Table 3.
Overview of current and historical Trypanosoma cruzi genotyping methods.
| Genotyping method | Method description | Example of genetic markers | Reproducibility b/w assays | Level of resolution | Reagent cost | Advantages | Disadvantages | Ref. |
|---|---|---|---|---|---|---|---|---|
| MLEE | Measures differences in electrophoretic mobilities of isoenzymes | ASAT, ALAT, PGM, ACON, MPI, ADH, MDH, ME, ICD, 6PGD, G6PD, GD, PEP, GPI | High | DTU level Intra-lineage |
Moderate | Easy visual interpretation Data amenable to numerical taxonomic analysis, for example, rates of similarity or genetic distance |
Requires large quantities of parasite lysate from live strains | [16,128,173,293,294,306] |
| RAPD | Short random sequence primers used to amplify unknown DNA fragments to create unique band patterns | N/A | Low | DTU level | Low | Can be performed directly on field samples No prior sequence knowledge needed Unlimited number of primers Data amenable to numerical taxonomic analysis |
Reproducibility issues Dominant markers may conceal heterozygosity Strain profiles may vary with DNA template amount and quality |
[40,41,45,46] |
| kDNA-RFLP | RFLP analysis of kinetoplast mHVR | mHVR | Low | Intra-lineage | Low | Hypervariable markers Can produce strain-specific profiles |
Requires isolation of kDNA from live parasites Strain profile inheritance may not be stable or correlate with nuclear typing Potential problems of contamination due to very high copy number |
[136] |
| kDNA hybridization | Analysis of mHVR by radioactive probe hybridization | mHVR | Low | DTU-level Intra-lineage |
Hypervariable markers Can produce strain-specific profiles |
DNA probes may cross-react b/w DTUs Potential problems of contamination due to very high copy number |
[307,308] | |
| Karyotyping (aCSDI) | Comparison of chromosome size variation by PFGE separation and radioactive probe hybridization | 1F8, cruzipan, FFAg6, Tc2, CA7.12, CA7.32, P19 | Moderate | DTU level | Moderate | Data amenable to numerical taxonomic analysis | Requires live strains Strain profiles may not be stable due to expansion/contraction of tandem repeats Prone to convergence b/w unrelated strains |
[144,154,155,309] |
| DNA fingerprinting | Analysis of variability in nuclear minisatellites by restriction digestion and probe hybridization | 33.15 | Low | Intra-lineage | Low | Hypervariable markers Can produce strain-specific profiles |
Requires live strains Reproducibility issues |
[156] |
| LSSP-PCR | Analysis of size polymorphisms in mHVR amplified by LSSPs | mHVR | Low | DTU level | Low | Highly sensitive Can be used to detect Trypanosoma cruzi in infected tissues without parasite isolation |
Reproducibility issues Potential problems of contamination due to very high copy number kDNA signatures may vary with DNA template amount and quality |
[310–312] |
| SSCP | Analysis of size polymorphisms in multicopy gene fragments | SL-IR, 24Sα rRNA, 18S rRNA, cruzipain, P7-P8 | Moderate | DTU level | Low | Requires limited technical expertise | Requires live strains DTU assignment based on presence/absence of amplicons; insensitive to potential mutations in novel strains Unknown intra-strain copy homology |
[25,313,314] |
| PCR product size polymorphism | Analysis of size polymorphisms in multicopy gene fragments | SL-IR, 24Sα rRNA, 18S rRNA, A10 | High | DTU level | Low | Can be performed directly on field samples Requires limited technical expertise |
DTU assignment based on presence/absence of amplicons; insensitive to potential mutations in novel strains Unknown intra-strain copy homology |
[41–43] |
