Polymerase Chain Reaction-based DNA Research Articles

A Study on Maternal Viral Load, CD4 Cell Counts and Time of Mother-to-Child Transmission of HIV-1 in Two Hospitals in Central Ethiopia

Background: Vertical transmission of HIV among women who have no access to ART is estimated to be 25% - 35% in Africa. Objective: The aim of the study was to assess the time of MTCT of HIV-1 the efficacy of ART on prevention of mother-to-child transmission (MTCT) of HIV-1 in two hospitals. Methods: Prospective cohort study was conducted from November 2011 to May 2012. A total of 162 participants, 81 mothers (24 non-breastfeeding and 57 breastfeeding) and 81 infants were involved in the study. Venous blood from mothers and heel blood (dried blood spot) from infants were collected and processed in Adama Regional laboratory. All dried blood spot specimens of infants reactive on ELISA test were assessed by DNA-based polymerase chain reaction tests (DNA PCR) to determine HIV infection. Data were entered and analysed using SPSS version 19 computer software. Results: Five infants were infected with HIV at the end of this study. The overall rate of vertical transmission of HIV was 6.2%. Maternal viral load at delivery was independently associated with both in-utero and intra-partum transmission (OR = 27.0, 95% CI, 3.5 - 210, p = 0.001). Maternal viral load at 6 weeks of birth and low infant birth weight was associated with intra-partum transmission among breastfeeding mothers (OR = 25.5, 95% CI, 1.14 - 572, p = 0.04) and (OR = 29.6, 95% CI, 3.2 - 273, p = 0.004), respectively. There were 40% MTCT of HIV among non ARV drug users and only 3.9% among those used ARV drugs during their current pregnancy. Conclusion: Strategies planned to reduce maternal viral load during pregnancy can be successful in substantially reducing vertical transmission of HIV. In addition, other contributing factors for MTCT of HIV-1 should be controlled.

HLA‐DR typing in polyarticular juvenile idiopathic arthritis: a study from a tertiary care hospital in northern India

Many studies of human leukocyte antigen (HLA) association with juvenile idiopathic arthritis (JIA) have reported conflicting results, which were probably related to ethnic differences. Moreover, in India, studies on HLA-DR typing on JIA, particularly polyarticular JIA, is lacking. The aim of our study was to reveal the frequency of HLA DR types in a cohort of polyarticular JIA in northern India. Fifty-two polyarticular JIA patients were included as per the recent International League of Associations for Rheumatology classification, 2001. HLA-DR typing was performed in 21 patients (18 rheumatoid factor [RF]+ and three RF-) by a DNA-based polymerase chain reaction method for the determination of HLA alleles using sequence specific primers (SSP). The results were compared with that of 23 healthy controls of the same age and sex. HLA-DR4 was present in five cases (23%) in the diseased group while only in one case (4.3%) in the control group with a relative risk of 5.47, but when compared with only RF+ polyarticular JIA, HLA-DR4 was found to be significantly high (27.7% vs. 4.43%; P < 0.05) with a relative risk of 6.3. Further, DR4, DR1, DR2, DR9, DR10 were also non-significantly high in these patients with relative risk of 3.2 for DR9 and 1.8 for DR10. In contrast, HLA-DR6 was seen only in 5.5% of polyarticular JIA cases, whereas it was present in 39% of controls (P < 0.05), a showing negative association. HLA-DR4 codes for susceptibility to RF+ polyarticular JIA with a six-fold risk, whereas HLA-DR6 offers protection.

Donor-Derived Metastatic Melanoma in a Liver Transplant Recipient Established by DNA Fingerprinting

