**4. Uses of NSG and dPCR in the diagnosis of the causal agents of NTDs**

NTDs are caused by several types of common and rare pathogens. A common concern with conventional testing methods is the limitation in the range of pathogens that can be detected and the lack of sensitivity for their diagnosis. Obstacles such as incomplete knowledge of natural history make it difficult to understand the ecology and pathogenesis of rare and neglected diseases. Emerging technologies, including NGS and dPCR, provide opportunities to accelerate the diagnosis and development of treatments for these diseases [60, 61]. The application of NGS in Leishmania isolates has allowed the characterization of populations through the identification and analysis of variations. Information on population structure can reveal important insights into disease dynamics and identify genetic backgrounds associated with parasite virulence and ecology [62]. The metagenomic analysis of the Leishmania vectors revealed the microbiota present in them, these studies will allow us to understand how the microbiota interacts with the parasite vectors and to develop tools for biological control [63]. Also by means of NGS, it has been observed that some HLA class I and class II genes could be involved in the predisposition of cutaneous leishmaniasis [64].

Mycetoma is one of the neglected tropical diseases, characterized by painless subcutaneous inflammation, multiple paranasal sinuses, and discharge containing aggregates of the infectious organism known as pimples. Studies of host genetic variation in mycetoma susceptibility by NGS will allow the identification of new treatments for mycetoma and will also improve the ability to stratify 'at risk' individuals, allowing the possibility of developing preventive and personalized clinical care strategies in the future [65]. The application of NGS in the study of malaria has greatly contributed to a better understanding of Plasmodium biology as well as host−parasite interactions [66].

*Trypanosoma cruzi*, the etiological agent of Chagas disease, represents a challenge due to its repetitive nature. Only three of the parasite's six recognized discrete typing units (DTUs) have their draft genomes published, and, therefore, analyzes of genome evolution in the taxon are limited, thus the assembly of short NGS reads can be applied for the detection of highly repetitive genomes [67]. Also, single nucleotide polymorphisms (SNPs) have been identified in the protein sequences of *T. cruzi* [68] and studies have been carried out with an EcoHealth approach [69]. These results may lead to a better understanding of Chagas disease and will provide further development of biomarkers for the prognosis, diagnosis, and development of drugs for the treatment of Chagas disease.

Comparisons of *Treponema pallidum* genomic sequences using NGS have revealed a modular structure of several genomic loci. This diversification of *T. pallidum* genomes appears to be facilitated by genome recombination events within the strain [70]. On the other hand, unbiased sequencing of the Zika virus genome obtained by NGS from the cerebrospinal fluid of one patient revealed that no virus mutations associated with anatomical compartments were detected [71] and NGS has also been used in different experimental and epidemiological settings to understand how the adaptive evolution of dengue variants shapes the dengue epidemic and disease severity through its transmission [72].

ddPCR has been shown to be more accurate than qPCR; therefore, it has been finely modified to detect low-abundance nucleic acids, which might be more suitable for clinical diagnosis [59]. Human strongyloidiasis is one of the neglected tropical diseases caused by infection with soil-transmitted helminth *Strongyloides stercoralis*. Conventional stool examination, a method commonly used for diagnosis of

*S. stercoralis*, has low sensitivity, especially in the case of light infections. However, the use of ddPCR showed high sensitivity and specificity for the detection of *S. stercoralis* in stool samples. This technique can help improve diagnosis, especially in cases of mild infection. In addition, the ddPCR technique could be useful for the detection of patients before starting immunosuppressive drug therapy and the follow-up after treatment of strongyloidiasis [73]. The usefulness of the ddPCR platform in the detection of *T. cruzi* infection has also been evaluated. The clinical sensitivity and specificity of the assay were both 100%, with perfect agreement between positive and negative qPCR and ddPCR results in the clinical samples tested. However, the fact of not performing a calibration curve in ddPCR offers an advantage for its use in the diagnosis of *T. cruzi* [74]. Moreover, RT-ddPCR in dengue diagnosis could help harmonize DENV quantification results and improve field findings, such as identifying a DENV titer threshold that correlates with disease severity [75, 76]. The use of ddPCR to absolutely quantify human malaria parasites successfully detects *Plasmodium falciparum* and *Plasmodium vivax*, and the sensitivity of ddPCR to detect *P. falciparum* is significantly higher than qPCR [77].

NGS and ddPCR have recently shown great potential for pathogen detection, however, in a comparative study between these techniques, the results were subject to their respective limitations and strengths, the ddPCR method being more useful for rapid detection of common isolated pathogens, while the mNGS test is more appropriate for diagnosis where classical diagnostic methods (microbiological or molecular) fail to identify the causative pathogens [78]. NGS is a new technology that holds the promise of improving our ability to diagnose, interrogate, and track infectious diseases. For its part, the third generation of the PCR; ddPCR can be used to directly quantify and clonally amplify DNA, the latter has been widely used in the detection


*Metabarcoding and Digital PCR (dPCR): Application in the Study of Neglected Tropical Diseases DOI: http://dx.doi.org/10.5772/intechopen.106272*


