Scholarly article on topic 'Detection of toxigenic Clostridium difficile by Loop-Mediated Isothermal Amplification (LAMP)'

Detection of toxigenic Clostridium difficile by Loop-Mediated Isothermal Amplification (LAMP) Academic research paper on "Health sciences"

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Academic research paper on topic "Detection of toxigenic Clostridium difficile by Loop-Mediated Isothermal Amplification (LAMP)"

16th ¡CID Abstracts/International Journal of Infectious Diseases 2IS (2014) 1-460

Type: Poster Presentation

Final Abstract Number: 59.039 Session: Diagnosis Date: Saturday, April 5,2014 Time: 12:45-14:15 Room: Ballroom

Type: Poster Presentation

Final Abstract Number: 59.040 Session: Diagnosis Date: Saturday, April 5, 2014 Time: 12:45-14:15 Room: Ballroom

Evaluation &validation of a highly multiplexed LATE-PCR single-tube assay for M(X)DR-TB »m k

M. deVos1-*, J. Rice2, L.Rice2, B. Kreishwirth3, N. Kurepina3, E.M. Streicher1, R.M.Warren1, P.D. vanHelden1, L. Wangh2

1 Stellenbosch University, Tygerberg, South Africa

2 Brandeis University, Waltham, MA, USA

3 UMDNJ, New Jersey, USA

Background: In 2006, the World Health Organization (WHO) and the Stop TB partnership called for strengthening of diagnostic services and highlighted the need for the development of rapid diagnostics to fight the tuberculosis pandemic. In 2011, WHO estimated that approximately 630,000 (5.3%) of the 12 million TB cases had multiple drug resistant (MDR)-TB, while more than 80 countries have reported cases of extremely drug-resistant (XDR)-TB. Only a small fraction of reported cases (<20%) were correctly diagnosed and even fewer were treated according to WHO standards. In response the WHO endorsed the Genotype® MTBDRplus (version 1.0) line probe assay (LPA) in 2008 and the Xpert®MTB/RIF assay in 2010. But these tests only provide evidence for resistance to isoniazid and/or rifampicin. There continues to be a critical need for a more comprehensive convenient diagnostic technology. The highly multiplexed LATE-PCR assay for M(X)DR-TB described here was designed to meet that need.\

Our goal was to firmly establish that a highly multiplexed Linear-After-the-Exponential (LATE) PCR single closed-tube assay can simultaneously detect and distinguish multiple mutations in multiple gene targets that are known to confer resistance to isoniazid, rifampicin, ethambutol, ofloxacin, amikacin, kanamycin and capreomycin.

Methods & Materials: In this initial study, DNA from clinical isolates with different rpoB, katG, embB, inhA promoter, gyrB, gyrA and rrs genotypes were selected from a DNA bank housed at Stellenbosch University. Each DNA samples was amplified and the singled-stranded DNA products were scanned for mutations at endpoint using the same mixture of Lights-On/Lights-Off Probes. The resulting fluorescent signatures were compared to that of H37Rv, a pan-susceptible "wildtype" strain.

Results: Each clinical isolate harbouring a unique mutation had its own, highly reproducible fluorescent signature distinct from that of H37Rv, as well as all other isolates with different mutations.

Conclusion: This study achieved the intended transfer of the Brandeis University technology to Stellenbosch University. This study also demonstrates that this single tube multiplexed assay can simultaneously distinguish the different mutations that confer resistance to rifampicin, isoniazid, ethambutol, fluoroquinolones, aminoglycosides and ethionamide in less than three hours.

(Supported by NIH Grant R01 A1099532)

Classification of tuberculous meningitis using Marais Criteria

M.G. Espanol1, M.A. Hache Marliere1-*, C. Pena2, H. Coradin2, V. Gonzalez Pantaleon1


1 Universidad Iberoamericana Unibe, Santo Domingo, Dominican Republic

2 Hospital Infantil Dr. Robert Read Cabral, Santo Domingo, Dominican Republic

Background: Tuberculous meningitis (TBM) diagnosis is difficult, being the worst prognostic form of extrapulmonary tuberculosis. Diagnostic delays contribute significantly to mortality and neurologic sequelae.

Methods & Materials: We performed an observational, descriptive and transversal study applying new consensus criteria for the definition of Meningeal Tuberculosis diagnosis and British Medical Research Council stage prognosis. In the study period, 68 patients were identified for tuberculous meningitis, 40 were excluded, and the final sample consisted of 28 cases

Results: The diagnostic group "Possible" was the most common with 35.7%, followed by Definite with 28.6%, Probable 21.4%, and 14.3% were excluded as Non-tuberculous meningitis in which an alternative diagnosis was established or considered dual disease. 60.71% were less than 5 years of age. 78.6% were male. 80.77% patients had symptoms for more than 5 days. 88.46% patients had focal neurological deficits. 64% patients showed alteration of consciousness. 64.3% underwent neuroimaging, which 50% demonstrated hydrocephalus. 75% of patients were in Stage III of the prognostic British Medical Research Council classification. 7.1% died.

