Polymerase Chain Reaction Amplification Of Genes Research Articles

Whole-Genome Sequencing Enables Molecular Characterization of Non-Clonal Group 258 High-Risk Clones (ST13, ST17, ST147 and ST307) among Carbapenem-Resistant Klebsiella pneumoniae from a Tertiary University Hospital Centre in Portugal.

The carbapenem-resistant Enterobacterales (CRE) strains have been identified by the World Health Organization as critical priority pathogens in research and development of diagnostics, treatments, and vaccines. However, recent molecular information about carbapenem-resistant K. pneumoniae (CRK) epidemiology in Portugal is still scarce. Thus, this study aimed to provide the molecular epidemiology, resistome, and virulome of CRK clinical strains recovered from a tertiary care hospital centre (2019–2021) using polymerase chain reaction (PCR) and the advanced molecular technique whole-genome sequencing (WGS). PCR amplification of carbapenemase genes was performed in 437 carbapenem-resistant K. pneumoniae strains. The most frequent carbapenemases were: KPC-3 (42%), followed by OXA-181 (20%), GES-5 (0.2%), and NDM-1 (0.2%). Additionally, 10 strains (2%) coproduced KPC-3 and OXA-181, and 1 strain coproduced KPC-3 and OXA-48 (0.2%). The genomic population structure of 68 strains characterized by WGS demonstrated the ongoing dissemination of four main high-risk clones: ST13, ST17, ST147, and ST307, while no clones belonging to the European predominant clonal groups (CG15 and CG258) were found. Moreover, we describe one K. pneumoniae ST39-KL62 that coproduced the NDM-1 carbapenemase and the extended-spectrum beta-lactamase CTX-M-15, and one K. pneumoniae ST29-KL54 GES-5 and BEL-1 coproducer. Furthermore, a high prevalence of iron siderophores were present in all CRK strains, with several strains presenting both colibactin and the hypermucoviscosity phenotype. Thus, the data presented here highlight an uncommon molecular epidemiology pattern in Portugal when compared with most European countries, further supporting the emergence and dissemination of nonclonal group 258 hypervirulent multidrug high-risk clones and the need to promote in-depth hospital molecular surveillance studies.

Resistant Clostridium perfringens Isolates from Freshwater Fishes of Dal Lake, Kashmir: A Public Health Concern

The present investigation was aimed to study the antimicrobial profile of Clostridium perfringens isolates in fish samples against 10 commonly used antibiotics. A total of 120 fish samples (60 each of Cyprinus carpio var. communis and Schizothorax niger) caught from different sites of the Dal Lake, Srinagar, India. Using species-specific 16S rRNA gene polymerase chain reaction amplification, 37 (30.8%) fish samples (21 C. carpio var. communis and 16 S. niger) out of 120 were found to be positive for C. perfringens. Antimicrobial resistance study of these positive C. perfringens isolates was done by disc diffusion method. The results revealed a similar pattern of antibiotic resistance in the two fish species where the isolates were sensitive to amoxicillin/clavulanic acid, chloramphenicol and ciprofloxacin while resistant to amikacin, ceftriaxone, metronidazole, norfloxacin and tetracycline. The resistance to antibiotics in C. perfringens isolates from fish is an alarming sign because these broad spectrum antibiotics are commonly used for treatment of the diseases caused by pathogen in humans and livestock.

South Korea’s COVID-19 Infection Status: From the Perspective of Re-positive Test Results After Viral Clearance Evidenced by Negative Test Results

Coronavirus disease (COVID-19) started to occur in South Korea by means of inflow of the virus from abroad, when a case from Wuhan, China, was first confirmed on January 19, 2020. Although South Korea has drastically reduced the number of new confirmed cases and is stabilizing the situation with its exemplary disease prevention policies, there remains a problem. These are cases that had shown negative results to polymerase chain reaction (PCR) (gene amplification) tests as the COVID-19 virus had become undetectable but turned re-positive after a short period. The Central Clinical Committee determined that these re-positive cases after COVID-19 viral clearance are due to the limits of the test method; it is considered that the genetic material of the "dead virus" remaining in a recovered patient's body is amplified during the test process. Comprehending the above evidence, re-positive cases of COVID-19 are not infectious; the virus is not even reactivated. However, further research is required as we lack research results on this subject. Until we can be sure, social distancing and other such policies should be maintained.

