Advantages and Limitations of Microscopy and Molecular Detections for Diagnosis of Soil-transmitted Helminths: An Overview

Summary

World Health Organization (WHO) reported that over 1.5 billion people are infected by soil-transmitted helminths (STH) worldwide in sub-Saharan Africa, the United States of America, China, and East Asia. Heavy infections and polyparasitism are associated with higher morbidity rates, and the patients are exposed to increased vulnerability to other diseases. Therefore, accurate diagnosis followed by mass treatment for morbidity control is necessary.STH diagnosis commonly involves the microscopic observation of the presence of the STH eggs and larvae in the faecal samples. Furthermore, molecular approaches are increasingly utilised in monitoring and surveillance as they show higher sensitivity. Their capability to differentiate hookworm species is an advantage over the Kato-Katz technique. This review discusses the advantages and limitations of microscopy and various molecular tools used for STH detection.

Introduction

Infections caused by soil-transmitted helminths (STH) are often neglected in tropical countries, as declared in the London Declaration of Neglected Tropical Diseases (NTD) 2012. However, STH infection is highly prevalent in under-developed countries and results in chronic illness (Werkman et al., 2018; Basuni et al., 2011). The resulting impacts on the education and economy of the affected nations remain unquantified (Parija et al., 2017). There are four main human-infecting STH species, specifically the roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura), and hookworms (Necator americanus and Ancylostoma sp.) (Ngwese et al., 2020; Werkman et al., 2018). Although the threadworm (Strongyloides stercoralis) is not included on the list of NTD by World Health Organization (WHO), its geographical distribution overlaps with other STH. It has high poverty-related morbidity (Ngwese et al., 2020).

WHO reported that over 1.5 billion people are infected by STH worldwide, with their significant occurrence in sub-Saharan Africa, the United States of America, China, and East Asia (WHO, 2020). The infections caused by STH remain endemic in these regions, with the most impoverished sub-Saharan Africa and several regions in South-East Asia suffering a more significant impact (Parija et al., 2017; Pullan et al., 2014). A. lumbricoides is the most common STH where it has infected around 1.2 billion people, followed by T. trichiura, A. duodenale and N. americanus, with 740 ‒ 795 million people infected (Hailegebriel et al., 2020). The prevalence of S. stercoralis is underestimated globally due to the low sensitivity of diagnostic tests and the inefficiency of case reporting in high-incidence countries (Mora & Meseeha, 2022). S. stercoralis is widespread globally except in the far north and far south regions, where it is commonly found in warm and moist areas with poor sanitary conditions (Mora & Meseeha, 2022).

Human infection by A. lumbricoides results in the common tropical infection ascariasis (Shiraho et al., 2016). Adult A. lumbricoides causes upper gastrointestinal bleeding, small bowel obstruction, volvulus and intussusception, peritonitis, and gastric ascariasis with possible perforation (Jourdan et al., 2018). A T. trichiura infection is commonly asymptomatic as the clinical disease depends on parasite burden (Viswanath et al., 2022). However, it can be symptomatic, accompanied by abdominal pain, painful passage of stools and abdominal discomfort. Trichuris Dysentery Syndrome signs such as anaemia could be present in severe cases (Viswanath et al., 2022; Jourdan et al., 2018). Individuals infected with S. stercoralis may experience irritation at the site on the skin where the larvae penetrated, followed by irregular localised oedema or urticaria (Nutman, 2017). S. stercoralis is associated with dermatological and gastrointestinal morbidity in children (WHO, 2020). On the other hand, hookworms rarely cause severe anaemia or significant protein loss in the host. Thus, the annual death is lower compared to other STH infections. However, hookworm infections impose long-term effects such as malnutrition and delayed cognitive development, particularly in children (Bethony et al., 2006).

WHO recommends annual and biannual regional mass drug administration (MDA) program in endemic populations by delivering 400 mg single dose albendazole or 500 mg mebendazole (Stracke et al., 2019). The MDA is an effort to deworm high-risk population groups such as pre-school-aged children (PSAC), school-aged children (SAC), and women of reproductive age (WRA) (Manuel et al., 2021). Since morbidity is more frequent in children than adults, adults are usually excluded from MDA programs except for hookworm infection in pregnant women (Werkman et al., 2018). The necessity of deworming in an area or country is determined by population-level parasitological studies, which evaluate the prevalence and severity of infection (Manuel et al., 2021).

