To see the other types of publications on this topic, follow the link: Multiplex PCR, Co-infection, Sequencing.
Journal articles on the topic 'Multiplex PCR, Co-infection, Sequencing'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Multiplex PCR, Co-infection, Sequencing.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
& et al., Salah. "MULTIPLEX SYBR GREEN ASSAY FOR CORONAVIRUS DETECTION USING FAST REAL-TIME RT-PCR." IRAQI JOURNAL OF AGRICULTURAL SCIENCES 51, no. 2 (April 26, 2020): 556–64. http://dx.doi.org/10.36103/ijas.v51i2.982.
Full textAbstract:
This study was aimed to provide a local database for detection of coronavirus (CoV) species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green reverse transcriptase real-time PCR (rRT-PCR) technique. A total of 500 samples was collected from individuals suffering from upper and/or lower respiratory tract diseases for testing of 4 CoV species (229E, OC43, NL63, and HKU1). RNA extracted, amplified and subsequent the positive samples sequencing. The results showed melting curve analysis (Tm) of the specific amplicons (79.73±0.36) and 9% positive for CoVs and some of them have other co-infection such as influenza virus 26.67%, and TB 11.11%. On the other hands, the CoVs were detected 4.62% in upper respiratory samples and 20.39% with lower respiratory samples. Sequencing results pointed out two isolates were CoV-NL63 and four isolates were CoV-229E, with first record accession number MN086823.1 and MN086824.1, respectively in GenBank. In conclusion, this rRT-PCR showed the rapid and efficient detection of CoVs with few copies number. This allows being used for the diagnosis of CoVs along with other respiratory viruses in a multiplex assay to reduce processing time. Subsequent applied nested RT-PCR to overcome the low viral load.
2
Song, Yuli, Chengxu Liu, Maureen McTeague, Ann Vu, Jia Yia Liu, and Sydney M. Finegold. "Rapid identification of Gram-positive anaerobic coccal species originally classified in the genus Peptostreptococcus by multiplex PCR assays using genus- and species-specific primers." Microbiology 149, no. 7 (July 1, 2003): 1719–27. http://dx.doi.org/10.1099/mic.0.26227-0.
Full textAbstract:
Here, a rapid and reliable two-step multiplex PCR assay for identifying 14 Gram-positive anaerobic cocci (GPAC) species originally classified in the genus Peptostreptococcus (Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus octavius, Anaerococcus prevotii, Anaerococcus tetradius, Anaerococcus vaginalis, Finegoldia magna, Micromonas micros, Peptostreptococcus anaerobius, Peptoniphilus asaccharolyticus, Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus ivorii and Peptoniphilus lacrimalis) is reported. Fourteen type strains representing 14 GPAC species were first identified to the genus level by multiplex PCR (multiplex PCR-G). Since three of these genera (Finegoldia, Micromonas and Peptostreptococcus) contain only a single species, F. magna, M. micros and P. anaerobius, respectively, these organisms were identified to the species level directly by using the multiplex PCR-G. Then six species of the genus Anaerococcus (A. hydrogenalis, A. lactolyticus, A. octavius, A. prevotii, A. vaginalis and A. tetradius) were further identified to the species level using multiplex PCR assays (multiplex PCR-Ia and multiplex PCR-Ib). Similarly, five species of the genus Peptoniphilus (Pn. asaccharolyticus, Pn. harei, Pn. indolicus, Pn. ivorii and Pn. lacrimalis) were identified to the species level using multiplex PCR-IIa and multiplex PCR-IIb. The established two-step multiplex PCR identification scheme was applied to the identification of 190 clinical isolates of GPAC species that had been identified previously to the species level by 16S rRNA sequencing and phenotypic tests. The identification obtained from multiplex PCR assays showed 100 % agreement with 16S rDNA sequencing identification, but only 65 % (123/190) agreement with the identification obtained by phenotypic tests. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of GPAC species. It will permit a more accurate assessment of the role of various GPAC species in infection and of the degree of antimicrobial resistance in each of the group members.
3
Ramteke, Manoj P., Kuldeep J. Patel, Mukul Godbole, Maulik Vyas, Kunal Karve, Anuradha Choughule, Kumar Prabhash, and Amit Dutt. "CRE: a cost effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons." F1000Research 4 (June 23, 2015): 160. http://dx.doi.org/10.12688/f1000research.6663.1.
Full textAbstract:
Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a novel and cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five KRAS and EGFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.
4
Ramteke, Manoj P., Kuldeep J. Patel, Mukul Godbole, Maulik Vyas, Kunal Karve, Anuradha Choughule, Kumar Prabhash, and Amit Dutt. "CRE: a cost effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons." F1000Research 4 (March 8, 2016): 160. http://dx.doi.org/10.12688/f1000research.6663.2.
Full textAbstract:
Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five KRAS and EGFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.
5
Babiker, Ahmed, Heath L. Bradley, Victoria D. Stittleburg, Autum Key, Colleen Kraft, Jesse Waggoner, and Anne Piantadosi. "64. Metagenomic Sequencing to Identify Alternative Infections and Co-infections in Persons Under Investigation for covid-19." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S163—S164. http://dx.doi.org/10.1093/ofid/ofaa439.374.
Full textAbstract:
Abstract Background Broad testing for respiratory viruses among persons under investigation (PUI) for SARS-CoV-2 is performed inconsistently, limiting our understanding of alternative infections and co-infections in these patients. Here, we used unbiased metagenomic next-generation sequencing (mNGS) to assess the frequencies of 1) alternative viral infections in SARS-CoV-2 RT-PCR negative PUIs and 2) viral co-infections in SARS-CoV-2 RT-PCR positive PUIs. Methods A convenience sample set was selected from PUIs who were tested for SARS-CoV-2 in the Emory Healthcare system during the first 2 months of the pandemic from 02/26-04/23/20. Laboratory results were extracted by chart review; Flu/RSV and multiplex respiratory pathogen PCRs had been performed at the discretion of treating physicians. Excess nasopharyngeal swab samples were retrieved within 72 hours of collection and underwent RNA extraction and SARS-CoV-2 testing by triplex RT-PCR. mNGS was performed by DNAse treatment, random primer cDNA synthesis, Nextera XT tagmentation, and high-depth Illumina sequencing. Reads underwent taxonomic classification by KrakenUniq, as implemented in viral-ngs. Results 53 PUIs were included, 30 negative and 23 positive for SARS-CoV-2 by RT-PCR. Among SARS-CoV-2 negative PUIs, 28 (93%) underwent clinical testing for alternative infections, and 8 (29%) tested positive for another respiratory virus. In all cases, mNGS identified the same virus (Table 1). In another 3 PUIs, mNGS identified two viruses that were not tested for and one that was missed by routine testing. No SARS-CoV-2 was detected by mNGS among RT-PCR negative PUIs. Among SARS-CoV-2 RT-PCR positive PUIs, 18 (69%) underwent clinical testing for co-infections, and none were detected. mNGS did not identify any viral co-infections but did detect SARS-CoV-2 in all 23 PUIs. Table 1: Molecular and Metagenomic Testing of Persons Under Investigation Conclusion Unbiased mNGS offers the powerful opportunity to streamline testing for PUIs by assessing for SARS-CoV-2 and alternative infections simultaneously; this technique can also be used to identify co-infections, but none were observed in our study population. Interestingly, many PUIs had no infection identified on routine testing or mNGS, which may reflect inadequate sampling, rapid virus clearance, or a non-viral process. Disclosures All Authors: No reported disclosures
6
Yamamoto, Lidia, Antonio G. Amorim Filho, Joelma A. Queiroz, Mario H. B. de Carvalho, Jonatas C. Rodrigues, Kelly A. Kanunfre, Rossana P. V. Francisco, and Thelma Suely Okay. "Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections." Archives of Pathology & Laboratory Medicine 144, no. 1 (June 20, 2019): 99–106. http://dx.doi.org/10.5858/arpa.2018-0544-oa.
Full textAbstract:
Context.— Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. Objective.— To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. Design.— Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. Results.— Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. Conclusions.— The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.
7
Kanjilal, Sanjat, Tracey A. Cho, and Anne Piantadosi. "Diagnostic Testing in Central Nervous System Infection." Seminars in Neurology 39, no. 03 (June 2019): 297–311. http://dx.doi.org/10.1055/s-0039-1688441.
Full textAbstract:
AbstractPatients with central nervous system (CNS) infection experience very high levels of morbidity and mortality, in part because of the many challenges inherent to the diagnosis of CNS infection and identification of a causative pathogen. The clinical presentation of CNS infection is nonspecific, so clinicians must often order and interpret many diagnostic tests in parallel. This can be a daunting task given the large number of potential pathogens and the availability of different testing modalities. Here, we review traditional diagnostic techniques including Gram stain and culture, serology, and polymerase chain reaction (PCR). We highlight which of these are recommended for the pathogens most commonly tested among U.S. patients with suspected CNS infection. Finally, we describe the newer broad-range diagnostic approaches, multiplex PCR and metagenomic sequencing, which are increasingly used in clinical practice.
8
Alanazi, Abdullah D., Abdulaziz S. Alouffi, Mohamed S. Alyousif, Mohammad Y. Alshahrani, Hend H. A. M. Abdullah, Sobhy Abdel-Shafy, Nichola Eliza Davies Calvani, Maryam Ansari-Lari, Alireza Sazmand, and Domenico Otranto. "Molecular Survey of Vector-Borne Pathogens of Dogs and Cats in Two Regions of Saudi Arabia." Pathogens 10, no. 1 (December 31, 2020): 25. http://dx.doi.org/10.3390/pathogens10010025.
