Detection of Submicroscopic Soil-Transmitted Helminth Infections from Fecal Samples in Nangapanda, Ende, Using Real-Time Polymerase Chain Reaction

Soil-transmitted helminth (STH) infections ( Necator americanus (hookworm), Ancylostoma duodenale (hookworm), and Ascaris lumbricoides) can lead to anemia, malnutrition, and iron defici n y. Traditionally, STH infections have been diagnosed using microscopy to detect eggs in human fecal samples. However, there are several limitatio ns of this method. The aim of this research was to detect the percenta g of submicroscopic STH infections from human feca l samples (children, 5–18 years old) in Nangapanda, Ende, usi ng the real-time polymerase chain reaction (PCR) me thod. The fecal samples were collected in two time periods, which w ere before and after treatment, using 400 mg of Alb endazole. There were 242 samples in total, but only 45 negative sam ples from microscopic detection were tested with re al-time PCR. The DNA samples were isolated and amplified wih primers of internal transcribed spacer (ITS-1 and ITS-2) r egion of rDNA. The detection of samples with real-time PCR g enerated an amplification curve in VIC, FAM, and Te xas Red fluorophore. Three samples (6.7%) in pre-treatment were low load of DNA ( N. americanus and A. lumbricoides) (Ct > 35). Four samples (9.1%) were low load of DNA ( N. americanus) (Ct > 35) in post-treatment. Five samples (11.4%) were moderate load of DNA ( A. lumbricoides) (30 < Ct < 35) in post-treatment. This study showed that real-time PCR could detect submicroscopic infections from specifi c species of hookworm and A. lumbricoides.


Introduction
Soil-transmitted helminth (STH) infections from Ascaris lumbricoides, Trichuris trichiura, and hookworms (Ancylostoma duodenale and Necator americanus) are highly prevalent in tropic and subtropic regions, especially in rural areas, as a result of poor sanitary conditions [1]. In 2003 until 2010 there are one billion people have contacted STH infections, and 58.1 million of them might have been caused by A. lumbricoides. Close to 26.6 million could have been caused by T. trichiura, and more than 59.9 million by hookworms [2].
These infections can lead to morbidity; however, infection severity can depend on the species with which humans have come in contact [3]. Parasites are transmitted when their eggs in human feces reach the environment which are then ingested, or when their larvae penetrate the skin [4]. Indonesia is a tropical country where low-rate sanitation is found in many areas, which contributes to the development of STH infections. Nangapanda, Nusa Tenggara Timur (NTT), is a semi-urban area with high humidity and environmental temperature. This area has a high risk of parasitic infections based on preliminary surveys in 2005 and 2006 [1].
Necator americanus infections can cause nekatoriasis, whereas A. duodenale infections can cause ankilostomiasis. These worms live in the large intestine, and they consume nutrients. Mild infections can cause malnutrition, whereas severe infections and chronic helminth infections can cause anemia. A report by Keiser & Utzinger (2008) showed that Albendazole (400 mg) was used to treat hookworm infections with a cure rate of approximately 78.4%, and for A. lumbricoides infections by approximately 93.9% [5]. A. lumbricoides infections occur predominantly because of its ova, which are able to survive in different environments for prolonged periods. Reinfection occurs often because its ova is abundant in the soil [6]. To decrease infections, health education and sanitation improvement are severely needed, particularly in endemic areas. A. lumbricoides infections can become chronic, which results in the host losing nutrients. This occurs mainly in children and individuals whose immune systems are weak [7]. Severe A. lumbricoides infections can lead to death.
Microscopic examination of fecal samples is still the gold standard to detect the presence of eggs in STH. However, the eggs of A. lumbricoides do not appear until 40 days after infection. The egg production stage lasts approximately 2-3 months (60-90 days). In some cases, there are only male worms in the intestine. When this happens, it is difficult to diagnose because the egg in the feces cannot be found. In some hookworm cases, it can be easy to break shell eggs because of chemical solutions, including ether. Hookworms are hard to differentiate using the microscopy method. This method might result in misdiagnosis, which can lead to improper and late treatment [6]. Detection of antibodies can be used in STH infections, but this test is not able to distinguish between infections [7].
Molecular approaches (methods based on nucleic acid) accurately detect infections caused by parasites. Real-time polymerase chain reaction (PCR) is able to identify parasites in new infections or dead larvae in the human intestinal lumen with a low load of DNA. By conducting the more accurate method, the early detection of an STH infection can be done successfully. A specific and sensitive diagnosis method to detect STH infections in humans is important for understanding the epidemiology, treatment, and strategy for controlling transmission [8].
The principle of the PCR technique is to amplify product-specific DNA fragments rapidly in vitro. The conventional PCR technique requires longer time processing, and has a high risk of contamination. The principle of real-time PCR is to amplify the DNA template and collect data in the exponential growth phase. Real-time PCR can be used as singleplex or multiplex. In singleplex PCR, only one flourophore and a pair of primers are used in a single reaction. In multiplex PCR, multiple flourophores and primers are used to enable the detection of several different targets [9]. A target gene in internal transcribed spacer (ITS) were used for molecular detection of hookworm and A. lumbricoides. The ITS gene is a nonfunctional RNA in ribosomal RNA (rRNA), acting as a precursor transcript, which has high-mutation rates [10]. The ITS region of rDNA can be used to distinguish species within the same group. Basuni et al. (2011) used 77 fecal samples to determine the sensitivity of real-time PCR compared to the microscopic method in the detection of STH infection. For real-time PCR, the sensitivity was 62.3%; however, microscopic detection of positive samples infected with STH had a sensitivity only 3.9%. Their results showed that real-time PCR's sensitivity is higher than the microscopic method's sensitivity [7]. Research on the submicroscopic infection was not discussed in that study, so further research is needed. The aim of this study was to determine the percentage of submicroscopic infection of hookworm and A. lumbricoides using the real-time PCR method.

