Introduction
Soil-transmitted helminths (STH) are intestinal parasites that affect the poorest and most vulnerable populations and are the most common infections worldwide with an estimated 1.5 billion infected people or 24% of the world’s population. The causative agents are Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, and hookworms. Hookworm infection is caused by the nematodes Necator americanus and Ancylostoma duodenale. N. americanus is the predominant species causing hookworm infections in humans. Infection occurs by penetration of larvae from the soil through skin in world areas with deficient sanitation systems. In children, serious infection affects their physical growth and cognitive development; it causes iron-deficiency anaemia because the worms feed on the host’s tissues, especially blood, leading to loss of iron and proteins. They also induce a reduction in labour performance of adults. In pregnant women, the anaemia may be responsible for bearing children with low birthweight (PAHO 2025; WHO 2023).
The diagnosis of hookworm infection is difficult, because many cases are asymptomatic and the signs and symptoms may be very mild or undetectable. Coproparasitological techniques are the most used, for example: direct examination with 0.85% physiological saline solution and Lugol’s iodine and the Kato and Kato-Katz (KK) techniques, the latter being the gold standard method. These techniques have several limitations; the sensitivity is low in minimal parasite loads, the eggs of N. americanus and A. duodenale are morphologically indistinguishable, the eggs can be overclarified with the KK and are very fragile and can break, making them undetectable and generating false negatives (Dacal et al. Reference Dacal, Köster and Carmena2020; Fleitas et al. Reference Fleitas, Vargas, Caro, Almazan, Echazú, Juárez, Cajal, Krolewiecki, Nasser and Cimino2021). Other diagnostic tests are immunological techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot, which allow the detection of the patient’s antibodies against the parasite, but the available tests are not very specific up to now, due to cross-reactions with other helminths (Dacal et al. Reference Dacal, Köster and Carmena2020; Yang et al. Reference Yang, Yang, Shi, Gao and Wang2016). Recently, multiplex peptide microarrays have been reported for screening parasite exposure in endemic areas. Although peptides with the least cross-reactivity are selected, there is a need to validate the sero-reactivity (Vengesai et al. Reference Vengesai, Naicker, Midzi, Kasambala, Mduluza-Jokonya, Rusakaniko, Mutapi and Mduluza2022).
An alternative for the diagnosis of N. americanus is the polymerase chain reaction (PCR), which detects the parasite’s DNA in stools. Several PCR protocols have been described for the amplification of the different DNA sequences of N. americanus, among them: Internal Transcribed Spacer 1 (ITS-1) (Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014), Internal Transcribed Spacer 2 (ITS-2) (Basuni et al. Reference Basuni, Muhi, Othman, Verweij, Ahmad, Miswan, Rahumatullah, Aziz, Zainudin and Noordin2011; Cunningham et al. Reference Cunningham, Stothard, Osei-Atweneboana, Armoo, Verweij and Adams2018; Llewellyn et al. Reference Llewellyn, Inpankaew, Nery, Gray, Verweij, Clements, Gomes, Traub and McCarthy2016; Romstad et al. Reference Romstad, Gasser, Monti, Polderman, Nansen, Pit and Chilton1997; Verweij et al. Reference Verweij, Pit, van Lieshout, Baeta, Dery, Gasser and Polderman2001, Reference Verweij, Brienen, Ziem, Yelifari, Polderman and van Lieshout2007), 18S ribosomal RNA encoding gene (Sanprasert et al. Reference Sanprasert, Kerdkaew, Srirungruang, Charuchaibovorn, Phadungsaksawasdi and Nuchprayoon2019), Internal Transcribed Spacer 2 plus 28S ribosomal RNA-encoding gene (ITS2-28S) (de Gruijter et al. Reference de Gruijter, van Lieshout, Gasser, Verweij, Brienen, Ziem, Yelifari and Polderman2005; Ngui et al. Reference Ngui, Lim and Chua2012; Romstad et al. Reference Romstad, Gasser, Monti, Polderman, Nansen, Pit and Chilton1997), subunit 1 mitochondrial cytochrome C oxidase (COI) (Zhan et al. Reference Zhan, Li, Xiao, Zheng and Hawdon2001). Recently, multiplex PCR formats that include N. americanus have been developed, both ‘in-house’ (Azzopardi et al. Reference Azzopardi, Hardy, Baker, Bonnici, Llewellyn, McCarthy, Traub and Steer2021; Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014) and in commercial kits (Autier et al. Reference Autier, Gangneux and Robert-Gangneux2021; Dacal et al. Reference Dacal, Köster and Carmena2020).
The specific primers and the reaction conditions of each PCR technique must be adapted to the laboratories where they will be used, since genetic variability could be found in parasites from different endemic areas and the PCR equipment and reagents are different in each case. Therefore, they may not work in areas other than those for which they were created (Marquet et al. Reference Marquet, Mora, Incani, Jesus, Méndez, Mujica, Trosel and Ferrer2023). In this work, different PCR amplification targets for molecular detection of N. americanus were compared in order to standardize the diagnostic protocols with the target that produces the best results and apply these techniques to detect N. americanus DNA in stool samples from individuals in endemic areas.
Materials and methods
Control samples
For the standardization and evaluation of the analytical sensitivity of the PCRs, a pool of DNAs extracted from five stool samples with positive coprology results (direct examination with 0.85% physiological saline solution and Lugol’s iodine, merthiolate-iodine-formalin (MIF), and the Kato-Katz technique) was used as the source of parasite DNA (positive control). These samples were from patients with hookworm eggs detected by coprology (those with the highest number of eggs per gram of stool), plus five negative control samples without any parasites. The samples were examined immediately upon being received at a clinical laboratory, in the morning. A portion of each sample was preserved in MIF for further evaluation by microscopy and another portion in ethanol for evaluation by PCR. To determine the analytical specificity of the PCRs, DNA samples from other parasites (n=41) were used, such as: Ascaris lumbricoides (5), Trichuris trichiura (3), Enterobius vermicularis (2), Strongyloides stercoralis (7), Schistosoma mansoni (3), Fasciola hepatica (2), Taenia solium (4), Taenia saginata (3) (obtained from stool samples of patients with that single parasite, as determined by coprological techniques), Toxocara canis, (5) (obtained from canine stool samples with that single parasite), Trypanosoma cruzi (3), Leishmania chagasi (2) (obtained from human blood samples with that single parasite), and Toxoplasma gondii (2) (obtained from cerebrospinal fluid samples of patients with that single parasite). Human DNA samples were also used (obtained from blood sample of five patients without parasites). Stool collection containers were given to patients (both healthy controls and those suspected of having different parasitic diseases), as well as to individuals in the community, and they were informed of the entire procedure so that they could take the sample to the laboratory or the community control post as soon as possible.
Experimental samples
One hundred and eighty stool samples from Piaroa indigenous people in the Tierra Blanca community, Rincón Escondido, Pijiguaos Parish, Cedeño Municipality, Bolívar State, Venezuela (Geographic coordinates 6°42′36″N, 66°06′46″W), an area endemic for hookworm infection according to coproparasitological diagnosis, were analyzed using coproparasitological techniques and standardized PCRs. All individuals in the population (180 individuals) were studied. As for control samples, each individual agreed to participate with prior authorization by means of an informed consent. Similarly, they agreed that the samples would be used, in addition to their diagnosis, for research purposes. Stool collection containers were given to individuals in the community, and they were informed of the entire procedure so that they could take the sample to the checkpoint (as soon as possible) of a health day held in the community. The samples were examined by coproparasitological microscopy techniques immediately upon being received at a health fair held in the community. Immediately, a portion of each sample was preserved in MIF for further evaluation by microscopy and another portion in ethanol for evaluation by PCR.