Full textAbstract:
Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood samples were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive samples were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.
9
Ljubin-Sternak, Sunčanica, Anamarija Slović, Maja Mijač, Mirna Jurković, Dubravko Forčić, Irena Ivković-Jureković, Tatjana Tot, and Jasmina Vraneš. "Prevalence and Molecular Characterization of Human Bocavirus Detected in Croatian Children with Respiratory Infection." Viruses 13, no. 9 (August 31, 2021): 1728. http://dx.doi.org/10.3390/v13091728.
Full textAbstract:
Human bocavirus (HBoV) 1 is considered an important respiratory pathogen, while the role of HBoV2-4 in clinical disease remains somewhat controversial. Since, they are characterized by a rapid evolution, worldwide surveillance of HBoVs’ genetics is necessary. This study explored the prevalence of HBoV genotypes in pediatric patients with respiratory tract infection in Croatia and studied their phylogeny. Using multiplex PCR for 15 respiratory viruses, we investigated 957 respiratory samples of children up to 18 years of age with respiratory tract infection obtained from May 2017 to March 2021 at two different hospitals in Croatia. Amplification of HBoV near-complete genome or three overlapping fragments was performed, sequenced, and their phylogenetic inferences constructed. HBoV was detected in 7.6% children with a median age of 1.36 years. Co-infection was observed in 82.2% samples. Sequencing was successfully performed on 29 HBoV positive samples, and all belonged to HBoV1. Croatian HBoV1 sequences are closely related to strains isolated worldwide, and no phylogenetic grouping based on mono- or co-infection cases or year of isolation was observed. Calculated rates of evolution for HBoV1 were 10−4 and 10−5 substitutions per site and year. Recombination was not detected among sequences from this study.
10
Ahmad, Suhail, and Eiman Mokaddas. "Diversity of Nontuberculous Mycobacteria in Kuwait: Rapid Identification and Differentiation of Mycobacterium Species by Multiplex PCR, INNO-LiPA Mycobacteria v2 Assay and PCR Sequencing of rDNA." Medical Principles and Practice 28, no. 3 (2019): 208–15. http://dx.doi.org/10.1159/000498910.
Full textAbstract:
Objective: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. Materials and Methods: DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.
11
Hwang, Yang-Ha, Wonsoo Son, Yong-Won Kim, Dong-Hun Kang, Hyun-Ha Chang, Youn-Kyoung Goo, Yeonchul Hong, and Dong-Il Chung. "A Retrieved Sparganum of Spirometra erinaceieuropaei from a Korean Man during Mechanical Thrombectomy." Korean Journal of Parasitology 58, no. 3 (June 26, 2020): 309–13. http://dx.doi.org/10.3347/kjp.2020.58.3.309.
Full textAbstract:
Human sparganosis is a zoonotic disease caused by infection and migration of the plerocercoid of Spirometra spp. Although sparganosis were reported from most parts of the body, the sparganum parasitizing inside cerebral artery is remarkably uncommon. We report a case of cerebral intravascular sparganosis in an elderly patient with acute ischemic stroke who was diagnosed by retrieving sparganum during mechanical thrombectomy. Finally, the parasites were identified as Spirometra erinaceieuropaei using multiplex PCR and cox1 gene sequencing.
12
Elmanakhly, Arwa R., and Amani Kholy. "550. Carbapenem--Resistant E. coli and A. baumannii Among Catheter-Related Blood Stream Infection Patients in Egyptian ICUs." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S262. http://dx.doi.org/10.1093/ofid/ofz360.619.
Full textAbstract:
Abstract Background The spreading of E. coli and A. baumannii in hospitals is a growing concern due to increased resistance to carbapenems and Fluoroquinolones. The present study aimed to specifically evaluate the presence of mutations in the gyrA and parC genes in Egyptian ICU and their correlation with carbapenem-resistant genes E. coli and A. baumannii isolates from patients in 4 tertiary care hospital in Egypt. Methods A total of 120 A. baumannii and E. coli clinical isolates were isolated from ICU patients in 4 tertiary hospitals in Egypt. The bacterial isolates were identified by VITEK-2 (Bio Merieux, France). Antimicrobial susceptibility testing was performed according to CLSI guidelines. Phenotypic detection of carbapenemase activity was done by carba-NP test, followed by molecular identification of carbapenemase encoding genes blaNDM, blaOXA-48 and blaKPC by multiplex PCR. The quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were amplified by singleplex PCR followed by reverse and forward sequencing to detect the gene mutation. The DNA sequences were compared with the sequences of wild type of these genes available in GenBank database. Then, the obtained DNA sequences and their amino acid sequences were analyzed using bioinformatics tools. Results All isolates showed a high level of resistance among tested antimicrobial agents (cephalosporins, aminoglycosides, carbapenems, penicillins) that ranged from 36% to 100%. Carba-NP detected 43.59% of the carbapenem-resistant isolates. Multiplex PCR detected that 17.95%, 46.15% and 2.56% of isolates were harboring blaKPC, blaNDM and blaOXA-48 respectively. PCR and sequencing technique showed combined gene mutation in 8 carbapenem-resistant E. coli and A. baumannii isolates. The specific substitutions observed in gyrA were Cys173Arg, Cys174 Gly, Asp80Val, Tyr178ASP, Tyr84Gly, Glu85Lys, Ser172Leu and Asp176Asn. While, the specific substitutions observed in parC were point mutation 62 Arg, Phe60Leu, Ils66Val, Gln76Lys. Point mutation 62 Arg was observed in two A. baumannii isolates, whereas Ser172Leu mutation was observed in two E. coli isolates. Conclusion The presence of carbapenem resistance genes in combination with single and multiple mutations in QRDR causes the presence of highly resistant E. coli and A. baumannii isolates in the Egyptian hospitals. Disclosures All authors: No reported disclosures.
13
Suryani, Utari Hartati, Andri Rezano, and Yunia Sribudiani. "Polymerase Chain Reaction Multiplication for the Detection of Bacterial Isolates Causing Neonatal Sepsis." Open Access Macedonian Journal of Medical Sciences 8, A (September 30, 2020): 623–28. http://dx.doi.org/10.3889/oamjms.2020.5343.
Full textAbstract:
BACKGROUND: Neonatal sepsis is a clinical syndrome caused by the presence of bacteria in the blood accompanied by symptoms and systemic signs of infection that occurs in the first 4 weeks of life after birth. The process of identifying pathogenic microorganisms is essential in determining the clinical condition in neonatal sepsis.
AIM: The study was aimed to develop a multiplex polymerase chain reaction (PCR) method to identify bacterial isolates that cause neonatal sepsis in Indonesia with the main target of optimization of an initial design and PCR optimization.
METHODS: This research is an explorative in vitro study for the optimization of an initial design and PCR methods for the detection of the main bacteria that cause sepsis neonatorum in Indonesia, namely, bacteria Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa. The study was conducted at the Biomolecular Laboratory of the Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia.
RESULTS: Sequencing carried out and continued with Basic Local Alignment Search Tool (BLAST) sequencing results, it appears that the PCR product that has been produced conforms with the optimization targets that were previously set when doing the primer design. The level of homology found in all four species based on the results of BLAST in a sequence is as follows: K. pneumoniae 94%, P. aeruginosa 99%, E. cloacae 100%, and E. coli 100%.
CONCLUSION: PCR multiplex method using design primers and conventional PCR analysis methods (using agarose gel) can be used to detect four species of bacteria that cause neonatal sepsis, namely, K. pneumoniae, P. aeruginosa, E. cloacae, and E. coli.
14
Pérez-Lago, Laura, Marta Herranz, Iñaki Comas, María Jesús Ruiz-Serrano, Paula López Roa, Emilio Bouza, and Darío García-de-Viedma. "Ultrafast Assessment of the Presence of a High-Risk Mycobacterium tuberculosis Strain in a Population." Journal of Clinical Microbiology 54, no. 3 (December 30, 2015): 779–81. http://dx.doi.org/10.1128/jcm.02851-15.
Full textAbstract:
A persistent 8-year infection by a BeijingMycobacterium tuberculosisstrain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission—despite opportunities—and challenged some of the advantages associated with Beijing strains.
15
Claus, Marlise Pompeo, Michele Lunardi, Amauri Alcindo Alfieri, Rodrigo Alejandro Arellano Otonel, Lara Munique Ferracin, Maria Helena Pelegrinelli Fungaro, and Alice Fernandes Alfieri. "A bovine teat papilloma specimen harboring Deltapapillomavirus (BPV-1) and Xipapillomavirus (BPV-6) representatives." Brazilian Archives of Biology and Technology 52, spe (November 2009): 87–91. http://dx.doi.org/10.1590/s1516-89132009000700012.
Full textAbstract:
The common occurrence of multiple papillomavirus infections has been shown in several studies involving the human host. However, investigations with the aim of identifying mixed papillomavirus infections in cattle have been conducted only recently. In the current work we describe a co-infection with two different bovine papillomavirus (BPV) types that was identified in a bovine teat papilloma. The skin wart was obtained from a cow belonging to a Brazilian beef herd. A PCR assay was carried out with the FAP primer pair, which amplifies a partial segment of the L1 gene (approximately 478 bp), and the amplicon was submitted to direct sequencing. Because nucleotide sequences with satisfactory quality scores were not obtained, the amplicon was cloned and further sequencing, involving ten selected clones, was performed. The sequence analysis of the cloned inserts revealed the presence of two different BPV types. BPV-1 (Deltapapillomavirus genus) was detected in six clones, while BPV-6 (Xipapillomavirus genus) was detected in four clones. This finding confirms the presence of BPV co-infection associated with cutaneous papillomatosis in cattle.
