Introduction:
Helminthes, including Nematodes, Trematodes, and Cestodes, are associated with substantial morbidity and economic losses worldwide. Almost one-sixth of the world’s population is infected with helminthes, with an estimated 15 billion individuals, particularly in low socio-economic regions, suffering from Soil-transmitted Helminthes (STHs). Novel techniques are now being developed and tested for the diagnosis of helminthic infections, a few of which are given below.
Concentration-based techniques:
FLOTAC
FLOTAC is a novel centrifugal flotation technique that was recently developed for fecal egg count by combining flotation, centrifugation, and translation using a novel FLOTAC apparatus. Up to 1 gram of stool is required, which has the sensitivity of 2 eggs/gram (24 times higher amount of stool than the Kato-Katz technique), thus resulting in higher sensitivity. The major advantage of this technique is multivalency, so that it can be used for the detection of different entities simultaneously, but it is on the slightly high cost side. Studies have FLOTAC is better than the classic copromicroscopic techniques in terms of sensitivity, accuracy, and precision in detection and quantification of eggs, larvae, and oocysts of helminthes in human and animal feces.
Mini-FLOTAC
It is a simplified form of FLOTAC. It has the advantage over FLOTAC in terms of no need for a centrifuge. Mini-FLOTAC has been used in helminthic infection diagnosis, and studies have shown that it detects a higher number of helminthic infections than the Formol-ether Concentration Method or direct smear, and has a sensitivity of around 90%, which is quite good in the case of parasites.
Serology-based techniques:
Antigen detection methods
Although Rapid Diagnostic Tests (RDTs) have shown huge success in antigen detection, cross-reactions could be problematic. Use of bioinformatics tools to identify Soil Transmitted Helminthes (STH) biomarkers should be encouraged and developed into an antigen-antibody test for the diagnosis of STH. Among new methods, antigen detection in stool will be useful for diagnosis, which can be developed as a rapid immunochromatographic dipstick test.
Coproantigen detection
Coproantigen detection could have the potential to be used in a Point of Care immunoassay-based test. Recently, BA-1 excretory-secretory product of Ascaris lumbricoides, was investigated as a coproantigen marker of infection by ELISA and showed sensitivity of 91.5% and specificity of 95.3%. Traditional techniques may eventually be replaced by the detection of adult worms and egg antigens in serum and urine due to their efficacy.
Circulating antigen assay (CAA
Circulating adult worm and egg antigens in serum or urine may have the potential to replace traditional methods of helminthic infection detection.
Circulating Anodic Antigen and Circulating Cathodic Antigen, two recent advancements in the detection of Schistosoma circulating worm antigen, have shown promise in point-of-care diagnosis. Luminescent quantitative Up-Conversion Phosphor (UCP) reporter particles and a quick, easy-to-use Lateral Flow test methodology are used in CAA. The ultra-sensitive quantification test UCP-LF CAA can identify every Schistosoma species in urine, blood, or other fluids. Very mild infections in both serum and urine have been demonstrated to be detectable by CAA detection.
As a point-of-care diagnostic tool, CCA is used to find active S. mansoni. S. mansoni has demonstrated 100% sensitivity when employed in conjunction with serology, as opposed to 95.7% for serology and 91.3% for CCA by itself.
Antibody detection
Falcon Assay Screening Test ELISA (FAST-ELISA)
The Falcon Assay Screening Test ELISA (FAST-ELISA) is a modified form of the traditional enzyme-linked immunosorbent assay (ELISA) which employs synthetic or recombinant peptides as antigens. The peptides are representative of particular regions (epitopes) of a pathogen or protein of interest, but the method has the same limitations as most serology-based tests: antibodies produced against a peptide from one parasite protein may cross-react with proteins from other species, antibodies raised against a peptide may react in some assays but not in others, and some regions of a peptide may be more immunogenic than others. The method has been used in the past to study Taeniasis, Schistosomiasis, and Fasciolasis.
Luciferase Immuno Precipitation System (LIPS)
LIPS is a modified ELISA-based test where serum containing antibody specific to the antigen can be identified by measuring light production. It has successfully been applied in the identification of Strongyloides stercoralis. In recent years, a recombinant antigen named NIE has been used to develop ELISA (NIE-ELISA) and NIE-LIPS. Recently, using recombinant Strongyloides antigen (NIE), a new test was developed based on LIPS and demonstrated 97% sensitivity and 100% specificity. In this test, no cross-reactions were seen in the serum of a Filaria-infected patient.
Molecular-based techniques/Nucleic acid-based technique:
Loop-mediated isothermal amplification (LAMP)
Using a sophisticated mechanism of autocycling strand displacement DNA synthesis, LAMP was developed as a novel method with high sensitivity, specificity, and rapidity under isothermal conditions (60-65â—¦C). Impressive progress has been made in the LAMP assay in the detection of helminthes like Schistosoma japonicum, Schistosoma mansoni, Schistosoma hematobium, Ascaris lumbricoides, Trichuris trichiura, Wuchereria bancrofti, Brugia malayi, B. timori, Loa loa, Taenia solium, and T. saginata. It can detect even a low worm burden. LAMP is cheaper than real-time PCR and can be used as a diagnostic tool for field testing.
Luminex Xmap technology
Luminex has emerged as a new potential approach for the diagnosis of helminths. It is a bead-based xMAP (Multianalyte Profiling) system that combines flow cytometry, fluorescent microspheres, lasers, and digital processingIt was discovered that the Luminex assay may use a relatively small volume to identify several species or distinct genotypes of the same organism in the same reaction.
Droplet Digital PCR (ddPCR)
Recent developments in nucleic acid testing have shown that ddPCR is a more sensitive and accurate method than qPCR. The accuracy of this method and the effectiveness of the target DNA quantification are significantly increased by the fact that it is carried out in tens of thousands of nanoliter-sized droplets. It has demonstrated remarkable sensitivity in detecting very low concentrations of A. lumbricoides. Additionally, it was used to diagnose S. japonicum in clinical samples from humans and animals and was able to measure the degree of infection in terms of gene copy number. In general, it has been shown to be a highly helpful technique for detecting STH infections, particularly in settings with low infection intensity.
Recombinase Polymerase Amplification (RPA)
RPA is a novel technique currently undergoing research and development. It is an isothermal amplification technique used under low temperature (40 degrees). It has been applied in the diagnosis of both intestinal and urinary Schistosomiasis, and field evaluation has shown it to be superior to microscopy, serology in terms of sensitivity. It has been shown to detect low levels of S. haematobium and S. japonicum and works by amplification of the Dra1 DNA region of S. haematobium. It is applicable in resource-poor settings, and there is no need for thermocyclers, electrophoresis, and Gel Documentation units. This new technique has now been integrated with a chip and Lateral Flow device, making it an easy point-of-care diagnostic tool.
Protein-based approaches:
Omic tests like Proteomics in the future can prove to be a very useful tool for helminth diagnosis, as they can identify proteins which act as biomarkers. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) are recently developed novel proteomic technologies. They have been used in the study of serum biomarkers in Cysticercosis, Fasciolasis.
miRNA-based diagnostic tests are being developed as a novel technique as a sensitive biomarker for the diagnosis of Strongyloides infection in humans. With the information of genomic sequences, miRNA can be identified by deep sequencing or by bioinformatics approaches.
References:
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