Metastatic melanoma is a donor-derived malignancy that has rarely been reported in liver allograft recipients. We present a case of a transmitted donor-derived melanoma to a liver allograft recipient in whom the diagnosis was established by polymerase chain reaction-based DNA fingerprinting. A 52-year-old African-American man underwent a successful orthotropic liver transplant for alcohol-induced cirrhosis. One year after the orthotropic liver transplant, he presented at our institution with diffuse abdominal pain, and a computed tomography scan of the abdomen and chest showed innumerable masses diffusely involving the liver and multiple subcutaneous nodules in the abdominal and chest wall. A liver biopsy confirmed the diagnosis of metastatic melanoma. The origin of melanoma was traced to the donor by DNA fingerprinting of the native liver, the donor liver, and the donor gallbladder. Chemotherapy was initiated with temozolomide (75 mg/m² daily) and thalidomide (50 mg daily), to which he responded within 8 weeks with radiologic improvement in metastatic lesions. Tacrolimus was switched to sirolimus because of renal insufficiency as well as reported effectiveness against melanoma. Our patient survived for 9 months after the diagnosis of metastatic melanoma. He ultimately died of brain metastases. Donor-derived metastatic melanoma is a rare cancer with the highest transmission and mortality rates, which requires better recognition. Prompt diagnosis of donor-derived melanoma is critical and can be achieved reliably with polymerase chain reaction-based DNA analysis. Management options after diagnosis include de-escalation of immunosuppression, with or without urgent organ removal or retransplant. The roles of chemotherapy, immunotherapy, and radiotherapy require further study.

Quantification of Live 'Candidatus Liberibacter asiaticus' Populations Using Real-Time PCR and Propidium Monoazide.

Huanglongbing (HLB) is a devastating citrus disease. It is associated with a phloem-restricted bacterium, 'Candidatus Liberibacter asiaticus', and primarily transmitted by Asian citrus psyllid in Florida. Because Liberibacter cannot be cultured, early diagnosis of HLB relies on DNA-based polymerase chain reaction (PCR), including real-time quantitative (q)PCR. Although estimating genomes from live bacteria (GLB) is critical for HLB research, PCR does not distinguish between live and dead cells and, thus, does not estimate GLB in hosts. Propidium monoazide (PMA), a novel DNA-binding dye, has been successfully used on many bacterial pathogens to effectively remove DNA from dead cells but there is no report of its use on uncultured bacteria. In this study, PMA-qPCR protocols were first optimized to work with plant and psyllid samples, respectively. Both TissueLyser treatment and plant tissue were demonstrated to have an insignificant impact on the GLB detected by PMA-qPCR. Finally, a standard curve for GLB determination was successfully established between PMA-qPCR results and microscopic counts and then applied in two studies with different greenhouse plant samples. This rapid qPCR method provides a more accurate way to determine GLB in HLB hosts which, in turn, should benefit disease epidemiology studies and serve as a crucial component in HLB management.

Identification of HESX1 mutations in Kallmann syndrome

To determine whether HESX1 mutations are present in patients with idiopathic hypogonadotropic hypogonadism (IHH)/Kallmann syndrome (KS). Polymerase chain reaction-based DNA sequencing was performed on 217 well-characterized IHH/KS patients. Putative missense mutations were analyzed by sorting intolerant from tolerant (SIFT) and Clustal Ω. Academic medical center. Two hundred seventeen patients with IHH/KS and 192 controls. Deoxyribonucleic acid was extracted from patients and controls; genotype/phenotype comparisons were made. Deoxyribonucleic acid sequence of HESX1, SIFT analysis, and ortholog alignment. Two novel heterozygous missense mutations (p.H42Y and p.V75L) and previously reported heterozygous missense mutation p.Q6H in HESX1 were identified in 3 of 217 patients (1.4%). All were males with KS. Both p.Q6H and p.H42Y were predicted to be deleterious by SIFT, whereas p.V75L was conserved in 8 of 9 species. No other IHH/KS gene mutations were present. HESX1 mutations may cause KS in addition to more severe phenotypes. Our findings expand the phenotypic spectrum of HESX1 mutations in humans, thereby broadening its role in development.

Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction

Molecular biological methods for the detection of periodontitis-associated bacteria based on DNA amplification have many advantages over classical culture techniques. However, when it comes to assessing immediate therapeutic success, e.g. reduction of viable bacteria, DNA-based polymerase chain reaction is unsuitable because it does not distinguish between live and dead bacteria. Our objective was to establish a simple RNA-based method that is easily set up and allows reliable assessment of the live bacterial load. We compared conventional quantitative real-time PCR (qPCR), propidium monoazide-qPCR and reverse transcription qPCR (RT-qPCR) for the detection of periodontal pathogens after antibiotic treatment in vitro. Applicability was tested using clinical samples of subgingival plaque obtained from patients at different treatment stages. The bacterial load was remarkably stable over prolonged periods when assessed by conventional qPCR, while both propidium monoazide intercalation as well as cDNA quantitation showed a decline according to decreasing numbers of viable bacteria after antibiotic treatment. Clinical samples of subgingival plaque were directly subjected to DNase I treatment and RT without previous extraction or purification steps. While the results of the DNA- and RNA-based methods are comparable in untreated patients, the classical qPCR frequently detected substantial bacterial load in treated patients where RT-qPCR no longer indicates the presence of those pathogens. The disagreement rates ranged between 4 and 20% in first visit patients and 8-50% in the group of currently treated patients. We propose to use RNA-based detection methods to verify the successful eradication of periodontal pathogens.

VKORC1 C1173T and VKORC1 G-1639A Gene Polymorphisms in Turkish Behçet’s Patients with Ocular and Non-ocular Involvement

Background: Behçet’s disease (BD) is a multisystemic vasculitis with unknown etiology. Vitamin K epoxide reductase complex subunit 1 (VKORC1) is the key enyzme in the formation of active vitamin K that is a cofactor of various coagulation factors. Polymorphisms of the VKORC1 may affect the levels of active forms of vitamin K-dependent coagulation proteins and the tendency to thrombosis. The current study aimed to evaluate the role of VKORC1 gene polymorphisims in ocular and non-ocular Behçet’s disease.Methods: VKORC1 C1173T (rs 9934438) and G-1639A (rs 9923231) gene polymorphisms were evaluated by real-time polymerase chain reaction-based DNA analysis. The frequency of alleles and distribution of genotypes were assessed by the chi-squared test. Genotype distribution and Hardy–Weinberg equilibrium were tested with the χ2 test for quality of fit.Results: The distribution of GG, GA and AA and CC, CT, TT genotypes and the frequency of G,A and C,T alleles were not found to be different between patients and controls (p = 0.5651; p = 0.335 respectively), as well as patients with or without eye involvement (p = 0.9267; p = 0.384 respectively).Conclusion: VKORC1 polymorphisms seem not to be related with the thrombotic state of systemic and ocular Behçet’s disease.

A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.

Accuracy and reliability of Dreissena spp. larvae detection by cross-polarized light microscopy, imaging flow cytometry, and polymerase chain reaction assays

The expansion of Dreissena spp. mussels into the western United States has generated an increased need for reliable early detection methods, especially for larvae (veligers), which are a primary transport vector and an indicator of spawning adults. Cross-polarized light microscopy (CPLM) currently provides the most reliable means for detecting quagga and zebra mussel (Dreissena spp.) larvae in plankton samples. In this study a double-blind experiment was undertaken to assess the current reliability of 3 different methods for detecting Dreissena spp. larvae in plankton samples. Methods included CPLM, imaging flow cytometry (IFC), and DNA-based polymerase chain reaction (PCR) assays. We distributed 216 reference samples consisting of concentrated plankton spiked with known numbers of Dreissena spp. larvae to 19 laboratories for analysis. Results indicated that presence/absence detection CPLM was the most reliable (96.3% accuracy), IFC analysis was next most reliable (91.7% accuracy), and PCR was the least reliable (75.8% accuracy). The most prevalent type of error associated with all the methods was false negatives, suggesting that all methods are more likely to fail to detect the presence of larvae rather than to falsely indicate their presence.

Development of a Real-Time Pcr Assay for Detection of Planktonic Red King Crab (Paralithodes camtschaticus(Tilesius 1815)) Larvae

ABSTRACT The Alaskan red king crab (Paralithodes camtschaticus) fishery was once one of the most economically important single-species fisheries in the world, but is currently depressed. This fishery would benefit from improved stock assessment capabilities. Larval crab distribution is patchy temporally and spatially, requiring extensive sampling efforts to locate and track larval dispersal. Large-scale plankton surveys are generally cost prohibitive because of the effort required for collection and the time and taxonomic expertise required to sort samples to identify plankton individually via light microscopy. Here, we report the development of primers and a dual-labeled probe for use in a DNA-based real-time polymerase chain reaction assay targeting the red king crab, mitochondrial gene cytochrome oxidase I for the detection of red king crab larvae DNA in plankton samples. The assay allows identification of plankton samples containing crab larvae DNA and provides an estimate of DNA copy number present i...