16
Chagas, Domitila B., Francielle Liz Monteiro, Lariane da S. Barcelos, Matheus Iuri Frühauf, Leonardo C. Ribeiro, Marcelo de Lima, Silvia de O. Hübner, and Geferson Fischer. "Black queen cell virus and Nosema ceranae coinfection in Africanized honey bees from southern Brazil." Pesquisa Veterinária Brasileira 40, no. 11 (November 2020): 892–97. http://dx.doi.org/10.1590/1678-5150-pvb-6678.
Full textAbstract:
ABSTRACT: Bees are fundamental in several aspects, especially in relation to plant biodiversity and pollination. Recently, immense losses are being faced in the number of Brazilian colonies, mainly in southern states of the country, which has a strong beekeeping activity. There are indications that, among the reasons for the losses, pathogens that affect the health of bees may be involved. Among them, the microsporidium Nosema and the black queen cell virus (BQCV) stand out for their prevalence. In this study, 92 colonies of 17 apiaries from southern Brazil were evaluated for infection by Nosema ceranae, Nosema apis and BQCV. Nucleic acid extractions and cDNA synthesis were performed from adult bee samples, followed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and multiplex PCR. Eight BQCV positive samples were subjected to sequencing. The results showed that N. ceranae and BQCV are circulating in the Southern region of the country, which may be the reason for the loss of colonies. N. apis was not found. N. ceranae was found in 57.6% (53/92) of the colonies and BQCV in 32.6% (30/92). Co-infection was found in 25% (23/92) of the colonies studied, a factor that is suggested to be reducing the hosts’ longevity due to the synergistic action of the pathogens. The samples submitted to sequencing indicated similarity of 96.8 to 100% between them, in addition to strong similarity with sequences from Asia, United States, Germany and Peru. This study reports the circulation of N. ceranae and BQCV in apiaries in southern Brazil, in addition to being the first phylogenetic analysis of the Brazilian BQCV sequence.
17
Kumar, P., M. Jain, A. K. Goel, S. Bhadauria, S. K. Sharma, D. V. Kamboj, L. Singh, T. Ramamurthy, and G. B. Nair. "A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, Eastern India." Journal of Medical Microbiology 58, no. 2 (February 1, 2009): 234–38. http://dx.doi.org/10.1099/jmm.0.002089-0.
Full textAbstract:
A total of 32 Vibrio cholerae isolates were collected during a recent large cholera outbreak in Eastern India. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. Two multiplex PCR assays confirmed the presence of various toxigenic and pathogenic genes – ace, ctxAB, hlyA, ompU, ompW, rfbO1, rtx, tcp, toxR and zot – in all of the isolates. Sequencing of the ctxB gene from the isolates revealed a novel mutation in the gene. Sequencing also confirmed the presence of altered cholera toxin B of the classical biotype in all of the El Tor isolates, suggesting infection of isolates by classical CTXΦ. The molecular diversity of V. cholerae isolates studied by enterobacterial repetitive intergenic consensus sequence PCR, BOX-PCR and randomly amplified polymorphic DNA analysis uniformly showed the clonal relationship among the outbreak V. cholerae O1 isolates. The results of this study suggest that cholera-causing V. cholerae strains are constantly evolving in epidemic areas, highlighting the potential of the emergence of more virulent strains.
18
BULLMAN, S., J. O'LEARY, D. CORCORAN, R. D. SLEATOR, and B. LUCEY. "Molecular-based detection of non-culturable and emerging campylobacteria in patients presenting with gastroenteritis." Epidemiology and Infection 140, no. 4 (May 18, 2011): 684–88. http://dx.doi.org/10.1017/s0950268811000859.
Full textAbstract:
SUMMARYFrom January 2009 to May 2010, 436 faecal samples from patients with diarrhoeal illness in Southern Ireland were identified asCampylobactergenus-positive by an automated multiplex PCR; however, 204 (46·8%) of these samples were culture-negative for campylobacters. A combination ofCampylobacter-specific uniplex PCR and 16S rRNA sequencing confirmed the presence ofCampylobacterDNA in 191 (93·6%) of the culture-negative samples. Species-specific PCR identifiedC. jejuni(50·7%)C. ureolyticus(41%) andC. coli(5·7%) as the most prevalent species whileC. fetus,C. upsaliensis,C. hyointestinalisandC. lariaccounted for 10% of culture-negative samples; mixedCampylobacterspp. were detected in 11% of samples. We conclude that non-culturableCampylobacterspp. are responsible for a considerable proportion of human enteritis and the true incidence of infection is likely to be significantly underestimated where conventionalCampylobacterculture methods are used in isolation.
19
Yepes, Luz Marcela, Elizabeth Cieniewicz, Björn Krenz, Heather McLane, Jeremy R. Thompson, Keith Lloyd Perry, and Marc Fuchs. "Causative Role of Grapevine Red Blotch Virus in Red Blotch Disease." Phytopathology® 108, no. 7 (July 2018): 902–9. http://dx.doi.org/10.1094/phyto-12-17-0419-r.
Full textAbstract:
Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch’s postulates and revealed the causative role of GRBV in red blotch disease.
20
Eberlein, Jens, Thomas Harrison, Ian McKittrick, Megan Wemmer, Laura Griffin, Brady P. Culver, Laura Johnson, and Brian A. Kudlow. "Characterization of B- and T-Cell Immune Repertoires By Anchored Multiplex PCR and Next-Generation Sequencing." Blood 128, no. 22 (December 2, 2016): 4896. http://dx.doi.org/10.1182/blood.v128.22.4896.4896.
Full textAbstract:
Abstract The adaptive immune system is involved in various disease conditions including cancer, chronic infection, autoimmune disease and transplant rejection. Adaptive immunity is mediated by B and T lymphocytes, which are activated upon antigen binding to antigen receptors expressed on their surface. Therefore, the spectrum of these antigen receptors, or immune repertoire (IR), provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions. Next-generation sequencing (NGS) of antigen receptor genes is a valuable tool in the study of disease states and responses to various interventions. Traditional amplicon-based NGS assays use opposing primers for targeted amplification of rearranged antigen receptor genes. Thus, large primer panels are required to capture the extensive combinatorial diversity exhibited by the IR. Quantification from such assays requires a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Here, we describe an Anchored Multiplex PCR (AMP)-based NGS assay to analyze the IR, employing a minimal set of gene-specific primers in conjunction with molecular barcodes (MBCs) to reduce amplification bias. AMP uses MBCs ligated to cDNA ends and gene-specific primers for amplification, enabling immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Furthermore, this facilitates CDR3 sequence capture from highly fragmented RNA inputs. We validated the quantitative reproducibility and sensitivity of AMP-based B- and T-cell IR assays using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples. Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%. Furthermore, our data indicate that clonal diversity in sequencing data is driven by input quantity, total T-cell number, and, to a lesser degree, mRNA quality. Therefore, AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire. This enables sensitive clone tracking and measurement of lymphocyte diversity from fragmented RNA samples. Disclosures Eberlein: ArcherDX, Inc.: Employment. Harrison:ArcherDX, Inc.: Employment. McKittrick:ArcherDX, Inc.: Employment. Wemmer:ArcherDX, Inc.: Employment. Griffin:ArcherDX, Inc.: Employment. Culver:ArcherDX, Inc.: Employment. Johnson:ArcherDX, Inc.: Employment. Kudlow:ArcherDX, Inc.: Employment.
21
DE ARAÚJO, VITOR ANTÔNIO L., MARIANA C. BOITÉ, ELISA CUPOLILLO, ANA MARIA JANSEN, and ANDRÉ LUIZ R. ROQUE. "Mixed infection in the anteater Tamandua tetradactyla (Mammalia: Pilosa) from Pará State, Brazil: Trypanosoma cruzi, T. rangeli and Leishmania infantum." Parasitology 140, no. 4 (December 20, 2012): 455–60. http://dx.doi.org/10.1017/s0031182012001886.
Full textAbstract:
SUMMARYSome Trypanosoma and Leishmania species are multi-host parasites whose distribution overlaps in several parts of the Brazilian Amazon basin. Despite being a common trait among wild mammals, mixed infections and their consequences for the host's health and parasite transmission are still a poorly known phenomenon. Here we describe a triple mixed infection – Trypanosoma cruzi, T. rangeli and Leishmania infantum – in a bone marrow sample from an anteater Tamandua tetradactyla captured in a house backyard from the endemic Abaetetuba municipality in the Amazon basin. T. cruzi was also isolated from blood samples. The mini-exon multiplex PCR characterization detected the infection by T. rangeli and T. cruzi (TcI genotype), while L. infantum infection was confirmed by an ITS-PCR followed by amplicon sequencing. This is the first description of T. rangeli isolation from bone marrow and the first report of L. infantum infection in xenarthrans. The implications of this finding are discussed considering the influence of mixed infections in the role of this mammal species as a putative reservoir host of these 3 trypanosomatid species.
22
Sashina, T. A., O. V. Morozova, N. V. Epifanova, and N. A. Novikova. "IDENTIFICATION OF ROTAVIRUS I- AND E-GENOTYPES BY MULTIPLEX PCR METHOD." Problems of Virology, Russian journal 64, no. 3 (June 20, 2019): 140–44. http://dx.doi.org/10.18821/0507-4088-2019-64-3-140-144.
