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Objective To report PCR results and vaccination status of rabbits with rabbit haemorrhagic disease following an investigation into sudden or unexpected death. Materials and methods PCR testing for RHDV2 and RHDV1 was performed on rabbit liver samples at two laboratories. Laboratory A reported results as positive or negative; Laboratory B reported results quantitatively as RNA copies per mg liver, categorised as negative, inconclusive or positive. The vaccination status of rabbits with both histopathological features of rabbit haemorrhagic disease and positive PCR test results were collated. Results PCR results matched histopathological findings in 188 of 195 (96%) cases. Seven individuals showed equivocal results, all of which had histopathological features of RHD but three tested PCR-negative and four results conflicted between laboratories. RHDV2 was the serotype detected in all PCR-positive cases. Histological features of rabbit haemorrhagic disease and PCR test results were positive in 125 rabbits; 51 unvaccinated, 56 in-date with Nobivac Myxo-RHD and 13 vaccinated against RHDV2 - although nine of these were vaccinated within 10 days of death. Clinical significance PCR testing complements histopathology in cases of sudden death in rabbits by confirming the diagnosis and identifying virus serotype, but there can be false negatives. Although RHDV2 is currently prevalent in UK pet rabbits, vaccination against both RHDV1 and RHDV2 is recommended. Failures of RHDV2 vaccine are infrequent.

In November 2018, Butte County, California, was decimated by the Camp Fire, the deadliest wildfire in state history. Over 150,000 acres were destroyed, and at its peak, the fire consumed eighty acres per minute. The speed and intensity of the oncoming flames killed scores of people, and weeks before the fire was contained, first responders began searching through the rubble of 18,804 residences and commercial buildings. As with most mass disasters, conventional identification modalities (e.g., fingerprints, odontology, hardware) were utilized to identify victims. The intensity and duration of the fire severely degraded most of the remains, and these approaches were useful in only 22 of 84 cases. In the past, the remaining cases would have been subjected to conventional DNA analysis, which may have required months to years. Instead, Rapid DNA technology was utilized (in a rented recreational vehicle outside the Sacramento morgue) in the victim identification effort. Sixty‐nine sets of remains were subjected to Rapid DNA Identification and, of these, 62 (89.9%) generated short tandem repeat profiles that were subjected to familial searching; essentially all these profiles were produced within hours of sample receipt. Samples successfully utilized for DNA identification included blood, bone, liver, muscle, soft tissue of unknown origin, and brain. In tandem with processing of 255 family reference samples, 58 victims were identified. This work represents the first use of Rapid DNA Identification in a mass casualty event, and the results support the use of Rapid DNA as an integrated tool with conventional disaster victim identification modalities.

A developmental validation was performed to demonstrate reliability, reproducibility, and robustness of the ANDE Rapid DNA Identification System for processing of crime scene and disaster victim identification (DVI) samples. A total of 1705 samples were evaluated, including blood, oral epithelial samples from drinking containers, samples on FTA and untreated paper, semen, bone, and soft tissues. This study was conducted to address the FBI’s Quality Assurance Standards on developmental validation and to accumulate data from a sufficient number of unique donors and sample types to meet NDIS submission requirements for acceptance of the ANDE Expert System for casework use. To date, no Expert System has been approved for such samples, but the results of this study demonstrated that the automated Expert System performs similarly to conventional laboratory data analysis. Furthermore, Rapid DNA analysis demonstrated accuracy, precision, resolution, concordance, and reproducibility that were comparable to conventional processing along with appropriate species specificity, limit of detection, performance in the presence of inhibitors. No lane‐to‐lane or run‐to‐run contamination was observed, and the system correctly identified the presence of mixtures. Taken together, the ANDE instrument, I‐Chip consumable, FlexPlex chemistry (a 27‐locus STR assay compatible with all widely used global loci, including the CODIS core 20 loci), and automated Expert System successfully processed and interpreted more than 1200 unique samples with over 99.99% concordant CODIS alleles. This extensive developmental validation data provides support for broad use of the system by agencies and accredited forensic laboratories in single‐source suspect‐evidence comparisons, local database searches, and DVI.

East Coast fever is caused by Theileria parva, and poses serious concerns for dairy farmers owing to massive economic losses. In the current study, we compared three methods (DNA extraction kits, FTA-NaOH and FTA-TENT) of DNA extraction to identify the most economical and reliable method. A survey for T. parva prevalence was conducted in dairy cattle in Mbarara, Uganda. Cytochrome C oxidase subunit I (COI) and T. parva-p104 genes were amplified to compare the methods. FTA-NaOH-based polymerase chain reaction (PCR) yielded the best detection rate for both COI gene and p104 gene. Prevalence of T. parva was 45.0% and 83.3% at animal and farm-level, respectively. FTA-NaOH based-PCR is simple, highly sensitive and cost-effective tool for T. parva diagnosis in resource constrained settings.

Proteinuria is a condition in which an excessive amount of protein is excreted in urine. It is, among others, an indicator of kidney disease or risk of cardiovascular disease. Rapid and reliable diagnosis and monitoring of proteinuria is of great importance for both patients and their physicians. For that reason, a paper-based sensor for proteinuria diagnosis was designed, optimized, and validated utilizing smartphone-assisted signal acquisition. In the first step, a few commonly employed protein assays were optimized and compared in terms of analytical performance on paper matrix. The tetrabromophenol blue method was selected as the one providing a sufficiently low limit of detection (39 mg·L−1) on the one hand and appropriate long-term stability (up to 3 months) on the other hand. The optimized assay was employed for protein-to-creatinine ratio (PCR) determination on a single paper-based sensor. For both analytes the linear ranges were within the clinically relevant range. The analytical usefulness of the developed sensors was demonstrated by a PCR recovery study in artificial urine. The obtained PCR recoveries were from ca. 80 to 150%.

