Table 1
Microscopy-based diagnostic methods for soil-transmitted helminths diagnosis.
| Microscopy-based techniques | Procedure | Outcome | Sensitivity | Negative Predictive Value | Advantages | Limitation | References |
|---|---|---|---|---|---|---|---|
| Direct wet mount Microscopy | Preparation of stool sample with one drop of saline/iodine on a microscopic slide with a cover glass. | Egg detection | Sensitivity: A. lumbricoides 83.3% and Hookworm 85.7% Sensitivity: Hookworm: 37.9 %, A. lumbricoides 52% and T. trichiura 12.5% | Hookworm 97.5% and A. lumbricoides 98.8% | Low cost, easy and able to detect motile trophozoite | Low sensitivity | (Demeke et al., 2021; Dana et al., 2020; Else et al., 2020; Mengist et al., 2018 ; Nikolay et al., 2014; Endris et al., 2012) |
| Formol-ether concentration (FEC) | The stool sample is added to 7 mL of 10% formalin and the suspension is filtered through a sieve into a 15 mL conical centrifuge tube. Then, 4 mL diethyl ether is added to the formalin solution and centrifuged at 300 rpm for 1 min. The supernatant is discarded and the smear is prepared using a slide from the sediment. The sediment is screened under a microscope. | Egg detection | Sensitivity: A. lumbricoides 32.5% hookworm 64.2% and T. trichiura 75% Sensitivity: A. lumbricoides 81.4% hookworm 72.4% and T. trichiura 57.8% | A. lumbricoides 94.7%, hookworm 84.5% and T. trichiura 75% A. lumbricoides 93%, hookworm 97% and T. trichiura 91.5% | Low cost Essential for helminths ova detection | Need for centrifugation and does not detect unembryonated eggs of Ascaris spp. | (Hailu et al., 2022; Demeke et al., 2021; Else et al., 2020; Fenta et al., 2020; Sam et al., 2018; Endris et al., 2012) |
| Kato-Katz | The stool sample is pressed through a mesh screen to remove particles and an amount of sieved stool sample is placed into the hole of the template on a slide. Then, the template is removed and the remaining sample is covered with cellophane which is previously immersed with Glycerol-malachite green. The sample is screened under a microscope after 30 min and the eggs are counted. | Egg detection and egg quantification | Sensitivity: A. lumbricoides 93.1 %, hookworm 69% and T. trichiura 90.6% Sensitivity: A. lumbricoides 50%, hookworm 55.7% and T. trichiura 75% Sensitivity: A. lumbricoides 73%, hookworm 75.3% and T. trichiura 90.9% Sensitivity: A. lumbricoides 89.8%, hookworm 89.1% and T. trichiura 96.1% | A. lumbricoides 97.3%, hookworm 97.3% and T. trichiura 98% A. lumbricoides 96%, hookworm 81.5% and T. trichiura 99.8% | WHO recommended gold standard, lowcost, possible to determine the burden of infection | Low sensitivity, fails to detect infections of low intensity | (Khurana et al., 2021; Fenta et al., 2020; Cools et al., 2019; Moser et al., 2018; Endris et al., 2012) |
| McMaster | A known amount of stool is mixed with a saturated salt solution. The top layer of the solution is added to the reading chamber and the EPG count can easily be derived by multiplying the number of eggs under the marked areas by a simple conversion factor | Egg quantification | Sensitivity: A. lumbricoides 74.3%, hookworm 67.6% and T. trichiura94.6% Sensitivity: A. lumbricoides 74.3%, hookworm 92.3% and T. trichiura 94.9% | A. lumbricoides 82.4%, hookworm 62% and T. trichiura 47.8% | Inexpensive, easy, has been extensively used in human studies for estimation of anthelmintic cure rates. Highly sensitive for low-intensity soil-transmitted helminth infectionS | Need for a special counting chamber | (Khurana et al., 2021; Manuel et al., 2021; Else et al., 2020; Albonico et al., 2013; Albonico et al., 2012) |
| FLOTAC | The FLOTAC apparatus is a cylindrical-shaped device made of polycarbonate amorphous thermoplastic with two flotation chambers. An amount of stool sample is homogenized and filtered with water or saline. The filtrate is mixed with a floatation solution and is added to the chambers. The chambers are centrifuged and examined under a microscope. | Egg quantification | Sensitivity: A. lumbricoides 81.9%, hookworm 80.% and T. trichiura 96.8% Sensitivity: A. lumbricoides 79.7%, hookworm 92.4.% and T. trichiura 91% | A. lumbricoides 86.9%, hookworm 73.4% and T. trichiura 61.1% | Detection of different STHs simultaneously especially for low- intensity infections. | It is complex, a specific device is needed and requires centrifugation with two different rotors. | (Khurana etal., 2021; Manuel etal., 2021; Else etal.,2020; Knopp et al., 2014; Nikolay et al., 2014; Albonico et al., 2013; Cringoli et al.,2010) |