STH detection is commonly based on the microscopic diagnosis of the helminth eggs or larvae in human stool and occasional shedding of the eggs or larvae of the parasites (Glinz et al., 2010). However, the availability of well-trained professionals is less in low prevalence areas, often misdiagnosed (Khurana & Sethi, 2017; O’Connell & Nutman, 2016). The Kato-Katz thick stool smear is still recommended in the WHO 2030 roadmap as the standard diagnostic to detect and quantify the intensity of STH infections. However, the Kato-Katz technique is not recommended to diagnose Strongyloidiasis infections due to its lower sensitivity for STH with low intensity of infection, despite its ideal application for diagnosing moderate and high-intensity infection (M & HI) (Stuyver & Levecke, 2021). Besides, the sample stirring step decreases faecal egg count variation for hookworm and T. trichiura even though A. lumbricoides diagnosis is not affected (Bosch et al., 2021). Although the traditional coprological methods are reliable and widely used to detect parasite larvae, the need for enhanced sensitivity is growing as a measure for STH control (Papaiakovou et al., 2019). Therefore, it is crucial to develop new diagnostics to monitor, control and eliminate STH (Knopp et al., 2014)

With the increasing emphasis on diagnostic accuracy, molecular methods are gaining attention as alternatives to overcome the drawbacks of microscopy-based techniques (Azzopardi et al., 2021). The advantages of molecular methods are they provide higher sensitivity and specificity than microscopy and are constantly improving due to new technologies in genomics and bioinformatics(Papaiakovou et al., 2019).On the other hand, FECPAK^G2 is a newly developed quantitative method for the microscopic quantification and detection of STH eggs in a human stool(Ayana et al., 2018). FECPAK^G2 involves a special microscope equipped with an electronic camera to capture the image of the sample (Boelow et al., 2022). The image is stored and shared through cloud storage to provide a reference for settings with the limited resource (Moser et al., 2018). Therefore, based on the current research in this area, this review discusses the advantages and limitations of the various molecular tools used for STH detection compared to the microscopy-based methods.

Methodology

For this review, we used PubMed, Google Scholar, Science Direct, ResearchGate, World Organization Health (WHO) website web to do a web searching using the following keywords: “Diagnosis soil-transmitted helminths” OR “S. stercoralis” OR “T. trichiura” OR “hookworm, OR “A. lumbricoides” OR “A. duodenale” OR “N. americanus” OR “nematodes” OR “roundworm” OR “whipworm” OR “helminth” OR “soil-transmitted helminths” OR “worm burden” OR “Kato-Katz” OR “McMaster” OR “ FLOTAC” OR “Real-Time PCR” OR “PCR” OR “LAMP”. Our searching covered articles published between 2000 and 2022.

Advantages and limitations: Microscopy-based Techniques

Microscopy-based techniques are simple and low-cost, but their sensitivity is affected by several factors, including intermittent excretion of parasite ova, low infection intensity that limits the detection, inappropriate transportation or sample storage (Khurana et al., 2021; Papaiakovou et al., 2019). Table 1 summarises several microscopy-based diagnostic methods for STH diagnosis.

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)

The direct wet mount examination of moist stool samples has the advantage of detecting the motile trophozoite stage of the protozoan species (Demeke et al., 2021). Direct wet mount microscopy is widely used in Africa, particularly in Ethiopia, to diagnose parasitic intestinal infections (Demeke et al., 2021; Mengist et al., 2018). Meanwhile, the formol–ether concentration (FEC) method helps detect STH eggs in a large sample of fixed stool (Speich et al., 2014). Nevertheless, it shows substandard accuracy in detecting threadworm larvae (Glinz et al., 2010), and hookworm eggs were easily damaged while processing the samples because the eggshell is very thin and fragile (Panggabean et al., 2017).