Ethical approval
The project was approved by the Committee of Bioethics of the Institute of Biomedical Research of the University of Carabobo (BIOMED-UC), following the guidelines for human and animal care from the Commission of Bioethics of the Ministry of Science and Technology and the ‘Operational Guidelines for Ethics Committees that Review Biomedical Research’ (TDR/PRD/ETHICS/2000.1). Each individual was informed about the project and the informed consent process. Those who agreed to participate signed the informed consent form beforehand and agreed that their samples could be used for research purposes, in addition to the diagnosis. The entire sample collection process was also explained to them. Informed consent was obtained from all adults; for children aged 1–6, consent was obtained from their parents or guardians; and for children over the age of 7, in addition to consent from their parents or guardians, their own assent was also obtained.
Coproparasitological techniques
The 180 experimental stool samples were analyzed by coproparasitological techniques of direct examination with 0.85% physiological saline solution and Lugol’s iodine, MIF, and the Kato-Katz technique (Botero and Restrepo Reference Botero and Restrepo2019).
Extraction of DNA
Four conventional DNA extraction methods were standardized: salt precipitation, phenol-chloroform, Chelex 100® Resin (Bio-Rad), and the commercial Wizard® Genomic DNA Purification Kit (Promega). The goal was to achieve a simple and reliable extraction procedure that would allow for the amplification of target sequences from N. americanus. Extraction using salt precipitation and phenol-chloroform techniques was performed as previously described (Sambrook and Russell Reference Sambrook and Russell2001). Extraction with Chelex 100® resin (Bio-Rad) and the Wizard® Quick Protocol kit was carried out according to the manufacturer’s instructions. All four techniques underwent some prior modifications for use with stool samples. In brief, 300 μL of ethanol-preserved faeces suspension was centrifuged for 1 min (5600 g) and the pellet washed with 1 mL Phosphate Buffered Saline (PBS). After centrifugation, the pellet was resuspended in 200 μL of freshly prepared lysis buffer (2% polyvinylpolypyrrolidone, 10% Sodium Dodecyl Sulfate (SDS), 0.5 M EDTA) and heated for 10 min at 100 °C. After proteinase K (Roche, Switzerland) treatment (2 h at 55 °C), DNA was extracted using the four techniques as usual (Sambrook and Russell Reference Sambrook and Russell2001 and manufacturer’s instructions).
After extraction using the saline precipitation and phenol-chloroform techniques, the DNA was dissolved in 25 μL of sterile water. For Chelex 100®, the DNA was in approximately 200 μL of supernatant, and for the Wizard Quick Protocol kit, it was suspended in 50 μL of sterile water. The extracted DNA samples were stored at –20 °C until use. All extraction methods were compared based on their yield, purity, and DNA integrity. The concentration and purity of the extracted DNAs were determined by the methods described by Sambrook and Russell (Reference Sambrook and Russell2001). The DNA concentration was measured using an Ultrospec 3000 UV/Visible spectrophotometer (Pharmacia Biotech, USA) by measuring the optical density at 280 and 260 nm, using the following formula: [DNA] = OD260 nm × D (dilution factor) × 50 μg/mL. The degree of purity was calculated using the OD260 nm/OD 280 nm ratio, and its integrity was assessed by electrophoresis. Moreover, the 180 community samples were extracted using the Wizard® Genomic DNA Purification Kit (Promega), and the quantity, purity, and integrity of the DNAs obtained from these extractions were all evaluated. Extraction controls in all cases were included, both positive (5) and negative stool samples (5).
DNA electrophoresis
DNA electrophoresis was performed according to the procedure described by Sambrook and Russell (Reference Sambrook and Russell2001). Electrophoresis was performed in 2% agarose gels, using TBE buffer 0.5× (Tris-Borate-EDTA) (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.0) and a MiniRun GE-100® horizontal electrophoresis system. A voltage of 100 V was used for 25 minutes. The gels were stained with Diamond™ Nucleic Acid Dye (Promega) (according to the manufacturer’s instructions). DNA bands were visualized under ultraviolet light using the Gel Doc 2000™ photodocumentation system (Bio-Rad, USA). The size of the DNA bands was estimated by comparing them to that of the 1 kb and 100 bp DNA markers (Promega).
Bioinformatic analysis
Bioinformatic analysis of the primer sequences used for amplification of the different targets (ITS1, ITS2, and ITS2-28S) was performed using the BLAST algorithm (Altschul et al. Reference Altschul, Gish, Miller, Myers and Lipman1990), comparing the primer sequences with sequences of these targets deposited in GenBank (National Center for Biotechnology Information (NCBI) 2025). The primer sequences analyzed were: NaITS1-F and NaITS1-R for ITS1 (Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014), Na58F and Na158R for ITS2 (Verweij et al. Reference Verweij, Brienen, Ziem, Yelifari, Polderman and van Lieshout2007), NA and NC2 for ITS2 (Romstad et al. Reference Romstad, Gasser, Monti, Polderman, Nansen, Pit and Chilton1997), and NC1 and NC2 for ITS2-28S (Romstad et al. Reference Romstad, Gasser, Monti, Polderman, Nansen, Pit and Chilton1997) (Table 1).
Characteristics of the primers for the different PCR amplification targets for Necator americanus
Table 1. Long description
The table consists of four columns and four data rows.Row 1: P C R target is I T S 1. Primers are Na I T S 1-F (Forward) 5 prime A T G C T T G G C A A G A G T C G T T T 3 prime and Na I T S 1-R (Reverse) 5 prime T G T T G G C G T C C A C A C A T A T T 3 prime. Product size is 330 b p. Reference is Phuphisut et al. 2014.Row 2: P C R target is I T S 2. Primers are Na 5 8 F (Forward) 5 prime C T G T T T G T C G A A C G G T A C T T G C 3 prime and Na 1 5 8 R (Reverse) 5 prime A T A A C A G C G T G C A C A T G T T G C 3 prime. Product size is 101 b p. Reference is Verweij et al. 2007.Row 3: P C R target is I T S 2 to 2 8 S. Primers are N A (Forward) 5 prime A T G T G C A C G T T A T T C A C T 3 prime and N C 2 (Reverse) 5 prime T T A G T T T C T T T T C C T C C G C T 3 prime. Product size is 250 b p. Reference is Romstad et al. 1997.Row 4: P C R target is I T S 2 to 2 8 S. Primers are N C 1 F (Forward) 5 prime A C G T C T G G T T C A G G G T T G T T 3 prime and N C 2 R (Reverse) 5 prime T T A G T T T C T T T T C C T C C G C T 3 prime. Product size is Na 420 b p and Ad 310 b p. Reference is Romstad et al. 1997.Footnote: b p equals base pairs.
Row 1:
- P C R target: I T S 1.
- Primers: Forward Na I T S 1-F 5 prime 5 prime A T G C T T G G C A A G A G T C G T T T 3 prime and Reverse Na I T S 1-R 5 prime T G T T G G C G T C C A C A C A T A T T 3 prime.
- Product size: 330 b p.
- Reference: Phuphisut et al. 2014.
Row 2:
- P C R target: I T S 2.
- Primers: Forward Na 5 8 F 5 prime C T G T T T G T C G A A C G G T A C T T G C 3 prime and Reverse Na 1 5 8 R 5 prime A T A A C A G C G T G C A C A T G T T G C 3 prime.
- Product size: 101 b p.
