|Year : 2022 | Volume
| Issue : 3 | Page : 32-35
Role of gene sequencing in the diagnosis, tracking and prevention of parasitic diseases – A brief review
Monika Sivaradjy, Nonika Rajkumari
Department of Microbiology, JIPMER, Puducherry, India
|Date of Submission||16-Sep-2022|
|Date of Acceptance||29-Sep-2022|
|Date of Web Publication||11-Nov-2022|
Department of Microbiology, 2nd Floor, Institute Block, JIPMER, Dhanvantri Nagar, Puducherry - 605 006
Source of Support: None, Conflict of Interest: None
Parasitic infections and its burden are increasing worldwide and there are many unknown areas among the parasites for a long time which makes the eradication of most of the parasites impossible till now. The mechanism of how certain parasites evade human immune response and how they escape from the action of antiparasitic drugs were unclear. It is also difficult to maintain their culture in the laboratory which makes it difficult to identify to the species level most of the time. The field of sequencing has undergone many advances recently. The availability of entire genome sequences has revolutionised the study of infectious organisms, including parasites. It helps us to know the complete nucleotide sequence of even a complex genome at larger number. In the form of pilot genome sequencing studies, genomics approaches have been used to deal with the problem of tropical neglected parasitic illnesses for more than 20 years. In this, new technology like sequencing is coming up in a big way not only in the diagnosis but also targeted therapeutics and its control. Hence, different sequencing methods have been explored briefly in its role in parasitic diseases.
Keywords: Diagnosis, identification, parasites, sequencing
|How to cite this article:|
Sivaradjy M, Rajkumari N. Role of gene sequencing in the diagnosis, tracking and prevention of parasitic diseases – A brief review. J Acad Clin Microbiol 2022;24, Suppl S1:32-5
|How to cite this URL:|
Sivaradjy M, Rajkumari N. Role of gene sequencing in the diagnosis, tracking and prevention of parasitic diseases – A brief review. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Mar 22];24, Suppl S1:32-5. Available from: https://www.jacmjournal.org/text.asp?2022/24/3/32/360977
| Introduction|| |
The burden of parasitic infections is increasing worldwide and there are many unknown mysteries amongst the parasites for a long time which makes the eradication of most of the parasites impossible till now. Furthermore, the mechanism of how certain parasites evade the human immune response and how they escape from the action of antiparasitic drugs was unclear. It is also difficult to maintain their culture in the laboratory which makes it difficult to identify to the species level in most of the time. The field of sequencing has undergone many advances recently. The availability of entire genome sequences has revolutionised the study of infectious organisms, including parasites. It helps us to know the complete nucleotide sequence of even a complex genome at a larger number. In the form of pilot genome sequencing studies, genomics approaches have been used to deal with the problem of neglected tropical parasitic illness for more than 20 years. The genomic sequence of Plasmodium falciparum was released in 2002. The first reference genome sequences of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major were published in 2005. The emergence of next-generation sequencing (NGS) technology has opened up a slew of new possibilities for using genomics in parasitic disease research as well as the integration of other types of large-scale biological data. Comparative genome sequencing, transcriptomics, proteomics, metabolomics and epigenetics are amongst them. Although we have various sequencing strategies available currently, the genomic features of the parasites affect the sequencing strategies in many ways. This is mainly because of the variation in the size, nucleotide composition, level of polymorphism, content and distribution of repetitive elements of the parasitic genome. In this review, we are going to discuss the various sequencing methods which help us to understand them in a better manner with respect to the diagnosis, treatment and prevention of parasitic infections.
| Genomic Sequencing and Their Role in the Diagnosis, Tracking and Prevention of Parasitic Diseases|| |
The process of establishing the precise order of nucleotides within a DNA molecule is known as genomic sequencing. It refers to any method that determines the sequence of a single gene or operon, entire chromosome or the complete genome, including the order of the bases in a DNA strand. Various methods of genome sequencing are currently in use, out of which Sanger sequencing, shotgun sequencing, 454 sequencing (pyrosequencing), Illumina (Solexa) sequencing and bridge polymerase chain reaction (PCR) sequencing are the most commonly used genome sequencing technologies.