Conclusion: Classification of tuberculous meningitis allows early diagnosis and treatment. Tuberculous meningitis manifestations vary and TBM is usually diagnosed when brain damage has already occurred. Type: Poster Presentation

Final Abstract Number: 59.041 Session: Diagnosis Date: Saturday, April 5, 2014 Time: 12:45-14:15 Room: Ballroom


Detection of toxigenic Clostridium difficile by Loop-Mediated Isothermal Amplification (LAMP)


St. Joseph's Healthcare, Hamilton, ON, Canada

Background: Clostridium difficile-associated disease (CDAD) is a leading cause of nosocomial diarrhea in adults. Therefore, rapid and

16th ICID Abstracts/International Journal of Infectious Diseases 2IS (2014) 1-460

accurate reporting of toxigenic C. difficile is essential for improving patient outcomes and minimizing hospital-acquired disease. The PCR-based in-house and commercial methods available now to detect toxigenic C. difficile are expensive or require nucleic acid purification. We describe a multiplex real-time loop-mediated isothermal amplification method (LAMP) to detect toxigenic C. difficile without DNA purification and to presumptively identify the NAP1 strain directly from diarrheal stools.

Methods & Materials: Five-hundred and eighteen stools submitted for routine PCR testing of C. difficile was used. The DNA was extracted by mixing and boiling 100 |il of 1:10 dilution of stools with 100 |l of lysis solution for 15 min. Two microliters of the clear supernatant was used for LAMP reactions. LAMP method amplified and detected tcdC, cdtA, and \ DNA (IC). Primers used for amplification were designed using Genbank and PrimerExplorer V4 (Eiken Chemical Co., Ltd. Japan). DNA amplification was done at 59 0C for 60 min using Rotorgene 6500 (Qiagen) and a standard LAMP reaction. Amplification was detected by displacement of a fluorescent probe annealed to the quencher-labeled primer: Detection of Amplification using Reduced Quenching (DARQ).The limit of detection (LOD) was determined by using a C. difficile negative stool specimen spiked with known number of colony forming units of C. difficile ATCC43255.

Results: Out of 518 specimens 200 tested were positive and 307 were negative for tcdC by both methods. There were 11 discrepant specimens that were all negative by LAMP but positive by PCR with high CT values (>35). The test performance characteristics of LAMP method as compared to the PCR was as follows: sensitivity, 95%; specificity, 100%; NPV, 97%; PPV, 100%. The LOD was estimated to be 750 genome equivalents. The presence of both tcdC and cdtA presumptively identified 65 specimens to have the NAP1 strain by both methods.

Conclusion: The turn-around-time for LAMP-DARQ was 90 min as compared to 3.5 hours for the in-house PCR. The estimated cost per test for LAMP-DARQwas Cdn $4.00 and 60% cheaper than the PCR. The LAMP-DARQ is a cost-effective, sensitive, faster method than PCR to detect toxigenic C. difficile.

Type: Poster Presentation

Final Abstract Number: 59.042 Session: Diagnosis Date: Saturday, April 5,2014 Time: 12:45-14:15 Room: Ballroom

Cross-species multiplex microarray for m ^

serological detection of flavi-, phlebo- and " s '


N.B. Cleton1-*, M.P.G. Koopmans1, G.-J. Godeke2, J. Reimerink2, K. van Maanen3, J.A. Kortekaas4, R.A. Bowen5, C.B.E.M. Reusken1

1 Erasmus Medical Center and Center for Infectious Disease Control, Bilthoven, Netherlands

2 Center for Infectious Disease Control, Bilthoven, Netherlands

3 GD, Animal Health Services, Deventer, Netherlands

4 Central Veterinary Institute ofWageningen University Research, Lelystad, Netherlands

5 Colorado State University, Fort Collins, USA

Background: New animal and human diseases continue to emerge across the world, influenced by human and animal population densities, climate and globalization in travel and trade. Arboviruses form a specific group within these (re-)emerging threats and, due to their vector-borne and zoonotic nature, require extensive, complex and expensive surveillance and control schemes. They cause clinical diseases in both humans and animals, ranging from life threatening meningoencephalomyelitis and hemorrhagic fever to rash and crippling arthralgia. Diagnosis is based mostly on serology, as viremia is often short-lived. Further complicating diagnostics is the fact that clinical syndromes and geographical distribution overlap, and antibodies cross-react extensively within virus families in common serological tests. Our objective is to be able to detect, diagnose and monitor clinically significant arboviruses simultaneously in multiple species and with an approach easily adaptable to constantly changing demographics and syndromes. Therefore we develop a novel cross-species protein microarray for profiling of antibodies to six flaviviruses (DENV1-4, WNV, JEV, TBEV, USUV, YFV), three alphaviruses (CHIKV, ONNV, SINV) and one phlebovirus (RVFV).

Methods & Materials: Target antigens were selected and spotted onto nitrocellulose pads using a non-contact array spotter. Serum samples from humans (180), horses (80), sheep (160), chickens (10) and other bird species (15) with virologically and/or serologically confirmed arboviral infections and control sera of non-exposed individuals were incubated in serial 2-fold dilutions followed by incubation with a species specific IgG, IgM or IgY specific Cy5-labeled conjugate. After quantifying signals using a scanarray scanner, data were analyzed in 'R'.

Results: Profiling of antibodies in human patients exposed to flaviviruses and alphaviruses showed highly discriminatory patterns of reactivity with sensitivities and specificities ranging from 87%-100%. Additionally, vaccinated individuals could be distinguished from non-vaccinated individuals. Initial results also showed high sensitivity and specificity of 100% for sheep infected with RVFV and horses infected with JEV, while WNV showed some cross-reactivity with JEV and USUV antigens in horses. Further testing is ongoing to determine the usefulness of this system for multiple bird species.