Significance Populations living in remote rural communities would benefit from rapid, highly sensitive molecular, DNA-based diagnostics to inform the correct and timely treatment of infectious diseases. Such information is also becoming increasingly relevant in global efforts for disease elimination, where the testing of asymptomatic patients is now seen as being important for the identification of disease reservoirs. However, healthcare workers face practical and logistical problems in the implementation of such tests, which often involve complex instrumentation and centralized laboratories. Here we describe innovations in paper microfluidics that enable low-cost, multiplexed DNA-based diagnostics for malaria, delivered, in a first-in-human study, in schools in rural Uganda., Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in

Aims This study was designed to analyse the prevalence of class 1 and class 2 integron integrase genes among antibiotic-resistant coliform bacteria isolated from paper currency circulating in Pakistan. Methods and results A total of 500 individual currency notes were collected from different food vending sites at Lahore, Pakistan. Bacterial population were identified by biochemical and PCR techniques. Antimicrobial susceptibility testing was performed by disc diffusion assay. The highest bacterial population on currency was found from street vendors and butcher shops. Escherichia coli was found to be the most prevalent coliform bacteria followed by Klebsiella sp. and Enterobacter sp. PCR amplification of antimicrobial resistance gene showed the presence of ampC, blaTEM , blaNDM-1 , qnrA, tet(A) and tet(B) genes among coliform isolates. A total of 47 integron integrase bearing strains of coliform bacteria were analysed. Sequence analysis showed the presence of dfrA1-aadA1, dfrA1, dfrA5, dfrA7, aadA1, aadA4 cassette arrays in class 1 integron and dfrA1-sat2-aadA1 in class 2 integrase genes. Conclusion Circulating currency was heavily contaminated with antimicrobial-resistant coliform bacteria bearing class 1 and class 2 integron integrase genes. Significance and impact of the study This study describes a potential threat of severe bacterial infections due to improper hand hygiene and community sanitation when dealing with the currency notes.

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.

α-Amylase is a starch-hydrolyzing enzyme (EC 3.2.1.1) indispensable for germination of cereal seeds, but it is also expressed during the ripening stage. Previous studies demonstrated that the enzyme is activated in developing rice seeds under extremely hot weather and triggers a loss of grain quality by hindering the accumulation of storage starch in the endosperm. Since inactive or, preferably, heat-labile α-amylases are preferable for breeding premium rice, we developed a method for rapid screening of inactive and temperature-sensitive mutants of the enzyme by combining the random mutagenesis by error-prone PCR and an on-filter activity test of the recombinant enzyme expressed by Escherichia coli. This technique was applied to a major α-amylase in the developing seed, Amy3D, and the activity of the isolated mutant enzymes was verified with both the bacteria-expressed recombinant proteins and the extract from the endosperm overexpressing each of them. Then, we identified several substitutions leading to loss of the activity of amino acid residues (Leu28, Asp112, Cys149, Trp201, Asp204, Gly295, Leu300 and Cys342), as well as a variety of heat-sensitive substitutions of Asp83, Asp187 and Glu252. Furthermore, variations of the heat-labile enzymes were created by combining these heat-sensitive mutations. The effects of the respective mutations and their relationship to the structure of the enzyme molecule are discussed.

Currently, diagnosis of influenza is performed either through tedious polymerase chain reaction (PCR) or through rapid antigen detection assays. The rapid antigen detection assays available today are highly specific but not very sensitive, and most importantly, lack the ability to show if the strain of influenza detected is susceptible to antiviral agents, such as Tamiflu and Relenza. The ability to rapidly determine if a patient has an infectious disease and what type of treatment the infection will respond to, would significantly reduce the treatment decision time, shorten the impact of symptoms, and minimize transfer to others. In this study, a novel, point-of-care style μPAD (microfluidic paper-based diagnostic) for influenza has been developed with the ability to determine antiviral susceptibility of the strain for treatment decision. The assay exploits the enzymatic activity of surface proteins present on all influenza strains, and potential false positive responses can be mitigated. A sample can be added to the device, distributed to 4 different reagent zones, and development of the enzymatic substrate under different buffer conditions takes place on bottom of the device. Analysis can be performed by eye or through a colorimetric image analysis smartphone application.

A novel colorimetric/fluorescence bimodal lab-on-paper cyto-device was fabricated based on concanavalin A (Con A)-integrating multibranched hybridization chain reaction (mHCR). The product of mHCR was modified PtCu nanochains (colorimetric signal label) and graphene quantum dot (fluorescence signal label) for in situ and dynamically evaluating cell surface N-glycan expression. In this strategy, preliminary detection was carried out through colorimetric method, if needed, then the fluorescence method was applied for a precise determination. Au-Ag-paper devices increased the surface areas and active sites for immobilizing larger amount of aptamers, and then specifically and efficiently captured more cancer cells. Moreover, it could effectively reduce the paper background fluorescence. Due to the specific recognition of Con A with mannose and the effective signal amplification of mHCR, the proposed strategy exhibited excellent high sensitivity for the cytosensing of MCF-7 cells ranging from 100 to 1.0×10(7) and 80-5.0×10(7) cellsmL(-1) with the detection limit of 33 and 26 cellsmL(-1) for colorimetric and fluorescence, respectively. More importantly, this strategy was successfully applied to dynamically monitor cell-surface multi-glycans expression on living cells under external stimuli of inhibitors as well as for N-glycan expression inhibitor screening. These results implied that this biosensor has potential in studying complex native glycan-related biological processes and elucidating the N-glycan-related diseases in biological and physiological processes.