Full textAbstract:
Introduction. In recent years the presence of reassortant rotavirus strains is increasingly mentioned in the world due to the application of the full-genome based classification system. Information on the circulation of such strains in the territory of Russia is limited. The aim of this work was the development of the approach for determination of genotypes of segments encoding VP6 (I) and NSP4 (E) to reveal reassortant strains. Material and Methods. Rotavirus-positive samples were studied by means of nucleotide sequencing and multiplex PCR. Phylogenetic analysis was conducted using the Bayesian approach. Results. Three alleles of the VP6 gene (I1-1, I2-IV, I2-VII) and seven alleles of the NSP4 gene (E1-I, E1-III, E2-VI, E2-VII, E2-X, E2-XII, E3) were detected on the base of nucleotide sequences of Nizhny Novgorod rotaviruses. Taking into account these results, the oligonucleotide primers specific to genotypes I1, I2, I3 and E1, E2, E3 were designed. Optimal conditions for multiplex PCR were chosen. The method was tested using the strains collected in Nizhny Novgorod in 2018. The diversity of I and E genotypes was determined and various combinations with G and P genotypes were identified. Discussion. G9-P[8]-I1-E1 rotaviruses were predominant (32.7 %) and G2-P[4]-I2-E2 rotaviruses were in second place (29.1 %). Strains with genotypes G4-P[8]-I1-E2, G3-P[8]-I2-E2 and G2-P[4]-I2-E1 were detected sporadically. They had genes of two rotavirus genogroups, so can be considered to be reassortant. Conclusion. The proposed approach is a useful tool for the characterization of rotaviruses in the conditions of the beginning of vaccination against rotavirus infection in Russia.
23
TRACHSEL, D., P. DEPLAZES, and A. MATHIS. "Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA." Parasitology 134, no. 6 (February 9, 2007): 911–20. http://dx.doi.org/10.1017/s0031182007002235.
Full textAbstract:
SUMMARYA multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers forEchinococcus multilocularis(amplicon size 395 bp) were species-specific as assessed byin silicoanalysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA fromE. granulosusorTaeniaspp. was not possible. The primers designed forE. granulosusalso amplified DNA (117 bp) fromE. vogeli, and those designed forTaeniaspp. amplified products (267 bp) from species ofMesocestoides,DipylidiumandDiphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the describedE. granulosusgenotypes.Taeniaspp. can be identified by direct sequencing of the 267 bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.
24
Wieczorek, Przemysław, and Aleksandra Obrępalska-Stęplowska. "Multiplex RT-PCR Reaction for Simultaneous Detection of Tomato Torrado Virus and Pepino Mosaic Virus Co-Infecting Solanum Lycopersicum." Journal of Plant Protection Research 53, no. 3 (July 1, 2013): 289–94. http://dx.doi.org/10.2478/jppr-2013-0043.
Full textAbstract:
Abstract The tomato (Solanum lycopersicum L.) is cultivated all over the world and is a vegetable of significant economic importance. However, an increased production of the vegetable is directly connected with an elevated occurrence of pathogens limiting the production efficiency of the vegetable. Both, Tomato torrado virus and Pepino mosaic virus have been found to be serious disease factors. When not controlled, these viruses can significantly decrease tomato cultivation. In this article, we report a multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocol for simultaneous detection of both, Tomato torrado virus (ToTV) and Pepino mosaic virus (PepMV) in virus infected plants. The assay was designed to specifically amplify the conserved regions of genomic ribonucleic acid (RNA) of both viruses. Moreover, the glycerandehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control of amplification to exclude false-negative assay results. High-resolution melt analysis of generated RT-PCR products was additionally performed to increase sensitivity and double-check the specificity of the reaction without the need of subsequent complementary deoxyribonucleic acid (cDNA) sequencing
25
Lasham, Annette, Peter Tsai, Sandra J. Fitzgerald, Sunali Y. Mehta, Nicholas S. Knowlton, Antony W. Braithwaite, and Cristin G. Print. "Accessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual TP53 Transcripts." Cancers 12, no. 3 (March 24, 2020): 769. http://dx.doi.org/10.3390/cancers12030769.
Full textAbstract:
TP53, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53’s alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5’ and 3’ ends, but because they have a common central region of 618 bp, the individual TP53 transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major TP53 transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms.
26
Girdhar, Anurag, Jagdish Chinnappa, Feroze Ganaie, Vandana Govindan, Kadahalli Ravikumar, and Geetha Nagaraj. "Bacterial Profile of Middle Ear Fluid with Recurrent Acute Otitis Media Infection Using Culture Independent 16S rDNA Gene Sequencing." Journal of Pediatric Infectious Diseases 14, no. 03 (December 11, 2018): 108–15. http://dx.doi.org/10.1055/s-0038-1675786.
Full textAbstract:
Background Recurrent otitis media is one of the common infections of childhood. The causative bacterial pathogen is one of the major risk factors of recurrent infection. With limited availability of Indian data, we performed this study to identify the bacterial pathogens. Materials and Methods Otitis media cases were diagnosed based on clinical criteria. Thirty-six middle ear fluid (MEF) samples were collected by tympanocentesis and cultured for pathogens. Seventy-eight per cent of the cases had three previous episodes of otitis media in the past 6 months; the remaining 22% had four episodes in the preceding 6 months. At the time of sample collection, all patients were on antibiotic coverage. Genomic DNA was extracted from MEF samples using Qiagen DNA mini Kit. The 16s rDNA polymerase chain reaction (PCR) and quantitative multiplex (qmPCR) for Streptococcus pneumoniae was performed on these samples. Streptococcus pneumoniae–positive samples were serotyped using PCRSeqTyping. Results None of the 36 samples showed growth by conventional culture. The 16s rDNA PCR identified bacterial pathogens in 33 samples. Four samples gave mixed reads. The organisms identified were Neisseria spp. other than Neisseria meningitidis (n = 7), N. meningitidis (n = 8), Lactococcus spp. (n = 5), S. pneumoniae (n = 2), Pseudomonas aeruginosa (n = 2), Haemophilus influenzae (n = 1), Salmonella infantis (n = 1), Staphylococcus epidermidis (n = 1), Staphylococcus auricularis (n = 1), and Streptococcus sp. (n = 1). The qmPCR detected the presence of S. pneumoniae in six samples. PCRSeqTyping was able to identify Serotype 19A in two samples positive for S. pneumoniae. Conclusion The study demonstrates the usefulness of 16s rDNA PCR protocol to identify the bacterial pathogens in MEF by a culture-independent method. Neisseria spp. were the predominant species identified followed by Lactococcus spp. and S. pneumoniae. Detection of pneumococci by 16s rDNA PCR correlated well with qmPCR-based detection and PCRSeqTyping.
27
Anokhin, Vladimir V., Liliia B. Bakhteeva, Gulshat R. Khasanova, Svetlana F. Khaiboullina, Ekaterina V. Martynova, Richard L. Tillett, Karen A. Schlauch, Vincent C. Lombardi, and Albert A. Rizvanov. "Previously Unidentified Single Nucleotide Polymorphisms in HIV/AIDS Cases Associate with Clinical Parameters and Disease Progression." BioMed Research International 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/2742648.
Full textAbstract:
The genetic background of an individual plays an important role in the progression of HIV infection to AIDS. Identifying previously unknown or uncharacterized single nucleotide polymorphisms (SNPs) that associate with disease progression may reveal important therapeutic targets and provide a greater understanding of disease pathogenesis. In the present study, we employed ultra-high multiplex PCR on an Ion Torrent next-generation sequencing platform to sequence 23 innate immune genes from 94 individuals with HIV/AIDS. This data was used to identify potential associations of SNPs with clinical parameters and disease progression. SNPs that associated with an increased viral load were identified in the genes for the interleukin 15 receptor (IL15RA), toll-like receptor 7 (TLR7), tripartite motif-containing protein 5 (TRIM5), and two killer-cell immunoglobulin-like receptors (KIR2DL1andKIR2DL3). Additionally, SNPs that associated with progression from HIV infection to AIDS were identified in two 2′-5′-oligoadenylate synthetase genes (OAS2andOAS3). In contrast, other SNPs identified inOAS2andOAS3genes, as well as in theTRIM5andKIR2DS4genes, were associated with a slower progression of disease. Taken together, our data demonstrates the utility of ultra-high multiplex PCR in identifying polymorphisms of potential clinical significance and further,identifies SNPs that may play a role in HIV pathogenesis.
28
Furness, Caroline L., Marcela B. Mansur, Victoria J. Weston, Sarah Jenkinson, Frederik W. van Delft, Lyndal Kearney, Ian Titley, et al. "The Sub-Clonal Complexity of STIL-TAL1 T-ALL." Blood 124, no. 21 (December 6, 2014): 3788. http://dx.doi.org/10.1182/blood.v124.21.3788.3788.
