Development of a deep amplicon sequencing method to determine the species composition of piroplasm haemoprotozoa

Chaudhry, Umer; Ali, Qasim; Rashid, Imran; Shabbir, Muhammad Zubair; Ijaz, Muhammad; Abbas, Muhammad; Evans, Michael J.; Ashraf, Kamran; Morrison, Ivan; Morrison, Liam J.; Sargison, Neil

doi:10.1016/j.ttbdis.2019.101276

Cited by 28 publications

(40 citation statements)

References 35 publications

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“…In conclusion, we describe for the first time the use of metabarcoded DNA sequencing using an Illumina MiSeq platform to quantify P. falciparum and P. vivax , and demonstrate its accuracy on malaria-positive samples. Our results provide a proof of concept study for the use of the method in disease surveillance, similar to its application in the study of haemoprotozoan parasites of livestock (Chaudhry et al, 2019) and dogs (Huggins et al, 2019). This work was undertaken to explore the possibilities for the application of this high throughput method to determine the dynamics of co-infections, disease biology and epidemiology in Plasmodium parasites, and has applications in monitoring the changes in parasite populations after the emergence and spread of antimicrobial drug resistance (Shaukat et al, 2019).…”

Section: Discussionmentioning

confidence: 67%

“…After the purification, a second-round PCR was performed by using sixteen forward and twenty-four reverse barcoded primers. The barcoded forward (N501 to N516) and reverse (N701 to N724) primers (10 uM each) were previously described by Chaudhry et al (2019). The primers were used in a manner that repetition of same forward and reverse sequences did not occur in the different samples.…”

Section: Methodsmentioning

confidence: 99%

“…The overall scheme of the data handling and bioinformatics analysis is described in Figure 2B. MiSeq data were handled with our own bioinformatics pipeline (Chaudhry et al, 2019). Briefly, MiSeq separates all sequence reads during post-run processing using the barcoded indices and to generate FASTQ files.…”

Section: Methodsmentioning

confidence: 99%

“…Sensitive diagnostic tools are needed to detect clinically and subclinically infected patients (Echeverry et al, 2016). High-throughput genomic resources have potential applications in the surveillance, diagnosis, control, and treatment of haemoprotozoan diseases, as well as to study the parasite biology (Chaudhry et al, 2019; Shaukat et al, 2019). Determination of sequence variations in the hypervariable 18S rDNA cistron can discriminate between Plasmodium species (Agudelo et al, 2013; Haanshuus et al, 2013; Lee et al, 2015; Lefterova et al, 2015), and overcome limitations of traditional microscopic and immunochromatographic methods for the diagnosis of this group of parasites at species level.…”

Section: Introductionmentioning

confidence: 99%

“…Determination of sequence variations in the hypervariable 18S rDNA cistron can discriminate between Plasmodium species (Agudelo et al, 2013; Haanshuus et al, 2013; Lee et al, 2015; Lefterova et al, 2015), and overcome limitations of traditional microscopic and immunochromatographic methods for the diagnosis of this group of parasites at species level. Molecular methods including qPCR, species-specific PCR, nested PCR, and multiplex PCR have been described (Canier et al, 2013; Cunha et al, 2009; Das et al, 1995; Echeverry et al, 2016; Haanshuus et al, 2013; Steenkeste et al, 2009), but these are low throughput, hence relatively expensive (Chaudhry et al, 2019). These methods depend on the use of species-specific probes and primers, meaning that only the tested species are identified, hence are limited in their ability to describe true circulating species diversity (Moody, 2002).…”

Section: Introductionmentioning

confidence: 99%

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A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

Wahab

Shaukat

Ali

et al. 2019

Preprint

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Various PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have been recently developed for the first time a novel high throughput ‘haemoprotobiome’ metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species and to assess the accuracy of proportional quantification. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. Metabarcoded DNA sequencing showed better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.

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Section: Discussionmentioning

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

Wahab

Shaukat

Ali

et al. 2019

Preprint

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An appraisal of oriental theileriosis and the Theileria orientalis complex, with an emphasis on diagnosis and genetic characterisation

et al. 2019

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Oriental theileriosis, a tick-borne disease of bovids caused by members of the Theileria orientalis complex, has a worldwide distribution. Globally, at least 11 distinct genotypes of T. orientalis complex, including type 1 (chitose), type 2 (ikeda), type 3 (buffeli), types 4 to 8, and N1-N3, have been described based on the sequence of the major piroplasm surface protein (MPSP) gene. Of these 11 genotypes, mainly ikeda and chitose are known to be pathogenic and cause considerable morbidity (including high fever, anaemia, jaundice and abortion), production losses and/or mortality in cattle. Mixed infections with two or more genotypes of T. orientalis is common, but do not always lead to a clinical disease, posing challenges in the diagnosis of asymptomatic or subclinical forms of oriental theileriosis. The diagnosis of oriental theileriosis is usually based on clinical signs, the detection of piroplasms of T. orientalis in blood smears, and/or the use of serological or molecular techniques. This paper reviews current methods used for the diagnosis of T. orientalis infections and the genetic characterisation of members of the T. orientalis complex, and proposes that advanced genomic tools should be established for investigations of these and related haemoparasites.

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Ticks and tick-borne diseases of bovines in a smallholder livestock context: The Pakistani example

Ghafar

Gasser

Abbas

et al. 2021

Advances in Parasitology

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Development of a deep amplicon sequencing method to determine the species composition of piroplasm haemoprotozoa

Cited by 28 publications

References 35 publications

A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

An appraisal of oriental theileriosis and the Theileria orientalis complex, with an emphasis on diagnosis and genetic characterisation

Ticks and tick-borne diseases of bovines in a smallholder livestock context: The Pakistani example

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