scholarly journals Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claire Y. T. Wang ◽  
Emma L. Ballard ◽  
Zuleima Pava ◽  
Louise Marquart ◽  
Jane Gaydon ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Plasmodium Falciparum ◽  
18S Rrna ◽  
Drug Efficacy ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Qpcr Assay ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Blood Samples

Abstract Background Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. Methods A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. Results The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. Conclusions The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

Investigations on First Confirmed Outbreak of Ovine Theileriosis (Theileria luwenshuni) from Maharashtra State, India

2020 ◽  
Author(s):  
V.S. Dhaygude ◽  
K. Kundu ◽  
B.P. Kamdi ◽  
U.R. Bagal ◽  
S.B. Bhosale ◽  
...  
Keyword(s):  
18S Rrna ◽  
Clinical Signs ◽  
Specific Treatment ◽  
Small Ruminants ◽  
18S Rrna Gene ◽  
Pcr Detection ◽  
Nucleotide Sequencing ◽  
Rrna Gene ◽  
Blood Samples ◽  
Theileria Luwenshuni

Background: Clinical theileriosis of small ruminants is tick-borne disease caused by Theileria lestoquardi, Theileria uilenbergi and Theileria luwenshuni. Theileria annulata, the causative agent of bovine tropical theileriosis in cattle, can also infect sheep but does not cause any significant illness. It is one of the economically important diseases. There are no reports of ovine clinical theileriosis from Maharashtra state and there is paucity of information on its epidemiology. This paper reports first confirmed outbreak of ovine theileriosis based on clinical signs, microscopic examination, PCR and sequencing in the Maharashtra State of India. Methods: Whole blood samples from 22 ailing sheep were collected and subjected to hematological examination. Blood smears stained with Leishman’s stain were examined under 100X objective of the microscope. The blood samples from sheep found positive by microscopic method were subjected to PCR detection of 18S rRNA gene of hemoprotozoa and then for nucleotide sequencing and sequence analysis.Conclusion: Samples from 14 out of 22 sheep were found positive for piroplasms of Theileria spp by light microscopy. All positive samples were further confirmed by PCR detection of 18S rRNA gene of hemoprotozoa. PCR amplification yielded expected product of 1750 bp for all samples. BLAST and phylogenetic analysis of one sample revealed high sequence homology with T. luwenshuni reported from India and other countries. Characteristic clinical signs like fever, progressive anaemia, laboured breathing, lymphadenopathy, debility and non-responsiveness to antibiotic therapy were recorded. The animals responded to specific treatment against theileriosis. It is the first ever confirmed report of ovine theileriosis in Maharashtra state of India and hence reported.

scholarly journals Molecular Detection of Tick-Borne Agents in Cats from Southeastern and Northern Brazil

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 106
Author(s):  
Marcos Rogério André ◽  
Ana Cláudia Calchi ◽  
Maria Eduarda Chiaradia Furquim ◽  
Isabela de Andrade ◽  
Paulo Vitor Cadina Arantes ◽  
...  
Keyword(s):  
18S Rrna ◽  
Clinical Signs ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Molecular Identity ◽  
Northern Brazil ◽  
Rrna Gene Sequencing ◽  
First Time ◽  
Theileria Spp

Even though the epidemiology of tick-borne agents (TBA) in dogs has been extensively investigated around the world, the occurrence, vectors involved, and molecular identity of these agents in cats remains elusive in many regions. Among TBA, Ehrlichia, Anaplasma, Babesia, Cytauxzoon, and Hepatozoon are responsible for diseases with non-specific clinical signs in cats, making essential the use of molecular techniques for accurate diagnosis and proper treatment. The present work aimed to investigate the occurrence and molecular identity of tick-borne agents (Ehrlichia, Anaplasma, Babesia/Theileria, Cytauxzoon, and Hepatozoon) in cats from southeastern (states of São Paulo (SP) and Minas Gerais (MG)) and northern (state of Rondônia (RO)) Brazil. For this purpose, 390 blood samples were collected from domiciled cats in MG (n = 155), SP (n = 151), and RO(n = 84) states, submitted to DNA extraction and PCR assays for Ehrlichia spp. (dsb gene), Anaplasma spp. (rrs gene), piroplasmids (18S rRNA gene), and Hepatozoon spp. (18S rRNA gene), sequencing, and phylogenetic inferences. The overall positivity for Anaplasma spp., Ehrlichia spp., Babesia/Theileria spp., Cytauxzoon spp., and Hepatozoon spp. were 7.4% (12.3% (MG) and 6.6% (SP)), 2% (4.5% (MG) and 0.6% (SP)), 0.7% (0.6% (MG), 0.6% (SP) and 1.2% (RO)), 27.2% (41.9% (MG), 24.5% (SP) and 4.8% (RO), and 0%, respectively. The phylogenetic analysis grouped the obtained sequences with ‘Candidatus Anaplasma amazonensis’, A. platys, B. vogeli, and Cytauxzoon sp. previously detected in wild felids from Brazil. qPCR specific for E. canis based on the dsb gene confirmed the molecular identity of the detected ehrlichial agent. The present study expanded the list and geographical distribution of hemoparasites in cats. ‘Candidatus Anaplasma amazonensis’, recently detected in sloths from northern Brazil, was described for the first time in cats. This is the first report of piroplasmids infecting cats in northern Brazil. Coinfection by Cytauxzoon and other TBA (Ehrlichia, Anaplasma, and B. vogeli) reported in the present study raises the need for veterinary practitioners’ awareness of cats parasitized by multiple TBA.