Molecular epidemiology of clinically high-risk Pseudomonas aeruginosa strains: Practical overview.

In recent years, numerous outbreaks of multidrug-resistant Pseudomonas aeruginosa have been reported across the world. Once an outbreak occurs, besides routinely testing isolates for susceptibility to antimicrobials, it is required to check their virulence genotypes and clonality profiles. Replacing pulsed-field gel electrophoresis DNA fingerprinting are faster, easier-to-use, and less expensive polymerase chain reaction (PCR)-based methods for characterizing hospital isolates. P. aeruginosa possesses a mosaic genome structure and a highly conserved core genome displaying low sequence diversity and a highly variable accessory genome that communicates with other Pseudomonas species via horizontal gene transfer. Multiple-locus variable-number tandem-repeat analysis and multilocus sequence typing methods allow for phylogenetic analysis of isolates by PCR amplification of target genes with the support of Internet-based services. The target genes located in the core genome regions usually contain low-frequency mutations, allowing the resulting phylogenetic trees to infer evolutionary processes. The multiplex PCR-based open reading frame typing (POT) method, integron PCR, and exoenzyme genotyping can determine a genotype by PCR amplifying a specific insertion gene in the accessory genome region using a single or a multiple primer set. Thus, analyzing P. aeruginosa isolates for their clonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple-locus targeting methods with other methods that can identify specific virulence and antimicrobial genes. Software packages such as eBURST, R, and Dendroscope, which are powerful tools for phylogenetic analyses, enable researchers and clinicians to visualize clonality associations in clinical isolates.

Detection of mecA and panton-valentine leukocidin genes in methicillin resistant staphylococcus aureus isolated from various clinical samples in a tertiary care hospital

Detection of mecA and panton-valentine leukocidin genes in methicillin resistant staphylococcus aureus isolated from various clinical samples in a tertiary care hospital - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/2394-5478.2019.0014, Indian Journal of Microbiology Research-I

Epidemiology of Carbapenem-Resistant Enterobacteriaceae at a Long-term Acute Care Hospital.

BackgroundResidents of long-term acute care hospitals (LTACHs) are considered important reservoirs of multidrug-resistant organisms, including Carbapenem-resistant Enterobacteriaceae (CRE). We conducted this study to define the characteristics of CRE-infected/colonized patients admitted to an LTACH and the molecular characteristics of the CRE isolates.MethodsThis retrospective study was conducted to collect information on demographic and comorbid conditions in CRE-colonized/infected patients admitted to a 77-bed LTACH in Detroit between January 2011 and July 2012. Data pertaining to hospital-related exposures were collected for 30 days before positive CRE culture. Polymerase chain reaction (PCR) gene amplification, repetitive sequence–based PCR, and multilocus sequence typing (MLST) were performed on 8 of the CRE isolates.ResultsThe study cohort included 30 patients with CRE-positive cultures, 24 (80%) with infections, and 6 (20%) with colonization. The mean age of cohort was 69 ±12.41 years; 19 (63%) patients were ventilator-dependent, and 20 (67%) were treated with at least 1 antibiotic. Twenty-three (77%) patients had CRE detected following LTACH admission, and the median days from admission to CRE detection in these patients (interquartile range) was 25 (11–43). Seven more patients were already positive for CRE at the time of LTACH admission. Molecular genotyping and MLST of 8 CRE isolates demonstrated that all isolates belonged to the same strain type (ST258) and contained the blaKPC-3 sequence.ConclusionsThe majority of patients with CRE presented several days to weeks after LTACH admission, indicating possible organism acquisition in the LTACH itself. The genetic similarity of the CRE isolates tested could further indicate the occurrence of horizontal transmission in the LTACH or simply be representative of the regionally dominant strain.

Molecular Typing and Macrolide Resistance of Syphilis Cases in Manitoba, Canada, From 2012 to 2016.