The Kato-Katz technique is the most widely used to detect STH infection (Bosch et al., 2021), and it is recommended by WHO for surveillance and epidemiological field surveys of schistosome and STH infections due to its relative simplicity, pace, and low cost (Habtamu et al., 2011). Kato-Katz procedure is modifiable (Khurana et al., 2021), whereby the parasitic load is reported as eggs per gram of stool (EPG) (Azzopardi et al., 2021). The Kato-Katz thick smear is commonly used to detect Ascaris, Trichuris, and the hookworms in which helminth eggs are identified and enumerated by microscopy (Azzopardi et al., 2021). The sensitivity of the Kato-Katz method varies from 65.2 – 96.9 %, with a high specificity of 93.8 – 99.4 % (Azzopardi et al., 2021; Nikolay et al., 2014).

The McMaster (MM) method is another microscopy-based technique that requires a special counting chamber. McMaster is the standard coprological method. It is widely used to assess STH in veterinary parasitology and estimate anthelmintics’ cure rates in humans. Its ease of use in estimating faecal egg counts and suitability for unestablished laboratories make it a good choice for public health monitoring of human STH (Periago et al., 2015). On the other hand MM approach seems more suitable for standardisation due to its efficient multiplication factor and ability to detect all STH organisms simultaneously (Levecke et al., 2011).

The FLOTAC technique was first developed for veterinary parasitology before being applied for human parasitology (Cringoli et al., 2010; Utzinger et al., 2008). This technique has recently been proven highly responsive to the Kato-Katz approach for diagnosing hookworm, roundworm, and whipworm (Glinz et al., 2010). Since the FLOTAC technique may analyse stool load of 1 g or more, high multiplication factors are not required to determine faecal EPG (Tello et al., 2012; Glinz et al., 2010). In addition, the Mini-FLOTAC technique is a sensitive and straightforward method for determining the severity of helminth infections (Barda et al., 2013). The Mini-FLOTAC procedure does not require any centrifugation steps or costly facilities. Both fresh and fixed stool samples can be used for this procedure, and the whole process only takes 10 – 12 min (Barda et al., 2013). The faecal egg count and the number of helminth eggs are counted per species and multiplied by 10 (Dana et al., 2020).

The Baermann Technique (BM) is another microscopy-based technique that requires a glass funnel that used for the detection of nematode larvae in faecal samples and is based on the thermotropism and hydrotropism of the larvae (Carvaho et al., 2012). The Baermann Technique (BM) is a diagnostic method for detecting S. stercoralis (Knopp et al., 2008). Larvae detection and first-stage S. stercoralis larvae (L1) confirmation is carried out under a microscope at 100x magnification and 400x magnification respectively. Although the BM technique has low positivity rates of parasite detection, it shows moderate sensitivity in detecting S. stercoralis infection with a sensitivity of 28.3 % and specificity of 75.2 % (Hailegebriel et al., 2017; Knopp et al., 2014; Carvaho et al., 2012). FECPAK^G2 is developed to produce digital images of helminth eggs that have been concentrated into one microscopic field of view, and the images can be backed up online (Ayana et al., 2018). FECPAK^G2 achieved a detection sensitivity of 75.6 % for A. lumbricoides, 71.5 % for hookworm and 65.8 % for T. trichiura from human stools in a randomised controlled trial with increased sensitivity for moderate infection intensities (Moser et al., 2018). However, faecal FEC given by Mini-FLOTAC and FECPAK^G2 differed markedly and led to a false classification for the low load intensity samples, thus highlighting the need for method-specific infection intensity thresholds (Cools et al., 2019).

Principles and advantages of molecular-based techniques

The molecular approaches include conventional polymerase chain reaction (PCR), real-time PCR, digital PCR, loop-mediated isothermal amplification assay (LAMP) and, more recently, cell-free DNA detection and quantitative paper-based DNA reader. Various molecular diagnostic tests for STH diagnosis are summarised in Table 2.

