Specific DNA Probes Research Articles

Trypanosoma cruzi diversity in the Gran Chaco: Mixed infections and differential host distribution of TcV and TcVI

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% – 36/98) and a previously not detected high proportion of mixed infections (47.9% – 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% – 30/98) in human samples; while TcVI (42.8% – 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% – 32/98); and TcI/TcVI (14.3% – 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco.

Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample

Pathogenic Vibrio cholerae produces a cholera toxin which is the cause of a severe diarrheal disease called “Cholera”. Available detection methods, including standard bacteriological test and immuno-based detection, are specific to the suspected pathogenic V. cholerae O1 and O139, but they are not specific to the cholera toxin producible strain. This work combined the polymerase chain reaction (PCR) of cholera toxin gene, ctxA gene, and microcantilever-based DNA sensor to improve the sensitivity and specificity of detection. Gold coated microcantilever, 250µm long and 50µm wide, with an embedded polysilicon wire acting as a piezoresistive material was modified by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) for immobilization of specific DNA probe via avidin layer on the surface. The avidin and 5′ biotinylated single-stranded DNA (ssDNA) probe concentrations were optimized for the immobilization at 50µg/mL and 1µM, respectively. The hybridization between ssDNA probe on this DNA sensor and target DNA creates nanomechanical bending and resistance change of piezoresistive material inside the beam. This microcantilever-based DNA sensor offers a detection sensitivity of 3.25pg or 14nM of DNA template for ctxA gene detection. The lowest number of V. cholerae O1 in food sample with and without the enrichment process that the polymerase chain reaction (PCR) for ctxA gene combined with this DNA sensor can detect is 0.835 and 835cells/g, respectively. This detection sensitivity is 10 times higher than that of the conventional PCR method.

Application of loop-mediated isothermal amplification assay combined with lateral flow dipstick for detection of Plasmodium falciparum and Plasmodium vivax

Malaria is largely a preventable and curable disease. However, a delay or an inappropriate treatment can result in serious adverse outcomes for patient. Rapid, simple and cost-effective diagnostic tests that can be easily adapted and rapidly scaled-up at the field or community levels are needed. In this study, accelerated detection methods for the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) dihydrofolate reductase–thymidylate synthase were developed based on the loop-mediated isothermal amplification (LAMP) method. The developed methods included the use of species-specific biotinylated primers to amplify LAMP amplicons, which were then hybridized to specific FITC-labeled DNA probes and visualized on a chromatographic lateral flow dipstick (LFD). The total LAMP–LFD assay time was approximately 1.5h. The LAMP–LFD assays showed similar detection limit to conventional PCR assay when performed on plasmid DNA carrying the malaria dhfr-ts genes. The LAMP–LFD showed 10 folds higher detection limit than PCR when performed on genomic DNA samples from Pf and Pv parasites. The dhfr-ts LAMP–LFD assays also have the advantages of reduced assay time and easy format for interpretation of results.

Granulomatous hepatitis associated with chronic Borrelia burgdorferi infection: a case report

Although Lyme borreliosis has been linked to hepatitis in early stages of infection, the association of chronic Borrelia burgdorferi infection with hepatic disease remains largely unexplored. We present the case of a 53-year-old woman diagnosed with Lyme disease who developed acute hepatitis with elevated liver enzymes while on antibiotic treatment. Histological examination of liver biopsy tissue revealed spirochetes dispersed throughout the hepatic parenchyma, and the spirochetes were identified as Borrelia burgdorferi by molecular testing with specific DNA probes. Motile spirochetes were also isolated from the patient’s blood culture, and the isolate was identified as Borrelia burgdorferi sensu stricto by two independent laboratories using molecular techniques. These findings indicate that the patient had active, systemic Borrelia burgdorferi infection and consequent Lyme hepatitis, despite antibiotic therapy.

Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

Porcine circovirus type 2 (PCV2) has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR) for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

English

During the 2012 growing season, thirty three samples were collected from squash, pumpkin and muskmelon plants showing virus-like symptoms in Riyadh and Al-Madena regions of Saudi Arabia. Eleven of these samples were found positive for Zucchini yellow mosaic virus (ZYMV) by double antibody sandwich ELISA (DAS-ELISA). In the host range study for the five selected ZYMV isolates, 11 out of the 22 mechanically inoculated test plants were infected and showed variable symptoms. The amplification of viral DNA product through reverse transcription-polymerase chain reaction amplification (RT-PCR) using a primer pair specific for ZYMV, yielded fragments of approximately 1185 bp. Southern blot hybridization confirmed the results obtained through RT-PCR, using a specific DNA probe homologous to ZYMV. Nucleotide sequences for the coat protein gene from all five Saudi isolates of ZYMV indicated a similarity of 97.1-100.0% between them. Comparative analysis of the nucleotide sequences of coat protein gene from the Saudi isolates and other ZYMV isolates obtained from NCBI, showed a relatively high nucleotide sequence similarity that ranged between 92.0-98.8%. The highest similarity was found with Syria, Jordan, Iran, Hungarian, Austria, Slovenia and Germany isolates (97.1 to 98.8%). The nucleotide sequence data obtained for the five ZYMV isolates was deposited in the GenBank under the accession numbers JQ899263, JQ899264, JQ899265, JQ899266 and JQ899267. Key words: ZYMV, DAS-ELISA, RT-PCR, Hybridization, Sequence.

Molecular diagnosis of α-thalassemias by the colorimetric nanogold

A new application of gold nanoparticles (AuNPs) as a colorimetric method for gene detection of α-thalassemia1 (SEA deletion) is reported here for the first time. This technique is based on color changes from salt-induced aggregation of un-hybridized nanogold probes after hybridization with the target DNA. Specific DNA probes were synthesized, thiol modified and conjugated on the surface of AuNPs. The target DNA was amplified and hybridized with the AuNPs-immobilized probe. Salt solution (NaCl) was added to induce aggregation of the un-hybridized nanogold probes. The color changes were visualized either by the naked eye or by UV-vis spectrophotometry at 520 nm. By this nanogold colorimetric method samples carrying normal α-globin genes could be successfully identified from samples carrying α-globin genes causing α-thalassemia1 (SEA deletion), either as a carrier or disease form. Results demonstrated that the new colorimetric nanogold method is a definite gene diagnosis of α-thalassemia. It is accurate, simple, rapid, specific, sensitive, and cost effective. It is also a promising point-of-care testing (POCT) method for thalassemias and other genetic disorders. The new colorimetric nanogold is a method of choice for areas where access to sophisticated molecular diagnosis is limited.

The haplosporidian Bonamia exitiosa is present in Australia, but the identity of the parasite described as Bonamia (formerly Mikrocytos) roughleyi is uncertain

Protistan oyster parasites in the genus Bonamia have been observed in recent years infecting new hosts on five continents, with most of these parasites genetically similar to austral species Bonamia exitiosa and Bonamia roughleyi. Identification of the newly observed parasites as one or another of these described species has been complicated by the fact that B. exitiosa and B. roughleyi are phylogenetically indistinguishable at the small-subunit ribosomal DNA (SSU rDNA) level, with samples of B. roughleyi type material no longer available for genetic re-analyses using more informative internal transcribed spacer (ITS) region DNA sequences. To resolve this issue, we evaluated B. roughleyi in field collections of hosts Saccostrea glomerata and Ostrea angasi (as well as Crassostrea gigas) in New South Wales, Australia in 2006 and 2007, and re-analyzed histological samples from the original description of this parasite species using in situ hybridization. Despite (1) reports of the oyster disease putatively caused by B. roughleyi during the time of collections, (2) the observation of gross lesions characteristic of the disease, and (3) the observation of B. roughleyi cells in association with the lesions, we detected a Bonamia sp. by PCR in just 1/42 O. angasi (2.4%), and 1/608 S. glomerata (0.2%), the latter oyster of which is the type host. SSU rDNA sequences of the amplicons were nearly identical to those of B. exitiosa and B. roughleyi, and phylogenetic analysis of ITS region sequences placed them on a B. exitiosa clade. A Haplosporidium sp. sequence similar to that of H. costale was PCR-amplified from nearly half the S. glomerata and O. angasi, but no Haplosporidium sp. was observed histologically. Our inability to identify a Bonamia sp. sequence in association with the B. roughleyi observed histologically suggests that this parasite is not a Bonamia sp. at all, and should be regarded as B. roughleyi nomen dubium. We conclude that the Bonamia sp. that we and other investigators detected in southeastern Australian S. glomerata and O. angasi was B. exitiosa.

Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.

Sensitive, simultaneous quantitation of two unlabeled DNA targets using a magnetic nanoparticle-enzyme sandwich assay.

We report herein the development of a simple, sensitive colorimetric magnetic nanoparticle (MNP)–enzyme-based DNA sandwich assay that is suitable for simultaneous, label-free quantitation of two DNA targets down to 50 fM level. It can also effectively discriminate single-nucleotide polymorphisms (SNPs) in genes associated with human cancers (KRAS codon 12/13 SNPs). This assay uses a pair of specific DNA probes, one being covalently conjugated to an MNP for target capture and the other being linked to an enzyme for signal amplification, to sandwich a DNA target, allowing for convenient magnetic separation and subsequent efficient enzymatic signal amplification for high sensitivity. Careful optimization of the MNP surfaces and assay conditions greatly reduced the background, allowing for sensitive, specific detection of as little as 5 amol (50 fM in 100 μL) of target DNA. Moreover, this sensor is robust, it can effectively discriminate cancer-specific SNPs against the wild-type noncancer target, and it works efficiently in 10% human serum. Furthermore, this sensor can simultaneously quantitate two different DNA targets by using two pairs of unique capture- and signal-DNA probes specific for each target. This general, simple, and sensitive DNA sensor appears to be well-suited for a wide range of genetics-based biosensing and diagnostic applications.

Genotypic and phenotypic analysis of diarrheagenic Escherichia coli strains isolated from Brazilian children living in low socioeconomic level communities

BackgroundChildhood diarrheal diseases remain highly endemic in developing areas of Brazil. The importance of Escherichia coli among children with diarrhea in these areas was unknown. This study determined the prevalence of different E. coli categories in symptomatic and asymptomatic children from low socioeconomic level rural communities in southeastern Brazil.MethodsA total of 560 stool samples were collected from 141 children with diarrhea (< 10 years) and 419 apparently healthy controls who resided in 23 communities. E. coli isolates (n = 1943) were subjected to two multiplex PCRs developed for the detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and Shiga toxin-producing E. coli (STEC). Strains were also examined for the presence of EPEC, EAEC, and DAEC by assays of adhesion to HEp-2 cells and by hybridization with specific DNA probes.ResultsDiarrheagenic E. coli strains were isolated from 253 (45.2%) children, and were associated with diarrhea in children aged < 5 years (p < 0.001). EAEC (20.9%), DAEC (11.6%), EPEC (9.3%) were the most frequent pathotypes, followed by ETEC (2.7%), EIEC (0.5%), and STEC (0.2%). Depending of the assay, EPEC, EAEC, and DAEC (collectively termed enteroadherent E. coli) strains were isolated in 45% to 56% of diarrhea cases, a significantly higher incidence than in controls (P < 0.05). Individually, only DAEC showed significant association with diarrhea (p < 0.05), particularly in children aged 2–5 years.ConclusionThis study indicates that enteroadherent E. coli is an important cause of diarrhea in children living in low socioeconomic level communities in southeastern Brazil. Our results reveal that the PCR1 assay is an excellent tool for the identification of EAEC and DAEC.