The province of Manitoba, Canada, with a population of approximately 1.3 million, has been experiencing increased incidence of syphilis cases since 2015. In this study, we examined the detection of Treponema pallidum DNA in 354 clinical samples from 2012 to 2016, and determined molecular types and mutations conferring resistance to azithromycin in the polymerase chain reaction (PCR)-positive samples. T. pallidum DNA detection was done by PCR amplification of tpp47, bmp, and polA genes. Syphilis serology results were reviewed for the PCR-positive cases. Molecular typing of syphilis strains was done by analysis of the T, pallidum arp, tpr, and tp0548 gene targets as well as partial sequencing of the 23S rRNA gene for azithromycin resistance. Of the 354 samples tested, 74 individual cases were PCR positive. A result from the treponemal antibody chemiluminescent microparticle immunoassay test was positive in 72 of these cases and that from the Venereal Disease Research Laboratory testing was positive in 66. Mutations conferring resistance to azithromycin were found in all 74 PCR-positive samples. Molecular typing was completed on 57 PCR-positive samples, and 12 molecular types were identified with 14d/g found in 63.2%. Increased strain diversity was observed with 8 molecular types detected in 2016, whereas only 2 to 3 types were found in 2012 to 2014. A patient with 2 episodes of infection 9 months apart caused by different molecular strain types was also identified. The finding of an increase in genetic diversity in the strains in this study and an increase in macrolide resistance compared with previous Canadian reports highlighted the need for continued surveillance including strain characterization.

DETERMINATION AND DISTRIBUTION OF CRY1-TYPE GENES IN Bacillus thuringiensis ISOLATED FROM NORTH INDIA

Bacillus thuringiensis (Bt) is a spore-forming bacterium which produces insecticidal crystal protein in the sporulation phase. Polymerase chain reaction (PCR)-based identification of Bacillus thuringiensis toxin genes has become a routine step in most B. thuringiensis isolation and characterization initiatives. In the present study, eighteen native Bt isolates from diverse habitats of North India were taken for the presence of cry1 type genes. The distribution of cry1 gene families in native Bt isolates was examined by PCR amplification of genes with three sets of corresponding PCR primers. In native Bt isolates many variant bands were also observed in addition to expected bands on PCR amplification. Different sets of primers for the same gene give different results due to different sites of primer binding. Maximum number of isolates showed expected bands when primers designed by Ceron were used compared to other primers. However, with other primers, the numbers of variant bands was larger. The isolates, SK-13, 63 and 105 showed the maximum number of cry1-type genes, followed by SK-20, 28, 48, 88, 94, 301, 304 and 307, whereas SK-3 showed the presence of only 3 cry1-type genes. RFLP analysis of 1.6 kb fragment indicated the presence of variant bands from the reference strains. Several promising isolates with predicted toxicity towards lepidoptera have been observed in this study.

Detection of Mycoplasma agalactiae in Small Ruminants of Southeast Iran.

Agalactia is an infectious and contagious disease of small ruminants caused by Mycoplasma agalactiae (M. agalactiae). Although different microorganism strains contribute to this disease, M. agalactiae is known as the most prominent causative agent. Therefore, this study aimed to investigate the rate of M. agalactiae involvement in contagious agalactia in the southeast region of Iran. Sampling was performed from milk, conjunctiva, ear lesions, and joints exudate of suspicious sheep and goat flocks according to the reports of Iran Veterinary Organization. The presence of Mycoplasma and its species, namely M. agalactiae, was evaluated through microbial culture and polymerase chain reaction (PCR) techniques. The detected microorganisms were confirmed to be Mycoplasma and M. agalactiae by the PCR amplification of 16S rRNA and lipoprotein target genes. According to the findings of present study, 14.8% and 36.0% of the samples were diagnosed as positive for Mycoplasma by culture and PCR, respectively. Moreover, the incidence of M. agalactiae was determined as 6.1% using the specific PCR method. Therefore, it is recommended to identify the other species of Mycoplasma in small ruminant samples involved with contagious agalactiae disease.