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)

Molecular-based methods can detect and identify intestinal parasites in faecal samples (Rogers et al., 2021) from appropriate DNA targets (Verweij et al., 2001). Advances in the bioinformatics sequencing of nematode genomes with the availability of nematode sequence data have enabled the development of assays which target species-specific genomic regions (Khurana et al., 2021; Grant et al., 2019). The DNA sequences that are commonly used as targets for the detection of STH are ribosomal internal transcribed spacer (ITS) sequences, 18S or ribosomal sub-unit sequences, mitochondrial genes such as cytochrome oxidase I (COI), and repetitive sequences (Manuel et al., 2021; Pilotte et al., 2016). The first and second internal-transcribed spacer (ITS-1 and ITS-2, respectively) of ribosomal DNA (rDNA) provide genetic markers for the specific identification of parasites, especially A. duodenale and N. americanus (Gruijter et al., 2005). Ribosomal sequences are chosen as diagnostic targets since they are frequently found in nucleated organisms as easily identified moderate copy number tandem repeats (Pilotte et al., 2016). Mitochondrial genes, particularly cytochrome oxidase-I, are also used as DNA targets due to multiple copies of mitochondrial targets in most eukaryotic cells (Manuel et al., 2021; Pilotte et al., 2016). Non-coding repetitive sequences that are discovered in nearly all eukaryotes could also be used as targets for next-generation sequencing (NGS) owing to their high sensitivity and species-specific STH identification capability (Pilotte et al., 2016). Table 3 shows gene targets used in molecular diagnosis for STH detection.

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)

Stool Sample Storage and Preservation

The stool is currently the only validated sample for STH detection due to the biology of the STH infection (Hawkins et al., 2020). Therefore, stool sample storage and DNA extraction are crucial in any molecular assay to assure accuracy. Papaiakovou and co-workers (2018) compared the standard sample storage technique at 20 °C used in most laboratories with different sample preservation techniques. The methods include silica bead two-step desiccation, FTA cards, 5 % potassium dichromate, RNAlater, ethanol, Paxgene, and Formalternate. The comparison was conducted at 4 °C or 32 °C coupled with four collection times of 0 day, 1 day, 7 days and 60 days. In contrast, the control is kept at −20 °C without any preservatives with the same collection time. Stool samples spiked with N. americanus ova were used in this investigation. All preservation techniques were stable at 4 °C. Still, high stability was observed only for silica bead two-step desiccation, 5 % potassium dichromate, and FTA card-based preservation conducted at 32 °C, with no significant change in DNA concentration (Manuel et al., 2021; Papaiakovou et al., 2018).

Ayana and co-workers (2019) found that higher concentrations of A. lumbricoides, T. trichiura, and N. americanus DNA can be extracted if the stool samples were preserved with ethanol and RNA than those maintained with potassium dichromate, with no significant changes in sensitivity. In addition, Azzopardi and colleagues (2021) developed a scalable system to wash stool preserved in potassium dichromate. The washed stools could be stored at −30 °C for up to 15 weeks without compromising yield before DNA isolation. Briefly, 7.5 mL of the preserved faeces were poured into a 50 mL tube and centrifuged at 700 x g for 3 min to prevent worm egg lysis. The stools were rinsed with 50 mL of phosphate-buffered saline (PBS) and recentrifuged. The material was transferred to a 15 mL tube after a final 15 mL PBS wash followed by centrifugation. The supernatant was removed, and the washed stool pellets were stored at 4 °C for 4 weeks until the DNA extraction.

DNA Extraction

The diagnostic performance of DNA-based tests must be evaluated to warrant the quality of diagnostic testing (Cools et al., 2020; Hawkins et al., 2020). The method used for DNA extraction is crucial as it affects the outcome of the assessed tests. Thus, appropriate DNA extraction methodologies are required to increase the sensitivity and accuracy of detection (Sharifdini et al., 2015). Advancement in DNA extraction procedures for STH ova is progressing to develop processes that homogenise faecal samples and break up the outer layer of the egg to achieve high DNA yields (Manuel et al., 2021). Most laboratories prefer commercial kits because of their standardised procedures and relative convenience. However, commercially available kits such as Powersoil DNA Isolation Kit (MO Bio, Carlsbad, CA, USA), FastDNA Spin Kit for Soil (MP Biomedicals, Irvine, CA, USA), QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA) require pre-processing steps such as bead beating and/or lysis which has an important impact on kit sensitivity and DNA recovery (Manuel et al., 2021; Ayana et al., 2019).