Background In recent years, interest in universal newborn screening for congenital cytomegalovirus (cCMV) has intensified. consequently, reliable and simple methods of mass screening for cCMV, are essential. Herein, we present a novel approach of using polymerase chain reaction (PCR) in saliva with direct inoculation onto Guthrie paper. Our aim was to determine the diagnostic accuracy of real- time PCR in saliva rolled directly onto Guthrie paper in diagnosing cCMV infection. Objectives To evaluate the diagnostic accuracy of real-time PCR analysis of dried saliva on Guthrie paper as a future approach for mass screening of newborns in diagnosing cCMV infection. Study design This prospective, blinded study was performed in a tertiary cytomegalovirus (CMV) clinic from May 2018 through January 2019. Forty-two cCMV positive newborns and 41 without cCMV, were enrolled and tested for CMV using PCR from their saliva placed onto Guthrie paper and from their saliva using standard methods. Specificity and sensitivity of dried saliva PCR versus gold-standard methods were analyzed. Results Forty-two out of 42 (100 %) CMV positive infants showed positive PCR in the dried saliva on the Guthrie paper. All (100 %) controls exhibited negative PCR results. Congenital CMV infection was identified with a sensitivity of 100 % (95 % C.I. = 91.4%–100.00%) and specificity of 100 % (95 % C.I. = 91.4%–100.00%). Discussion CMV testing with saliva real-time PCR on Guthrie paper displayed a high sensitivity and specificity, rendering it a powerful screening test. The accuracy, simplicity of sampling, storage and transportation and the potential reliance on existing logistic resources, establishes this method as a candidate for cCMV universal screening programs.

A rapid and low-cost method of diagnosis is becoming important for detecting fetal inherited diseases, including single-gene disorders and chromosomal abnormalities. Here, we demonstrated an innovation that use paper-dried cord blood (PCB) as the starting material for PCR and whole genome amplification without any DNA extraction step at a very low cost. A novel PCR buffer named "DDB buffer" containing ammonium sulfate and glycerol were used instead of the conventional 10× PCR buffer. The amplicons were directly analyzed through microchip electrophoresis and whole genome sequencing. Inhibitory substances in filter paper were effectively inactivated using DDB buffer. Direct PCR amplification of DNA fragments ranging from 100 to 900 bp using filter paper spotted with 0.5 to 5 μL of cord blood and various anticoagulants was successful. We were able to determine fetal single-gene disorders and chromosomal diseases in all 46 chromosomes using PCB samples successfully. Compared with prenatal diagnosis using purified DNA, the proposed method is simple, fast, less prone to cross-contamination at minimal cost. Researchers and clinical and healthcare workers may employ this method for genetic diagnosis using cord blood samples with minimum laboratory resources. This method is very promising for a variety of genetic diagnosis applications in underserved communities at the point of need in developing areas. Graphical abstract.

