Intestinal parasitoses are one of the main causes of health-service consultations in developing countries and are an important cause of morbidity worldwide. Among these, infections with some protozoa are recognized as having significant consequences on health, particularly in children [1]. Direct consequences are usually gastrointestinal disorders, such as diarrhea, dysentery, abdominal pain, vomiting and lack of appetite and indirect impacts include synergistic negative effects on nutritional status, stunting and reduced educational achievement. For instance, every year there are more than 200 million symptomatic patients with giardiosis [2] and cryptosporidiosis which cause diarrhea in children younger than 5 years globally, only second after rotaviral enteritis [3]. Infections with Giardia duodenalis (syn. Giardia lamblia or Giardia intestinalis) Cryptosporidium spp. and Entamoeba histolytica are considered to be among the most common and important causes of parasite-related diarrhea in human populations. In contrast, the pathogenicities and symptoms associated with infections with Blastocystis spp. [3]. Intestinal protozoa are major causes of gastrointestinal infections in humans and are diagnosed, traditionally, using microscopy, the diverse concentration protocols usually involving formalin fixation. Microscopic analysis guided by morphological characteristics of parasites remains the gold standard for diagnosing such infections, often caused by members of the genera Giardia, Entamoeba or Cryptosporidium. Species and genotypes within each of these genera may differ in their pathogenesis, host range and epidemiology, which can only be elucidated using molecular methods [4]. However, other methods are available, including detection of parasite antigen in feces by ELISA or immunochromatograpy (e.g., snap tests) and molecular methods [5]. Given the excellent sensitivities and specificities achieved by molecular methods, detection of parasite-specific DNA by PCR is fast gaining popularity. Not only is PCR simple and potentially offers a more rapid turnaround time, but it also allows discrimination between morphologically indistinguishable parasites that are of different clinical relevance (such as Entamoeba dispar and E. histolytica). In addition, multiplex PCR enables samples to be investigated for several pathogens simultaneously and can also be used to determine relative copy numbers, although the potential for quantification is often not used in the diagnostic setting. However, due to the requirement for comparatively costly equipment and reagents, including some for which refrigeration is essential, such diagnostic assays have not been implemented in many diagnostic labs and are particularly underused in labs in less-developed regions. Nevertheless, where facilities and resources are available, multiplexed PCR-based methods that target the most relevant gastrointestinal parasites can provide fast, reliable results and could be implemented in routine clinical diagnostics [6]. However, comparison of commercial multiplex real-time PCR assays has indicated that performance may be variable and the required sensitivities and specificities, along with lab workflow, diagnostic algorithms and population should be considered, as well as the cost, when considering incorporating such assays into the diagnostic lab [7]. Although, in the past few decades molecular diagnostics of parasitic infections have been accepted as first-line diagnostics, laboratories tend to be reluctant of introducing nucleic acid based tests as they dread the validation of laboratory developed tests (LDTs) compliant with ISO1589 [8].

Aim of the Study

This cross-sectional study aims to Molecular detection of intestinal protozoa parasite infection in patients at Wasit province.

Study Design and Patients 

A cross-sectional study was conducted in Wasit province. The study included 100 stool samples (44 male and 56 female) were randomly collected from patients in Wasit province during January 2023 to May 2023. The samples examined were used to detect the presence of intestinal parasitic infestation while the study utilized a pre-tested structure, after collection, each stool sample was divided into two parts; the first used to diagnosed the parasite by microscopic examination of M.Z.N stain, wet mounts were stained with Lugol's iodine and the second was preserved in the laboratory freezer in -20°C for PCR for detection of parasite DNA. Cases were matched based on demographic parameters, such as age, gender, residence, malnutrition and source water.

Collection of Samples

During this study, 100 stool specimens were collected from both gender and different age groups of patients in clean specimen container tube. During the period from January 2023 till the end of May 2023, data were collected simultaneously, each stool sample was divided into two parts; the first used to diagnose the parasite by microscopic examination and the second was preserved in the laboratory freezer in -20°C for PCR for detection of parasite DNA [9].