Full textAbstract:
Abstract Introduction The STIL-TAL1 fusion is found in 16% cases of paediatric and adolescent T-ALL, making it one of the most common T-ALL subgroups. Our study considers this leukaemia subtype in the context of a complex ecosystem that is diverse, evolving and subject to selective pressures. We used single cell methods to understand the order of co-operating mutational events and the clonal evolution of mutations in genes that are re-iteratively targeted, such as PTEN. Methods Diagnostic DNA from five STIL-TAL1 positive T-ALL cases was exome sequenced using Agilent SureSelect Human all Exon kit plus Illumina paired end sequencing. Driver copy number alterations and NOTCH1/PTEN exon 7 mutation status had been identified in a previous study and candidate driver mutations for inclusion in single cell experiments were validated by sequencing or Q-PCR using custom assays. Where more than one mutation was present within the same exon of a candidate driver gene, cloning experiments were carried out to verify the independent mutation sequences. Material from xenograft transplants was available in three of the five cases to assess their clonal heterogeneity in the leukaemia initiating cell compartment. Single cell multiplex Q-PCR was used to examine the single cell genetics of the pre-defined mutation events. Briefly, single cells were sorted and lysed prior to multiplex specific (DNA) target amplification and Q-PCR using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm, UK). Copy number assays for the 1p33 deletion and custom assays for the patient specific STIL-TAL1 fusion breakpoints were used to confirm that the 1p33 deletion leading to this gene fusion was a clonal event. Results The only aberrant events common to all five samples were CKDN2A copy number loss and the 1p33 deletion that results in the STIL-TAL1 fusion. Exome sequencing revealed further mutations in known T-ALL drivers including NOTCH1, PTEN and PHF6 as well as candidate driver mutations in FREM2, PIK3CD, RPL14, BMPR1A and CDH18. Both NOTCH1 and PTEN demonstrated re-iterative inactivation and this was investigated in detail for PTEN. Case 1 had multiple PTEN exon 7 mutations and sub-clonal copy number loss. Case 2 had parallel frameshift mutations in PTEN exons 5 and 7. Case 3 contained an exon 8 mutation and multiple PTEN exon 7 mutations. In this case the three most frequent PTEN exon 7 indels were validated and tracked in a single cell multiplex Q-PCR experiment. This revealed a branching sub-clonal genetic architecture (see figure 1) in which all malignant cells at the proposed apex of the branching architecture harboured the STIL-TAL1 fusion and CDKN2A deletion with copy number losses of 4p, 6q and FREM2 and PTEN mutations occurring as sub-clonal events. PTEN indels 2 and 3 were found co-localised in the same sub-clone. Preliminary analysis of the paired mouse xenograft bone marrow did not detect PTEN exon 7 indels 1 – 3 in 84 single cells. However, bulk Sanger Sequencing analysis did identify the PTEN exon 8 mutation in the mouse. Ongoing work is in progress to determine whether single cells of the xenograft carry alternative PTEN exon 7 mutations detected in the diagnostic sample exome data and to characterise in which diagnostic sub-clone the PTEN exon 8 mutation resides. Conclusions This study demonstrates how exome sequencing and single cell multiplex Q-PCR can be used as complementary tools to understand the sub-clonal complexity of STIL-TAL1 T-ALL. PTEN inactivation is sub-clonal by single cell analysis, demonstrating the parallel evolution of multiple independent PTEN inactivated sub-clones, highlighting PTEN inactivation as a key event in this T-ALL subgroup. In a wider cohort of 20 patients collected by our group at least 50% had PTEN inactivation as assessed by sequencing of exon 7 and copy number data alone. Results indicate a strong evolutionary pressure selecting for mutational events that result in inactivation of the PTEN-PI3Kinase pathway. These events occur via multiple mechanisms, including copy number loss and truncating mutations, which are not limited to the known T-ALL hotspot in exon 7. Current work is focussing on using a similar approach to examine the clonal evolution of NOTCH1 mutations in STIL-TAL1 T-ALL samples in diagnostic and xenograft samples of cases 4 and 5. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
29
Rawangchue, Thanakorn, and Sivapong Sungpradit. "Clinicopathological and molecular profiles of Babesia vogeli infection and Ehrlichia canis coinfection." July-2020 13, no. 7 (2020): 1294–302. http://dx.doi.org/10.14202/vetworld.2020.1294-1302.
Full textAbstract:
Background and Aim: Canine babesiosis, a tick-borne parasitic disease, is caused by the hemoprotozoa, Babesia vogeli, and Babesia gibsoni. Infection with these parasites, which is endemic globally, leads to life-threatening immunosuppression in dogs. The merozoites invade the red blood cells (RBCs) of infected dogs. Ehrlichia canis, an intracellular bacterium that infects monocytes, is transmitted by the same tick species (Rhipicephalus sanguineus) during blood consumption and coinfection with B. vogeli and E. canis has been reported. Although the hematology and biochemistry of canine babesiosis have been studied, more studies are needed to develop a better understanding of the hematobiochemical and molecular profiles associated with cases of single infection and coinfection of canine babesiosis in Thailand. This study aimed to investigate the hematological, biochemical, and molecular profiles of B. vogeli infection and E. canis coinfection. Materials and Methods: The study included 33 B. vogeli–positive blood samples and 11 E. canis–coinfected blood samples. To exclude coinfection with Hepatozoon canis and Anaplasma platys, only dogs with B. vogeli infection and B. vogeli–E. canis coinfection were included in the study. A multiplex polymerase chain reaction (PCR) assay was conducted to detect B. vogeli, E. canis, and H. canis, and a conventional PCR assay was conducted for the detection of A. platys. Besides, the PCR assay and sequencing, comprehensive data analysis was conducted, including a microscopic blood parasite examination and hematological and biochemical data analysis. Results: The comparison of the hematobiochemical data between the B. vogeli–positive and E. canis coinfection groups identified that there were statistically significant differences in the RBC parameters, including RBC count, hemoglobin concentration, hematocrit, and RBC distribution width (p=0.001). Neither B. vogeli infection nor coinfection with E. canis was associated with the sex, breed, recorded clinical signs, geographic origin of the dog and also B. vogeli 18S rRNA gene sequencing results. Conclusion: Coinfection with E. canis increased the severity of babesiosis. The pathogenic mechanisms underlying this infection, such as destruction of RBCs, require further investigation. This study may enhance diagnosis, treatment, and prevention of canine babesiosis.
30
Abdel Aleem, Engy E., Radwa M. Taha, and Faiza A. Fattouh. "Biodiversity and full genome sequence of potato viruses Alfalfa mosaic virus and potato leaf roll virus in Egypt." Zeitschrift für Naturforschung C 73, no. 11-12 (November 27, 2018): 423–38. http://dx.doi.org/10.1515/znc-2018-0033.
Full textAbstract:
Abstract Solanum tuberosum (potato) is the second most important vegetable crop in Egypt. It is locally consumed, manufactured or supplied for export to Europe and other Arab countries. Potato is subject to infection by a number of plant viruses, which affect its yield and quality. Potato virus Y (PVY), potato leaf roll virus (PLRV), and Alfalfa mosaic virus (AMV) were detected in major potato-growing areas surveyed. Multiplex-RT-PCR assay was used for the detection of these three viruses in one reaction using three specific primer pairs designed to amplify genomic parts of each virus (1594 bp for PLRV, 795 bp for AMV, 801 bp for PVY). All three viruses were detected in a single reaction mixture in naturally infected field-grown potatoes. Multiplex RT-PCR improved sensitivity necessary for the early detection of infection. Incidence of single, double, or triple infection has been recorded in some locations. Full-length sequencing has been performed for an Egyptian FER isolate of PLRV. Through phylogenetic analysis, it was shown to occupy the same clade with isolate JokerMV10 from Germany. Complete nucleotide sequence of an Egyptian FER isolate of AMV and phylogenetic analysis was also performed; we propose that it is a new distinct strain of AMV belonging to a new subgroup IIC. This is the first complete nucleotide sequence of an Egyptian isolate of AMV. Genetic biodiversity of devastating potato viruses necessitates continuous monitoring of new genetic variants of such viruses.
31
Sidrim, José Júlio Costa, Ana Karoline Freire Costa, Rossana Aguiar Cordeiro, Raimunda Sâmia Nogueira Brilhante, Fernanda Edna Araújo Moura, Débora Souza Collares Maia Castelo-Branco, Manoel Paiva de Araújo Neto, and Marcos Fábio Gadelha Rocha. "Molecular methods for the diagnosis and characterization ofCryptococcus: a review." Canadian Journal of Microbiology 56, no. 6 (June 2010): 445–58. http://dx.doi.org/10.1139/w10-030.
Full textAbstract:
Cryptococcosis is a fungal infection caused by yeasts of the genus Cryptococcus , with Cryptococcus neoformans and Cryptococcus gattii as the primary pathogenic species. This disease is a threat to immunocompromised patients, especially those who have AIDS. However, the disease has also been described in healthy individuals. The tests used to identify these microorganisms have limitations that make final diagnosis difficult. However, currently there are specific gene sequences that can be used to detect C. neoformans and C. gattii from clinical specimens and cultures. These sequences can be used for identification, typing, and the study of population genetics. Among the main identification techniques are hybridization, which was the pioneer in molecular identification and development of specific probes for pathogen detection; PCR and other PCR-based methods, particularly nested PCR and multiplex PCR; and sequencing of specific genomic regions that are amplified through PCR, which is especially useful for diagnosis of cryptococcosis caused by unconventional Cryptococcus sp. Concerning microorganism typing, the following techniques have shown the best ability to differentiate between fungal serotypes and molecular types: PCR fingerprinting, PCR–RFLP, AFLP, and MLST. Thus, the accumulation of data generated by molecular methods can have a positive impact on monitoring resistant strains and treating diseases.
32
Fyfe, Janet A. M., Caroline J. Lavender, Paul D. R. Johnson, Maria Globan, Aina Sievers, Joseph Azuolas, and Timothy P. Stinear. "Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of Mycobacterium ulcerans in Clinical and Environmental Samples." Applied and Environmental Microbiology 73, no. 15 (May 25, 2007): 4733–40. http://dx.doi.org/10.1128/aem.02971-06.