Detection of Leishmania in cutaneous lesions is possible by visualization of amastigotes. Detection of Leishmania DNA by PCR presents greater sensitivity, and PCR has been used to diagnose cutaneous leishmaniosis in humans using noninvasive clinical specimens.Study I: to determine if Leishmania DNA could be efficiently extracted and amplified from archived Diff-QuikSamples from cutaneous lesions of 54 dogs.Study I: Leishmania-qPCR was performed on 19 glass slides (from nine dogs) with cytologically visible amastigotes. Fifteen slides with no visible amastigotes, obtained from 12 dogs seronegative for Leishmania by ELISA, served as controls. Study II: Leishmania-qPCR was performed on glass slides and FPI from cutaneous lesions compatible with clinical leishmaniosis in 33 dogs.Study I: all slides with visible amastigotes had positive qPCR, whereas all control slides yielded negative results. Study II: of 13 dogs definitively diagnosed with clinical leishmaniosis, eight had visible amastigotes on cytology, whereas Leishmania-qPCR was positive on 11 glass slides and 13 FPI. Leishmaniosis was ruled out by standard methods in 20 dogs, four of which yielded positive qPCR on FPI and/or glass slides.Leishmania-DNA can be detected efficiently by qPCR from cutaneous cytological specimens and FPI to diagnose Leishmania infection in dogs with cutaneous lesions suggestive of CanL.Détection de Leishmania dans les lésions cutanées est possible par visualisation des amastigotes. La détection de l’ADN de Leishmania par PCR présente une meilleure sensibilité et la PCR a été utilisée pour diagnostiquer la leishmaniose cutanée de l'homme à l'aide de techniques cliniques non invasives.Etude I : déterminer si l’ADN de Leishmania peut être efficacement extraite et amplifiée de lames colorées au Diff-QuikEchantillons de lésions cutanées de 54 chiens. MATÉRIEL ET MÉTHODE: Etude I : la PCRq a été réalisée sur 19 lames (de 9 chiens) avec des amastigotes visibles à la cytologie. Quinze lames sans amastigote visible, obtenu de 12 chiens séronégatifs pour Leishmania par ELISA, ont servi de contrôle. Etude II : Une PCRq Leishmania a été réalisée sur des lames et FPI de lésions cutanées compatibles avec une leishmaniose clinique chez 33 chiens. RÉSULTATS: Etude I : toutes les lames avec amastigotes visibles avaient une PCRq positive tandis que les lames contrôles étaient négatives. Etude II : sur les 13 chiens diagnostiqués définitivement comme leishmaniose clinique, huit avaient des amastigotes visibles à la cytologie tandis que la PCRq Leishmania était positive sur 11 des lames et 13 FPI. LA leishmaniose a été exclue par méthodes standard chez 20 chiens, dont quatre présentaient une PCRq positive sur FPI et/ou lames.L’ADN de Leishmania peut être détectée efficacement par PCRq de cytologies cutanées et FPI pour le diagnostic de leishmaniose chez les chiens présentant des lésions cutanées évocatrices de CanL.INTRODUCCIÓN: la visualización de amastigotes permite la detección de Leishmania en lesiones cutáneas. La detección del DNA de Leishmania por PCR es más sensible, y la PCR se ha utilizado para diagnosticar la leishmaniasis cutánea en humanos utilizando muestras clínicas no invasivas. OBJETIVOS: Estudio I: determinar si el DNA de Leishmania se podría extraer y amplificar de manera eficiente a partir de preparaciones teñidas con Diff-Quik® archivadas de muestras citológicas de lesiones cutáneas caninas. Estudio II: evaluar el valor diagnóstico de una PCR cuantitativa (qPCR) de Leishmania en muestras citológicas teñidas y en impresiones de papel de filtro (FPI) obtenidas de lesiones cutáneas sugestivas de leishmaniasis canina (CanL). ANIMALES: muestras de lesiones cutáneas de 54 perros. MÉTODOS Y MATERIALES: Estudio I: se realizó qPCR para Leishmania en 19 portaobjetos de vidrio (de nueve perros) con amastigotes citológicamente visibles. Quince preparaciones sin amastigotes visibles, obtenidas de 12 perros seronegativos para Leishmania por ELISA, sirvieron como controles. Estudio II: se realizó qPCR para Leishmania en portaobjetos de vidrio y FPI de lesiones cutáneas compatibles con leishmaniasis clínica en 33 perros. RESULTADOS: Estudio I: todas las preparaciones con amastigotes visibles tuvieron qPCR positiva, mientras que todas las preparaciones de control dieron resultados negativos. Estudio II: de 13 perros diagnosticados definitivamente con leishmaniasis clínica, ocho tuvieron amastigotes visibles en la citología, mientras que qPCR para Leishmania fue positiva en 11 portaobjetos de vidrio y 13 FPI. La leishmaniasis se descartó mediante métodos estándar en 20 perros, cuatro de los cuales produjeron qPCR positiva en FPI y/o portaobjetos de vidrio. CONCLUSIONES E IMPORTANCIA CLÍNICA: la qPCR para DNA de Leishmania puede realizarse de forma efectiva a partir de muestras citológicas cutáneas y FPI para diagnosticar la infección por Leishmania en perros con lesiones cutáneas sugestivas de CanL.Leishmanien können in Hautveränderungen durch eine Sichtbarmachung der Amastigoten gefunden