Conventional PCR

Polymerase chain reaction (PCR)-based methods possess high sensitivity as parasite detection is possible with low DNA concentrations (Manuel et al., 2021; Verweij et al., 2001). A recent study by Fleitas et al., (2021) reported successful detection of S. stercoralis and hookworm in stool samples with 2 – 20 pg genomic DNA. The sensitivity for S. stercoralis and hookworms was 97.4 % and 90.3 %, while the specificity was 100 % and 87.6 %, respectively (Fleitas et al., 2021). Conventional PCR is also used with other downstream techniques, such as restriction fragment length polymorphism (RFLP), to identify different species of STH (George et al., 2016; Manuel et al., 2021). Based on the work conducted by George and colleagues (2015), a semi-nested PCR-RFLP assay of specific amplification of the internal transcribed spacer gene (ITS) 1, 2, and 5.8s was demonstrated to detect roundworm A. duodenale, and animal hookworm A. ceylanicum in children from a tribal community in Tamil Nadu, India. On the other hand, Ngui and co-workers (2012) used nested PCR to differentiate between N. americanus and Ancylostoma spp. in Malaysia with a specific gene target of ITS-2 and 28S ribosomal RNA. In Thailand, Phuphisut and colleagues (2014) employed triplex PCR to detect STH simultaneously in the same reaction, thereby reducing cost. Their study reported that the sensitivity for A. lumbricoides, N. americanus, and T. trichiura detection was 87 %, with a specificity of 83 % where a single copy of DNA was detectable. Nonetheless, agarose gel electrophoresis is required to visualise the results of PCR. The process is time-consuming compared to other molecular diagnostics, contributes to higher costs, and has a potential for contamination (Manuel et al., 2021; Gordon et al., 2011).

Real-time PCR

Quantitative real-time PCR (qPCR) or real-time PCR is suitable for detecting DNA targets in stools, even in low-prevalence settings (Papaiakovou et al., 2019). The dyes can be either nonspecific dyes, such as fluorescent DNA-binding dyes (e.g., SYBR Green I) or strand-specific probes (e.g., Taqman or Molecular Beacons) (Fraga, 2008). The detection and monitoring of hookworm N. americanus infections can be performed using SYBR Green qPCR, where the ITS-2 sequence of N. americanus is amplified for detection (Wang et al., 2012). Besides, probe-based assays such as TaqMan allow multiplexing and multi-parallel detection of multiple helminths from a single stool sample (Benjamin-chung et al., 2020). The higher sensitivity of real-time multiplex PCR compared to that of the Kato-Katz technique is associated with the direct detection of STH DNA in minimal quantities where DNA is less likely to degrade than STH ova (Aung et al., 2022; Benjamin-chung et al., 2020; Llewellyn et al., 2016). It can detect more low-intensity infections compared to the Kato-Katz technique with a limit of detection (LOD) of 10 copies (Basuni et al., 2011; Keller et al., 2020). A recent study conducted by Zendejas-Heredia et al., 2021 revealed that only qPCR was able to classify low-moderate infection cut-offs accurately and able to detect very low infection intensities. A summary of representative studies evaluating the performance of real-time PCR and other methods is provided in table 4.

Table 4.

A summary of representative studies evaluating the performance of real-time PCR and other methods.

