall, the yield was7.0% for cultures vs33.3% by SES. SES was positive in 89.5% and 94.7% of CRP and Procalcitonin positive cases respectively. SES was negative in 87.7% and 93.3% of CRP and procalcitonin negative cases respectively. Among CSF cell count positive and negative cases, SES was positive and negative in 30% and 81.2% cases respectively. SES results were available to us within 24 hours, as compared to 48 hours for traditional culture. Out of 19 SES positive cases, we escalated antimicrobials in 10 and de-escalated in 9. In 38 SES negatives, we de-escalated antimicrobials in 16 , escalated in 1 and made no change in remaining 21. In effect 36 /57cases (63.15%), underwent change in antimicrobial therapy based on SES results. Conclusion : In this retrospective analysis, we have seen a 4.75 fold increase in detection rates using SES, as compared to traditional culture. SES results seem to correlate with CRP and Procalcitonin. Most importantly, SES results elicited change in antimicrobial therapy in 63.15% of the cases. The fact that we de-escalated antibiotics and antivirals, with no adverse clinical course, shows that emerging molecular diagnostics like SES have good negative predictive value. We feel that molecular diagnostics in PICU setting, used in conjunction with traditional culture, may reduce antimicrobial usage and overall cost of therapy.

Identification of Virulent Capnocytophaga canimorsus Isolates by Capsular Typing.

ABSTRACTCapnocytophaga canimorsus is a dog oral commensal that causes rare but severe infections in humans. C. canimorsus was recently shown to be endowed with a capsular polysaccharide implicated in resistance to the innate immune system of the host. Here, we developed the first C. canimorsus capsular serotyping scheme. We describe nine different serovars (A to I), and this serotyping scheme allowed typing of 25/25 isolates from human infections but only 18/52 isolates from dog mouths, indicating that the repertoire of capsules in the species is vast. However, while only three serovars (A, B, and C) covered 88% of the human isolates tested (22/25), they covered only 7.7% of the dog isolates (4/52). Serovars A, B, and C were found 22.9-, 14.6-, and 4.2-fold more often, respectively, among human isolates than among dog isolates, with no geographical bias, implying that isolates endowed with these three capsular types are more virulent for humans than other isolates. Capsular serotyping would thus allow identification of virulent isolates in dogs, which could contribute to the prevention of these infections. To this end, we developed a PCR typing method based on the amplification of specific capsular genes.

Co-existence of blaOXA-23 and blaNDM-1 genes of Acinetobacter baumannii isolated from Nepal: antimicrobial resistance and clinical significance

BackgroundMolecular analysis of carbapenem-resistant genes in Acinetobacter baumannii, an emerging pathogen, is less commonly reported from Nepal. In this study we determined the antibiotic susceptibility profile and genetic mechanism of carbapenem resistance in clinical isolates of A. baumannii.MethodsA. baumannii were isolated from various clinical specimens and identified based on Gram staining, biochemical tests, and PCR amplification of organism specific 16S rRNA and blaOXA-51 genes. The antibiotic susceptibility testing was performed using disc diffusion and E-test method. Multiplex PCR assays were used to detect the following β-lactamase genes: four class D carbapenem hydrolyzing oxacillinases (blaOXA-51, blaOXA-23, blaOXA-24 and blaOXA-58). Uniplex PCRs were used to detect three class B metallo-β-lactamases genes (blaIMP, blaVIM and blaNDM-1), class C cephalosporin resistance genes (blaADC), aminoglycoside resistance gene (aphA6), and ISAba1 of all isolates. Insertion sequence ISAba125 among NDM-1 positive strains was detected. Clonal relatedness of all isolates were analyzed using repetitive sequence-based PCR (rep-PCR).ResultsOf total 44 analyzed isolates, 97.7% (n = 43) were carbapenem-resistant A. baumannii (CR-AB) and 97.7% (n = 43) were multidrug resistant A. baumannii (MDR-AB). One isolate was detected to be extremely drug resistant A. baumannii (XDR-AB). All the isolates were fully susceptible to colistin (MICs < 2 μg/ml). The blaOXA-23 gene was detected in all isolates, while blaNDM-1 was detected in 6 isolates (13.6%). Insertion sequence, ISAba1 was detected in all of blaOXA-23 positive isolates. ISAba125 was detected in all blaNDM-1 positive strains. The blaADC and aphA6 genes were detected in 90.1 and 40.1%, respectively. The rep-PCR of all isolates represented 7 different genotypes.ConclusionWe found high prevalence of CR-AB and MDR-AB with blaOXA-23 gene in a tertiary care hospital in Nepal. Systemic network surveillance should be established for monitoring and controlling the spread of these resistant strains.

Functional Responses of Salt Marsh Microbial Communities to Long-Term Nutrient Enrichment.