Benznidazole treatment in chronic children infected with Trypanosoma cruzi: Serological and molecular follow-up of patients and identification of Discrete Typing Units

A total of 221 children from two rural settlements in Northeast Argentina were examined for T. cruzi infection. Blood samples were taken for serology tests and PCR assays. In addition, T. cruzi Discrete Typing Units (DTUs) were determined by hybridization with specific DNA probes of the minicircle hypervariable regions (mHVR). Serological results indicated that 26% (57/215) were reactive against T. cruzi antigens. PCR analyses were performed on seropositive samples showing presence of parasite DNA in 31 out of 53 samples (58.5%). All seropositive children underwent specific chemotherapy with Benznidazole (5mg/kg/day) for a period of two months and were monitored two and five years after treatment. Overall the treatment was well tolerated and low side effects were observed. Serological conversion was observed at two years post -treatment in one child form Pampa Ávila and at five years in two children from Tres Estacas. However, at the end of the follow-up period, T. cruzi DNA could not be detected by PCR in samples from treated children, except in two cases. In addition, the results of hybridizations with specific DNA probes showed that DTU TcV was detected in 68% (21/31), TcVI in 7% (2/31) and TcV/VI in 3% (1/31) of the samples. Altogether, results of the follow-up of treated children showed a low rate of seroconversion; however trend toward seroconversion was evident at five years post-treatment. On the other hand, detection of T. cruzi DNA by PCR significantly decreased after Benznidazole treatment. The existence of data regarding serological and molecular follow-ups from controlled studies in the Chaco Region will be important for future treatment efforts against T. cruzi infection in this region. The results obtained in the present study represent a contribution in this regard.

The cAMP effector EPAC activates Elk1 transcription factor in prostate smooth muscle, and is a minor regulator of α1-adrenergic contraction

BackgroundProstate smooth muscle tone is regulated by α1-adrenoceptor-induced contraction and cAMP-mediated relaxation. EPAC is an effector of cAMP, being involved in smooth muscle relaxation and cell cycle control outside the lower urinary tract. Here, we investigated the expression and function of EPAC in human prostate tissues from patients undergoing radical prostatectomy.ResultsmRNA and protein expression of EPAC was detected in all prostate tissues by RT-PCR and Western blot analysis. Immunoreactivity was observed in stromal cells, and colocalized with immunofluorescence for α-smooth muscle actin and calponin. Under normal conditions, noradrenaline- or phenylephrine-induced contraction of prostate strips in the organ bath was not affected by the EPAC activator pCPT (SP-8-pCPT-2′-O-Me-cAMPS.NA) (30 μM). However, when the cyclooxygenase inhibitor indomethacin (50 μM) was added, EPAC activators pCPT and OME (8-CPT-2′-O-Me-cAMP.Na) (30 μM) significantly reduced contractions by low concentrations of phenylephrine. These effects were not observed on noradrenaline-induced contraction. OME and pCPT caused phosphorylation of the transcription factor Elk1 in prostate tissues. Elk1 activation was confirmed by EMSA (electrophoretic mobility shift assay), where OME and pCPT incresed Elk1 binding to a specific DNA probe.ConclusionsEPAC activation may reduce α1-adrenergic prostate contraction in the human prostate, although this effect is masked by cyclooxygenases and β-adrenoceptors. A main EPAC function in the human prostate may be the regulation of the transcription factor Elk1.

Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles

This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65°C). The DNA products with biotin were then conjugated to a first AuNP1 (55±2nm) through biotin–avidin binding. A second AuNP2 (30±1.5nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents.

Male-to-Female Sex Ratios of Abnormalities Detected by Fluorescence In Situ Hybridization in a Population of Chronic Lymphocytic Leukemia Patients