Full textAbstract:
ABSTRACT Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.
33
Li, Jin, Shunxiang Qi, Chen Zhang, Xiumei Hu, Hongwei Shen, Mengjie Yang, Ji Wang, Miao Wang, Wenbo Xu, and Xuejun Ma. "A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Sixteen Human Respiratory Virus Types/Subtypes." BioMed Research International 2013 (2013): 1–8. http://dx.doi.org/10.1155/2013/327620.
Full textAbstract:
There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20–200 copies per reaction when each viral type was assayed individually, 2000 copies with 9 premixed viral targets in the multiplexed assay in tube 1, and 200 copies with 8 premixed templates in tube 2. A total of 247 specimens were used to evaluate the two-tube assay, and the results were compared with those obtained from the Luminex xTAG RVP Fast assay. The discordant results were confirmed by sequencing or by the Seeplex RV15 ACE detection kit. There were no false positives, but six false negatives occurred with the two-tube assay. In conclusion, the two-tube assay is demonstrated to have great potential for routine surveillance of respiratory virus infection in China.
34
Dinh, Phat X. "Identification of porcine circovirus type 2 (PCV2), type 3 (PCV3) and porcine parvovirus (PPV) in swine by multiplex PCR test." Journal of Agriculture and Development 20, no. 03 (June 30, 2021): 11–17. http://dx.doi.org/10.52997/jad.2.03.2021.
Full textAbstract:
This study aimed to simultaneously detect three important viruses reported to be involved in the reproductive problems of sows. A multiplex PCR (mPCR) test was developed to provide rapid diagnosis of porcine circovirus type 2 and 3 (PCV2, PCV3) and to illustrate parvovirus (PPV) prevalence in sow herds. Three pairs of specific primers were designed to target PCV2 Cap gene, PCV3 Cap gene and PPV NS1 gene, with predicted mPCR products of 702 bp, 267 bp and 380 bp, respectively. The detection limit of mPCR was 100 copies/reaction per target gene. The mPCR was run against a panel of 94 swine serum samples whose infection status had been pre-determined by commercial real-time PCR kits. Sequencing of mPCR products performed with clinical serum samples accurately confirmed the results. Overall, the results indicated that the mPCR functioned accurately and specifically and matched 100% with the single-target real-time PCRs. The mPCR was developed successfully and can be used in routine diagnosis of PCV2, PCV3 and PPV.
35
Breitschwerdt, Edward B., Rosalie Greenberg, Ricardo G. Maggi, B. Robert Mozayeni, Allen Lewis, and Julie M. Bradley. "Bartonella henselae Bloodstream Infection in a Boy With Pediatric Acute-Onset Neuropsychiatric Syndrome." Journal of Central Nervous System Disease 11 (January 2019): 117957351983201. http://dx.doi.org/10.1177/1179573519832014.
Full textAbstract:
Background: With the advent of more sensitive culture and molecular diagnostic testing modalities, Bartonella spp. infections have been documented in blood and/or cerebrospinal fluid specimens from patients with diverse neurological symptoms. Pediatric acute-onset neuropsychiatric syndrome (PANS) is characterized by an unusually abrupt onset of cognitive, behavioral, or neurological symptoms. Between October 2015 and January 2017, a 14-year-old boy underwent evaluation by multiple specialists for sudden-onset psychotic behavior (hallucinations, delusions, suicidal and homicidal ideation). Methods: In March 2017, Bartonella spp. serology (indirect fluorescent antibody assays) and polymerase chain reaction (PCR) amplification, DNA sequencing, and Bartonella enrichment blood culture were used on a research basis to assess Bartonella spp. exposure and bloodstream infection, respectively. PCR assays targeting other vector-borne infections were performed to assess potential co-infections. Results: For 18 months, the boy remained psychotic despite 4 hospitalizations, therapeutic trials involving multiple psychiatric medication combinations, and immunosuppressive treatment for autoimmune encephalitis. Neurobartonellosis was diagnosed after cutaneous lesions developed. Subsequently, despite nearly 2 consecutive months of doxycycline administration, Bartonella henselae DNA was PCR amplified and sequenced from the patient’s blood, and from Bartonella alphaproteobacteria growth medium enrichment blood cultures. B henselae serology was negative. During treatment with combination antimicrobial chemotherapy, he experienced a gradual progressive decrease in neuropsychiatric symptoms, cessation of psychiatric drugs, resolution of Bartonella-associated cutaneous lesions, and a return to all pre-illness activities. Conclusions: This case report suggests that B henselae bloodstream infection may contribute to progressive, recalcitrant neuropsychiatric symptoms consistent with PANS in a subset of patients.
36
Chen, Huan, Jun Li, Shanshan Yan, Hui Sun, Chuyi Tan, Meidong Liu, Ke Liu, Huali Zhang, Mingxiang Zou, and Xianzhong Xiao. "Identification of pathogen(s) in infectious diseases using shotgun metagenomic sequencing and conventional culture: a comparative study." PeerJ 9 (June 29, 2021): e11699. http://dx.doi.org/10.7717/peerj.11699.
Full textAbstract:
Background Early and accurate diagnosis of microorganism(s) is important to optimize antimicrobial therapy. Shotgun metagenomic sequencing technology, an unbiased and comprehensive method for pathogen identification, seems to potentially assist or even replace conventional microbiological methodology in the diagnosis of infectious diseases. However, evidence in clinical application of this platform is relatively limited. Methods To evaluate the capability of shotgun metagenomic sequencing technology in clinical practice, both shotgun metagenomic sequencing and conventional culture were performed in the PCR-positive body fluid specimens of 20 patients with suspected infection. The sequenced data were then analyzed for taxonomic identification of microbes and antibiotic resistance gene prediction using bioinformatics pipeline. Results Shotgun metagenomic sequencing results showed a concordance of 17/20 compared with culture results in bacterial detection, and a concordance of 20/20 compared with culture results in fungal detection. Besides, drug-resistant types annotated from antibiotic resistance genes showed much similarity with antibiotic classes identified by susceptibility tests, and more than half of the specimens had consistent drug types between shotgun metagenomic sequencing and culture results. Conclusions Pathogen identification and antibiotic resistance gene prediction by shotgun metagenomic sequencing identification had the potential to diagnose microorganisms in infectious diseases, and it was especially helpful for multiple microbial co-infections and for the cases where standard culture approached failed to identify microorganisms.
37
Lebedeva, Svetlana, Elena Zerkalenkova, Olga Soldatkina, Michael Maschan, Alexey A. Maschan, Galina Novichkova, and Yulia Olshanskaya. "Novel KMT2A Partner Gene NUTM2A Revealed By Anchored Multiplex PCR in ALL." Blood 134, Supplement_1 (November 13, 2019): 5203. http://dx.doi.org/10.1182/blood-2019-126602.
Full textAbstract:
Objectives Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric and adult acute leukemias. Such rearrangements are more frequent in infants, in whom they are found in 60% - 80% of acute lymphoblastic leukemias (ALL). KMT2A-r group itself is genetically heterogeneous. KMT2A-r can occur with at least 94 partner genes previously described in «THE MLL RECOMBINOME <…> IN 2017». Detection of KMT2A-r is an essential part of AL initial diagnostics. Also, the accurate detection of all KMT2A-r types is crucial in order to perform minimal residual disease (MRD) monitoring, as it is clear now that MRD-based therapy adjustment has a very strong impact on outcome. Here we report a case of novel KMT2A partner in pediatric ALL - NUT family member 2A (NUTM2A). Methods The patient is 8 y.o. girl with T-cell acute lymphoblastic leukemia with pleural effusion. Bone marrow and pleural fluid aspirates were analyzed by G-banded karyotyping and FISH with KMT2A breakapart probe. Pleural fluid aspirates were also analyzed by real-time RT-PCR for 8 most common KMT2A rearrangements screening, long-distance inversed PCR and targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA). Sanger sequencing was used for validation. Results Conventional cytogenetics and FISH showed 47,XX,t(10;11)(q22;q23),+mar[5] karyotype with 100% KMT2A-rearranged nuclei. Gene fusion analysis identified novel fusion KMT2A-NUTM2A with exon 11 - exon 1 breakpoint junction. NUTM2A is a gene at 10q23.2. This gene is not fully described in the literature. Rearrangements of this gene were identified in endometrial stromal sarcomas (ESS) (Cheng-Han Lee et al. 2012) and small round cell sarcoma (SRCS) (Sugita et al. 2017). In case of ESS, this rearrangement results in an in-frame fusion between YWHAE and NUTM2A (or highly homologous gene NUTM2B), and in case of SRCS it results in an in-frame fusion between NUTM2A and CIC. To our knowledge, KMT2A-NUTM2A fusion in our study is the first case demonstrating NUTM2A rearrangement in hematological malignancies. Conclusions Here we for the first time show the novel KMT2A-NUTM2A fusion transcript, which was found in pediatric T-cell acute lymphoblastic leukemia. Anchored multiplex PCR is one of the most sensitive way to detect rare variants of KMT2A rearrangements. It also allows selecting patient-specific primers for further PCR detection of MRD. Disclosures No relevant conflicts of interest to declare.
38
Adel, Hawraa, Nada Madi, and Widad Al-Nakib. "1777. Metagenomic Approach for the Detection of Viruses in Stool Samples from Infants and Children with Acute Gastroenteritis in Kuwait." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S654—S655. http://dx.doi.org/10.1093/ofid/ofz360.1640.