Isolation and polymerase chain reaction detection of virulence invA gene in Salmonella spp. from poultry farms in Jos, Nigeria

Background: Salmonella serovars are one of the most common food-borne pathogens, and poultry consumption is responsible for the majority of routes of infection worldwide. There is a paucity of documented data regarding the prevalence of virulence determinant genes in Salmonella serovars in Nigeria. The aim of the study was to isolate Salmonella spp. in selected poultry farms in Jos Metropolis, Plateau State, Nigeria. Methodology: A total of eighty samples were conveniently collected from 18 commercial poultry. The samples were from poultry droppings, egg shells, workers' hands, and feeds. The samples were examined for the presence of Salmonella by standard microbiological techniques. The isolates were phenotypically confirmed using biochemical characterization and virulence gene determined by polymerase chain reaction (PCR). Results: The overall isolation percentage of Salmonella species was 28.75% (23/80). DNA extraction was carried out on the isolated 23 Salmonella isolates and 11 successfully quantified. Of the 11 isolates, ten (91.0%) successfully amplified using the invA gene-specific primers by PCR method. The result indicates the presence of Salmonella in poultry farms, and this posed a major concern for public health. Conclusion: The result showed that the use of PCR amplification of virulence genes in suspected Salmonella spp. from poultry farms proved to be efficient and could serve as an alternative rapid tool for the detection of Salmonella spp. Further large studies with the use of more virulence genes are needed to understand the Salmonella epidemiology in poultry farms that serves as a major protein source of the nation.

Identification and Antimicrobial Activity Detection of Lactic Acid Bacteria Isolated from Corn Stover Silage

A total of 59 lactic acid bacteria (LAB) strains were isolated from corn stover silage. According to phenotypic and chemotaxonomic characteristics, 16S ribosomal DNA (rDNA) sequences and recA gene polymerase chain reaction amplification, these LAB isolates were identified as five species: Lactobacillus (L.) plantarum subsp. plantarum, Pediococcus pentosaceus, Enterococcus mundtii, Weissella cibaria and Leuconostoc pseudomesenteroides, respectively. Those strains were also screened for antimicrobial activity using a dual-culture agar plate assay. Based on excluding the effects of organic acids and hydrogen peroxide, two L. plantarum subsp. plantarum strains ZZU 203 and 204, which strongly inhibited Salmonella enterica ATCC 43971T, Micrococcus luteus ATCC 4698T and Escherichia coli ATCC 11775T were selected for further research on sensitivity of the antimicrobial substance to heat, pH and protease. Cell-free culture supernatants of the two strains exhibited strong heat stability (60 min at 100°C), but the antimicrobial activity was eliminated after treatment at 121°C for 15 min. The antimicrobial substance remained active under acidic condition (pH 2.0 to 6.0), but became inactive under neutral and alkaline condition (pH 7.0 to 9.0). In addition, the antimicrobial activities of these two strains decreased remarkably after digestion by protease K. These results preliminarily suggest that the desirable antimicrobial activity of strains ZZU 203 and 204 is the result of the production of a bacteriocin-like substance, and these two strains with antimicrobial activity could be used as silage additives to inhibit proliferation of unwanted microorganism during ensiling and preserve nutrients of silage. The nature of the antimicrobial substances is being investigated in our laboratory.

Molecular characterization of Duck Plague virus isolated from Bangladesh

Duck plague (DP) is the most feared duck disease in the world. For isolation, identification, molecular detection and characterization of DP virus (DPV), a total of 94 samples were collected from commercial farms (n=6) and households (n=13) from Rajshahi (n=37), Netrokona (n=35) and Mymensingh (n=22) districts of Bangladesh. The samples were processed and inoculated into 11-13 days old embryonated duck eggs for virus propagation. Virus was identified using agar gel immunodiffusion test (AGIT) and passive hemagglutination (PHA) test, and was confirmed by polymerase chain reaction (PCR) targeting DNA polymerase and gC genes, followed by sequencing. Pathogenicity tests were performed using duck embryos, ducklings and ducks. Among the 94 samples, 17 isolates were confirmed as DPV by PCR amplification of partial DNA polymerase (446-bp) and gC genes (78-bp), respectively. One of the isolates (Anatid herpes 1 BAU DMH) was sequenced and found to be closely related with a Chinese variant of DPV (GenBank: JQ647509.1). Thus, we assume that both Bangladeshi and Chinese isolates of DPV may have a common ancestor. http://dx.doi.org/10.5455/javar.2015.b90

HER2 and GATA4 are new prognostic factors for early‐stage ovarian granulosa cell tumor—a long‐term follow‐up study