- Reference: Verweij et al. 2007.
Row 3:
- P C R target: I T S 2 to 2 8 S.
- Primers: Forward N A 5 prime A T G T G C A C G T T A T T C A C T 3 prime and Reverse N C 2 5 prime T T A G T T T C T T T T C C T C C G C T 3 prime.
- Product size: 250 b p.
- Reference: Romstad et al. 1997.
Row 4:
- P C R target: I T S 2 to 2 8 S.
- Primers: Forward N C 1 F 5 prime A C G T C T G G T T C A G G G T T G T T 3 prime and Reverse N C 2 R 5 prime T T A G T T T C T T T T C C T C C G C T 3 prime.
- Product size: Na 420 b p and Ad 310 b p.
- Reference: Romstad et al. 1997.
A footer note specifies that b p equals base pairs.
bp = base pairs.
Standardization of the PCR protocols
The four amplification reactions were carried out according to the protocols: (1) described by Phuphisut et al. (Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014) and modified by Frango-Ramos et al. (Reference Frango-Ramos, Delgado, Catalano-Donaire, Jesús-Arzapalo, Incani-Cotognini, Perteguer-Prieto and Ferrer-Jesús2021) for ITS1 (Primers NaITS1-F and NaITS1-R). (2) In the case of ITS2 (Primers Na58F and Na158R), the protocol described by Verweij et al. (Reference Verweij, Brienen, Ziem, Yelifari, Polderman and van Lieshout2007) was used but adapting qPCR to conventional PCR. (3) For the amplification of the ITS2 (Primers NA and NC2), the protocol described by Romstad et al. (Reference Romstad, Gasser, Monti, Polderman, Nansen, Pit and Chilton1997) was carried out. (4) In the case of ITS2-28S (Primers NC1 and NC2), the protocol described by Romstad et al. (Reference Romstad, Gasser, Monti, Polderman, Nansen, Pit and Chilton1997) was used (Table 1). In the four PCRs tested, various concentrations of reagents (magnesium chloride, Bovine Serum Albumin (BSA), Deoxynucleotide triphosphates (dNTPs), primers, and DNA polymerase) and reaction conditions (temperature and number of cycles) were tested in order to obtain the best results (Table 2). PCR products were visualized on 2% agarose gels and compared with molecular size markers. With PCR-NA/NC2 (ITS2-28S) and PCR-NC1/NC2 (ITS2-28S), no amplification was obtained at any DNA concentration tested or when the concentrations of reagents or reaction conditions were varied. Therefore, only the PCR-NaITS1F/NaITS1R (ITS1) and PCR-Na58/Na158R (ITS2) protocols were standardized and the optimal conditions were used for the PCR assays. In brief, the optimal conditions determined in the standardization processes were used for the application of PCR to all samples; these were for the PCR-NaITS1F/NaITS1R (ITS1): PCR buffer (Promega), 1.5 mM MgCl2, 0.125 mg/mL BSA, 0.1 mM dNTPs, 0.6 μM for each primer, DNA polymerase 0.75 U, and 10 ng of DNA were included. Amplification program consisted of 5 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C, and 1 min at 72 °C, with final elongation for 5 min at 72 °C. For the PCR-Na58/Na158R (ITS2), the conditions were: PCR buffer (Promega), 2.5 mM MgCl2, 0.125 mg/mL BSA, 0.2 mM dNTPs, 0.2 μM for each primer (Table 1), DNA polymerase 1.0 U, and 10 ng of DNA. Amplification program consisted of 5 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C, with final elongation for 5 min at 72 °C. Negative and positive control samples were included in each amplification run (Table 1).
Conditions used in the PCR techniques for the amplification of Necator americanus DNA in stool samples
Table 2. Long description
The table consists of four columns: D N A forward slash reagents, Evaluated conditions, Na I T S 1 optimal conditions, and Na 5 8 F forward slash Na 1 5 8 R optimal conditions.
* D N A: Evaluated from 100 ng to 1 fg. Na I T S 1 optimal is 100 ng to 10 pg; Na 5 8 F forward slash Na 1 5 8 R optimal is 100 ng to 1 ng.
* M g C l sub 2: Evaluated 1 to 2.5 m M. Na I T S 1 optimal is 1.5 m M; Na 5 8 F forward slash Na 1 5 8 R optimal is 2.5 m M.
* B S A: Evaluated 0.125 to 0.500 mg per m L. Both techniques optimal at 0.125 mg per m L.
* d N T P s: Evaluated 50 to 200 micro M. Na I T S 1 optimal is 100 micro M; Na 5 8 F forward slash Na 1 5 8 R optimal is 200 micro M.
* Primers: Evaluated 0.2 to 0.8 micro M. Na I T S 1 optimal is 0.6 micro M; Na 5 8 F forward slash Na 1 5 8 R optimal is 0.2 micro M.
* Taq polymerase: Evaluated 0.5 to 1.25 U. Na I T S 1 optimal is 0.75 U; Na 5 8 F forward slash Na 1 5 8 R optimal is 1.0 U.
* Annealing temperature: Evaluated 57, 60, and 63 degrees Celsius. Na I T S 1 optimal is 57 degrees Celsius; Na 5 8 F forward slash Na 1 5 8 R optimal is 60 degrees Celsius.
* Number of cycles: Evaluated 30, 35, 40. Both techniques optimal at 35 cycles.
* Analytical sensitivity: Evaluated 100 ng to 1 ag. Na I T S 1 optimal is 10 pg; Na 5 8 F forward slash Na 1 5 8 R optimal is 1 ng.
* Analytical Specificity: Evaluated against various helminth and human D N A. Both techniques achieved 100 percent specificity.
Determination of analytical sensitivity and specificity of PCR
Sensitivity was determined by making titration curves with different amounts of DNA (100 ng to 1 fg) to observe what was the minimum amount of DNA that produced amplification. The specificity was determined using DNA from different parasites (A. lumbricoides, T. trichiura, E. vermicularis, S. stercoralis, S. mansoni, F. hepatica, T. solium, T. saginata, T. canis, T. cruzi, L. chagasi and T. gondii) and human DNA from healthy individuals, in which amplification should not occur.
Comparison of the amplification targets of the N. americanus PCRs
The results of the different conditions tested for the four amplification targets were compared based on: the presence of the expected product, which was clear; the absence of secondary bands, with differences between positives and negatives; and reproducibility of the technique. Subsequently, the PCR techniques standardized with the targets providing the best results were applied to the experimental samples. Each amplification included a negative control (DNA-free) and a positive control. The PCR products were separated on 2% agarose gels, and the sizes of the amplified bands were compared with the molecular size marker.
To compare the results of standardized PCRs with those obtained by coproparasitological techniques, 2×2 tables were made and the diagnostic indices were determined: sensitivity, specificity, and positive and negative predictive values. To compare PCR techniques with coprology, the chi-square test (p-value) and the Kappa index (to assess agreement between tests) were used. p-values ≤0.05 were considered significant, and the Kappa value was estimated as follows: ≤0.20 poor agreement, 0.21 to 0.40 weak agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 good agreement, and 0.81 to 1.0 very good agreement.
PCR results were compared with a composite standard, consisting of the results of PCRs and coproparasitological techniques. In this case, an individual was considered positive if the presence of an egg was observed by any of the coproparasitological techniques and/or specific diagnostic band in any PCR, whereas to be negative, an individual needed to have all these tests performed and to have tested negative on all of them. Since coproparasitological techniques are imperfect standards due to false negative results and lower sensitivity, this approach was used to estimate diagnostic parameters in the absence of a gold standard. Statistical analysis was performed using STATA 15.1 (StataCorp, College Station, Texas, USA).