PIK3CA Mutations in Advanced Cancers: Characteristics and Outcomes

PIK3CA mutations are frequently diagnosed in diverse cancers and may predict response to PI3K/AKT/mTOR inhibitors. It remains unclear whether they are associated with other characteristics. We analyzed characteristics and outcome of 90 consecutive patients with diverse advanced tumors and PIK3CA mutations and 180 wild-type PIK3CA controls matched by tumor type, gender, and age referred to the Clinical Center for Targeted Therapy. PIK3CA and MAPK mutations (KRAS, NRAS, and BRAF) were analyzed using polymerase chain reaction-based DNA sequencing. The most frequent PIK3CA mutations were E545K (31/90, 34%), E542K (16/90, 18%) in exon 9, and H1047R (20/90, 22%) in exon 20. PIK3CA mutations compared to wild-type PIK3CA were associated with simultaneous KRAS (p=0.047) and MAPK mutations (p=0.03), but only MAPK mutations were confirmed as having an independent association in multivariate analysis. Rates of lung, bone, liver and brain metastases were similar in PIK3CA-mutant and wild-type patients. Patients with PIK3CA mutations treated on trials with PI3K/AKT/mTOR inhibitors had a higher partial/complete response (PR/CR) rate than wild-type PIK3CA patients treated with their best phase I therapy (10/56, 18% vs. 12/152, 8%; p=0.045), but not a prolonged progression-free survival. Patients with H1047R PIK3CA mutations had higher PR/CR rate with PI3K/AKT/mTOR inhibitors compared to wild-type PIK3CA patients treated with their best phase I therapy (6/16, 38% vs. 12/152, 8%; p=0.003). In conclusion, PIK3CA mutations in diverse cancers were not associated with clinical characteristics, but were correlated with MAPK mutations. PIK3CA mutations, especially, H1047R, were associated with attaining a PR/CR to PI3K/AKT/mTOR pathway inhibitors.

Sex identification of ancient DNA samples using a microfluidic device

A microfluidic device has been developed for the sex identification of ancient DNA samples and works by manipulating liquids within an environment of micrometer dimensions. In this work a range of microfluidic DNA extraction methods were evaluated for their compatibility with ancient DNA samples, and the use of streptavidin-coated super paramagnetic particles to isolate biotin-labeled abasic sites within damaged DNA was shown to be the most reproducible. Polymerase chain reaction-based DNA amplification was possible on the microfluidic device when less than 50 pg of template DNA was added. As a proof-of-principle, powdered bone samples were analysed using the integrated methodology developed. Following conventional capillary gel electrophoresis, two out of the three samples produced positive amplification results and were successfully identified as female. These sex identifications were corroborated by independent Amelogenin, anthropological and Y chromosome analysis. The work reported here is the first step in the development of a complete miniaturized microfluidic system that would enable on-site ancient DNA analysis.

The prevalence of digenic mutations in patients with normosmic hypogonadotropic hypogonadism and Kallmann syndrome

To determine the prevalence of digenic mutations in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS). Molecular analysis of DNA in IHH/KS patients. Academic medical center. Twenty-four IHH/KS patients with a known mutation (group 1) and 24 IHH/KS patients with no known mutation (group 2). DNA from IHH/KS patients was subjected to polymerase chain reaction-based DNA sequencing of the 13 most common genes (KAL1, GNRHR, FGFR1, KISS1R, TAC3, TACR3, FGF8, PROKR2, PROK2, CHD7, NELF, GNRH1, and WDR11). The identification of mutations absent in ≥188 ethnically matched controls. Both SIFT (sorting intolerant from tolerant) and conservation among orthologs provided supportive evidence for pathologic roles. In group 1, 6 (25%) of 24 IHH/KS patients had a heterozygous mutation in a second gene, and in group 2, 13 (54.2%) of 24 had a mutation in at least one gene, but none had digenic mutations. In group 2, 7 (29.2%) of 24 had a mutation considered sufficient to cause the phenotype. When the 13 most common IHH/KS genes are studied, the overall prevalence of digenic gene mutations in IHH/KS was 12.5%. In addition, approximately 30% of patients without a known mutation had a mutation in a single gene. With the current state of knowledge, these findings suggest that most IHH/KS patients have a monogenic etiology.