scholarly journals Design of primer pairs for species-specific diagnosis of Leishmania (Leishmania) infantum chagasi using PCR

2012 ◽  
Vol 21 (3) ◽  
pp. 304-307 ◽  
Author(s):  
Osvaldo José da Silveira Neto ◽  
Sabrina Castilho Duarte ◽  
Hérika Xavier da Costa ◽  
Guido Fontgalland Coelho Linhares
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Reference Sequence ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Specific Detection ◽  
Pcr Products ◽  
Specific Amplification ◽  
Pcr Assays ◽  
Species Specific

The objective of this study was to design and evaluate new primers for species-specific detection of L. infantum chagasi using PCR. Two combinations of primer pairs were established with the aim of obtaining specific amplification products from the L. infantum chagasi 18S rRNA gene. The combinations of the primer pairs and the respective sizes of the PCR products, based on the U422465 GenBank reference sequence of L. infantum chagasi, were: LCS1/LCS3 (259 bp) and LCS2/LCS3 (820 bp). It was concluded that the new PCR assays optimized using the primer pairs LCS1/LCS3 and LCS2/LCS3 were effective for specific detection of L. infantum chagasi, with analytical sensitivity to detect 1 pg/µL of DNA.

scholarly journals Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates

2018 ◽  
Author(s):  
Mayra Araguaia Pereira Figueiredo ◽  
Silvia Maria Di Santi ◽  
Wilson Gómez Manrique ◽  
Marcos Rogério André ◽  
Rosangela Zacarias Machado
Keyword(s):  
Nonhuman Primates ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Soluble Antigen ◽  
Blood Samples ◽  
Two Samples ◽  
Dot Elisa

Abstract The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum . PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples.

scholarly journals Molecular Identification and Genotyping of Babesia canis in Dogs from Mesh-kin Shahr County, Northwestern Iran

2021 ◽  
Author(s):  
Majid Khanmohammadi ◽  
Reza Z Olfaghari-Emameh ◽  
Mehdi Arshadi ◽  
Elham Razmjou ◽  
Poorya Karimi
Keyword(s):  
18S Rrna ◽  
Gene Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Babesia Species ◽  
Canine Babesiosis ◽  
Sequence Alignments ◽  
Blood Samples ◽  
Standard Methods ◽  
Northwestern Iran

Background: Canine babesiosis is one of the mainly worldwide-distributed tick-borne haemoprotozoan parasitic dis-eases in dogs. Methods: A total of 43 blood samples were randomly collected from naturally infected dogs in seven villages from different geographical areas of Meshkin Shahr, Ardabil Province, Iran. The presence of Babesia species detected with standard methods including parasitological and gene sequencing techniques targeting the 18S rRNA gene. Results: Our results revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with Babesia. All four Babesia-infected dogs were confirmed B. canis by the molecular-based method. Sequence alignments comparison of the B. canis genotypes A and B, it was revealed that all B. canis isolates belonged to genotype B. Conclusion: This study provides essential data for subsequently define the critical importance of the molecular studies in management and prevention of the canine babesiosis in Iran.  

scholarly journals Molecular Screen of Trichoderma Isolates

1970 ◽  
Vol 17 ◽  
pp. 117-122
Author(s):  
Amna Ali ◽  
Rukhsana Bajwa ◽  
Nasir Mehmood ◽  
Rasheda Jabeen
Keyword(s):  
Gene Targeting ◽  
Genomic Dna ◽  
18S Rrna ◽  
Rflp Analysis ◽  
Stem Bark ◽  
18S Rrna Gene ◽  
Negative Control ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Trichoderma Species