Characterization by gender and age of individuals from Tierra Blanca community
The characterization of the study population was carried out according to gender and age, and the results were obtained using PCR-NaITS1F/NaITS1R (ITS1).
Results
Coproparasitological analysis
The coproparasitological test revealed 37/180 (20.6%) positive samples for hookworm eggs, ranging from 20 to 680 eggs/g feces (epg) (Table 3). The five samples with the highest number of eggs per gram of stool (ranging from 560 to 680 epg) were selected for DNA extraction technique standardization and PCR standardization.
Comparison of the positive results of coprological techniques with those of standardized PCR techniques
Table 3. Long description
The table consists of four columns: Coproparasitological techniques, Coprology, P C R-Na I T S 1 Positives, and Na 58 F forward slash Na 158 R-P C R positives. Data is categorized by e p g (eggs per gram) levels.
* Negative (0 e p g): Coprology 143/180 (79.4 percent); P C R-Na I T S 1 133/180 (73.9 percent); Na 58 F forward slash Na 158 R-P C R 0/180 (0.0 percent).
* Positive 1–100 e p g: Coprology 9/180 (5 percent); P C R-Na I T S 1 4/180 (2.2 percent); Na 58 F forward slash Na 158 R-P C R 0/180 (0.0 percent).
* Positive 101–200 e p g: Coprology 8/180 (4.4 percent); P C R-Na I T S 1 5/180 (2.8 percent); Na 58 F forward slash Na 158 R-P C R 0/180 (0.0 percent).
* Positive 201–300 e p g: Coprology 6/180 (3.3 percent); P C R-Na I T S 1 4/180 (2.2 percent); Na 58 F forward slash Na 158 R-P C R 0/180 (0.0 percent).
* Positive 301–400 e p g: Coprology 5/180 (2.8 percent); P C R-Na I T S 1 4/180 (2.2 percent); Na 58 F forward slash Na 158 R-P C R 0/180 (0.0 percent).
* Positive 401–500 e p g: Coprology 4/180 (2.2 percent); P C R-Na I T S 1 4/180 (2.2 percent); Na 58 F forward slash Na 158 R-P C R 0/180 (0.0 percent).
* Positive 501–600 e p g: Coprology 3/180 (1.7 percent); P C R-Na I T S 1 3/180 (1.7 percent); Na 58 F forward slash Na 158 R-P C R 0/180 (0.0 percent).
* Positive greater than or equal to 601 e p g: Coprology 2/180 (1.1 percent); P C R-Na I T S 1 2/180 (1.1 percent); Na 58 F forward slash Na 158 R-P C R 2/180 (1.1 percent).
* Total Positives: Coprology 37/180 (20.6 percent); P C R-Na I T S 1 159/180 (88.3 percent); Na 58 F forward slash Na 158 R-P C R 2/180 (1.1 percent).
Epg (eggs per gram).
DNA extraction
The four DNA extraction techniques: Salt precipitation, phenol-chloroform, Chelex 100® Resin (Bio-Rad), and the commercial Wizard® Genomic DNA Purification Kit (Promega) were applied to the five stool samples selected. Several assays were performed for each technique, modifying certain conditions (precipitation and centrifugation times), and the quantity, purity, and integrity of the DNA obtained from these extractions were evaluated (Table 4). It was observed that with the Wizard® Genomic DNA Purification Kit (Promega) technique, a higher concentration of DNA, a better degree of purity, and better DNA integrity were obtained since very little degradation and large DNA were observed, while with the salt precipitation and phenol-chloroform techniques, DNA was obtained in small quantities and with some degradation. On the other hand, with the Chelex 100® resin technique, the yield was the lowest and the DNA was the most degraded. With the DNA extracted (using the Wizard® Genomic DNA Purification Kit, Promega) from the five selected faecal samples (Figure 1), a pool was made to be used as a positive control in the standardization of the PCR protocols.
Comparison of the four DNA extraction techniques: phenol-chloroform, salt precipitation, Chelex 100® Resin (Bio-Rad), and the commercial Wizard® Genomic DNA Purification Kit (Promega) using positive control samples
Table 4. Long description
The table consists of nine columns. The first column lists samples 001 through 005. The remaining eight columns are paired under four extraction methods, each providing D N A in micrograms and the R ratio (O D 260 nm over O D 280 nm).
* Phenol-chloroform: D N A yields range from 243 to 272 micrograms with R ratios between 1.49 and 1.62.
* Salt precipitation: D N A yields range from 147 to 173 micrograms with R ratios between 1.37 and 1.57.
* Chelex 100: D N A yields are the lowest, ranging from 46 to 68 micrograms with R ratios between 1.11 and 1.41.
* Kit (Promega): D N A yields are the highest, ranging from 296 to 333 micrograms with R ratios between 1.79 and 1.86.
Specific data for Sample 001: Phenol-chloroform (272, 1.57), Salt precipitation (172, 1.39), Chelex 100 (46, 1.11), Kit (333, 1.81).
Specific data for Sample 005: Phenol-chloroform (243, 1.49), Salt precipitation (153, 1.39), Chelex 100 (68, 1.24), Kit (318, 1.83).
DNA μg = total DNA extracted from stool samples.
R = OD260 nm/OD 280 nm ratio.
DNA extraction of positive control stool samples using the commercial Wizard® Genomic DNA Purification Kit (Promega) in 2% agarose gel electrophoresis. Stained with Gel Diamond™ Nucleic Acid Dye (Promega); (M) Marker 100 bp Promega®, (1–5) DNA control samples.
Bioinformatics analysis of the primers
The primers of the four PCR protocols studied allow the amplification of the ITS1, ITS2, and ITS2-28S sequences of N. americanus ribosomal DNA. Analysis using the GenBank BLAST algorithm (NCBI 2025) showed 100% identity of the primers with respect to the target sequences deposited in GenBank. However, it is important to note that the sequences deposited in GenBank (52 for ITS1, 100 for ITS2, and 53 for 28S) are mostly of N. americanus from countries in Asia and Africa, with only two from the Americas (one from Brazil and one from Guatemala). On the other hand, comparing the ITS1 and ITS2 regions of the sequences from different countries revealed similarities between 78.21% and 100% for ITS1, allowing up to 21.79% variability, while the ITS2 regions showed similarities between 82.75% and 100%, allowing up to 17.25% variability. Given the 100% sequence identity of all primers with respect to the target sequences (found in GenBank), the four selected PCR protocols were evaluated.
Standardization of PCR protocols
PCR-NaITS1F/NaITS1R (ITS1) was standardized in a previous study (Frango-Ramos et al. Reference Frango-Ramos, Delgado, Catalano-Donaire, Jesús-Arzapalo, Incani-Cotognini, Perteguer-Prieto and Ferrer-Jesús2021). But since several years have passed, several of the reagents tested are from different brands and it was decided to change the final reaction volume from 25 μL to 10 μL (we are adapting all PCRs to 10 μL final volume), a new standardization of this protocol was carried out. For the standardization of the four PCR protocols, different amounts of DNA were evaluated. The optimal amount of DNA ranged between 100 nanograms (ng, 10-9 g) and 10 picograms (pg, 10-12 g) for PCR-NaITS1F/NaITS1R (ITS1) and between 100 ng and 1 ng for PCR-Na58F/Na158R (ITS2). Consequently, 10 ng was taken as the amount of DNA to make subsequent determinations. With PCR-NA/NC2 (ITS2-28S) and PCR-NC1/NC2 (ITS2-28S), no amplification was obtained at any DNA concentration tested or when the concentrations of reagents or reaction conditions were varied. Therefore, only the PCR-NaITS1F/NaITS1R (ITS1) and PCR-Na58F/Na158R (ITS2) protocols were standardized by generating titration curves for each reagent and testing different hybridization temperatures and number of cycles. The reagent concentrations tested and optimal conditions for each PCR protocol are described in Table 2 and the amplification bands can be seen in Figure 2.