Granulosa cell tumors (GCTs) carry a risk of recurrence also at an early stage, but reliable prognostic factors are lacking. We assessed clinicopathological prognostic factors and the prognostic roles of the human epidermal growth factor receptors (HER 2–4) and the transcription factor GATA4 in GCTs. We conducted a long-term follow-up study of 80 GCT patients with a mean follow-up time of 16.8 years. A tumor-tissue microarray was immunohistochemically stained for HER2–4 and GATA4. Expression of HER2–4 mRNA was studied by means of real time polymerase chain reaction and HER2 gene amplification was analyzed by means of silver in situ hybridization. The results were correlated to clinical data on recurrences and survival. We found that GCTs have an indolent prognosis, with 5-year disease-specific survival (DSS) being 97.5%. Tumor recurrence was detected in 24% of the patients at a median of 7.0 years (range 2.6–18 years) after diagnosis. Tumor stage was not prognostic of disease-free survival (DFS). Of the molecular prognostic factors, high-level expression of HER2, and GATA4, and high nuclear atypia were prognostic of shorter DFS. In multivariate analyses, high-level coexpression of HER2 and GATA4 independently predicted DFS (hazard ratio [HR] 8.75, 95% CI 2.20–39.48, P = 0.002). High-level expression of GATA4 also predicted shorter DSS (HR 3.96, 95% CI 1.45–12.57, P = 0.006). In multivariate analyses, however, tumor stage (II–III) and nuclear atypia were independent prognostic factors of DSS. In conclusion HER2 and GATA4 are new molecular prognostic markers of GCT recurrence, which could be utilized to optimize the management and follow-up of patients with early-stage GCTs.

Attenuated live vaccine (Bartha-K16) caused pseudorabies (Aujeszky’s disease) in sheep

Pseudorabies (PR, Aujeszky's disease) is an acute, contagious viral disease that affects a wide range of domestic and wild species. In the present study, an outbreak of PR in sheep induced by attenuated live vaccine (Bartha-K16) was diagnosed and analyzed in China. The presence of PR virus (PRV) in brain samples from infected sheep was confirmed by gC gene polymerase chain reaction amplification and PRV-like particle observation by electron microscopy. The molecular characterization of the PRV was performed with homology analysis and phylogenetic tree construction based on the gC gene. This is the first description of an outbreak of PR induced by a live attenuated vaccine (Bartha-K16), indicating that this vaccine is unsuitable for use in sheep.

Molecular Diagnosis and Ocular Imaging of West Nile Virus Retinitis and Neuroretinitis

To describe the ocular features of West Nile virus (WNV) infection proven by serology and molecular diagnostic techniques. Prospective case series. Fifty-two patients who presented to the uveitis clinic with ocular inflammatory signs and history of fever preceding ocular symptoms between January 2010 and January 2012 were enrolled for laboratory diagnosis. Serum samples were collected from 30 healthy controls from the same geographic area. Patients were tested for all endemic infectious diseases that can cause ocular inflammation by serology or molecular diagnostics. When patients had positive antibodies for WNV, serum/plasma samples were tested by real-time reverse transcription (RT) polymerase chain reaction (PCR) and RT loop-mediated isothermal gene amplification assays. The PCR product was subjected to nucleotide sequencing. Fundus fluorescence angiography (FFA), optical coherence tomography (OCT), and indocyanine green angiography were performed. Visual prognosis was analyzed. Clinical signs (retinitis, neuroretinitis, and choroiditis) and ocular complications (decrease in vision). A total of 37 of 52 patients (71%) showed positive results for at least 2 laboratory tests for WNV. Fundus examination revealed discrete, superficial, white retinitis; arteritis; phlebitis; and retinal hemorrhages with or without macular star. The FFA revealed areas of retinal inflammation with indistinct borders, vascular and optic disc leakage, vessel wall staining, or capillary nonperfusion. Indocyanine green angiography confirmed choroidal inflammation in 1 of the patients who was diabetic. The OCT scan of the macula revealed inner retinal layer edema in active inflammation and retinal atrophy in late stage. At the final visit, 43% of patients had visual acuity better than 6/12. In addition to previously reported clinical signs, retinitis, neuroretinitis, and retinal vasculitis were seen in this population. Atrophy of the inner retinal layer was seen on OCT after resolution of inflammation. Visual prognosis was good in patients with focal retinitis and poor in patients with occlusive vasculitis. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Application of peptide nucleic acid (PNA)-PCR clamping technique to investigate the community structures of rhizobacteria associated with plant roots