Since the start of the P. falciparum (malaria) genome project in 1996, a wide range of additional parasite genomes has been sequenced, resulting in a paradigm shift in parasite biology research. The Carucci laboratory has developed microarray DNA chips for P. falciparum chromosomes 2, 3, 12 and 14, with plans to expand to the whole malaria genome over the next two years. These microarrays can be used to investigate pharmacological impacts on parasite development, drug-resistance mechanisms, antigenic variation processes and cell invasion genes. The mitochondrial genome (mt) has continually provided a rich source of new markers for systematic and epidemiological studies. The mtDNA genomics of helminths, particularly lung flukes, liver flukes and intestinal flukes, has advanced significantly in the previous decade. Until recently, sequencing the genomes of mt was a difficult task. The traditional strategy of combining long-range PCR with subsequent primer walking has been used. The radical change brought about by the third-generation sequencing technologies has paved the way for NGS technologies, which has prompted proposals for more straightforward integrated pipelines for sequencing complete mt genomes which are more cost effective and time efficient. NGS technologies enable high-throughput sequencing, assembly and annotation in a short amount of time. The intestinal fluke's total mt genome sequence is 14,118 bytes long, making it the shortest trematode mt genome sequenced to date. In a study conducted by Biswal et al., the mtDNA for the intestinal fluke was reported for the first time. This mtDNA NGS data will help to investigate the Fasciolidae taxonomy further in detail. It would also be useful for comparing mitochondrial genomes and systematic research on trematode parasites. Prior idea about the target sequence is needed for Sanger sequencing. The main disadvantage of Sanger sequencing is that only short sequence lengths can be determined. Certain protozoan species such as for example Cryptosporidium parvum and Cryptosporidium hominis cannot be easily differentiated by the available conventional methods, similarly, Entamoeba histolytica and Entamoeba dispar are also very difficult to differentiate morphologically.,,Hence, when more than one species infect a single host, they can be misdiagnosed. Metagenomics along with next-generation sequencing is the best way to confirm the species in these situations., The main advantage of this method when compared to Sanger sequencing is that prior microbial knowledge of the sample is not required and also it allows for faster microbial assessment and recovery of novel species., For metagenomic profiling, two techniques have been used which include deep amplicon metagenomics, also known as meta profiling/amplicon-based sequencing, which entails PCR amplification of a target gene marker before NGS sequencing. The second approach is the shotgun metagenomic analysis includes shredding DNA sequences into tiny portions or fragments and then sequencing the total nucleic acid present in a sample., Although we have different metagenomics techniques available, the application of these methods over protozoan parasites is very much limited., The huge size of the genome, increased variability of certain genes and their presence in multiple copies are the main reasons for this. For example, Toxoplasma gondii (110 copies), Cryptosporidium parvum (5 copies) and Acanthamoeba castellanii (600 copies) all have multiple gene copy counts in the 18S small subunit ribosomal DNA gene. Repetition of noncoding DNA sequences also has been observed in eukaryotes which also contributes for the limited application of MNGS amongst them. Due to all these above reasons, only an accurate and standard protocol for the metagenomic profiling of the protozoans could not be developed. A third-generation NGS called SMRT (single-molecule real-time sequencing) delivers substantially faster and longer read lengths (1000–15,000 bp) than other sequencing platforms, allowing for nucleotide modification detection and highly precise DNA sequence, which other sequencing platforms often do not provide., However, because of the lack of universal primers for protozoans, more research is needed to develop primers that would ensure that all parasite genomes in a sample are properly represented if targeted sequencing is to be employed. It is therefore recommended to use the shotgun whole-genome approach to examine both known and unknown protozoan diversity. The filarial nematode Brugia malayi, one of the causes of lymphatic filariasis, was the first helminth parasite genome to be sequenced. In the ensuing 10 years, we have gained access to high-quality genome data for 81 parasitic nematode species. In the helminth post-genomic revolution, expanded genomic data have collided with improved proteomic technologies, resulting in a two-way relationship, in which genome sequencing data have helped to improve protein sequence identification, while proteomic data have similarly helped to improve genome annotations. Genetic sequencing played an important role in the identification of many nematode species, to spot the genetic variations and also to identify the functional genes in various nematodes. Shotgun metagenomic sequencing of cerebrospinal fluid has recently been used to confirm multiple protozoal and helminthic infections, including four cases of Balamuthia mandrillaris-induced granulocytic amoebic encephalitis, one case of Taenia solium neurocysticercosis and four cases of Angiostrongylus cantonensis-induced meningitis. Flaherty et al. recently described a pan-parasitic targeted amplicon deep sequencing technique with potential diagnostic usefulness. This strategy used a pan-eukaryotic primer pair that targeted a portion of the 18S rDNA gene with restriction enzyme cut sites found only in vertebrates., This approach identified all protozoa and helminths tested, including the most common parasites found in human blood. The main disadvantage of this method was the limit of detection which was comparable to most traditional PCR tests.