Detection of N. americanus DNA in stool control samples using the four PCR evaluated at optimal conditions, in 2% agarose gel electrophoresis of PCR products stained by gel Diamond™ Nucleic Acid Dye (Promega) (M) Marker 100 bp Promega®, (1–4) PCR-NaITS1F/NaITS1R (ITS1), (5–8) PCR-NC1/NC2 (ITS2-28S), (9–12) PCR-NA/NC2 (ITS2-28S), (13–16) PCR-Na58F/Na158R (ITS2). C+1 (positive control 1), C+2 (positive control 2), C-1 (negative control 1), C-2 (negative control 2).
Figure 2. Long description
The photo displays an agarose gel with seventeen lanes labeled at the top.
* On the far left, lane M contains a 100 b p DNA ladder with markers ranging from 100 to 500 b p.
* Lanes 1 through 4 are grouped under P C R Na I T S 1. Lanes 1 and 2 (C plus 1 and C plus 2) show bright bands at 330 b p, while lanes 3 and 4 (C minus 1 and C minus 2) show no bands.
* Lanes 5 through 8 are grouped under P C R N C 1 forward slash N C 2. No distinct bands are visible in these lanes.
* Lanes 9 through 12 are grouped under P C R N A forward slash N C 2. No distinct bands are visible in these lanes.
* Lanes 13 through 16 are grouped under P C R Na 58 forward slash Na 158. Lanes 13 and 14 (C plus 1 and C plus 2) show bright bands at 101 b p, while lanes 15 and 16 (C minus 1 and C minus 2) show no bands.
Faint primer dimer clouds are visible at the bottom of most lanes.
Analytical sensitivity and specificity of the PCRs standardized
Once the PCRs were standardized, the analytical sensitivity and specificity were determined. In the 2% agarose gel, it was possible to observe that the DNA was capable of producing amplification in a range of concentrations from 100 ng to 10 pg for PCR-NaITS1F/NaITS1R (ITS1), with the analytical sensitivity of the technique being 10 pg (Table 2). For the PCR-Na58F/Na158R (ITS2), amplification bands were obtained with 100 ng to 1 ng. The analytical sensitivity was 1 ng. To determine the specificity of the two PCR protocols, DNA from different parasites was used under the same optimal reaction conditions of each PCR and it was shown that there was only amplification of the N. americanus DNA sample, indicating a specificity of 100% for both PCR protocols (Table 2).
Application of standardized PCRs to experimental samples
Once the standardization process of the selected PCR protocols was completed, DNA extraction was carried out using the previously standardized method on the 180 stool samples from individuals of the Tierra Blanca community for molecular diagnosis. Of the 180 samples analyzed by PCR-NaITS1F/NaITS1R (ITS1), 159 (88.3%) were positive and 21 (11.7%) were negative (Figure 3). Using PCR-Na58F/Na158R (ITS2), only 2 (1.1%) samples were positive and 178 (98.9%) were negative. This is a completely unexpected result, which, after numerous repetitions and evaluations, all conducted in accordance with good laboratory practices, was confirmed and can be attributed to several causes, which are detailed in the discussion.
Detection of N. americanus DNA in stool samples of individuals from Tierra Blanca community using PCR-NaITS1F/NaITS1R (ITS1), by Diamond™ Nucleic Acid (Promega) stained 2% agarose gel electrophoresis of PCR products. (M) Marker 100 bp Promega®. (1) Positive control, (2–15) samples from each individual, (16) negative control.
Figure 3. Long description
A photo of an electrophoresis gel with a dark background and bright horizontal bands.
* On the far left, a vertical ladder labeled M for marker shows distinct bands with b p (base pair) labels on the Y axis at 100, 200, 300, 400, and 500.
* Lane 1 contains a bright band aligned between the 300 and 400 b p markers, serving as the positive control.
* Lanes 2 through 8 show similar bright bands at the same horizontal level, indicating positive D N A detection.
* Lane 9 is empty, showing no band.
* Lanes 10 through 15 show bright bands consistent with the positive control level.
* Lane 16 is empty, serving as the negative control.
* The bands across the positive lanes are uniform in size, appearing slightly below the 400 b p mark.
Diagnostic evaluation of the standardized PCR protocols
To compare the results of standardized PCRs with those obtained by coproparasitological techniques, the diagnostic indexes were determined for all tests employed using a composite standard, consisting of the combined result of PCRs and coproparasitological tests, since coproparasitological techniques are imperfect standards due to false negative results and lower sensitivity. The best diagnostic indexes was obtained by PCR-NaITS1F/NaITS1R (ITS1) with sensitivity of 93.5%, followed by coproparasitological techniques with a sensitivity of 21.8%, while the sensitivity of PCR-Na58F/Na158R (ITS2) was only 1.2%. The specificity of all tests performed was 100% (Table 5). The prevalence of N. americanus infection in the Tierra Blanca community was 88.3% determined by PCR-NaITS1F/NaITS1R (ITS1), 20.6% determined by coproparasitological tests, and 1.1% determined by PCR-Na58F/Na158R (ITS2), while using the composite standard, the prevalence was 94.4% (Table 5). The agreement between the techniques with respect to the composite standard was good for PCR-NaITS1F/NaITS1R (ITS1) (Kappa index 0.62) and poor for the coproparasitological tests (Kappa index 0.03) and PCR-Na58F/Na158R (ITS2) (Kappa index 0.0) (Table 5). These results are unexpected, particularly the sensitivity of the PCR-Na58F/Na158R (ITS2) test; therefore, they were confirmed several times and may be due to various factors described in the discussion.
Diagnostic indexes of coproparasitological techniques and PCRs
Table 5. Long description
The table consists of four columns: Indices, Coproparasitological techniques, P C R-Na I T S 1, and Na 58 F / Na 158 R-P C R.* Sensitivity (95% C I): Coproparasitological techniques at 21.8% (16.2â28.6); P C R-Na I T S 1 at 93.5% (88.8â96.3); Na 58 F / Na 158 R-P C R at 1.2% (0.3â4.2).* Specificity (95% C I): All three methods show 100% (72.2â100).* Positive predictive value (95% C I): All three methods show 100%, with varying confidence intervals: (90.6â100), (97.6â100), and (34.2â100) respectively.* Negative predictive value (95% C I): Coproparasitological techniques at 7% (3.8â12.4); P C R-Na I T S 1 at 47.6% (28.3â67.6); Na 58 F / Na 158 R-P C R at 5.6% (3.1â10.0).* Prevalence: Coproparasitological techniques at 37/180 (20.6%); P C R-Na I T S 1 at 159/180 (88.3%); Na 58 F / Na 158 R-P C R at 2/180 (1.1%).* p-value: Coproparasitological techniques at 0.0978; P C R-Na I T S 1 at 0.7301; Na 58 F / Na 158 R-P C R at less than 0.0001.* Kappa index: Coproparasitological techniques at 0.03 (-0.05â0.11); P C R-Na I T S 1 at 0.62 (0.40â0.84); Na 58 F / Na 158 R-P C R at 0.0 (-0.04â0.04).* Composite prevalence: 170/180 (94.44).