Molecular Testing

The Multiplex PCR primers for detection Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, Blastocystis spp., Isospora sp., Balantidium coli and Dientamoeba fragilis based on small subunit ribosomal RNA gene were designed in this study using NCBI-Genbank and primer 3 plus design. These primers were provided from Scientific Resercher. Co. Ltd, Iraq. 

Multiplex PCR technique was performed for detection Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, Blastocystis sp., Isospora sp., Balantidium coli and Dientamoeba fragilis based on small subunit ribosomal RNA gene from Human stool samples. This method was carried out according to method described as following steps: 

• DNA Extraction: DNA from water samples were extracted by using Presto™ Stool DNA Extraction Kit and done according to company instructions

• Genomic DNA Estimation: The extracted genomic DNA from stool samples was checked by using Nanodrop spectrophotometer (THERMO. USA) that check and measurement the purity of DNA through reading the absorbance in at (260 /280 nm)

• Multiplex PCR Master mix Preparation: The mPCR master mix was prepared by using (GoTaq Green PCR Master Mix) and this master mix done according to company instructions

• PCR Thermocycler Conditions: Initial Denaturation (95ºC for 5 min), Denaturation (95ºC for 30 sec) Annealing (58ºC for 30 sec) Extension (72ºC for 1 min), Final extension (72ºC for 5 min) Hold (4°C Forever)

• Multiplex PCR product analysis: The mPCR products of was analyzed by agarose gel electrophoresis

Statistical Analysis 

Statistical analysis was carried out using of ANOVA by the SPSS program [10].

The study included examination of 100 stool samples of patient by Multiplex Polymerase chain reaction PCR. In this section, the results of the Multiplex PCR test were demonstrated. Table 1 shows that 60% of the samples were E. histolytica followed by 23% Cryptosporidium and then 20% for G. lamblia. Regarding Isospora, there were 7 cases (7%) detected Table 1.

Table 2 shows the association of age with the presence of E. histolytica by Multiplex PCR. There was no significant association (p-value = 0.830) between being in a specific age group and testing positive for this parasite. About 60% of those ages 16-30 years and 60% of 31-45 years old appeared to be positive by PCR test. Also, there was 65% with less than 15 years old.

Table 1: Frequency Distribution of Parasites Detected By Multiplex PCR in 100 Samples

Table 2: Association of Age Groups with the Presence of E. Histolytica by Multiplex PCR

Table 3: Association of Age Groups with the Presence of G. Lamblia by Multiplex PCR

Table 4: Association of age groups with Presence of Cryptosporidium by Multiplex PCR

Table 5: Association of Age Groups with the Presence of Blastocystis by Multiplex PCR

Table 3 found no significant association between age groups and the presence of G. lamblia by Multiplex PCR test with a p-value = 0.799. One-quarter of patients who were less than 15 years old were positive followed by 23% of those aged between 46-60 and 20% of those (31-45) years.

The diagnosis of Cryptosporidium by Multiplex PCR was also not related to age distribution among the patients (p-value = 0.455). Around a third (33.3%) of those belonging to the age group (31-45) years were infected with this parasite and 30.8 % of the age group (46-60) years were also infected followed by 28.6% of those above 61 years Table 4.

Table 5 shows that 28.6% of samples aged above 61 years old had positive Multiplex PCR results of blastocysts followed by 20% for age groups (16-30) years, then 15.4% for (46-60) years. That means no significant difference between positive and negative cases according to age groups (p-value = 0.876).

In Table 6 there is no significant association between those diagnosed with Isospora and age groups (p-value= 0.608).

Table 6: Association of Age Groups with the Presence of Isospora by Multiplex PC

Figure 1: Agarose Gel Electrophoresis Showing Multiplex PCR Analysis of Small Subunit Ribosomal RNA Gene for the Detection of Entamoeba Histolytica, Giardia Intestinalis, Cryptosporidium Parvum, and Blastocystis Sp. from Human Stool Samples. Lane M Represents the DNA Marker (2000–100 Bp), While Lanes 1–24 Show Positive PCR Products for E. Histolytica (526 Bp), G. Intestinalis (615 Bp), C. Parvum (728 Bp), and Blastocystis Sp. (414 Bp), Respectively.