| PCR-RFLP | RFLP analysis of multicopy gene fragments | HSP60, GPI, COII, GP72, 1F8, histone H3, ITS, TcSC5D | High | DTU level | Moderate | Can be performed directly on field samples Requires limited technical expertise |
DTU assignment based on presence/absence of SNPs; insensitive to potential mutations in novel strains | [94,158,315] |
| Nucleotide sequencing: nuclear loci (nMLST) | SNP analysis of nuclear housekeeping gene fragments | TcMSH2, DHFR-TS, TR, LYT1, Met-II, Met-III, TcAPX, TcGPX, TcMPX, HMCOAR, PDH, GTP, STTP2, RHO1, GPI, SODA, SODB, LAP | High | DTU level (intra-lineage) | High | Data amenable to MLST analysis Data highly reproducible, portable and transferable b/w laboratories |
Requires live strains Level of intra-lineage resolution dependent upon analysis of multiple loci |
[60,61,298,316] |
| Nucleotide sequencing: maxicircle loci (mtMLST) | SNP analysis of mitochondrial gene fragments | 12S rRNA, 9S rRNA, Cytb, MURF1, ND1, COII, ND4, ND5, ND7 | High | [DTU-level] Intra-lineage |
High | Data amenable to MLST analysis Data highly reproducible, portable and transferable b/w laboratories |
Requires live strains Potential phylogenetic incongruence with nuclear loci Identifies three maxicircle classes (TcI, TcII and TcIII–VI); not specific to all six DTUs |
[54,317,318] |
| Nucleotide sequencing: minicircle regions | SNP analysis of mHVR | mHVR | High | DTU level Intra-lineage |
High | Hypervariable markers Can produce strain-specific profiles |
Strain profile may not be DTU specific; minor sequence classes shared b/w DTUs | [137,138] |
| FFLB | Analysis of size polymorphisms in multicopy gene fragments | 28Sα rRNA, 18S rRNA | High | DTU level | High | Can be performed directly on field samples | Unable to differentiate hybrid lineages (TcV and TcVI) | [319] |
| HRM | Analysis of amplicon melting temperatures generated by real-time PCR | SL-IR, 24Sα rRNA | High | DTU level | Moderate | Data rapidly generated in real time | Requires live strains Difficult to standardize b/w laboratories Requires specialized laboratory infrastructure |
[320] |
| MLMT | Analysis of size polymorphisms of microsatellite repeat regions | 10101(CA)a, 11283(TA)b, 7093(TA)b, TcUn4, mclf10, 10187(CA)(TA), 6855(TA)(GA), 10359(CA), 8741(TA), 10187(TTA), 7093(TA)c | Moderate | DTU level Intra-lineage |
High | Neutrally evolving, co-dominant, hypervariable markers Can produce strain-specific MLGs |
Requires live strains Prone to homoplasy Data interpretation highly subjective |
[53,70,78,89,133,134,157,321] |
| Amplicon sequencing | Analysis of millions of sequencing reads generated by Illumina deep sequencing | TcGP63, ND5 | High | DTU level Intra-lineage Parasite multiclonality |
Very high | Can detect intra-host parasite multiclonality and genetic diversity | Requires live strains Prone to loss of clonal diversity from parasite isolation Requires bioinformatics expertise, computational infrastructure and comparatively high cost reagents |
[289] |
aCSDI: Absolute chromosomal size difference index; DTU: Discrete typing unit; FFLB: Fluorescent fragment length barcoding; GPI: Glucose-6-phosphate isomerase; HRM: High-resolution melting; HSP60: Heat shock protein 60; kDNA: Kinetoplast DNA; LSSP: Low stringency single specific primer; mHVR: Minicircle hypervariable region; MLEE: Multilocus enzyme electrophoresis; MLG: Multilocus genotype; MLMT: Multilocus microsatellite typing; mtMLST: Maxicircle multilocus sequence typing; nMLST: Nuclear multilocus sequence typing; PFGE: Pulsed-field gel electrophoresis; RAPD: Random amplification of polymorphic DNA; RFLP: Restriction fragment length polymorphism; SL-IR: Spliced-leader intergenic region; SNP: Single nucleotide polymorphism; SSCP: Single-stranded DNA conformation polymorphism.