* Sensitivity (95 percent C I): Coproparasitological techniques at 21.8 percent (16.2–28.6); P C R-Na I T S 1 at 93.5 percent (88.8–96.3); Na 58 F / Na 158 R-P C R at 1.2 percent (0.3–4.2).
* Specificity (95 percent C I): All three methods show 100 percent (72.2–100).
* Positive predictive value (95 percent C I): Coproparasitological techniques at 100 percent (90.6–100); P C R-Na I T S 1 at 100 percent (97.6–100); Na 58 F / Na 158 R-P C R at 100 percent (34.2–100).
* Negative predictive value (95 percent C I): Coproparasitological techniques at 7.0 (3.8–12.4); P C R-Na I T S 1 at 47.6 percent (28.3–67.6); Na 58 F / Na 158 R-P C R at 5.6 percent (3.1–10.0).
* Prevalence: Coproparasitological techniques at 37 over 180 (20.6 percent); P C R-Na I T S 1 at 159 over 180 (88.3 percent); Na 58 F / Na 158 R-P C R at 2 over 180 (1.1 percent).
* p-value: Coproparasitological techniques at 0.0978; P C R-Na I T S 1 at 0.7301; Na 58 F / Na 158 R-P C R at less than 0.0001.
* Kappa index: Coproparasitological techniques at 0.03 (-0.05–0.11); P C R-Na I T S 1 at 0.62 (0.40–0.84); Na 58 F / Na 158 R-P C R at 0.0 (-0.04–0.04).
* Composite prevalence: 170 over 180 (94.44).
Results of PCR NaITS1F/NaITS1R (ITS1) for detection of Necator americanus by gender and age in individuals from Tierra Blanca community
One hundred and eighty individuals, of whom 56.7% (102/180) and 43.3% (78/180) were female and male, respectively, with an age range of 1–75 years, were included in this study. The most frequent age group encountered was 1–15 with 38.9% (70/180) (Table 6). Samples from 88.3% (159/180) of individuals were positive for PCR NaITS1F/NaITS1R (ITS1). Although there were more positive results in women and in the 1–15 age group, there were no statistically significant differences. Significant differences were only found in the 46–60 age group (Table 6).
Results of the PCR NaITS1F/NaITS1R (ITS1) for detection of Necator americanus by gender and age in individuals from Tierra Blanca community, Bolívar State, Venezuela
Table 6. Long description
The table consists of six columns: Category, Total n (%), P C R N a I T S 1 F / N a I T S 1 R (I T S 1) Positive n (%), P C R Negative n (%), O R (95% C I), and p-value.* Total: 180 individuals (100%), with 159 (88.3%) positive and 21 (11.7%) negative.* Gender Category:- Female (Reference): 102 total (56.7%), 90 positive (56.6%), 12 negative (57.1%), O R 1.0.- Male: 78 total (43.3%), 69 positive (43.4%), 9 negative (42.9%), O R 1.0 (0.4â2.5), p 0.963.* Age Category (years):- 1â15 (Reference): 70 total (38.9%), 63 positive (39.6%), 7 negative (33.3%), O R 1.0.- 16â30: 40 total (22.2%), 36 positive (22.6%), 4 negative (19.0%), O R 1.0 (0.3â3.7), p 1.000.- 31â45: 33 total (18.3%), 30 positive (18.9%), 3 negative (14.3%), O R 1.1 (0.2â3.7), p 0.884.- 46â60: 22 total (12.2%), 16 positive (10.1%), 6 negative (28.6%), O R 0.3 (0.1â1.0), p 0.042.- Greater than or equal to 61: 15 total (8.3%), 14 positive (8.8%), 1 negative (4.8%), O R 1.6 (0.2â13.7), p 0.688.
* Total (n): 180 (100%) total samples, with 159 (88.0%) positive and 21 (12.0%) negative.
* Gender Category:
- Female (Reference): 102 (56.7%) total, 90 (56.6%) positive, 12 (57.1%) negative, O R 1.0.
- Male: 78 (43.3%) total, 69 (43.4%) positive, 9 (42.9%) negative, O R 1.0 (0.4–2.5), p 0.963.
* Age (years) Category:
- 1–15 (Reference): 70 (38.9%) total, 63 (39.6%) positive, 7 (33.3%) negative, O R 1.0.
- 16–30: 40 (22.2%) total, 36 (22.6%) positive, 4 (19.0%) negative, O R 1.0 (0.3–3.7), p 1.000.
- 31–45: 33 (18.3%) total, 30 (18.9%) positive, 3 (14.3%) negative, O R 1.1 (0.2–3.7), p 0.884.
- 46–60: 22 (12.2%) total, 16 (10.1%) positive, 6 (28.6%) negative, O R 0.3 (0.1–1.0), p 0.042.
- Greater than or equal to 61: 15 (8.3%) total, 14 (8.8%) positive, 1 (4.8%) negative, O R 1.6 (0.2–13.7), p 0.688.
* Reference category.
Discussion
Human hookworm infection is among the most significant neglected tropical diseases in terms of morbidity, with approximately 500 million people infected in the tropics. Globally, hookworm infections tend to be more prevalent in rural areas, where favourable environmental conditions, such as temperature, soil moisture, and soil type, coincide with poverty and inadequate sanitation infrastructure (Servián et al. Reference Servián, Repetto, Zonta and Navone2022). Patients infected with N. americanus may have low parasite loads, so the low sensitivity of parasitological and the low specificity of immunological methods, due to cross-reactions with other helminths, do not allow an adequate diagnosis of the disease (Dacal et al. Reference Dacal, Köster and Carmena2020; Fleitas et al. Reference Fleitas, Vargas, Caro, Almazan, Echazú, Juárez, Cajal, Krolewiecki, Nasser and Cimino2021), as it happened in this study where coproparasitological techniques only allowed the detection of 20.6% of infected individuals from the Tierra Blanca community, a result much lower than that obtained by PCR-NaITS1F/NaITS1R (ITS1).
Some researchers have used molecular biology methods for the diagnosis of hookworm, for its high sensitivity and specificity (Basuni et al. Reference Basuni, Muhi, Othman, Verweij, Ahmad, Miswan, Rahumatullah, Aziz, Zainudin and Noordin2011; de Gruijter et al. Reference de Gruijter, van Lieshout, Gasser, Verweij, Brienen, Ziem, Yelifari and Polderman2005; Llewellyn et al. Reference Llewellyn, Inpankaew, Nery, Gray, Verweij, Clements, Gomes, Traub and McCarthy2016; Ngui et al. Reference Ngui, Lim and Chua2012; Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014; Romstad et al. Reference Romstad, Gasser, Monti, Polderman, Nansen, Pit and Chilton1997; Sanprasert et al. Reference Sanprasert, Kerdkaew, Srirungruang, Charuchaibovorn, Phadungsaksawasdi and Nuchprayoon2019; Verweij et al. Reference Verweij, Pit, van Lieshout, Baeta, Dery, Gasser and Polderman2001, Reference Verweij, Brienen, Ziem, Yelifari, Polderman and van Lieshout2007). But all PCR protocols require prior standardization, adaptation, and evaluation of the reaction conditions for their use and must be validated in each laboratory (Ferrer, Reference Ferrer2015). The first step for a successful PCR protocol is proper DNA extraction. Therefore, in this study, four DNA extraction protocols were standardized and compared using faecal samples. The best protocol was the commercial Wizard® Genomic DNA Purification Kit (Promega), which provided the highest yield, purity, and integrity of the extracted DNA. Using commercial DNA extraction kits avoids the use of hazardous organic solvents and is faster to perform and yields high-quality DNA, as demonstrated in this study and by other authors (Ayana et al. Reference Ayana, Cools, Mekonnen, Biruksew, Dana, Rashwan, Prichard, Vlaminck, Verweij and Levecke2019; Devyatov et al. Reference Devyatov, Davydova, Luparev, Karseka, Shuryaeva, Zagainova and Shipulin2023).