werde. Die Feststellung von Leishmania DNA mittels PCR bedeutet eine größere Sensibilität. Außerdem wurde die PCR verwendet, um kutane Leishmaniose bei Menschen mittels nichtinvasiver klinischer Proben zu diagnostizieren.Studie I: festzustellen, ob ausreichend Leishmania DNA aus archivierten Diff-Quick® gefärbten zytologischen Präparaten aus kutanen Veränderungen extrahiert und amplifiziert werden konnte. Studie II: eine Evaluierung der diagnostischen Leistung einer quantitativen Leishmania (q) PCR aus gefärbten Zytologiepräparaten sowie aus Abdrücken auf Filterpapier (FPI), die aus Hautveränderungen stammten, die verdächtig für eine canine Leishmaniose (CanL) waren.Es wurden Proben von Hautveränderungen von 54 Hunden verwendet.Studie I: Es wurde ein Leishmania-qPCR von 19 Objektträgern (von neun Hunden) mit zytologisch sichtbaren Amastigoten durchgeführt. Fünfzehn Objektträger ohne sichtbare Amastigoten, die von 12 Hunden stammten, die mittels ELISA seronegativ auf Leishmania waren, dienten als Kontrollen. Studie II: Ein Leishmania-qPCR wurde von Objektträgern und FPI kutaner Läsionen von 33 Hunden, die mit klinischer Leishmaniose kompatibel waren, durchgeführt.Studie I: alle Objektträger mit sichtbaren Amastigoten zeigten eine positive qPCR, während die Kontrollen ein negatives Ergebnis lieferten. Studie II: von den 13 Hunden, die definitiv mit einer Leishmaniose diagnostiziert worden waren, zeigten acht sichtbare Amastigoten in der Zytologie, während der Leishmania-PCR von 11 Objektträgern und 13 FPI positiv war. Eine Leishmaniose war bei 20 Hunden mit Standardmethoden ausgeschlossen worden, von denen vier eine positive qPCR von FPI und/oder Objektträgern lieferten.Leishmania-DNA kann effektiv mittels qPCR aus kutanen zytologischen Hautproben und aus FPI festgestellt werden, um eine Leishmania Infektion bei Hunden mit Hautveränderungen, die auf CanL hinweisen zu diagnostizieren.背景: 皮膚病変におけるリーシュマニアの検出は、無鞭毛期虫体の視覚化によって可能となる。 PCRによるリーシュマニアDNAの検出はより高い感度を示し、PCRは非侵襲性臨床標本を用いて人の皮膚リーシュマニア症を診断するために使用されてきた。 目的: 研究I:研究Iの目的は、犬の皮膚病変から得られた細胞学的標本のアーカイブされたDiff-Quik®染色スライドからLeishmania DNAを効率的に抽出および増幅できるかどうかを決定することである。研究II:研究IIの目的は、染色した細胞学的標本および犬リーシュマニア症(CanL)を示唆する皮膚病変から得られたfilter paper impressions (FPI)に対するリーシュマニア定量的(q)PCRの診断性能を評価することである。 被験動物: 54頭の犬の皮膚病変からのサンプル。 材料と方法: 研究I:Leishmania-qPCRを、細胞学的に観察可能な無鞭毛期虫体を含む19枚のスライドガラス(9頭の犬から)によって実施した。 ELISAによってリーシュマニアに対して血清陰性の12頭の犬から得られた、目に見える無鞭毛期虫体のない15枚のスライドを対照として用いた。研究II:33頭の犬において、リーシュマニアqPCRを、臨床的にリーシュマニア症と適合性のある皮膚病変からのスライドガラスおよびFPIに対して実施した。 結果: 研究I:目に見える無鞭毛期虫体を含む全てのスライドがqPCR陽性であったのに対し、全対照スライドは陰性結果をもたらした。研究II:臨床リーシュマニア症と確定診断された13頭の犬のうち、8頭が細胞診で目に見える斑点があったのに対し、Leishmania-qPCRは11枚のスライドガラスと13枚のFPIで陽性を示した。リーシュマニア症は、20頭の犬において標準的な方法で除外され、そのうちの4頭はFPIおよび/またはスライドガラス上でqPCR陽性を示した。 結論と臨床的重要性: リーシュマニアDNAは、CanLを示唆する皮膚病変を有する犬におけるリーシュマニア感染を診断するために、皮膚細胞診標本およびFPIからqPCRによって効率的に検出することができる。.背景: 通过发现无鞭毛体可以检测皮肤病变中的利什曼原虫。 PCR检测利什曼原虫DNA具有更高的灵敏度,并且无创临床样本的PCR检测已被用于诊断人类皮肤利什曼病。 目的: 研究I:确认是否能从已被Diff-Quik®染色的犬皮肤病变细胞学载玻片中,有效提取和扩增利什曼原虫DNA。研究II:染色细胞学和滤纸印迹(FPI)均采自疑似犬利什曼病(CanL)的皮肤病变,评估利什曼原虫定量(q)PCR方法对这些样本的诊断效果。 动物: 54只犬的皮肤病变样本。 方法和材料: 研究I:细胞学检查可见无鞭毛体的19张载玻片(来自9只犬),对其进行利什曼原虫-qPCR检测;从 12只犬身上获得的15张载玻片样本作为对照,细胞学检查未见无鞭毛体,经ELISA检测利什曼原虫血清阴性。研究II:33只症状符合利什曼病的犬,用利什曼原虫-qPCR法检测其皮肤病变载玻片和FPI。 结果: 研究I:所有可见无鞭毛体的载玻片,其qPCR检测均为阳性,而所有对照载玻片的检测结果均为阴性。 研究II: 13只确诊利什曼病的患犬中,8只细胞学上可见无鞭毛体,而利什曼原虫-qPCR检测中11张载玻片和13份FPI呈阳性。用标准方法排除利什曼病的20只犬中,经qPCR检测FPI和/或载玻片,有4只呈阳性。 结论和临床价值: 用qPCR技术检测皮肤细胞学和FPI中的利什曼原虫DNA,可有效诊断皮肤病变疑似CanL的利什曼原虫感染。.A detecção de Leishmania em lesões cutâneas é possível pela visualização de amastigotas. A detecção de DNA de Leishmania por PCR possui maior sensibilidade, e a PCR tem sido utilizada para diagnosticar leishmaniose cutânea em humanos utilizando espécimens clínicos não invasivos.Estudo I: determinar se o DNA de Leishmania pode ser eficientemente extraído e amplificado de lâminas coradas em Diff-QuikAmostras cutâneas de lesões de 54 cães. MÉTODOS E MATERIAIS: Estudo I: Leishmania-qPCR foi realizado em 19 lâminas de vidro (de nove cães) com amastigotas citologicamente visíveis. Quinze lâminas sem amastigotas visíveis, obtidas de 12 cães soronegativos para Leishmania por ELISA serviram como controle. Estudo II: Leishmania-qPCR foi realizado em lâminas de vidro e em FPI de lesões cutâneas compatíveis com leishmaniose clínica em 33 cães.Estudo I: todas as amostras com amastigotas visíveis apresentaram qPCR positivo, enquanto todas as lâminas controle tiveram resultados negativos. Estudo II: dos 13 cães diagnosticados definitivamente com leishmaniose clínica, oito apresentaram amastigotas visíveis na citologia, enquanto Leishmania-qPCR foi positivo em 11 lâminas de vidro e 13 FPI. Descartou-se a leishmaniose pelos métodos convencionais em 20 cães, dos quais quatro apresentaram qPCR positiva na FPI e/ou nas lâminas de vidro. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: O DNA de Leishmania pode ser eficientemente detectado por qPCR de espécimens citológicos cutâneos e FPI para diagnosticar uma infecção por Leishmania em cães com lesões cutâneas sugestivas de CanL.