No	Country	Study population	Sensitivity		References
			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%
				Agar plate culture (APC) : S. stercoralis 28.0%
2	Fiji	40 individuals	Sensitivity qPCR compare to Kato- Katz: A. lumbricoides 75% and hookworms 66.7%		(Azzopardi et al., 2021)
3	Tanzania	1636 individuals	Sensitivity: T. trichiura 94.4%, A. lumbricoides 85.1%, and hookworm 85.2%	Kato-Katz: T. trichiura 78.2%, A. lumbricoides 38.5% and hookworm 39.7%	(Keller et al., 2020)
4	Bangladesh	2,799 children	Sensitivity: A. lumbricoides 79%, hookworm 93%, and T. trichiura 90%	Double-slide Kato-Katz: A. lumbricoides 49%, hookworm 32% and T. trichiura 52%	(Benjamin-chung et al., 2020)
5	Tanzania	320 individuals	Sensitivity: T. trichiura 89.1%, hookworm 72.7% and A. lumbricoides 87.5%	Kato-Katz: T. trichiura 83.6%, hookworm 43.0% and A. lumbricoides 53.8%	(Barda et al., 2020)
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%
				FECPAKG2 A. lumbricoides 58.9%, T. trichiura 59.8% and hookworm 52.4%
7	Timor Leste	571 children (860 sample)	Sensitivity: A. lumbricoides 94.1%, hookworm 75.7%, and T. trichiura 53.1%	Sodium nitrate flotation (SNF): A. lumbricoides 68.1%, hookworm 66.9% and T. trichiura 81.3%	(Clarke et al., 2018)
8	Philippines	263 school children	Sensitivity: A. lumbricoides 60.8%, and T. trichiura 38.8%	Kato-Katz: A. lumbricoides 20.5%, and T. trichiura 23.6%	(Mationg et al., 2017)
9	Kenya	796 individuals	Sensitivity: A. lumbricoides 98% and N. americanus 98%	Kato-Katz: A. lumbricoides 70% and N. americanus 32%	(Easton et al., 2016)
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%

Real-time PCR can be used in combination with other techniques for better detection. For example, PCR-Luminex, high-resolution melt curve analysis, and multiplexed-tandem. PCR-Luminex is high throughput multiplex PCR combined bead assay involving the binding of PCR products to carboxylated Luminex beads with biotinylated primers and amine-modified probes as an alternative to real-time PCR (Taniuchi et al., 2011). The advantages of PCR-Luminex are higher sensitivity in detection than microscopy, shorter time for technical handling due to the exclusion of several testing modalities such as larvae ova and cysts concentration step, specific staining, or immunoassays, and provide the capacity to perform their tests in-house. The 83 – 100 % sensitivity and specificity were reported based on 319 clinical specimens (Taniuchi et al., 2011). Next, real-time PCR coupled with high-resolution melt curve (HRM) analysis is used to detect different hookworm species, whereby this assay can detect as low as 0.01 ng/mL DNA (Ngui et al., 2012). Furthermore, multiplexed-tandem qPCR (MT-PCR) is a rapid and semi-automated tool that is more user-friendly than conventional multiplex qPCR in identifying and quantifying STH eggs in samples. It displays higher sensitivity than faecal microscopy (Stracke et al., 2019).

Digital PCR

Digital PCR (dPCR) is closely related to qPCR as it involves the same amplification reagents. A fluorescent readout indicates adequate amplification of target molecules due to using intercalating agents or hydrolysis-based probe reporters (Salipante & Jerome, 2020). Thus, digital PCR is advantageous over real-time PCR with good repeatability and reproducibility due to the PCR-inhibitory substances which lower the risk of inhibition (Sreejith et al., 2018). Soto and co-workers (2017) demonstrated high sensitivity of dPCR whereby the DNA concentrations of 5 A. lumbricoides eggs in reclaimed water equivalents were detected (Soto et al., 2017). Droplet dPCR (ddPCR) is a form of dPCR based on oil-water emulsion technology for partitioning the reaction mixture, which is a potential surveillance tool for detecting STH in environmental samples in endemic areas (Manuel et al., 2021). Despite the diagnostical merits, the implementation of dPCR is challenged by the high cost required to establish the setups.