In Venezuela, there is no published work on the use of PCR for the diagnosis of N. americanus, only a work by our group (Frango-Ramos et al. Reference Frango-Ramos, Delgado, Catalano-Donaire, Jesús-Arzapalo, Incani-Cotognini, Perteguer-Prieto and Ferrer-Jesús2021), so it was unknown which amplification target could give the best results for the molecular detection of circulating parasites in the setting selected for the present study. Consequently, the main objective of this work was to compare four PCR protocols with different targets for the molecular detection of N. americanus and to standardize the PCR technique that would provide the best diagnostic results.
The four PCRs evaluated were chosen because they have been the most widely used worldwide for the molecular detection of N. americanus. Also, the bioinformatics analysis of all the primers of the four PCRs studied showed in all cases 100% identity of the primers with respect to the target sequences deposited in GenBank. However, when attempting to standardize the four PCRs, amplification was only obtained in two of them: the PCR-NaITS1F/NaITS1R (ITS1) and PCR-Na58F/Na158R (ITS2) protocols. Optimal reagent quantities were established, generally lower than those used by the original authors and others who have used these protocols (Basuni et al. Reference Basuni, Muhi, Othman, Verweij, Ahmad, Miswan, Rahumatullah, Aziz, Zainudin and Noordin2011; Cunningham et al. Reference Cunningham, Stothard, Osei-Atweneboana, Armoo, Verweij and Adams2018; Llewellyn et al. Reference Llewellyn, Inpankaew, Nery, Gray, Verweij, Clements, Gomes, Traub and McCarthy2016; Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014; Verweij et al. Reference Verweij, Brienen, Ziem, Yelifari, Polderman and van Lieshout2007), improving the quality and reducing the costs of the reactions.
With PCR-NA/NC2 (ITS2-28S) and PCR-NC1/NC2 (ITS2-28S), no amplification was obtained at any DNA concentration tested nor when the concentrations of reagents or reaction conditions were varied, although apparently the primers were all suitable for their target sequence. However, it is important to note that the sequences deposited in GenBank (52 for ITS1, 100 for ITS2, and 53 for 28S) are mostly of N. americanus from countries in Asia (mostly) and Africa, with only two from the Americas (one from Brazil and one from Guatemala). The ITS1 and ITS2 regions of the sequences from these different countries revealed similarities between 78.2% and 100% for ITS1, allowing up to 21.8% variability, while the ITS2 regions showed similarities between 82.8% and 100%, allowing up to 17.2% variability. Some authors have found differences in the ITS2 sequence of N. americanus from Africa compared to those from Asia (Romstad et al. Reference Romstad, Gasser, Nansen, Polderman and Chilton1998). In spite of the human health importance of hookworms, there have been only a small number of studies of genetic variability within N. americanus. We registered only one in South America, but using another amplification target, a 395-bp region of the mitochondrial cytochrome c oxidase subunit 1 gene, they found haplotypic diversity of N. americanus from six villages in Colombia (Hu et al. Reference Hu, Rabelo, Schindler, Roberts and Gasser2008). Another study revealed genetic variability in the mitochondrial genome of N. americanus from Togo (Africa) compared to another from China (Asia) (Hu et al. Reference Hu, Chilton, Abs El-Osta and Gasser2003). On the other hand, possible variability of N. americanus ITS2 and ITS2-28S can lead to primers failing because the primer binding regions were designed in variable regions with no complete complementarity and this could generate weak and unstable junctions to the DNA template, resulting in no detectable products. Further studies are needed to confirm this assumption. In another STH, Strongyloides stercoralis, different results for various amplification targets have been reported, perhaps attributable to genetic variability in the 18S rDNA gene (Marquet et al. Reference Marquet, Mora, Incani, Jesus, Méndez, Mujica, Trosel and Ferrer2023).
With respect to the analytical sensitivity of the PCR-NaITS1F/NaITS1R (ITS1) for the amplification of the ITS1 of N. americanus, an amplification range that goes from 100 ng to 10 pg was obtained, indicating that the minimum amount of DNA that is amplified by the PCR technique is approximately equivalent to three eggs, since it has been estimated that an N. americanus egg has between 2.16 and 4.32 pg of DNA (Pilotte et al. Reference Pilotte, Papaiakovou, Grant, Bierwert, Llewellyn, McCarthy and Williams2016). Conversely, the analytical sensitivity of PCR-Na58F/Na158R (ITS2) was one hundred times smaller, 1 ng, approximately equivalent to 300 eggs. However, it is important to note that the conversion of analytical sensitivity to egg equivalents using a range of 2.16–4.32 pg DNA/egg (Pilotte et al. Reference Pilotte, Papaiakovou, Grant, Bierwert, Llewellyn, McCarthy and Williams2016) was derived from laboratory-maintained worm populations and may not apply to field-derived samples with variable egg integrity. The PCR-NaITS1F/NaITS1R (ITS1) has good analytical sensitivity and is suitable for molecular diagnosis, and is similar to that obtained by other authors (Hassan et al. Reference Hassan, Noor Badi, Mohd-Shaharuddin, Wan Yusoff, Lim, Chua, Sidi Omar, Chang, Majid and Ngui2022), while the PCR-Na58F/Na158R (ITS2) has low sensitivity, as it would require samples with a large number of eggs in the stools to be able to give a positive result. The analytical specificity of both PCRs was 100%, since they only amplify DNA of N. americanus, as expected, due to the results of previous work with these targets (Basuni et al. Reference Basuni, Muhi, Othman, Verweij, Ahmad, Miswan, Rahumatullah, Aziz, Zainudin and Noordin2011; Llewellyn et al. Reference Llewellyn, Inpankaew, Nery, Gray, Verweij, Clements, Gomes, Traub and McCarthy2016; Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014; Verweij et al. Reference Verweij, Brienen, Ziem, Yelifari, Polderman and van Lieshout2007) and because in the bioinformatics analysis the primers only showed similarity to the target sequences of N. americanus. Unfortunately, in Venezuela it is very difficult to obtain more closely related parasites such as Ancylostoma duodenale; it would be important for further work to incorporate Ancylostoma species to better estimate specificity.
When applying the two standardized PCRs to all samples from individuals in the Tierra Blanca community (experimental samples), the results were very different, since the PCR-NaITS1F/NaITS1R (ITS1) detected a large number of positives, while the PCR-Na58F/Na158R (ITS2) only detected two positives (corresponding to the samples with the highest number of eggs/g of faeces). This is related to the low analytical sensitivity of this PCR, but it contrasts with the results of other authors using this same PCR. In fact, it is one of the most widely used PCRs worldwide, although it has been used mainly with samples from individuals in Asia and Africa, coming from the countries where the sequences deposited in GenBank originate (Basuni et al. Reference Basuni, Muhi, Othman, Verweij, Ahmad, Miswan, Rahumatullah, Aziz, Zainudin and Noordin2011; Llewellyn et al. Reference Llewellyn, Inpankaew, Nery, Gray, Verweij, Clements, Gomes, Traub and McCarthy2016; Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014; Verweij et al. Reference Verweij, Brienen, Ziem, Yelifari, Polderman and van Lieshout2007). Perhaps the N. americanus circulating in the Venezuelan community presented in the work exhibits genetic variability in the primer sequences, and the junctions are weak and unstable, causing a lack of amplification in samples where the amount of parasite DNA is low. Further studies are needed to confirm these assumptions.