Point-of-care Nucleic acid testing (POCNAT) has become an attractive technique for DNA identification in resource-limited settings, offering a rapid system for urgent clinical applications. In this study, a chemiluminescence-based lateral flow biosensor (CL-LFB) was developed for the quantitative analysis of DNA, without labeling and amplification. The developed biosensor employs a two-step hybridization, a primary hybridization of 5'-biotinylated detector probe to the target DNA and a secondary hybridization of the resulting complex with the immobilized capture probe. Quantitative analysis of DNA was provided via HRP-catalyzed reaction with the chemiluminescense substrate followed by imaging with a complementary metal-oxide-semiconductor (CMOS) digital camera. The assay performance was investigated using a synthetic target, 16S rRNA gene (775 bp) and the whole genome derived from Escherichia coli (E.coli). A detection limit of 1.5 pM for the synthetic target and 0.4 ng/ml for 16S rRNA gene was obtained. In spite of LFBs limitations for the detection of large DNA fragments, the proposed assay provided a low-cost, fast, and sensitive tool for PCR-free diagnosis of small and larger fragments of nucleic acids.

A paper-based DNA biosensor was developed for food authenticity testing using dairy products as a model. DNA from milk-based samples was isolated, and species-specific DNA sequences were amplified and identified by the biosensor using specific DNA probes. The properties of gold nanoparticles were exploited for visual detection. The biosensor was applied for detection of three species, namely cow, sheep and goat, while as low as 1.6 fmol for cow and goat, and 3.1 fmol for sheep PCR product were detected. Moreover, adulteration down to 0.01% could be detected, based on binary mixtures of cows', ewes' and goats' milk yogurt, containing 0.01 to 5% of cows' yogurt in ewes' and goats' yogurts, respectively. The proposed paper-based DNA biosensor offered 10 times higher detectability than other methods, good specificity and reproducibility, and could be applied easily for the detection of other adulterated food products, such as meat, olive oil and legumes.

We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. The efficiency of filter paper-based spin columns was evaluated for purification of nucleic acids from various sources. Following protocols of commercial kits, we found filter paper to be a useful binding material for purification of nucleic acids, including plant genomic DNA, plant total RNA, PCR products, and DNA from agarose gels. However, filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Protocols for the use of filter paper recharged spin columns or homemade spin columns for low throughput purification of plant genomic DNA and total RNA with unused commercial kit buffers or less expensive homemade buffers are presented.

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards. DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction. For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.

Previously (Glushakova et al. 2017), a cellulose-based cationic (Q) paper derivatized with quaternary ammonium groups was shown to be a convenient platform to collect, preserve, and store nucleic acids (NAs) derived from mosquito vectors infected with pathogens for surveillance. NAs bind electrostatically to Q-paper, but the quantity of NA bound depends on the paper’s binding capacity. To optimize the original technology for mosquito surveillance, factors that affected NA absorbance on Q-paper were evaluated. Sixteen variations of Q-paper were prepared with modifications of the derivatizing reagents and derivatization temperature. The binding capacities of these variations were determined first with 1,3,5-benzenetricarboxylic (BTCA), then viral RNA (purified or in infected mosquito samples) was used for validation. For this, samples with Zika (ZIKV) and chikungunya (CHIKV) RNA or virus-infected Aedes aegypti mosquito bodies were applied to sixteen Q-paper variants. Washing the paper samples with water versus elution with aqueous salt (1 M) gave samples that were analyzed for viral RNA by a PCR-based direct Luminex hybridization assay. The comparison ranked the Q-paper binding capacities from the lowest to the highest. The Q-paper with the highest RNA binding capability was further validated with ZIKV- and CHIKV-infected mosquito saliva.

Waterborne microbial pathogen detection via nucleic acid analysis on portable microfluidic devices is a growing area of research, development, and application. Traditional polymerase chain reaction (PCR)-based nucleic acid analysis detects total extracted DNA, but cannot differentiate live and dead cells. A propidium monoazide (PMA) pretreatment step before PCR can effectively exclude DNA from nonviable cells, as PMA can selectively diffuse through compromised cell membranes and intercalate with DNA to form DNA–PMA complex upon light exposure. The complex strongly inhibits the amplification of the bound DNA in PCR, and thus, only cells with intact cell membranes are detected. Herein, this study reports the development of a microfluidic device to carry out PMA pretreatment ‘on-chip’. Chip design was guided by computer simu-lations, and prototypes were fabricated using a high-resolution 3D printer. The optimized design utilizes split and recombine mixers for initial PMA-sample mixing and a serpentine flow channel containing her-ringbone structures for dark and light incubation. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was successfully demonstrated.

Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards with buccal swabs and compared the results with those obtained with DNA extracted using the EZ1 DNA Investigator Kit. Concordant profiles were obtained for all samples. Our protocol includes simple punch, wash, and PCR steps, reducing cost and hands-on time in the laboratory. Furthermore, it facilitates automation of DNA sequencing.