Distorted sex ratios occur in hematologic disorders. For example, chronic lymphocytic leukemia (CLL) displays disproportionate sex ratios with a large male excess. However, the underlying genetics for these disparities are poorly understood, and gender differences for specific cytogenetic abnormalities have not been carefully investigated. We sought to provide an initial characterization of gender representation in genetic abnormalities in CLL by using fluorescence in situ hybridization (FISH). We confirm the well known skewed male-tofemale (M/F sex ratio) of ~1.5 in our CLL study population, but also determine the genotypic M/F sex ratio values corresponding to specific FISH DNA probes. Genetic changes in CLL detectable by four FISH probes were statistically compared with respect to gender. Initial FISH evaluations of 4698 CLL patients were retrospectively examined and new findings of the genotypic M/F sex ratios for these probes are reported. This study represents the largest CLL survey conducted in the United States using FISH probes. The CLL database demonstrated that FISH abnormalities (trisomy 12, 13q14.3 deletion and 17p13.1 deletion) probes had skewed M/F ratios of ~1.5. Also, by statistical analysis it was shown that ATM gene loss (11q22.3q23.1 deletion) solely or with other abnormalities was considerably higher in males with an M/F ratio of 2.5 and significantly different from M/F ratios of 1.0 or 1.5. We hypothesize that interactions involving these autosomal abnormalities (trisomy 12, and deletions of 11q22.3, 13q14.3, and 17p13.1), and the sex chromosomes may provide the genetic basis for the altered phenotypic M/F ratio in CLL.

Automated Quantitative RNA in Situ Hybridization for Resolution of Equivocal and Heterogeneous ERBB2 (HER2) Status in Invasive Breast Carcinoma

Patient management based on HER2 status in breast carcinoma is an archetypical example of personalized medicine but remains hampered by equivocal testing and intratumoral heterogeneity. We developed a fully automated, quantitative, bright-field in situ hybridization technique (RNAscope), applied it to quantify single-cell HER2 mRNA levels in 132 invasive breast carcinomas, and compared the results with those by real-time quantitative PCR (qPCR) and Food and Drug Administration-approved methods, including fluorescence in situ hybridization (FISH), IHC, chromogenic in situ hybridization, and dual in situ hybridization. Both RNAscope and qPCR were 97.3% concordant with FISH in cases in which FISH results were unequivocal. RNAscope was superior to qPCR in cases with intratumoral heterogeneity or equivocal FISH results. This novel assay may enable ultimate HER2 status resolution as a reflex test for current testing algorithms. Quantitative in situ RNA measurement at the single-cell level may be broadly applicable in companion diagnostic applications.

The occurrence of haplosporidian parasites, Haplosporidium nelsoni and Haplosporidium sp., in oysters in Ireland

The phylum Haplosporidia is a group of obligate protozoan parasites that infect a number of freshwater and marine invertebrates. Haplosporidian parasites have caused significant mortalities in commercially important shellfish species worldwide. In this study, haplosporidia were detected in Pacific oysters Crassostrea gigas originating in Ireland and were subsequently identified independently in laboratories both in Ireland and in Spain as Haplosporidium nelsoni. In Ireland, H. nelsoni plasmodia were also observed in the heart tissue of a single Ostrea edulis. A range of techniques including heart smear screening, histology, standard polymerase chain reaction (PCR), direct sequencing and in situ hybridisation with an H. nelsoni specific DNA probe were carried out to confirm diagnosis. This is the first reporting of H. nelsoni in oysters in Ireland and this is the first reporting of the detection of this haplosporidian in O. edulis. In Ireland, another haplosporidian was also observed in a single O. edulis during heart smear screening. PCR and DNA sequencing were carried out and indicated the presence of a Haplosporidium sp., most likely Haplosporidium armoricanum. The low prevalence and intensity of infection of both haplosporidian species in Irish C. gigas and in particular O. edulis may indicate that their presence is inconsequential.