When calculating the diagnostic indexes for the two PCR tests, it was necessary to use a ‘composite standard’, since coprology, the ‘gold standard’, for most studies is an ‘imperfect standard’ for other studies because it is less sensitive than PCR. This strategy has already been used by many authors to calculate diagnostic indexes of PCR for N. americanus and other parasites (Azzopardi et al. Reference Azzopardi, Hardy, Baker, Bonnici, Llewellyn, McCarthy, Traub and Steer2021; Ferrer et al. Reference Ferrer, Villegas, Mughini-Gras, Hernández, Jiménez, Catalano and Incani2020; Fleitas et al. Reference Fleitas, Vargas, Caro, Almazan, Echazú, Juárez, Cajal, Krolewiecki, Nasser and Cimino2021; Köller et al. Reference Köller, Hahn, Altangerel, Verweij, Landt, Kann, Dekker, May, Loderstädt, Podbielski and Frickmann2020). All diagnostic indexes were higher and better in the PCR-NaITS1F/NaITS1R (ITS1) than in PCR-Na58F/Na158R (ITS2). PCR-Na58F/Na158R (ITS2) assay was adapted from a qPCR protocol to conventional PCR, and although in our laboratory we have adapted several qPCR assays to conventional PCR, and they have worked very well. However, this could be a factor contributing to the differences in sensitivity. For this reason, the observed differences cannot be attributed solely to target choice. Some of the discrepancy may derive from assay format, chemistry, or optimization constraints rather than from the biological suitability of the target region itself. In most studies comparing PCR techniques with coprology techniques, PCR yields more positive results due to its high sensitivity compared to the low sensitivity of coprology, a fact acknowledged by all researchers. Of course, sequencing the PCR products from PCR-positive samples with negative coprology results would be advisable; this was not possible for this study, but it will be done for future research. Although it might be thought that the large number of positive results obtained with the PCR-NaITS1F/NaITS1R (ITS1) could be due to PCR contamination, this is unlikely since reagent mixtures are prepared in separate laminar flow hoods with different pipettes than those where the extracted DNA is added. DNA extractions are also performed in separate laminar flow hoods with different pipettes. Filter tips are always used for the pipettes. Negative controls are used in all reactions, both in the DNA extractions and in each PCR session and have always yielded negative results. Positive controls are added in separate laminar flow hoods, located far from where the samples are added, and with different pipettes. Moreover, the results of the PCR-NaITS1F/NaITS1R (ITS1) are the most accurate in relation to the epidemiological reality, since rural areas such as the community Tierra Blanca (an indigenous community where most of its inhabitants walk barefoot almost all the time) are characterized by having favourable conditions for the persistence of human hookworm, such as humid climate and sandy soils with abundant tree and shrub vegetation and with houses in open clearings of the jungle. The population description (Table 6) shows that the majority are children and young people who tend to have more contact with the soil. No statistically significant differences were found by gender, although the positivity rate was higher among females, the difference was not statistically significant, perhaps because there were more females than males. Therefore, the probability of infection was almost the same by gender. One interesting finding was that statistical significance was observed in the 46–60 age group. If we look at the positivity rates, they decreased with increasing age, which makes sense because older individuals generally practice better hygiene (for example, they are more likely to wear shoes and spend less time on the ground). The children and young people in rural communities generally don’t wear shoes and play on the ground (PAHO 2025). Perhaps the last age group did not show significant differences due to the smaller number of samples.
Despite several limitations, coproparasitological microscopy is the common ‘gold standard’ for these parasites; that’s what all published articles mention when comparing techniques. Many articles also mention that it’s an imperfect gold standard; there’s no single technique that can be a suitable gold standard in this case (Argy et al. Reference Argy, Nourrisson, Aboubacar, Poirier, Valot, Laude, Desoubeaux, Pomares, Machouart, Le Govic, Dalle, Botterel, Bourgeois, Cateau, Leterrier, Le Pape, Morio and Houze2022; Oliva et al. Reference Oliva, Clemente, Menegotto, Varani, Bruno, Gargiulo, Petrullo, Farina and Raglio2025). Of course, this analytical strategy (use of a composite standard) makes the reported diagnostic indexes difficult to interpret fully and may favour the diagnostic indexes of the assays; this is a limitation of this approach and can affect the interpretation of the reported performance estimates. However, this is what most authors use when comparing coproparasitological techniques with molecular techniques. What we can estimate, even without calculating indexes, just with the test results, is that the PCR-NaITS1F/NaITS1R (ITS1) is more sensitive than coprological tests. Recently, multiplex PCR formats that include N. americanus have been developed, both ‘in-house’ (Azzopardi et al. Reference Azzopardi, Hardy, Baker, Bonnici, Llewellyn, McCarthy, Traub and Steer2021; Köller et al. Reference Köller, Hahn, Altangerel, Verweij, Landt, Kann, Dekker, May, Loderstädt, Podbielski and Frickmann2020; Phuphisut et al. Reference Phuphisut, Yoonuan, Sanguankiat, Chaisiri, Maipanich, Pubampen, Komalamisra and Adisakwattana2014) and in commercial kits (Autier et al. Reference Autier, Gangneux and Robert-Gangneux2021; Dacal et al. Reference Dacal, Köster and Carmena2020; Köller et al. Reference Köller, Hahn, Altangerel, Verweij, Landt, Kann, Dekker, May, Loderstädt, Podbielski and Frickmann2020). Published articles on the evaluation of these multiplex PCRs found false-negative results in 54% of samples from N. americanus. In fact, the authors recommend the use of the commercial kit for the detection of several helminths except for hookworms (Autier et al. Reference Autier, Gangneux and Robert-Gangneux2021). The multiplex PCR formats developed ‘in-house’ do not pose the same problem because when applied in different areas, positive controls from such areas are used, and if no amplification is obtained, that protocol will not be applied. However, commercial kits contain DNA controls from the areas where they were manufactured (generally from Asia), and when the positive controls test positive, they assume their use with confidence in other areas, with somehow false negative results.
This is the first study about molecular diagnosis of N. americanus in Venezuela and the possible genetic variability in PCR targets is only a possible hypothesis, so it is very important to carry out sequencing studies of the ribosomal DNA of these parasite populations, in order to clarify the sequences of N. americanus circulating in Venezuela and in Latin America. This would help to design primers specific to the region, allowing appropriate diagnosis and treatment follow-up.
Conclusions
This work suggests the importance of evaluating the amplification targets to be used in molecular diagnosis of N. americanus, as well as other conditions of the PCR protocols and the need for adequate standardization using samples from the regions where they will be used so that the PCRs become reliable and reproducible, and can be applied in individual diagnosis as well as support in epidemiological studies. Also, it was demonstrated by the use of both the coproparasitological test and PCR-NaITS1F/NaITS1R (ITS1) that the community Tierra Blanca has a high prevalence of N. americanus.
Financial support
This work was supported by Universidad de Carabobo, Project DIPISA-PG-2017-004.
Competing interests
None.
Ethical standard
The project was approved by the Committee of Bioethics of the Institute of Biomedical Research (University of Carabobo, BIOMED-UC). Informed consent was signed by the individuals who supplied the stool samples and they agreed that their samples could be used for research.