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

It is well established that the contamination sources of cholera causing bacteria, Vibrio cholerae, are water and food, but little is known about the transmission role of the fomites (surfaces that can carry pathogens) commonly used in households. In the absence of appropriate nutrients or growth conditions on fomites, bacteria have been known to assume a viable but non-culturable (VBNC) state after a given period of time. To investigate whether and when V. cholerae O1 assumes such a state, this study investigated the survival and viable quantification on a range of fomites such as paper, wood, glass, plastic, cloth and several types of metals under laboratory conditions. The fomites were inoculated with an outbreak strain of V. cholerae and its culturability was examined by drop plate count method at 30 min intervals for up to 6 h. For molecular detection, the viable/dead stain ethidium monoazide (EMA) which inhibits amplification of DNA from dead cells was used in combination with real-time polymerase chain reaction (EMA-qPCR) for direct quantitative analyses of viable V. cholerae at 2, 4, 6, 24 h and 7 day time intervals. Results showed that V. cholerae on glass and aluminum surfaces lost culturability within one hour after inoculation but remained culturable on cloth and wood for up to four hours. VBNC V. cholerae on dry fomite surfaces was detected and quantified by EMA-qPCR even 7 days after inoculation. In conclusion, the prolonged survival of V. cholerae on various household fomites may play vital role in cholera transmission and needs to be further investigated.

PCR and DNA sequencing are currently the diagnostic methods of choice for detection of Blastocystis spp. and their suptypes. Fresh or frozen stool samples have disadvantages in terms of several aspects such as transportation, storage, and existence of PCR inhibitors. Filter paper technology may provide a solution to these issues. The aim of the present study was to detect Blastocystis spp. and their subtypes by employing two different preservation methods: conventional frozen stool (FS) and dried stool spots on filter paper (DSSFP). Concentration and purity of DNA, sensitivity of PCR, and DNA sequencing results obtained from the two methods were also compared. A total of 230 fecal samples were included and separated into two parts: one part of the fecal samples were directly frozen and stored at ?20 °C. The remaining portion of the specimens were homogenized with saline and spread onto the filter papers as thin layer with a diameter of approximately 3 cm. After air-dried, the filter papers were stored at room temperature. DSSFP samples were collected by scraping from the filter papers. DNA were extracted by EURx Stool DNA Extraction Kit from both samples. Concentration and purity were measured with Nano-Drop, then PCR and sequencing were conducted for detection of Blastocystis spp. and its genotypes. Pure DNA was obtained with a A260/A280 ratio of 1.7–2.2 in both methods. DNA yield from FS was 25–405 ng/?l and average DNA concentration was 151 ng/?l, while these were 7–339 and 122 ng/?l for DSSFP, respectively. No PCR inhibition was observed in two methods. DNA from DSSFP were found to be stable and PCR were reproducible for at least 1 year. FS-PCR- and DSSFP-PCR-positive samples were 49 (21.3 %) and 58 (25.3 %), respectively (p = 0.078). The 43 specimens were concordantly positive by both FS-PCR and DSSFP-PCR. When the microscopy was taken as the gold standard, sensitivity of DSSFP-PCR and FS-PCR was 95.5 and 86.4 %, while specificity of both tests was 99.4 and 98.3 %, respectively. DNA sequencing results of 19 microscopically confirmed cases were strictly identical (concordance 100 %) in both methods, and ST2:6, ST3:8, ST4:3, and ST6:2 were the detected subtypes. Among the 230 fecal samples, the most predominant subtypes were ST3, ST2, ST4, and ST1 by both FS and DSSFP methods. Concordance of DNA sequencing results obtained from the two methods was noted to be 90.7 %. To our knowledge, this is the first study that demonstrates DNA extraction from DSSFP is more sensitive and effective than the FS method for diagnosis of Blastocystis spp. and their subtypes by PCR and DNA sequencing. © 2016, Springer-Verlag Berlin Heidelberg.

Robustness of comprehensive DNA- and RNA-based assays at diagnosis of acute myeloid leukemia using blood and bone marrow stored on filter cards

We have developed a new method for unprocessed biological specimens as templates directly into the TaqMan assay. Saliva was needed to be put on a water-soluble paper and dried, because foreign substances, such as a filter paper, hinder fluorescence detection through the assay. Genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) polymorphisms was, subsequently, performed by TaqMan PCR assay using dried saliva in the present investigation. The optimized technique was tested on 114 samples of alcoholic patients. The PCR-RFLP methods with purified DNA from blood samples were employed for validation of the assay. Upon validation, complete concordance was observed between the two independent results. These results highlight the ability of TaqMan PCR assays using dried saliva on water-soluble paper in genotyping of ADH1B and ALDH2 genes. Our results showed a rapid, simple, reliable, and cost-effective method for SNP genotyping of mutations in ADH1B and ALDH2 genes. This will be very useful for large-scale association studies in various fields. [Original].

The aim of the present study is to evaluate the probiotic attributes of Bacillus subtilis AMS6 isolated from fermented soybean (Churpi). This isolate exhibited tolerance to low pH (pH 2.0) and bile salt (0.3%), capability to autoaggregate and coaggregate. AMS6 also showed highest antibacterial activity against the pathogenic indicator strain Salmonella enterica typhimurium (MTCC 1252) and susceptibility towards different antibiotics tested. The isolate was effective in inhibiting the adherence of food borne pathogens to Caco-2 epithelial cell lines, and was also found to be non-hemolytic which further strengthen the candidature of the isolate as a potential probiotic. Further studies revealed B. subtilis AMS6 showed cellulolytic activity (0.54±0.05 filter paper units mL(-1)) at 37°C. The isolate was found to hydrolyze carboxymethyl cellulose, filter paper and maize (Zea mays) straw. The maize straw digestion was confirmed by scanning electron microscopy studies. The isolate was able to degrade filter paper within 96h of incubation. A full length cellulase gene of AMS6 was amplified using degenerate primers consisting of 1499 nucleotides. The ORF encoded for a protein of 499 amino acids residues with a predicted molecular mass of 55.04kDa. The amino acids sequence consisted of a glycosyl hydrolase family 5 domain at N-terminal; Glycosyl hydrolase catalytic core and a CBM-3 cellulose binding domain at its C terminal. The study suggests potential probiotic B. subtilis AMS6 as a promising candidate envisaging its application as an animal feed additive for enhanced fiber digestion and gut health of animal.