GA-map™ IBD Test - Potential New Tool for IBD Prediction and Management

In addition to genetic factors, several recent discoveries have shown that the gut microbiota seems to play an important role in the etiology of IBD (1). As a high through-put, low cost alternative to 454 pyro-sequencing, the GA-map™ technology platform has been develop to profile the composition of the gut microbiota (2). We have used this technology to develop a specific IBD test that offers promise of being an effective additional diagnostic tool in IBD disease prediction and management. A first version of the assay has been tested on treatment naïve IBD patients and symptomatic non-IBD patients and a healthy population. A population of 187 treatment naïve patients symptomatic of IBD identified by primary care physicians and referred to colonoscopy were used in this study. In addition, a reference population of 84 healthy individuals who did not undergo colonoscopy where also included. IBD were determined in all symptomatic patients by colonoscopy. Fecal samples were collected (before colonoscopy in symptomatic IBD patients), and DNA extracted after a mechanical lysis. The GA-map™ IBD test was performed essentially as described in (2), but with specific IBD related DNA probes (48 - 121 different probes) and using Luminex MagPix system for detection and quantification of labeled DNA probes (indicative of presence of different bacteria). The signals from each DNA probe in each sample was subject to PCA analysis and model was developed based on leave-on-out cross-validation. When comparing the healthy population to patients diagnosed with IBD through colonoscopy, the GA-map™ IBD test was able to correctly predict IBD with a 94% sensitivity and 86% specificity, and 90% accuracy. When comparing non-IBD (patients symptomatic for IBD, but confirmed non-IBD by colonoscopy) the numbers were 78%, 70% and 74%, respectively. The prediction capability of the GA-map™ test seems to be influenced by age and type of disease, as UC patients in the age-group 15 - 35 years showed the best prediction of disease with 88% sensitivity and 63% specificity when compared to symptomatic non-IBD. These results show that the GA-map™ IBD test can be used to distinguish healthy individuals from IBD patients with high accuracy. In addition, the test can be used to specify which symptomatic patients have IBD with 78% certainty. This shows potential to use the test to rule out symptomatic patients that do not need to undergo colonoscopy, thereby saving both payers and patients of costs, recourses and discomfort. The relatively poor specificity of the test could be an indication that symptomatic patients where IBD was not seen by colonoscopy, could be an early sign of developing gut related complications. This prospect will be followed up in a new study.

Detection of chromosomal aberrations in multiple myeloma with fluorescence in situ hybridization

To detect of chromosomal abnormalities in multiple myeloma (MM) patients with fluorescence in situ hybridization (FISH). FISH was performed in 20 MM patients using 5 specific DNA probes. The difference in chromosomal abnormalities was compared by FISH and other routine cytogenetic tests. Eighteen of the 20 patients showed chromosomal abnormalities (90%). The positive rates of t(14q32), del(13q14), dup(1q21), and p53 gene were 65% (13 in 20), 55% (11 in 20), 25% (5 in 20), and 15%(3 in 20), respectively. The abnormal rate of the conventional chromosome examination was 15% only. FISH is more sensitive than traditional chromosomal tests and can be used as an index in prognostic evaluation for MM. Del(13q14) and t(14q32) are the most common chromosomal abnormalities in MM patients.

Proof-of-Principle of Onchocerciasis Elimination with Ivermectin Treatment in Endemic Foci in Africa: Final Results of a Study in Mali and Senegal

BackgroundMass treatment with ivermectin controls onchocerciasis as a public health problem, but it was not known if it could also interrupt transmission and eliminate the parasite in endemic foci in Africa where vectors are highly efficient. A longitudinal study was undertaken in three hyperendemic foci in Mali and Senegal with 15 to 17 years of annual or six-monthly ivermectin treatment in order to assess residual levels of infection and transmission, and test whether treatment could be safely stopped. This article reports the results of the final evaluations up to 5 years after the last treatment.Methodology/Principal FindingsSkin snip surveys were undertaken in 131 villages where 29,753 people were examined and 492,600 blackflies were analyzed for the presence of Onchocerca volvulus larva using a specific DNA probe. There was a declining trend in infection and transmission levels after the last treatment. In two sites the prevalence of microfilaria and vector infectivity rate were zero 3 to 4 years after the last treatment. In the third site, where infection levels were comparatively high before stopping treatment, there was also a consistent decline in infection and transmission to very low levels 3 to 5 years after stopping treatment. All infection and transmission indicators were below postulated thresholds for elimination.Conclusion/SignificanceThe study has established the proof of principle that onchocerciasis elimination with ivermectin treatment is feasible in at least some endemic foci in Africa. The study results have been instrumental for the current evolution from onchocerciasis control to elimination in Africa.