The contamination of plant organelle (mitochondria and plastid) genes in the DNA extraction step becomes a major drawback in investigating the community structures of bacteria associated with plant samples. This is because organelle small subunit ribosomal RNA (SSU rRNA) genes are easily amplified by polymerase chain reaction (PCR) with a set of universal primers for bacteria. To suppress the PCR amplification of the organelle SSU rRNA genes, a peptide nucleic acid (PNA)-PCR clamping technique was applied for selective amplification of bacterial SSU rRNA genes. The PNA oligomers, which had sequences that were complementary to mitochondria and plastid SSU rRNA genes, were designed to overlap the region in the 1492r primer-binding site. PCR with the PNA oligomers significantly suppressed the amplification of the organelle SSU rRNA genes from spinach and cucumber roots. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that the conventional amplification without PNA oligomers generated the predominant T-RFLP fragments derived from mitochondria and plastids, whereas there was little detection of the rhizobacterial fragments. In contrast, several other T-RFLP fragments derived from rhizobacteria were detected in the products amplified with PNA oligomers, thereby enabling us to differentiate the community structures in spinach and cucumber roots. Thus, application of PNA-PCR clamping was considered to be effective and is a useful technique to amplify the rhizobacterial SSU rRNA genes from selectively extracted DNA containing plant mitochondria and plastid genes.

Phenotypic and Genotypic Methods for Detection of Extended Spectrum β Lactamase Producing Escherichia coli and Klebsiella pneumoniae Isolated from Ventilator Associated Pneumonia

Ventilator Associated Pneumonia (VAP) is one of the common nosocomial infections associated with high morbidity due to multidrug resistant pathogens. Rapid spread of resistance to broad spectrum beta-lactams in pathogenic strains causes antibiotics ineffectiveness and increased severity of illness. The CTX-M is the most dominant Extended Spectrum β Lactamase (ESBL) among Enterobacteriaceae in many regions of the world. The aim of the study was to identify the occurrence of ESBL and detect the genes responsible for ESBL production by conventional Polymerase Chain Reaction (PCR) method. This prospective study included patients, clinically diagnosed as VAP. Endotracheal aspirates (EA) were collected and cultured by quantitative method. The bacterial isolates were identified as per standard methods. Isolates resistant to 3(rd) generation cephalosporins were screened for ESBL production by disk approximation method and combination disc diffusion method. Isolates confirmed as ESBL producers were subjected to genotyping by conventional PCR. Statistical analysis was done by using MS Excel sheet. Descriptive statistics like percentage was done in the study. Among the isolates from 428 patients who developed VAP, 144 isolates belonged to the Enterobacteriaceae family (Klebsiella pneumoniae 87 and Escherichia coli 57). A total of 66 isolates (28 Klebsiella pneumoniae and 38 Escherichia coli) were confirmed as ESBL producer by disc approximation method and 63 isolates by double disc combination method. In the present study by conventional PCR bla CTX-M was the common gene in 48.5% strains followed by 22.22% bla SHV and 14.81% bla TEM. The genotypic methods using specific PCR amplification of resistance genes seems to have 100% specificity and sensitivity in detection of ESBL when compared to phenotypic methods which lacks the constant sensitivity.

PAH concentration gradients and fluxes through sand cap test cells installed in situ over river sediments containing coal tar