| Mini-FLOTAC | A weighed stool sample is homogenized with 5% formalin, filtered, and the filtrate is added to the flotation solution. The suspension is loaded into the two flotation chambers. Screened under a microscope after 5-10 min. | Egg quantification | Sensitivity: A. lumbricoides 42.1 %, hookworm 70.8.% and T. trichiura 85.6% Sensitivity: A. lumbricoides 75.5%, hookworm 79.2.% and T. trichiura76.2% | Detection of different STHs simultaneously | It is complex and a specific device is needed. Less sensitive for the diagnosis of A. lumbricoides | (Khurana et al., 2021; Manuel et al., 2021; Else et al., 2020; Cools et al., 2019; Barda et al., 2013; Nikolay et al., 2014) | |
| Baermann Technique | A 10 g of stools is place over few layers of gauze inserted into a cup filled with tap water. The apparatus is expose to artificial light directed at the funnel's bottom. Then 50 ml of the liquid is collected from the bottom of funnel in a plastic tube after 2 hours and centrifuged at 500 g for 2 minutes. A water suction pump use to drain the supernatant. | Larvae detection | Sensitivity: S. stercoralis 16% Sensitivity: S. stercoralis 20.7% Sensitivity: 28.3% and specificity of 75.2% | Highest sensitivities for S. stercoralis and detected larvae can be identified more easily Nearly 4 times higher sensitivity than FEC | Time consuming, large quantity of stool required, fresh sample required. | (Khurana etal., 2021 ;Hailegebriel et al., 2017; Knopp et al., 2014; Carvaho et al., 2012; Machicadoet al., 2012; Knopp et al., 2008) | |
| FECPAKG2 | The FECPAKG2 platform, which includes a cassette, focuses helminth eggs into a single microscopic field of vision, which is captured and saved on a computer, allowing the eggs to be counted. | Egg quantification | Sensitivity: A. lumbricoides 75.6%, hookworm 71.5%, and T. trichiura 65.8% | Detection of different STH simultaneously, simple procedure with a result within an hour | Internet connection is required | (Manuel et al., 2021; Else et al., 2020; Cools et al., 2019; Moser et al., 2018) |
Table 2
Molecular-based diagnostic methods for soil-transmitted helminths diagnosis.
| Molecular based techniques | Target | STH | Sensitivity and specificity / Rate of Detection | Advantages | Disadvantages | References | |
|---|---|---|---|---|---|---|---|
| Conventional PCR | Singleplex | Cytochrome Oxidase | A. duodenale and N. americanus | 1. Rapid, technically straightforward, and highly sensitive 2. Able to identify | Results must be visualized through gel electrophoresis | (Fleitas et al., 2021; George et al., 2015; Ngui et al., 2012; Nilforoush et al., 2007; J J Verweij et al., 2001; Zhan et al., 2001) | |
| Semi nested PCR | ITS-2,28S | N. americanus | Sensitivity: 94% Specificity:100% | individuals with mixed infections 3. Workable for fresh | |||
| Nested PCR | ITS-1 | S. stercoralis | or preserved fecal samples | ||||
| Semi-nested PCR-RFLP | ITS-1, ITS 2, 5.8s regions | A. duodenale and N. americanus | 4. Low cost | ||||
| Multiplex PCR | ITS-1,18S | A. lumbricoides and N. americanus S. stercoralis | Sensitivity: S. stercoralis 97.4% and hookworm 90.3% | ||||
| Real-Time PCR | Singleplex | 18S, ITS-2 | N. americanus, A. duodenale, S. stercoralis | Sensitivity: S. stercoralis 88.9% and hookworm 78.9% | 1. Screen multiple samples simultaneously 2. Higher sensitivity for all STH | 1. High usage of expensive consumables 2. Not feasible in resource-poor settings | (Othman et al., 2020; Benjamin-chung et al., 2020; Stracke et al., 2019; Cunningham et al., 2018; Schaer et al., 2013; Wang et al., 2012; Ngui etal., 2012; Taniuchi et al., 2011) |
| Multiplex | ITS-1, ITS-2,18S | A. lumbricoides, S stercoralis A. duodenale and N. americanus | 3. Fully automated and gel electrophoresis is not required 4. Provides quantified results | 3. A high-tech laboratory is required 4. Achieving maximized qPCR efficiency is challenging | |||