Methods
The Study Area. Preliminary studies in 2005 and 2006 found that Nangapanda is an endemic area for soil-transmitted helminth infections (A. lumbricoides, hookworms, and T. trichiuria) [11], which made Nangapanda the right location in which to conduct this study. Nangapanda is a district in Ende, Flores Island, NTT, Indonesia. It is located in the coastal marine area, and consists of 22,000 people. The temperature range is 23 °C to 33.5 °C, and humidity is approximately 86 to 95%. Nangapanda is a semi-urban area, and the most common occupations are fishing and farming. Some of them work in the public sector [1].
Sample size. Fecal samples were collected from two different sources, pre-treatment and post-treatment, which were taken from children 5 to 18 years old. For the sample, the status of the STH infections was previously completed. The inclusion criteria of the samples in this particular study is microscopically negative of all STH infections both before and after treatment using Albendazole (400 mg). The randomization using www.randomizer.org was applied to get 90 samples (paired samples of 45 participants before and after treatment) ( Figure 1).

DNA isolation.
Parasitic DNA was isolated from approximately 0.1 g of fecal sample using the QIAamp DNA-assay spin-column method according to manufacture's instructions, and partly modified by Verweij et al. 2001 [12]. In the spin-column extraction method, DNA molecules selectively bind with the silica surface in the presence of salts and under certain pH conditions. The nucleic acids are absorbed into the silica gel surface with high concentrations of salt, whereas other carbohydrates and proteins are not absorbed, and they are eliminated. In the extraction process, a lysis buffer and purification buffers were used [13]. A lysis buffer contains PhHV as an internal control. The internal control used phocine herpes virus (PhHV). Some experiments indicate that internal control can detect false-negatives [14]. Ethanol precipitation was used on the DNA because DNA is not soluble in ethanol. Wash solution (washing buffer) was used to wash the DNA of other contaminants, such as proteins or polysaccharides (glycogen/starch), to attach the DNA to the silica membrane of the spin column. An elution buffer was used to release the DNA that was bonded to the silica filter. Then, the extracted DNA was stored at 4 °C in a sterile microtube.

Results and Discussion
There were 96 wells in one plate. There were 90 wells for the samples (45 samples both pre-and post-treatment with the same ID) One and four wells were as negative and positive control, respectively, for each worm (N. americanus (Na), A. duodenale (Ad), A. lumbricoides (Al), and S. stercoralis (Ss)). Eleven samples in the first running of real-time PCR were known to have inhibition because there was no amplification curve on the internal control. The cycle threshold (Ct) value was zero, which indicated that there was no amplification. Replication was conducted on eleven samples, and started at the isolation stage. Complete data was obtained from the new amplification curve with the Ct value of each fluorophore.
Inhibitors are as a major parameters to measure the failure of amplification (i.e., complexpolysaccharides, fats, and proteins). The DNA extraction and amplification process of those 11 samples was repeated. Amplification of PhHV as internal control was detected at the expected Ct value, ranged between 24.99 to 38.91 in all the samples. One sample was excluded from analysis because no amplification curve was detected. Eighty-nine samples were eligible for further analysis.
Internal control is an oligonucleotide, which is designed based on the primer and probe of the DNA target. PhHV is an internal control, which was mixed with the lysis buffer during the process of DNA extraction. Internal control can monitor the DNA extraction process from inhibitors [14]; therefore, it was used to perform replication. Negative controls (H 2 O) were used as contamination control. No contamination was found during the amplification process. The positive controls reflected the amplification of specific species. The amplification of the positive controls indicated that primers specifically amplify the gene target.  Table 3.
The total STH infections in post-treatment were high. This indicated that the treatment of Albendazole 400 mg can excrete the eggs to leaving its host; however, there is some helminth eggs that will remained in the host. This indicated that treatment without accompanied health practice was still the same so that the rate of infections were high [17]. there are a few worms, it is difficult to detect the worms using the microscopic method. For A. lumbricoides, it takes 2-3 months (60-90 days) to produce eggs, which means that eggs will not appear in feces until 40 days after infection [6].