Full textAbstract:
Abstract Background Metagenomics techniques are target-independent tools that enable the identification of uncommon disease etiologies and genomic characterization of all microorganisms present in a sample in less time and at a lower cost than previous sequencing techniques. In this study, we developed a metagenomic approach using next-generation sequencing technology to identify known and unknown viruses in stool samples from infants and children with acute gastroenteritis in Kuwait. Methods We have investigated 84 stool samples from infants and children aged one month to 10 years old with signs and symptoms of gastroenteritis who attended Mubarak Al-Kabeer and Al-Amiri hospitals in Kuwait from January to December 2017 using both multiplex real-time PCR and metagenomics sequencing (Illumina Miseq instrument) methods. Results The metagenomics analysis of viral sequences verified that human adenovirus was the leading cause of gastroenteritis among infants and children in Kuwait, and was detected in 23% of the samples, rotavirus A was detected in 16% of the samples, and the combined infection of human adenovirus and rotavirus was detected in 7% of the samples. Also, newly discovered viruses known to cause gastroenteritis were identified; astrovirus MLB2 and primate bocaparvovirus-1 were detected in 5% of the samples. Also, each of the following new viruses was detected in 2% of the samples; aichivirus A, cardiovirus, parechovirus A, astrovirus VA4, cosavirus F, and bufavirus-3. On the other hand, multiplex real-time PCR showed that the combined infection of human adenovirus and rotavirus was the leading cause of gastroenteritis among infants and children in Kuwait, which was detected in 27% of the samples. However, the rotavirus was the second most common cause of diarrhea, which was detected in 20% of the samples. And the human adenovirus alone was detected in 18% of the samples. Our results showed a 69% agreement between both methods. By applying the Cohen’s Kappa statistics for a measure of agreement, the result gave fair agreement between the two methods (k = 0.388, P = 0.0). Conclusion Our findings revealed the capability of a metagenomic approach to detect many viruses causing gastroenteritis in stool samples from infants and children in Kuwait. Disclosures All authors: No reported disclosures.
39
Dey, Shuvra Kanti, Nadim Sharif, Omar Sadi Sarkar, Mithun Kumar Sarkar, Ali Azam Talukder, Tung Phan, and Hiroshi Ushijima. "Molecular epidemiology and surveillance of circulating rotavirus among children with gastroenteritis in Bangladesh during 2014–2019." PLOS ONE 15, no. 11 (November 30, 2020): e0242813. http://dx.doi.org/10.1371/journal.pone.0242813.
Full textAbstract:
Acute gastroenteritis is one of the major health problems in children aged <5 years around the world. Rotavirus A (RVA) is an important pathogen of acute gastroenteritis. The burden of rotavirus disease in the pediatric population is still high in Bangladesh. This study investigated the prevalence of group A, B, and C rotavirus (RAV, RBV, RCV), norovirus, adenovirus (AdV) and human bocavirus (HBoV) infections in children with acute gastroenteritis in Bangladesh from February 2014 to January 2019. A total of 574 fecal specimens collected from children with diarrhea in Bangladesh during the period of February 2014-January 2019 were examined for RAV, RBV and RCV by reverse transcriptase- multiplex polymerase chain reaction (RT- multiplex PCR). RAV was further characterized to G-typing and P-typing by RT-multiplex PCR and sequencing method. It was found that 24.4% (140 of 574) fecal specimens were positive for RVA followed by AdV of 4.5%. RBV and RCV could not be detected in this study. Genotype G1P[8] was the most prevalent (43%), followed by G2P[4] (18%), and G9P[8] (3%). Among other genotypes, G9P[4] was most frequent (12%), followed by G1P[6] (11%), G9P[6] (3%), and G11P[25] (3%). We found that 7% RVA were nontypeable. Mutations at antigenic regions of the VP7 gene were detected in G1P[8] and G2P[4] strains. Incidence of rotavirus infection had the highest peak (58.6%) during November to February with diarrhea (90.7%) as the most common symptom. Children aged 4–11 months had the highest rotavirus infection percentage (37.9%). By providing baseline data, this study helps to assess efficacy of currently available RVA vaccine. This study revealed a high RVA detection rate, supporting health authorities in planning strategies such as introduction of RVA vaccine in national immunization program to reduce the disease burden.
40
Obranic, Sonja. "Molecular diagnostics in the clinical microbiology laboratory." Molecular and experimental biology in medicine 2, no. 2 (October 5, 2019): 1–8. http://dx.doi.org/10.33602/mebm.2.2.1.
Full textAbstract:
Molecular diagnostics is broadly available in clinical microbiology laboratories worldwide, especially for the detection and identification of difficult-to-cultivate microorganisms. The field of clinical microbiology has experienced significant changes over the past decade due to extensive molecular biology research that resulted in novel molecular diagnostics technologies. These new technologies are being introduced in clinical microbiology laboratories with the aim of improving sensitivity, specificity, accuracy and time-to-diagnosis, ensuring valuable data for effective infectious disease clinical management, infection control and surveillance. They have a potential to greatly improve general healthcare, but also present certain challenges, mainly regarding the cost and the proper definition of test ordering and interpretation. This review will discuss the current and potential application of next-generation sequencing, digital PCR and syndromic multiplex molecular assays in clinical microbiology.
41
Kim, Min-Jeong, Kyu-Min Kim, Jeong-Ih Shin, Jong-Hun Ha, Dong-Hae Lee, Jeong-Gyu Choi, Jin-Sik Park, et al. "Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis." Canadian Journal of Infectious Diseases and Medical Microbiology 2021 (February 22, 2021): 1–13. http://dx.doi.org/10.1155/2021/8844306.
Full textAbstract:
Background. Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. Methods. In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. Results. Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. Conclusions. The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea.
42
Gaebler, Christian, Julio C. C. Lorenzi, Thiago Y. Oliveira, Lilian Nogueira, Victor Ramos, Ching-Lan Lu, Joy A. Pai, et al. "Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir." Journal of Experimental Medicine 216, no. 10 (July 26, 2019): 2253–64. http://dx.doi.org/10.1084/jem.20190896.
Full textAbstract:
HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.
43
Ramchandar, Nanda, Jennifer Foley, Claudia Enriquez, Stephanie Osborne, Antonio Arrieta, Prithvi Sendi, Balagangadhar Totapally, et al. "342. Clinical Utility of a Next Generation Sequencing Test for Pathogen Detection in Pediatric Central Nervous System Infections." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S241. http://dx.doi.org/10.1093/ofid/ofaa439.537.
Full textAbstract:
Abstract Background Pediatric central nervous system (CNS) infections are potentially life-threatening and may incur significant morbidity. Identifying a pathogen is important, both in terms of guiding therapeutic management, but also in characterizing prognosis. However, standard care testing by culture, serology, and PCR is often unable to identify a pathogen. We examined use of next generation sequencing (NGS) of cerebrospinal fluid (CSF) in detecting an organism in children with CNS infections. Methods We prospectively enrolled children with CSF pleocytosis and suspected CNS infection admitted to 3 tertiary pediatric hospitals. After standard care testing had been performed, the remaining CSF was submitted for analysis by NGS. Results We enrolled 70 subjects over a 12-month recruitment period. A putative organism was isolated from CSF in 24 (34.3%) subjects by any diagnostic modality. NGS of the CSF samples identified a pathogen in 20 (28.6%) subjects. False positive results by NGS were identified in 2 patients. There were no cases in which NGS alone identified a pathogen. In 4 cases, a putative organism was recovered by standard care testing of the CSF, but not by CSF NGS. CSF culture recovered a putative organism in 12 cases (12.1%). A CSF PCR multiplex panel was utilized for 51 subjects. An organism was detected in 15 of these (29.4%). Using a reference composite of standard care testing, we determined the sensitivity and specificity of CSF NGS to be 83.3% (95% CI, 62.6–95.3%) and 91.3% (95% CI, 79.2–97.6%) respectively. Conclusion Sequencing of CSF has the potential to rapidly and comprehensively identify infection with a single test. Further studies are needed to determine the optimal use of NGS for diagnosis of CNS infections. Disclosures All Authors: No reported disclosures
44
Jain, M., P. Kumar, and A. K. Goel. "Emergence of Tetracycline ResistantVibrio choleraeO1 Biotype El Tor Serotype Ogawa with ClassicalctxBGene from a Cholera Outbreak in Odisha, Eastern India." Journal of Pathogens 2016 (2016): 1–6. http://dx.doi.org/10.1155/2016/1695410.
Full textAbstract:
In September 2010, a cholera outbreak was reported from Odisha, Eastern India.V. choleraeisolated from the clinical samples were biochemically and serologically confirmed as serogroup O1, biotype El Tor, and serotype Ogawa. Multiplex PCR screening revealed the presence of various genes, namely,ompW,ctxB,zot,rfbO1,tcp,ace,hlyA,ompU,rtx,andtoxR,in all of the isolates. The isolates were resistant to co-trimoxazole, nalidixic acid, polymyxin B, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). Minimum inhibitory concentration of tetracycline decreased in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), suggesting the involvement of efflux pumps. PCR analysis confirmed the presence of class I integrons as well as SXT elements harbouring antibiotic resistance genes in all isolates. Sequencing revealed the presence ofctxBgene of classical biotype in all the isolates. The isolates harboured an RS1-CTX prophage array with El Tor typerstRand classicalctxBon the large chromosome. The study indicated that theV. choleraeEl Tor variants are evolving in the area with better antibiotic resistance and virulence potential.