| Multi parallel | Repetitive sequence | A. lumbricoide,T. trichiura, A. duodenale and N. americanus | Sensitivity: A. lumbricoides 79%, hookworm 93%, and T. trichiura 90% | 5. Inhibition may occur 6. Triplication is required to ensure accuracy | |||
| PCR-Luminex | ITS-1,2 | N. americanus, A. lumbricoides, A. duodenale, and S. stercoralis | |||||
| Multiplex- tandem PCR- qPCR | β-tubulin | A. lumbricoides, T. trichiura, A. duodenale, and N. americanus | Rate of detection: MT-PCR ranged from 90.64% (A. lumbricoidess) to 100.00% (T. trichiura). | ||||
| SYBR Green quantification | ITS-2 | N. americanus | Rate of detection: 22.69% | ||||
| High Resolution Melting (HRM) | 18S, ITS-1,2 | A. lumbricoides, T. trichiura, A. duodenale, and N. americanus | Sensitivity: 80 % | ||||
| ITS-2 | N. americanus, Ancylostoma. spp. | Sensitivity: 100% | |||||
| dPCR | ITS-1 | A. lumbricoides | Rate of detection: 20 eggs in 10 L of reclaimed water | 1. Rapid 2. Precise 3. Sensitive 4. Useful in the detection of drug-resistant or high-pathogenicity subpopulations 5. Able to detect low amounts of A. lumbricoides eggs | The machines and reagents are costly | (Stuyver L.J et al., 2021; Soto et al., 2017) | |
| LAMP | SmartAmp2 | β-tubulin | A. lumbricoides, T. trichiura, N. americanus | 1. Great sensitivity and specificity 2. Low cost 3. Simple procedure | A precise and straightforward DNA extraction process is required to avoid the polyphenols found in faecal samples that inhibits DNA polymerase. | (Ngari et al., 2020; Rashwan et al., 2017; Shiraho et al., 2016) | |
| LAMP | ITS-1 | A. lumbricoides | Sensitivity: 96.3% Specificity: 61.5% | ||||
| ITS-2 | |||||||
| quantitative paper-based DNA reader (qPDR) | Singleplex Mini-PCR | β-tubulin | T. trichiura, | 1. Low cost 2. Simple procedure 3. Faster | 1. Required validation 2. Need to develop portable stool DNA extraction | (Manuel et al., 2021; Wang et al., 2018) | |
| Cell-free DNA | 18S rRNA, Cox1 | S. stercoralis | 1. Low cost 2. Simple procedure 3. Faster | Viability and different clearing times of STH eggs because eggs tend to be damaged and are lysed rapidly | (Manuel et al., 2021; Gorgani-Firouzjaee et al., 2018; Weerakoon et al.,2016) |
Table 3
Gene targets used for molecular diagnosis of soil-transmitted helminths infections.
| STH | Target gene | References |
|---|---|---|
| Strongyloides stercoralis | Cytochrome oxidase I Repetitive sequence 18S ITS-1 ITS-1, 5.8s, ITS-2 | (Basuni et al., 2011; Moghaddassani et al., 2011 ; Verweij et al., 2009; Nilforoush et al., 2007) |
| Ascaris lumbricoides | Repetitive sequence ITS-1 ITS-1, ITS-2, 5.8 s | (Pilotte et al., 2016; George et al., 2016; Mejia et al., 2013) |
| Trichuris trichiura | Repetitive sequence ITS-1, ITS-2, 5.8 s ITS-1 | (George et al., 2016; Pilotte et al., 2016; Mejia et al., 2013) |
| Ancylostoma spp. | Repetitive sequence ITS-1, ITS-2, 5.8 s ITS-2 Cytochrome oxidase I | (Pilotte et al., 2016; George et al., 2015; Mejia et al., 2013; Zhan et al., 2001) |
| Necator americanus | Repetitive sequence ITS-1, ITS-2, 5.8 s ITS-2 Cytochrome oxidase I | (Pilotte et al., 2016; George et al., 2015; Basuni et al., 2011; Zhan et al., 2001) |
Table 4
A summary of representative studies evaluating the performance of real-time PCR and other methods.
| qPCR | Other methods | ||||
|---|---|---|---|---|---|
| 1 | Ethiopia | 844 school children | Sensitivity: S. stercoralis 73.9% | Formol-Ether concentration Technique (FECT): S. stercoralis 5.2% | (Hailu et al., 2022) |
| Spontaneous tube sedimentation technique (STST): S. stercoralis 10.3% | |||||
| Baermann concentration technique (BCT): S.stercoralis 26.4% | |||||
| 6 | Brazil, Ethiopia, Lao PDR, and Tanzania | 645 children | Sensitivity: A. lumbricoides 90.0%, T. trichiura 94.7% and hookworm 91.9% | Single Kato-Katz: A. lumbricoides 71.9%, T. trichiura 88.1% and hookworm 72.6% Duplicate Kato-Katz: A. lumbricoides 73.0%, T. trichiura 90.9% and hookworm 75.3% | (Cools et al., 2019) |
| Mini-FLOTAC: A. lumbricoides 63.3%, T. trichiura 91.5% and hookworm 73.9% | |||||
| 10 | Côte d’Ivoire | 256 individuals | Sensitivity: S. stercoralis 76.8% | Koga agar plate: S. stercoralis 21.4% | (Becker et al., 2015) |
| Baermann: S. stercoralis 37.5% |