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 Table of Contents  
REVIEW ARTICLE
Year : 2022  |  Volume : 24  |  Issue : 3  |  Page : 32-35

Role of gene sequencing in the diagnosis, tracking and prevention of parasitic diseases – A brief review


Department of Microbiology, JIPMER, Puducherry, India

Date of Submission16-Sep-2022
Date of Acceptance29-Sep-2022
Date of Web Publication11-Nov-2022

Correspondence Address:
Nonika Rajkumari
Department of Microbiology, 2nd Floor, Institute Block, JIPMER, Dhanvantri Nagar, Puducherry - 605 006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_15_22

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  Abstract 


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 2022 Dec 8];24, Suppl S1:32-5. Available from: https://www.jacmjournal.org/text.asp?2022/24/3/32/360977




  Introduction Top


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.[1] 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.[2] The first reference genome sequences of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major were published in 2005.[2] 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.[3] 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.[4] 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.[4] 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 Top


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.[5]

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.[5] 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.[6] 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.[6] 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.[7] 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.[8],[9],[10]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.[11],[12] 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.[11],[12] 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.[12],[13] Although we have different metagenomics techniques available, the application of these methods over protozoan parasites is very much limited.[14],[15] 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.[16] Repetition of noncoding DNA sequences also has been observed in eukaryotes which also contributes for the limited application of MNGS amongst them.[17] 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.[18],[19] 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.[12] The filarial nematode Brugia malayi, one of the causes of lymphatic filariasis, was the first helminth parasite genome to be sequenced.[20] In the ensuing 10 years, we have gained access to high-quality genome data for 81 parasitic nematode species.[20] 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.[6] 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.[21] 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.[21],[22] 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.[21]

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.[20] 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.[5] 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.[5] 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.[6] 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 Top


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

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Widmer G, Sullivan S. Genomics and population biology of Cryptosporidium species. Parasite Immunol 2012;34:61-71.  Back to cited text no. 9
    
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Feng Y, Ryan UM, Xiao L. Genetic diversity and population structure of Cryptosporidium. Trends Parasitol 2018;34:997-1011.  Back to cited text no. 10
    
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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.  Back to cited text no. 11
    
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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.  Back to cited text no. 12
    
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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.  Back to cited text no. 17
    
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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.  Back to cited text no. 19
    
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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.  Back to cited text no. 21
    
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