ABSTRACTEnvironmental nutrient enrichment from human agricultural and waste runoff could cause changes to microbial communities that allow them to capitalize on newly available resources. Currently, the response of microbial communities to nutrient enrichment remains poorly understood, and, while some studies have shown no clear changes in community composition in response to heavy nutrient loading, others targeting specific genes have demonstrated clear impacts. In this study, we compared functional metagenomic profiles from sediment samples taken along two salt marsh creeks, one of which was exposed for more than 40 years to treated sewage effluent at its head. We identified strong and consistent increases in the relative abundance of microbial genes related to each of the biochemical steps in the denitrification pathway at enriched sites. Despite fine-scale local increases in the abundance of denitrification-related genes, the overall community structures based on broadly defined functional groups and taxonomic annotations were similar and varied with other environmental factors, such as salinity, which were common to both creeks. Homology-based taxonomic assignments of nitrous oxide reductase sequences in our data show that increases are spread over a broad taxonomic range, thus limiting detection from taxonomic data alone. Together, these results illustrate a functionally targeted yet taxonomically broad response of microbial communities to anthropogenic nutrient loading, indicating some resolution to the apparently conflicting results of existing studies on the impacts of nutrient loading in sediment communities.IMPORTANCE In this study, we used environmental metagenomics to assess the response of microbial communities in estuarine sediments to long-term, nutrient-rich sewage effluent exposure. Unlike previous studies, which have mainly characterized communities based on taxonomic data or primer-based amplification of specific target genes, our whole-genome metagenomics approach allowed an unbiased assessment of the abundance of denitrification-related genes across the entire community. We identified strong and consistent increases in the relative abundance of gene sequences related to denitrification pathways across a broad phylogenetic range at sites exposed to long-term nutrient addition. While further work is needed to determine the consequences of these community responses in regulating environmental nutrient cycles, the increased abundance of bacteria harboring denitrification genes suggests that such processes may be locally upregulated. In addition, our results illustrate how whole-genome metagenomics combined with targeted hypothesis testing can reveal fine-scale responses of microbial communities to environmental disturbance.

Comprehensive analyses using next-generation sequencing and immunohistochemistry enable precise treatment in advanced gastric cancer

In advanced gastric cancer (AGC), most clinical trials are designed on the basis of protein expression or gene amplification of specific genes. Recently, next-generation sequencing (NGS) allowed us to comprehensively profile the tumor gene status. This study aimed to elucidate the profiling between gene alterations and protein expression in AGC to aid in future clinical trials on AGC. Formalin-fixed, paraffin-embedded tumor samples from 121 stage III/IV gastric cancer patients were examined for protein expression of tyrosine kinase receptors (RTKs; ERBB2, EGFR, c-MET, and FGFR2) using immunohistochemistry (IHC). Furthermore, 409 cancer-related genes were sequenced to detect mutations and copy number variations using NGS. Most ERBB2 overexpression (IHC 3+) cases (80.0%) had ERBB2 amplification and did not have other RTK amplification or oncogene mutations. However, one-fourth of MET overexpression cases (25.0%) had ERBB2 alterations. EGFR and FGFR2 overexpression cases had ERBB2 alterations or other gene alterations such as KRAS or PIK3CA. On the other hand, most of the four RTK amplification cases (88.2%) were mutually exclusive with each amplification. However, RTK amplification did not simply correlate with protein overexpression, whereas cases with RTK high-level amplification had protein overexpression and rarely showed other co-existing gene alterations. AGC involves a complicated arrangement of protein expression and gene alterations. Comprehensive analyses of NGS and IHC will be necessary to design the optimal therapy for treating the appropriate population of patients in future clinical trials.

A Rapid, low cost, and efficient method for isolation of high quality mitochondrial DNA from Oryza sativa

A rapid and inexpensive protocol for isolation of mitochondrial DNA from Oryza sativa with negligible genomic DNA contamination is developed without use of density-gradients materials. Mitochondria were isolated from rice seedlings in an in-house lysis buffer containing sucrose followed by DNase I treatment to remove nuclear DNA. Modified CTAB method was used to isolate mitochondrial DNA from isolated mitochondria. The presence of mitochondrial DNA was confirmed by using selective amplification of mtDNA specific genes. PCR amplification was observed in all genes except β-actin gene. In addition, Sanger sequencing and gene mapping to reference gene sequences in public database was performed to confirm the presence of mitochondrial DNA. The mapping analysis showed 99.71% similarity with mitochondrial DNA. The protocol demonstrated high specificity and yielded high purity mitochondrial DNA. It is concluded that the protocol described here will be beneficial for scientific communities by providing a cheap and robust mitochondrial DNA isolation protocol for potential applications.