45
Novikova, N. A., T. A. Sashina, L. A. Solntsev, N. V. Epifanova, A. Yu Kashnikov, L. V. Pogodina, I. N. Okun, and O. N. Knyagina. "MANIFESTATIONS OF EPIDEMIC PROCESS OF ROTAVIRUS INFECTION IN NIZHNY NOVGOROD IN PRE-VACCINATION PERIOD." Journal of microbiology epidemiology immunobiology, no. 5 (October 28, 2017): 46–52. http://dx.doi.org/10.36233/0372-9311-2017-5-46-52.
Full textAbstract:
Aim. Study the manifestations of epidemic process of rotavirus infection in Nizhny Novgorod in pre-vaccination period to evaluate the possible effect on morbidity for the rotavirus vaccine application introduction. Materials and methods. Rotavirus morbidity data were analyzed for the 12-year period (2005 - 2016), as well as its age and season distribution. Rotavirus genotyping was carried out using multiplex PCR and partial sequencing of VP4 and VP7 genes. Results. RVI morbidity in Nizhny Novgorod was shown to be at a moderate level when specific prophylaxis was not applied, multi-year dynamics for morbidity reflects the all-Russian state. 2015 - 2016 were characterized by intensification of the epidemic process in age groups of organized children (3 - 6 and 7 - 14) and adults. Season increase included December-May. seasonal morbidity maximums in different age groups took place during different months. Genetic structure of Nizhny Novgorod population PV-A during this time was presented by 10 types with G9P[8] (44,4%) dominating. Conclusion. Growth of RVI morbidity in Nizhny Novgorod in 2015 - 2016 and changes in age and season manifestations of the infection took place under the condition of change of the dominating genotype PV-A (G4P[8] to G9P[8]).
46
Kiyuka, Patience K., Charles N. Agoti, Patrick K. Munywoki, Regina Njeru, Anne Bett, James R. Otieno, Grieven P. Otieno, et al. "Human Coronavirus NL63 Molecular Epidemiology and Evolutionary Patterns in Rural Coastal Kenya." Journal of Infectious Diseases 217, no. 11 (March 21, 2018): 1728–39. http://dx.doi.org/10.1093/infdis/jiy098.
Full textAbstract:
Abstract Background Human coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime. Methods Nasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing. Results HCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching. Conclusions In this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.
47
NG, KAMELA CHARMAINE S., and WINDELL L. RIVERA. "Multiplex PCR–Based Serogrouping and Serotyping of Salmonella enterica from Tonsil and Jejunum with Jejunal Lymph Nodes of Slaughtered Swine in Metro Manila, Philippines." Journal of Food Protection 78, no. 5 (May 1, 2015): 873–80. http://dx.doi.org/10.4315/0362-028x.jfp-14-342.
Full textAbstract:
Food poisoning outbreaks and livestock mortalities caused by Salmonella enterica are widespread in the Philippines, with hogs being the most commonly recognized carriers of the pathogen. To prevent and control the occurrence of S. enterica infection in the country, methods were used in this study to isolate and rapidly detect, differentiate, and characterize S. enterica in tonsils and jejuna with jejunal lymph nodes of swine slaughtered in four locally registered meat establishments (LRMEs) and four meat establishments accredited by the National Meat Inspection Services in Metro Manila. A total of 480 samples were collected from 240 animals (120 pigs from each type of meat establishment). A significantly higher proportion of pigs were positive for S. enterica in LRMEs (60 of 120) compared with meat establishments accredited by the National Meat Inspection Services (38 of 120). More S. enterica–positive samples were found in tonsils compared with jejuna with jejunal lymph nodes in LRMEs, but this difference was not significant. A PCR assay targeting the invA gene had sensitivity that was statistically similar to that of the culture method, detecting 93 of 98 culture-confirmed samples. Multiplex PCR–based O-serogrouping and H/Sdf I typing revealed four S. enterica serogroups (B, C1, D, and E) and six serotypes (Agona, Choleraesuis, Enteritidis, Heidelberg, Typhimurium, and Weltevreden), respectively, which was confirmed by DNA sequencing of the PCR products. This study was the first to report detection of S. enterica serotype Agona in the country.
48
Rohde, Judith, Monir Majzoub-Altweck, Almuth Falkenau, Walter Hermanns, Eric R. Burrough, Mathias Ritzmann, and Julia Stadler. "Occurrence of dysentery-like diarrhoea associated with Brachyspira suanatina infection on a German fattening pig farm." Veterinary Record 182, no. 7 (February 1, 2018): 195. http://dx.doi.org/10.1136/vr.104705.
Full textAbstract:
The anaerobic intestinal spirochaete Brachyspira (B.) suanatina was first described in 2007 but since then no further isolates have been reported from pigs. Accordingly, when the species was validly published in 2016, the overall occurrence and clinical relevance in pigs were unknown. In a fattening farm in southern Germany, mucohaemorrhagic diarrhoea was observed in 60 per cent (750 animals) of the finisher pigs. A diagnostic workup including Brachyspira culture, Salmonella culture, Lawsonia intracellularis-specific, B. hyodysenteriae-specific and B. pilosicoli-specific multiplex PCR and postmortem examination of severely affected pigs was performed. Tests for Salmonella species, Lawsonia intracellularis and B. hyodysenteriae were all negative. Gross and microscopic lesions were in agreement with dysentery and spirochaetes could be demonstrated by silver staining in tissue samples of the caecum at the ileal papilla. B. suanatina was cultured from faeces or colon of all (five) animals sampled and identified using nox-RFLP, partial nox-gene-sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). According to the initial report from Scandinavia, B. suanatina can be isolated from birds and cross-species infection could be demonstrated infecting pigs with an avian isolate. Thus outdoor production as in the case presented here and international trade may pose a risk for infection of naive herds.
49
Posuwan, Nawarat, Sunchai Payungporn, Aunyaratana Thontiravong, Pravina Kitikoon, Alongkorn Amonsin, and Yong Poovorawan. "Prevalence of respiratory viruses isolated from dogs in Thailand during 2008-2009." Asian Biomedicine 4, no. 4 (August 1, 2010): 563–69. http://dx.doi.org/10.2478/abm-2010-0071.
Full textAbstract:
Abstract Background: A highly contagious respiratory disease in canines is infectious tracheobronchitis or kennel cough characterized by inflammation of the upper respiratory tract. The cause of kennel cough has been associated with multiple or complex agents such as canine adeno virus (CAV), canine influenza virus (CIV), canine distemper virus (CDV), and canine para influenzavirus (CPIV). Objective: Study the prevalence of canine respiratory viruses detected from in Thailand during 2008-2009. Methods: Nasal swab samples collected from 102 healthy dogs and 109 dogs with respiratory diseases. Then CAV, CIV, CDV, and CPIV were detected by in-house nested PCR and further confirmed by nucleotide sequencing. Results: Nested PCR showed that primers designed and used in this study yielded high specificity without any non-specific amplification. The prevalence of CAV, CIV, CDV and CPIV in healthy dogs was 0%, 2.94%, 2.94%, and 0.98%, whereas that found in dogs with respiratory diseases was 9.17%, 1.83%, 2.75%, and 11.93%, respectively. In healthy dogs, co-infection with CPIV + CDV was detected in only 0.98%. On the other hand, dogs with respiratory symptoms showed multiple infections with CAV + CIV in 1.83%, CIV + CPIV in 0.92%, CAV + CPIV in 1.83%, and CAV + CDV + CPIV in 0.92%. Conclusion: The prevalence data obtained from this study may be useful for outbreak preventions and to raise awareness of potential transmission of the newly emerged canine influenza virus to humans.
50
Ghasemian, Ehsan, Aleksandra Inic-Kanada, Astrid Collingro, Lamiss Mejdoubi, Hadeel Alchalabi, Darja Keše, Balgesa Elkheir Elshafie, Jaouad Hammou, and Talin Barisani-Asenbauer. "Comparison of genovars and Chlamydia trachomatis infection loads in ocular samples from children in two distinct cohorts in Sudan and Morocco." PLOS Neglected Tropical Diseases 15, no. 8 (August 9, 2021): e0009655. http://dx.doi.org/10.1371/journal.pntd.0009655.
Full textAbstract:
Trachoma is a blinding disease caused by repeated conjunctival infection with different Chlamydia trachomatis (Ct) genovars. Ct B genovars have been associated with more severe trachoma symptoms. Here, we investigated associations between Ct genovars and bacterial loads in ocular samples from two distinct geographical locations in Africa, which are currently unclear. We tested ocular swabs from 77 Moroccan children (28 with trachomatous inflammation-follicular (TF) and 49 healthy controls), and 96 Sudanese children (54 with TF and 42 healthy controls) with a Ct-specific real-time polymerase chain reaction (PCR) assay. To estimate bacterial loads, Ct-positive samples were further processed by multiplex real-time qPCR to amplify the chromosomal outer membrane complex B and plasmid open reading frame 2 of Ct. Genotyping was performed by PCR-based amplification of the outer membrane protein A gene (~1120 base pairs) of Ct and Sanger sequencing. Ct-positivities among the Moroccan and Sudanese patient groups were 60·7% and 31·5%, respectively. Significantly more Sudanese patients than Moroccan patients were genovar A-positive. In contrast, B genovars were significantly more prevalent in Moroccan patients than in Sudanese patients. Significantly higher Ct loads were found in samples positive for B genovars (598596) than A genovar (51005). Geographical differences contributed to the distributions of different ocular Ct genovars. B genovars may induce a higher bacterial load than A genovars in trachoma patients. Our findings emphasize the importance of conducting broader studies to elucidate if the noted difference in multiplication abilities are genovar and/or endemicity level dependent.