Figure 2: Agarose Gel Electrophoresis Showing Multiplex PCR Analysis Of The Small Subunit Ribosomal RNA Gene For The Detection Of Isospora Sp., Balantidium Coli, And Dientamoeba Fragilis From Human Stool Samples. Lane M Represents the DNA Marker Ladder (2000–100 Bp), While Lanes 1–34 and 1–33 Show Positive PCR Products for Isospora Sp. (530 Bp), Balantidium Coli (348 Bp), and Dientamoeba Fragilis (303 Bp), Respectively.

Percentage of Infection for parasites that cause Intestinal para sitic by PCR technique examination results were shown the highest infection with E. histolytca (60%) and followed by C. parvum (23%), G. lamblia (20%) and the lowest infection with Blastocystis hominis (17%), Isospora ( 7%). The current study agrees with Hraija et al. [11] in Wasit, Iraq and Abed et al. [12] in Salah al-Din and Alkhuzaey in Thi-Qar [13] also agree with Yakoob et al. [14] in Iraq and Ngosso and Namkinga [15] in Dar Es Salaam were detected 33.3% as E. histolytica by PCR higher than other parasites (Figure 1-2).

These disagree with Mathurin et al. in Cote d’Ivoire; they saw (36.93%) C. parvum is greater than another parasites and disagrees with, Ngosso and Namkinga in Dar Es Salaam they saw the highest parasites in total positive samples with G. lamblia (35.6%) and followed by E. histolytica, also disagree with study by Akram [16] in Thi-Qar was found the infection with G. lamblia higher than other parasites in total positive samples and followed by E. histolytica and differs with Parčina et al. in Germany they examined of 200 diarrheic stool samples by using RT-PCR and registered G. lamblia (69.0%) the highest infection. and multiplex PCR assay showed sensitivity of 90.91%, for G. intestinalis and Cryptosporidium spp., whereas multiplex PCR assay achieved sensitivity of 86.36% for the detection of E. histolytica by Bairami [17] in Iran. Disagree with Maas et al. [18] in The Netherlands shows the prevalence of intestinal protozoan infection according to patient’s age. The average age of the patients was 35 years, so they divided in to five groups for the purpose of study. The infection with the five parasites was more prominent in ≤15 years old with infection rates estimated as 65.0% for E. histolytica, (25.0%) for G. lamblia and (25.0%) C. parvum, (15.0%) Blastocystis hominis and (7.5%) Isospora. Subjects within 16-30 years age group have the lowest infection rates, 60.0%, 12.0%, 12.0%, 20.0% and 8.0% for E. histolytica, G. lamblia and C. parvum, Blastocystis hominis, Isospora, respectively. There was no association between the infection with the five parasites and age group agrees with Rahi and Majeed [19] in Wasit and disagreeing with Ten Hove et al. [20] in the Netherlands who found that Cryptosporidium infections were strongly associated with the age of the patients and my study also disagreeing with Mahmoud et al. [21] in Taif. We observed rate Blastocystis sp infestations in 11 (73.3%) from 82 samples in age group 1-40 years. And we see through study Masar Hadi [22], in Diyala the highest prevalence rate Blastocystis sp. In patients observed 27.7% in the age group 19-28 years, 22.7% in age group 8-18 years and finally the age groups 29-38 and 39-48 years old (18.1%).

Our study supports implementation of molecular tools into the parasitology diagnostic laboratory, even in under-resourced settings. Such tools are particularly important for detecting protozoa that are challenging to identify by microscopy. Molecular tools are also essential for distinguishing between morphologically identical species that are of differing clinical relevance. In addition, molecular and traditional techniques have overlapping, but complementary, roles in diagnosing protozoan infections resulting in clinical manifestations in patients. In conclusion, a multiplex PCR for the detection of intestinal protozoa is a technically feasible tool in a routine microbiological laboratory.

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