Table 3. Percentages for Submicroscopic STH Infections
Molecular detection using target genes accurately detects the number of worms and dead larvae inside human bodies [10]. Sensitivity and specification of real-time PCR is high, allowing for detection of low DNA load of parasites. It is a very efficient method to prevent dangerous diseases and serious health problems. The number of deaths because of A. lumbricoides and hookworm infection may be reduced [17].
The study using real-time PCR showed a higher detection rate of STH infections compared to other methods. This was because real-time PCR can detect small amounts of DNA [17]. The microscopic method is only able to detect infections in high Ct and moderate groups, whereas the molecular method successfully detects low Ct values. The examination using negative samples showed microscopically positive results, which were then checked by real-time PCR.
The Ct values that were categorized in low load of DNA indicated that the DNA contained in the sample was low. The high Ct values indicated that less genetic materials were in the samples; therefore, hookworm infection detection is not feasible by microscopic examination. The sensitivity of the microscopic method was low and highly dependent on a combination of exact laboratory procedures and the helminth species being examined, especially in terms of low DNA targets in the samples. Difficulties arose because of the low number of eggs or larvae in some samples, nevertheless the DNA of parasites were detected using real-time PCR with the acquisition of high Ct values. Failure detection (microscopically) of worm eggs in the sample can also be caused by the presence of dead larvae [17].
The study also found there was mixed infection between A. lumbricoides with hookworm N. americanus from post-treatment samples. Mixed infections can cause complex problems for patients, such as malnutrition, because of the existence of A. lumbricoides and blood loss from N. americanus, which are blood feeders. The amount of blood lost each day reached 5 mL for mild infections, which induced anemia in patients [18]. The infection is directly correlated with the intensity of species-specific infections [19]. Specific detection of N. americanus and A. duodenale was required to determine which species were more dominant because the effect of infection is caused by blood loss, which was higher in A. duodenale than in N. americanus. Ancylostoma duodenale can cause a daily blood loss ranged from 0.14 mL/worm to 0.26 mL/worm with an average of 0.2 mL/worm, whereas N. americanus ranged from 0.02 mL/worm to 0.07 mL/worm with an average of 0.04 mL/worm. This is because A. duodenale has a pair of hook-shaped teeth in its mouth, whereas N. americanus only has a cuttingplate structure. For A. duodenale, the hook structure can stick to and tear the intestinal wall [20].
Real-time PCR is very useful in detecting submicroscopic infections, especially when the intensity of the hookworm infection is low. It can be used to quantitatively determine the intensity of hookworm infections in an endemic area and can distinguish infections between N. americanus and A. duodenale [21].
The intensity of infections is determined by the value of Ct on real-time PCR. It reflects the amount of specific DNA parasites in feces. In microscopic techniques, the number of parasite DNA is expressed in eggs per gram (EPG). The real-time PCR method is more sensitive than microscopic methods, which might be proved by the ability to detect STH infections in low load of DNA. Real-time quantitative PCR can detect the intensity of A. lumbricoides infections in endemic areas, including Nangapanda. Real-time PCR should be used as a program to evaluate A. lumbricoides infections [9]. Nangapanda is a semi-urban area with poor sanitation; therefore, a high percentage of STH infections was found. STH infections occur through physical contact with soil contaminated by human feces. Therefore, the disposal of human waste is essential for the eradication of hookworm infections. It is necessary to include the provision of sanitary latrines as a place of human waste disposal. The right sanitation style/design could reduce helminth infections, delay transmission, and prevent reinfection [22]. The total samples in pre-and posttreatment for the chi-square test were 44. Next, the presence of submicroscopic STH infections was detected based on sex, age, and weight. The results for the frequency of submicroscopic STH infections based on age were divided into three groups. For weight, there were five groups. Using the chi-square test, the significant relationship between submicroscopic infections and demography data can be showed ( Table 4).
The level of worm infection, according to sex, age, and weight, is related to submicroscopic infection. The p-value was above 0.05, which indicated that there was no significant association between infection and sex. However, A. lumbricoides and N. americanus infections are more likely to occur on children especially boys, theoretically. In Nangapanda, the majority of the population are farmers. They are often exposed to soil in which the ova of A. lumbricoides or N. americanus is located, which increases their infection vulnerability [23].
The same result also shown between submicroscopic infection in the age group with no significant association. However, some studies have suggested that the highest intensity of STH infection occurred in age groups between 5-15 years old [24]. When age increases, infection decreases [25]. There is no significant association between submicroscopic infection and weight based on the p-value in the test results. Ezeamama et al. (2005) [25] stated that helminth infections can lower the life expectancy of children under 14 years old with an average weight 12-36 kg. There will be weight decline because of malnutrition caused by worm infection.

Conclusions
Real-time PCR can detect submicroscopic STH infections in samples from pre-treatment (6.7%) and post-treatment (20.4%). Mixed infections were detected between A. lumbricoides and N. americanus (2.3%). The submicroscopic STH infections found in three samples were grouped as low load of DNA (Ct > 35) while five samples were grouped to moderate load of DNA (30 < Ct < 35) for A. lumbricoides infections. Four samples were grouped as low load of DNA (Ct > 35) for N. Americanus infections. Sex, age, and weight were not associated significantly with submicroscopic STH infections.