Loop-mediated Amplification Assays

Loop-Mediated Amplification (LAMP) Assay is a one-step DNA amplification method with high specificity and sensitivity where a target sequence is amplified under isothermal conditions (Rashwan et al., 2017). LAMP assay was developed by Notomi et al. (2000) with the principle of auto-strand displacement DNA synthesis (Manuel et al., 2021). The assay only requires a water bath or thermal block to provide isothermal conditions (Manuel et al., 2021). LAMP reaction requires no specific reagents and can be performed in any general molecular biology facility (Notomi et al., 2015). This technique involves a DNA polymerase and a set of four specially designed primers that identify six distinct sequences on the target DNA (Notomi et al., 2000). LAMP has been suggested by Ngari and co-workers (2020) as potential rapid diagnostics for T. trichiura, both in laboratories and field setups, because of its convenience, low cost, high sensitivity, and specificity. Rashwan and co-worker (2017) recently developed a colourimetric isothermal assay using asymmetrical SmartAmp2 primer sets and reagents in LAMP to identify N. americanus, T. trichiura, and A. lumbricoides in human faecal samples. The assay involves a species-specific primer-specific target sequence on the β-tubulin gene for the detection of different species (Manuel et al., 2021)

Recent Technical Developments

A quantitative paper-based DNA reader (qPDR) is recently developed to quantify STH at the molecular level by measuring distance as readout. Thus, external readers are not required. Based on the unique interfacial interaction of a DNA intercalating dye SYBR Green I with native cellulose on a chromatographic paper, the distance-based quantification of minute amounts of double-stranded DNA as short as 6 min can be performed with qPDR. qPDR can be integrated with PCR with the aid of a smartphone-controlled portable thermal cycler to quantify a minute amount of genetic markers from adult worms of an STH. It has been successfully used to demonstrate that T. trichiura was expelled post-treatment in infected children living in the rural areas of Honduras (Wang et al., 2018).

Cell-free DNA assays are developed to detect DNA released into the bloodstream or appearing in the urine as cells pass through the glomerular barrier or found in other body fluids like sputum, saliva, or stool (Manuel et al., 2021). This method can also be used to detect STH through the cell-free DNA from the disintegration or decay of the parasites during different stages of their life cycle (Weerakoon & McManus, 2016). A study in Iran demonstrated successful detection of Strongyloides infection in immunosuppressed patients using cell-free DNA from serum (Gorgani-Firouzjaee et al., 2018). Another study conducted in Argentina showed successful detection of S. stercoralis using urine samples, and it was found to have a significant advantage over faecal examination (Lodh et al., 2016). Thus, this technique is worth to be explored further for STH detection (Manuel et al., 2021).

Limitations of molecular-based techniques

There are many limitations associated with molecular-based techniques. Stool samples contain highly variable components dependent on nutrition, gut flora, lifestyle, and environment of the patient (Schrader et al., 2012) that may inhibit molecular reaction (Khurana & Sethi, 2017). Inhibitors may include polysaccharides or chlorophyll originating from herbs and vegetables, bile salts, urea, glycolipids, haemoglobin and heparin (Schrader et al., 2012). The major consequence of partial or total inhibition of PCR reactions is decreased sensitivity or false-negative results, respectively (Schrader et al., 2012). Therefore, it is crucial to eradicating inhibitors through an established extraction protocol, achievable with many commercial nucleic acid extraction kits developed with inhibitor removal steps (Papaiakovou et al., 2019; Khurana & Sethi, 2017).

Besides, the shells of STH eggs are much more rigid than the cell walls of bacteria, thus causing a lower yield of nucleic acid (Khurana & Sethi, 2017). Several mechanical procedures (extensive heating, vortexing, blending and sonication), chemical procedures (alkaline supplementation, addition of lyticase, achromopeptidase or a higher amount of proteinase K) and a few combinations of mechanical and chemical procedures were evaluated for enhanced release of egg DNA. Still, none of them was promising (Kaisar et al., 2017). The bead-beating method before DNA extraction has been confirmed to destroy the eggshells and led to increased DNA yield in human faecal samples (Ayana et al., 2019; Kaisar et al., 2017). Although various types of beads, such as glass, garnet, zirconium, ceramic, and steel, have been investigated, their impact on detection sensitivity and DNA concentration has yet to be determined (Ayana et al., 2019).