Short-term performance of permeable sand cap test cells, installed over sediment containing liquid coal tar was monitored on the Grand Calumet River (Hammond, Indiana, USA). The sand cap test cells included two sand-only cells, two test cells containing a sand/peat mixed layer, two test cells containing a sand/organoclay mixed layer, and two sediment control cells. In each test cell, six monocyclic and twelve polycyclic aromatic hydrocarbons (MAHs and PAHs) were monitored over an 18 month period, and interfacial water flow was monitored periodically. Seepage velocities ranged from 3.8 cm per day into the sediments to 3.2 cm per day out of the sediments, with discharge out of the sediments being observed more often. A ferric iron test indicated that stratified oxic-anaerobic layers were formed in the caps. Within the sand caps, concentrations of MAHs and PAHs fluctuated with time, and this fluctuation was more significant near the bottom. Near the top, most of the MAHs and PAHs were attenuated above 95% in the first year of the study, but their attenuation rates decreased in the second year due to recontamination of the surface of the caps by the surrounding sediments. Functional genes involved in PAH degradation were detected by polymerase chain reaction (PCR) in upper and lower sections of the caps for each of the three treatments. Bacterial communities were characterized by PCR amplification of 16s rRNA genes and denaturing gradient gel electrophoresis (DGGE). The results indicate that the rate and direction of sediment porewater flow is an important factor for properly designing any remedial sand cap, and that biodegradation of many of the MAH and PAH compounds was likely a major removal mechanism leading to attenuation through the test cells.

Pacemaker endocarditis caused by Propionibacterium acnes: A case report

A 74-year-old man, with a permanent pacemaker placed 2 years ago for high-grade atrioventricular block, was admitted for worsening fatigue, confusion, and thrombocytopenic purpura without fever. White blood cell count and C-reactive protein were elevated, and echocardiography revealed a 6 × 3-cm echogenic mass surrounding the pacemaker leads. Multiple blood cultures were performed, and only 1 bottle grew Propionibacterium acnes at 93 hours. The patient underwent surgery, and 16S rRNA gene polymerase chain reaction amplification confirmed the presence of P. acnes in the removed vegetation. Patients with late-onset, device-related endocarditis often present with vague symptoms and fever may be absent, obscuring the clinical diagnosis. Blood cultures and transesophageal echocardiography are key diagnostic tests. As a slow-growing, low virulent, and common human skin germ, P. acnes can be wrongly considered as a blood culture contaminant.

Rapid molecular detection of metastatic head and neck squamous cell carcinoma as an intraoperative adjunct to sentinel lymph node biopsy

Clinical staging of early head and neck squamous cell carcinoma (SCCHN) is often inaccurate, leading to elective neck dissection to detect the 30% of patients with micrometastatic disease. Sentinel node biopsy accurately stages the regional lymphatics, but intraoperative pathology is only moderately sensitive, and final pathology takes several days to complete. To facilitate immediate neck dissection where necessary, we have identified several promising marker genes of SCCHN metastasis and developed a rapid, accurate, and automated quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) assay for intraoperative use. Prospective tissue collection, retrospective pathologic correlation with qRT-PCR. From a 40-gene marker screen, we quantified expression of 11 potential tumor genes using a test set of primary tumors (n = 32) and metastatic (n = 19) and benign (n = 10) lymph nodes. Eight patients' paired primary tumor and metastatic nodes were included. A validation set of 442 grossly tumor-negative nodes was evaluated for expression of the most promising markers, comparing metastasis detection by qRT-PCR with pathologic analysis (hematoxylin and eosin and immunohistochemistry). A novel multiplexed, automated, single-tube qRT-PCR assay was used to analyze more than 100 lymph nodes using a two-marker, 35-minute assay to determine its negative predictive value. Based on expression of 11 tumor-associated genes from the marker screen, the two most promising markers of SCCHN metastasis in the test set, pemphigus vulgaris antigen (PVA) and tumor-associated calcium signal transducer 1 (TACSTD1), also known as epithelial cell adhesion molecule (EpCAM), were selected. Development of a multiplexed qRT-PCR assay for the detection of metastasis compared favorably with pathologic analysis in the additional 442-node set. A rapid, multiplexed assay using PVA and TACSTD1 demonstrated excellent reproducibility, linearity, and accuracy (≈96% negative predictive value) for identifying positive (n = 40) and negative (n = 62) nodes in a validation subset. Detection of metastatic SCCHN using multiplexed qRT-PCR can be rapid, accurate, and automated and may enable sentinel node biopsy to be used for intraoperative decision-making. PCR amplification of tumor marker genes is an effective method of intraoperative molecular staging of SCCHN and could more appropriately guide application of neck dissection in pN+ SCCHN patients, sparing 60% to 70% of pN0 patients from unnecessary neck dissection. This technique may also be used for identifying residual neck disease posttreatment, using outpatient fine-needle aspiration biopsy specimens.