The use of Polymerase Chain Reaction (PCR) assays for pathogen detection in travelers' diarrhea (TD) field studies is limited by the on-site processing and storage requirements for fecal specimens. The objectives of this investigation were to i) characterize the pathogen distribution in deployed military personnel with TD using the TaqMan® Array Card PCR (TAC) on frozen stool and diarrheal smears on Whatman FTA Elute cards (FTA cards), and to ii) compare TAC detection of enteropathogen targets using smeared FTA cards and frozen stool, using TAC on frozen stool as the 'reference standard'. Stool samples, obtained from active duty personnel with acute TD enrolled in a field trial, were smeared onto FTA cards and stored at room temperature. A corresponding aliquot of stool was frozen in a cryovial. FTA cards and frozen stool samples were tested at a central lab, using a customized TAC for detection of TD pathogens. 187 paired frozen stool samples and smeared FTA cards were stored for a median of 712 days (IQR 396-750) before testing. Overall detection rates were 78.6% for frozen stool and 73.2% for FTA cards. Diarrheagenic Escherichia coli were the most common bacteria identified. Using the TAC results on frozen stool as the reference, the overall sensitivity and specificity of TAC on FTA cards was 72.9% and 98.0% respectively. TAC on FTA cards demonstrated a decrease in sensitivity with increasing frozen stool quantification cycle (Cq) (90.0% in FTA cards with a corresponding frozen stool Cq < 30, and 72.9% in samples with a corresponding frozen stool Cq < 35). Our findings support the use and further development of FTA cards in combination with a quantitative PCR assay for enteropathogen detection in TD field studies.

Paper documents in archives, libraries, and museums often undergo biodeterioration by microorganisms. Fungi and less often bacteria have been described to advance paper staining, so called “foxing” and degradation of paper substrates. In this study, for the first time, the fungal and bacterial diversity in biodeteriorated paper documents of Hellenic General State Archives dating back to the 19th and 20th century has been assessed by culture‐dependent and independent methods. The internally transcribed spacer (ITS) region and 16S rRNA gene were amplified by PCR from fungal and bacterial isolates and amplicons were sequenced. Sequence analysis and phylogeny revealed fungal phylotypes like Penicillium sp., Cladosporium sp., Penicillium citrinum, Alternaria infectoria, Alternaria alternata, Epicoccum nigrum, and Penicillium chrysogenum which are often implicated in paper deterioration. Bacterial phylotypes closely related to known biodeteriogenic bacteria such as Bacillus spp., Micrococcus spp., Kocuria sp. in accordance with previous studies were characterized. Among the fungal phylotypes described in this study are included well‐known allergens such as Penicillium spp., Alternaria spp., and Cladosporium spp. that impose a serious health threat on staff members and scholars. Furthermore, fungal isolates such as Chalastospora gossypii and Trametes ochracea have been identified and implicated in biodeterioration of historical paper manuscripts in this study for the first time. Certain new or less known fungi and bacteria implicated in paper degradation were retrieved, indicating that particular ambient conditions, substrate chemistry, or even location might influence the composition of colonizing microbiota.

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

Traditionary multiplex asymmetric polymerase chain reaction (PCR) can be applied to detect multiplex target organisms simultaneously, but complex optimizations of primer concentrations and staggered additions of primers are required to achieve equal amplification of multiplex genes. To overcome this shortcoming, we propose a novel method based on multiplex asymmetric PCR and paper-based nucleic acid diagnostics (PBNAD). In the asymmetric PCR, a universal primer was introduced to break the bottlenecks of low sensitivity and self-inhibition among different sets of primers. Amplification using the novel multiplex asymmetric PCR boosted the quantity of single-stranded amplicons, and the amplified products contained the same sequence at the 5' end. Therefore, only one gold nanoparticle-based signal probe was needed for the simultaneous detection of three genes using the PBNAD platform, and the detection signals could be observed with the naked eye. With this highly efficient, novel multiplex asymmetric PCR, as little as 1 pg/μL genomic DNA can be detected. This method can also be applied to genotyping for reliable epidemiological investigations. This proof-of-concept study highlights the potential of the PBNAD platform for cost- and labor-effective applications in the detection of pathogenic bacteria.

Background Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus. Methods Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing. Results For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%. Conclusion The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drugresistant TB in low-resource settings. Fil: Rabodoarivelo, Marie Sylvianne. University of Ghent; Bélgica Fil: Imperiale, Belén Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; Argentina Fil: Andrianiavomikotroka, Rina. Institut Pasteur de Madagascar; Madagascar Fil: Brandao, Angela. Fundación Oswaldo Cruz; Brasil Fil: Kumar, Parveen. All India Institute of Medical Sciences; India Fil: Singh, Sarman. All India Institute of Medical Sciences; India Fil: Ferrazoli, Lucilaine. Instituto Adolfo Lutz Núcleo de Tuberculose e Micobacteriosis Sao Paulo; Brasil Fil: Morcillo, Nora. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; Argentina Fil: Rasolofo, Voahangy. Institut Pasteur de Madagascar; Madagascar Fil: Palomino, Juan Carlos. University of Ghent; Bélgica Fil: Vandamme, Peter. University of Ghent; Bélgica Fil: Martin, Anandi. University of Ghent; Bélgica