Another challenge is obtaining worm eggs to be used as controls (Azzopardi et al., 2021). Many eggs at varying development stages for each STH species and labour-intensive processes for the purification, picking and placing of eggs are required for assured quality of reference (Cools et al., 2021; Baylis et al., 2019). An accessible distributed network connecting active researchers to update relevant research and reference material for further STH diagnostic development is necessary for information sharing (Papaiakovou et al., 2021; Emery et al., 2012). Besides a biobank or a global bank of eggs, larvae, and DNA must be made available to serve as a reference for wide implement qPCR to diagnose these STH (Azzopardi et al., 2021).

The lack of standardisation challenges the general adoption of qPCR diagnostics (Stuyver & Levecke, 2021). The existing methods to preserve stool samples, DNA extraction and qPCR protocols show varying efficiencies (Cools et al., 2021). Since immediate DNA isolation from fresh stool samples is impossible in the field, rapid freezing of the samples should be conducted to prevent DNA degradation from the nucleases found within faeces (Papaiakovou et al., 2018). Besides, internal amplification controls to determine the robustness of the DNA extraction process and standards of different STH species are required to establish qPCR protocols (Easton et al., 2016). Complete annotation of STH genomes is an ongoing process to use plasmids as standards where plasmids are potentially more cost-effective and feasible than worm material (Cools et al., 2021). Standardised validation and assessment of infection intensity using qPCR are also required as DNA concentrations are commonly calculated based on individual standard curves. Different standard curves are used as quantitative measures of infection intensity (Le B et al., 2022; Papaiakovou et al., 2018).

Operational Cost and Infrastructure

The accuracy of diagnostic tools plays a pivotal role in an epidemiological study, monitoring treatment efficacies in mass drug administration (MDA) programs and the possibility of drug-resistance development (Sanprasert et al., 2019). However, the choices of a diagnostic technique are commonly based on cost and simplicity, with little emphasis on their sensitivity (Bergquist et al., 2009). Microscopy methods are preferred over molecular-based techniques due to their low cost. Although molecular diagnostics such as qPCR provides sufficient sensitivity, specificity, and throughput, their higher costs hinder their comprehensive implementation in most endemic regions where widespread poverty and resources are limited (Wang et al., 2018).

In addition, sophisticated equipment required for molecular-based diagnosis is not particularly suitable for field surveys or rapid identification for urgent treatment due to the frangibleness of the machines and the longer time needed for diagnosis (Ngwese et al., 2020). Moreover, tropical communities of low socioeconomic status lack the required technology, resources, and expertise to access the requisite facilities (Weerakoon & McManus, 2016). Although the significant drawback of molecular-based techniques such as qPCR is their high cost, lowering their price to a competitive level is theoretically possible. However, an upgrade in technology, infrastructure, financial support and expertise is required to implement molecular-based techniques in endemic countries (O’Connell & Nutman, 2016; Stuyver & Levecke, 2021).

Conclusion

In conclusion, molecular-based methods are more sensitive, rapid, and specific for STH detection than the microscopy based-method. Among various molecular diagnostics, qPCR excels with its capability to diagnose mixed infections simultaneously in a short time. Besides, the high reproducibility of qPCR results obviates the need for multiple sampling and reagents reduction to save cost. However, there are currently no standard guidelines and procedures for DNA extraction and sample processing for STH samples before qPCR diagnosis. Therefore, attention from WHO and researchers is required to develop new standard guidelines to ensure a reliable and efficient alternative diagnostic to the conventional method. Thus, standardisation followed by the implementation of qPCR in STH epidemiology and control programs for case reporting is highly advised. In addition, a standard procedure is also necessary to achieve uniformity in faecal samples preservation, DNA extraction, and qPCR settings to assure results reproducibility in the studies. Moreover, the quality of diagnosis is limited as stool samples cannot be stored. Thus, the samples must be processed in a minimum time to assure the accuracy of diagnosis.

Microscopy is still reliable with advancements offered, such as the FECPAK^G2. However, this assay is less sensitive than single and two-slide Kato-Katz for human samples. Therefore, it is required to increase the sensitivity and egg recovery for FECPAKG2 to be a reliable diagnostic tool for microscopic detection, particularly for field applications.