Context: In the last few decades an ample array of molecular techniques has been introduced to obtain new disposition for the classification of Trichoderma species. Today the concern of scientists is either in the direction of gene targeting or ribotyping, the newest fingerprinting tool for genomic DNA that contain all or part of the genes coding for 18S rRNA in eukaryotes.Objectives: To take advantage of advanced molecular techniques for phylogenetic analysis of indigenous isolates of Trichoderma to comprehend our knowledge of this genus by supplementing the phenotypic identification.Materials and Methods:  Genomic DNA of twenty four isolates of Trichoderma species (T. harzianum, T. hamatum, T. koningii and T. pseudokoningii) were extracted by CTAB method and indicated band of ~15Kb on 0.8% agarose gel. Quality of DNA was determined by obtaining absorbance ratio (260/280) in the range of 1.7-1.9. Restriction fragment length polymorphism (RFLP) analyses were performed by using two restriction endonulease enzymes i.e., BamHI and HindIII. The BamHI represented results in the range of 500bp-750bp. 18S rRNA gene targeting was further carried out through optimization in ribotyping analysis.Results: The DNA bands of 24 isolates of Trichoderma species were compared with marker DNA bands and indicated the presence of genomic DNA intact band of ~15Kb. The ratio of absorbance 260/280nm (1.8-1.9 for pure DNA preparations) provided an estimate of the purity of the DNA RFLP analysis, along with the negative control of twenty four different isolates of Trichoderma species subjected to restriction using BamHI enzyme. The rRNA gene amplified band was observed at 600bp in the case of T. hamatum isolate (S. cumini stem bark, FCBP accession number 769) while in remaining isolates bands were in slightly smeared form. Furthermore, rRNA gene amplification conditions were optimized by altering different Tm and MgCl2 concentrations. Conclusion: The genomic DNA can serve as long term storage of information. Therefore advance molecular techniques can be used to study the variability in the genome of organism. RFLP are the initial steps for screening the genome of any organism.Key words: Trichoderma; DNA; RFLP; restriction endonuleases; 18S rRNA.DOI: 10.3329/jbs.v17i0.7117J. bio-sci. 17: 117-122, 2009

scholarly journals 16S rRNA gene and 18S rRNA gene diversity in microbial mat communities in meltwater ponds on the McMurdo Ice Shelf, Antarctica

Polar Biology ◽  
2021 ◽  
Author(s):  
Eleanor E. Jackson ◽  
Ian Hawes ◽  
Anne D. Jungblut
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
High Throughput Sequencing ◽  
Microbial Mats ◽  
Gene Diversity ◽  
Salinity Gradient ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Ice Shelf ◽  
The Impact

AbstractThe undulating ice of the McMurdo Ice Shelf, Southern Victoria Land, supports one of the largest networks of ice-based, multiyear meltwater pond habitats in Antarctica, where microbial mats are abundant and contribute most of the biomass and biodiversity. We used 16S rRNA and 18S rRNA gene high-throughput sequencing to compare variance of the community structure in microbial mats within and between ponds with different salinities and pH. Proteobacteria and Cyanobacteria were the most abundant phyla, and composition at OTU level was highly specific for the meltwater ponds with strong community sorting along the salinity gradient. Our study provides the first detailed evaluation of eukaryote communities for the McMurdo Ice Shelf using the 18S rRNA gene. They were dominated by Ochrophyta, Chlorophyta and Ciliophora, consistent with previous microscopic analyses, but many OTUs belonging to less well-described heterotrophic protists from Antarctic ice shelves were also identified including Amoebozoa, Rhizaria and Labyrinthulea. Comparison of 16S and 18S rRNA gene communities showed that the Eukaryotes had lower richness and greater similarity between ponds in comparison with Bacteria and Archaea communities on the McMurdo Ice shelf. While there was a weak correlation between community dissimilarity and geographic distance, the congruity of microbial assemblages within ponds, especially for Bacteria and Archaea, implies strong habitat filtering in ice shelf meltwater pond ecosystems, especially due to salinity. These findings help to understand processes that are important in sustaining biodiversity and the impact of climate change on ice-based aquatic habitats in Antarctica.

scholarly journals 18S rRNA gene sequences of leptocephalus gut contents, particulate organic matter, and biological oceanographic conditions in the western North Pacific

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsuyoshi Watanabe ◽  
Satoshi Nagai ◽  
Yoko Kawakami ◽  
Taiga Asakura ◽  
Jun Kikuchi ◽  
...  
Keyword(s):  
Organic Matter ◽  
Particulate Organic Matter ◽  
North Pacific ◽  
Western North Pacific ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Gut Contents ◽  
Rrna Gene ◽  
Marine Snow ◽  
Chlorophyll Maximum

AbstractEel larvae apparently feed on marine snow, but many aspects of their feeding ecology remain unknown. The eukaryotic 18S rRNA gene sequence compositions in the gut contents of four taxa of anguilliform eel larvae were compared with the sequence compositions of vertically sampled seawater particulate organic matter (POM) in the oligotrophic western North Pacific Ocean. Both gut contents and POM were mainly composed of dinoflagellates as well as other phytoplankton (cryptophytes and diatoms) and zooplankton (ciliophoran and copepod) sequences. Gut contents also contained cryptophyte and ciliophoran genera and a few other taxa. Dinoflagellates (family Gymnodiniaceae) may be an important food source and these phytoplankton were predominant in gut contents and POM as evidenced by DNA analysis and phytoplankton cell counting. The compositions of the gut contents were not specific to the species of eel larvae or the different sampling areas, and they were most similar to POM at the chlorophyll maximum in the upper part of the thermocline (mean depth: 112 m). Our results are consistent with eel larvae feeding on marine snow at a low trophic level, and feeding may frequently occur in the chlorophyll maximum in the western North Pacific.

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