Genomic sequencing also plays an important role in identifying the drug targets and mutations/deletions in a particular gene that leads to the development of resistance to antiparasitic drugs. With the availability of genomic data, researchers can now use genome-wide techniques to examine the genetics of anthelmintic resistance, comparing phenotypically different strains across the entire genome to find discrete areas that correlate with resistance. The apicoplast is an organelle found only in apicomplexan parasites. Despite having a 35-kb genome that largely contains housekeeping genes, the apicoplast has been confirmed as a preventive therapeutic target. Roos and his colleagues were able to uncover a number of genes that were anticipated to be located in the apicoplast by data mining the P. falciparum genomic sequence. Genomic sequencing also helped us to identify the gene targets for vaccines in certain parasitic diseases. The metacyclic promastigote stage invades and lives in the mammalian host as an amastigote form, genes produced at these phases of the life cycle could be used as vaccine targets for Leishmania species. Many ongoing studies are there which use various genomic sequencing and proteomics technologies to find out the vaccine targets for P. falciparum which commonly causes severe malaria.
| Conclusion|| |
Genomic sequencing plays a very important role in the diagnosis of various parasites that causes human infection with high accuracy; also it helps us to identify the potential drug target gene and vaccine target genes. Amongst all the genomic sequencing methods available, MNGS is found to be the most useful in identifying almost all pathogens. Antimicrobial resistance, pathogenicity, type and other information related to parasitic agents can be used to investigate epidemics based on mNGS data.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Winzeler EA. Advances in parasite genomics: From sequences to regulatory networks. PLoS Pathog 2009;5:e1000649.
Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al.
Genome sequence of the human malaria parasite Plasmodium falciparum
. Nature 2002;419:498-511.
Talavera-López C, Andersson B. Parasite genomics-time to think bigger. PLoS Negl Trop Dis 2017;11:e0005463.
Bartholomeu D, El-Sayed NM. Sequencing strategies for parasite genomes. Methods Mol Biol 2004;270:1-16.
Hall N. Meeting review: Royal society discussion meeting: Utilising the genome sequence of parasitic protozoa: 21-22 March 2001 the royal Society, 6 carlton house terrace, London SW1Y 5AG. Comp Funct Genomics 2001;2:257-62.
Su K. Genome sequencing. J Curr Res 2015;7:11260-3.
Slatko BE, Gardner AF, Ausubel FM. Overview of next-generation sequencing technologies. Curr Protoc Mol Biol 2018;122:e59.
Ricciardi A, Ndao M. Diagnosis of parasitic infections: What's going on? J Biomol Screen 2015;20:6-21.
Widmer G, Sullivan S. Genomics and population biology of Cryptosporidium
species. Parasite Immunol 2012;34:61-71.
Feng Y, Ryan UM, Xiao L. Genetic diversity and population structure of Cryptosporidium
. Trends Parasitol 2018;34:997-1011.
Alves LF, Westmann CA, Lovate GL, de Siqueira GM, Borelli TC, Guazzaroni ME. Metagenomic approaches for understanding new concepts in microbial science. Int J Genomics 2018;2018:2312987.
Mthethwa NP, Amoah ID, Reddy P, Bux F, Kumari S. A review on application of next-generation sequencing methods for profiling of protozoan parasites in water: Current methodologies, challenges, and perspectives. J Microbiol Methods 2021;187:106269.
Quince C, Walker AW, Simpson JT, Loman NJ, Segata N. Shotgun metagenomics, from sampling to analysis. Nat Biotechnol 2017;35:833-44.
Wang Y, Tian RM, Gao ZM, Bougouffa S, Qian PY. Optimal eukaryotic 18S and universal 16S/18S ribosomal RNA primers and their application in a study of symbiosis. PLoS One 2014;9:e90053.
Marzano V, Mancinelli L, Bracaglia G, Del Chierico F, Vernocchi P, Di Girolamo F, et al
.”Omic” investigations of protozoa and worms for a deeper understanding of the human gut “parasitome”. PLoSNeg Trop Dis 2017;11:e0005916.
Zahedi A, Gofton AW, Greay T, Monis P, Oskam C, Ball A, et al
. Profiling the diversity of Cryptosporidium
species and genotypes in wastewater treatment plants in Australia using next generation sequencing. Sci Total Environ 2018;644:635-48.
Huang Y, Chen SY, Deng F. Well-characterized sequence features of eukaryote genomes and implications for Ab initio gene prediction. Comput Struct Biotechnol J 2016;14:298-303.
Rhoads A, Au KF. PacBio sequencing and its applications. Genomics Proteomics Bioinformatics 2015;13:278-89.
Lee H, Gurtowski J, Yoo S, Nattestad M, Marcus S, Goodwin S, et al
. Third-generation sequencing and the future of genomics. BioRxiv 2016; p. 048603. doi: https://doi.org/10.1101/048603
McVeigh P. Post-genomic progress in helminth parasitology. Parasitology 2020;147:835-40.
Flaherty BR, Barratt J, Lane M, Talundzic E, Bradbury RS. Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing. Microbiome 2021;9:1.
Flaherty BR, Talundzic E, Barratt J, Kines KJ, Olsen C, Lane M, et al
. Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing. Microbiome 2018;6:164.