Protein or no protein? Using PCR for detecting allergens in foods

The latest amendment (2007/68/EC) of annex IIIa of the European Directive 2000/13/EC currently requires 14 groups of allergenic food ingredients to be mandatory labelled. The labelling does not refer to a certain compound of an allergenic food but the allergenic food itself. Thus, every method that has been demonstrated to reliably detect the target food is applicable. State of the art in the detection of allergenic foods has been the protein based Enzyme-Linked Immuno Sorbent Assay (ELISA) and meanwhile, numerous studies have demonstrated good correlation between protein based ELISA and DNA based Polymerase Chain Reaction (PCR) methods. Even in protein enriched isolates or concentrates DNA has been proven to be an alternative molecular marker for the presence of an allergenic food. Since 2005 the number of scientific publications on PCR for allergen detection has increased tremendously, of which real-time PCR with sequence specific fluorescent probes is considered state-of-the-art technology. Both ELISA and PCR exhibit strengths and limitations: ELISA are considered a quantitative methodology with high sensitivity at a level of 1-10 mg/kg. However, known cross-reactivity to phylogenetically closely related foods or ingredients thereof may lead to false-positive results. By contrast PCR offers unparalleled specificity to avoid complaints or potentially expensive food recalls due to false-positives. For PCR, a sensitivitiy below 10 mg/kg, which is considered sufficient in comparison to known clinical threshold data, is feasible. Moreover, real-time PCR allows multi-allergen screening in one run. In addition, first PCR methods with quantitative features have been published and more are expected in the near future. Thus, PCR may complement or even substitute ELISA depending on the allergenic food to detect.

The Association of Protease Activated Receptor 1 gene −506 I/D Polymorphism with Disease-Free Survival in Breast Cancer Patients

Recent data have shown that tumor development and dissemination may be regulated by procoagulant/anticoagulant axis. The aim of the present study was to search for an association of the protease activated receptor (PAR)1 gene -506 insertion/deletion (I/D), factor V Leiden (FVL), prothrombin (PT) G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms with disease-free survival (DFS) in breast cancer. Genotyping of -506 I/D in the promoter region of PAR1 gene was performed by single-strand conformation polymorphism analysis and sequencing. FVL, PT G20210A, and MTHFR C677T were also determined by the method of polymerase chain reaction-based DNA analysis. Data regarding patient's age, menopausal status, tumor size, lymph node status, disease stage, tumor grade, estrogen and progesterone receptor, c-erb B2 expression, PAR1 -506 I/D, MTHFR C677T, FVL, and PT G20210A polymorphisms were examined by the univariate and multivariate analyses. Recurrent disease occurred in 29 patients (19.6 %) within a median of 20 months. It was found that tumor size, lymph node status, tumor stage, tumor grade, c-erbB2 expression, and PAR1 -506 I/D polymorphism were associated with DFS when Kaplan-Meier method was applied (P<.05). By Cox proportional hazards model, the presence of allele D at -506 locus (P=.0249) and small tumor size (P=.0001) were significant favorable prognostic factor, but c-erbB2 expression was an adverse prognostic factor (P=.0049). Our study suggested the protective effect of the allele D at -506 locus of PAR1 gene on the recurrence of breast cancer.

Serum Detection of Epidermal Growth Factor Receptor Gene Mutations Using Mutant-Enriched Sequencing in Chinese Patients with Advanced Non-Small Cell Lung Cancer

Epidermal growth factor receptor gene (EGFR) mutations are among the best predictive markers of the efficacy of EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment. Mutations in the EGFR gene confer sensitivity to EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC). This study determined the concordance rate of EGFR mutations in serum samples and tumour tissue from Chinese patients with advanced NSCLC and compared two detection methods: mutant-enriched polymerase chain reaction-based DNA sequencing and non-enriched sequencing. The EGFR mutation status in serum was consistent with that in paired tumour samples, with a concordance rate of 93.1% for mutant-enriched sequencing. In serum samples, mutant-enriched sequencing demonstrated sensitivity and specificity of 77.8% and 100%, respectively, and was more sensitive than the non-enriched assay. Mutant-enriched sequencing in serum may provide a non-invasive and sensitive method for detecting EGFR mutation status in patients with unresectable NSCLC.

PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers.

BackgroundOncogenic mutations of PIK3CA, RAS (KRAS, NRAS), and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.MethodsTumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS), and BRAF mutations using polymerase chain reaction-based DNA sequencing.Results PIK3CA mutations were found in 54 (11%) of 504 patients tested; KRAS in 69 (19%) of 367; NRAS in 19 (8%) of 225; and BRAF in 31 (9%) of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%), uterine (7/28, 25%), breast (6/29, 21%), and colorectal cancers (18/105, 17%); KRAS in pancreatic (5/9, 56%), colorectal (49/97, 51%), and uterine cancers (3/20, 15%); NRAS in melanoma (12/40, 30%), and uterine cancer (2/11, 18%); BRAF in melanoma (23/52, 44%), and colorectal cancer (5/88, 6%). Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wt)PIK3CA (p = 0.001). In total, RAS (KRAS, NRAS) or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001). PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001) and in 20% of patients with RAS (KRAS, NRAS) or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS) or wtBRAF (p = 0.001).Conclusions PIK3CA, RAS (KRAS, NRAS), and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS) and BRAF mutations.

Molecular Techniques to Detect Biofilm Bacteria in Long Bone Nonunion: A Case Report

Biofilms cause chronic infections including those associated with orthopaedic hardware. The only methods that are Food and Drug Administration-approved for detecting and identifying bacterial infections are cultures and selected DNA-based polymerase chain reaction methods that detect only specific pathogens (eg, methicillin-resistant Staphylococcus aureus). New DNA-based technologies enable the detection and identification of all bacteria present in a sample and to determine the antibiotic sensitivities of the organisms. A 34-year-old man sustained an open tibia fracture. He experienced 3 years of delayed healing and episodic pain. In addition to his initial treatment, he underwent three additional surgeries to achieve fracture healing. During the last two procedures, cultures were taken and samples were tested with the IBIS T5000 and fluorescence in situ hybridization (FISH). In both cases, the cultures were negative, but the IBIS and FISH confirmed the presence of a biofilm within the tibial canal. Examinations of tissues from biofilm infections, by DNA-based molecular methods and by direct microscopy, have often found bacteria present despite negative cultures. Infections associated with orthopaedic hardware may be caused by bacteria living in biofilms, and these biofilm organisms are particularly difficult to detect by routine culture methods. Rapid DNA-based detection methods represent a potentially clinically useful tool in the detection of bacterial biofilms. The sensitivity and clinical impact of the technology has yet to be established.

Legionella in sugar factory process waters – Analysis and control in Nordic Sugar

This presentation will summarise the experiences gained from a Legionella project within Nordic Sugar which emphasises risk evaluation, modern DNA analysis methods, control strategies and water treatment methods. The project started in 2002 with yearly campaign screenings for Legionella spp and Legionella pneumophila. Initially an ISO Legionella method for quantification was used, but this was replaced with a modern, rapid DNA-based Real Time Polymerase Chain Reaction assay in 2005. The assay time for quantitative results using the Real Time PCR is approximately 3 hours compared to between 10 and 14 days for the ISO-method. All Nordic Sugar factories equipped with cooling towers are screened on a weekly or bi-weekly basis during every sugar campaign. Only one sugar factory has shown consistently positive results for Legionella pneumophila on a moderate level. A multiple biocide approach combined with a dispersing agent was successfully used to control the Legionella. In 2010 a non-chemical treatment method, Wallenius AOT-system was installed to eliminate an annual high cost for biocides. The system successfully controlled the L. pneumophila even when the cooling water was partially exchanged with fresh lake water.

NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

<i>NOTCH1</i> PEST domain mutation is an adverse prognostic factor in B‐CLL