Application of multiplex real-time polymerase chain reaction in diagnosis of intestinal parasitic infection among anemic pregnant women

Vivek Gupta; Quazi Syed Zahiruddin; Abhay Gaidhane; Prajakta Deshpande; Anil Tambekar

doi:10.4103/jdmimsu.jdmimsu_245_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 17 | Issue : 4 | Page : 881-886

Application of multiplex real-time polymerase chain reaction in diagnosis of intestinal parasitic infection among anemic pregnant women

Vivek Gupta¹, Quazi Syed Zahiruddin², Abhay Gaidhane², Prajakta Deshpande³, Anil Tambekar⁴
¹ Department of Pathology, Molecular Diagnostics and Research Laboratory, Government Institute of Medical Sciences, Greater Noida, Uttar Pradesh, India
² Department of Community Medicine, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra, India
³ Department of Biology, University of Dayton, Dayton, Ohio, United States
⁴ Department of R and D, Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra, India

Date of Submission	26-Jun-2021
Date of Acceptance	22-Mar-2022
Date of Web Publication	10-Feb-2023

Correspondence Address:
Dr. Vivek Gupta
Department of Pathology, Government Institute of Medical Sciences, Greater Noida -201 310, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jdmimsu.jdmimsu_245_21

Abstract

Background: Intestinal parasitic infection can cause anemia in pregnant women and is a major public health problem. Ascariasis, amebiasis, hookworm, giardiasis, and trichuriasis are common intestinal infestations causing anemia. The study detects them by using multiplex real-time polymerase chain reaction (PCR). Methods: Stool samples were collected, transported, DNA extraction was done followed by multiplex real-time PCR was performed using specific primers and probes for their detection. The results so obtained were compared with results of microscopy. Results: A total of 234 pregnant women were enrolled in the study, of which 82.5% were anemic. Among them, parasites were detected by multiplex real-time PCR such as Ascaris lumbricoides in 73%, Ancylostoma duodenale in 8%, Entamoeba histolytica in 17%, Giardia duodenalis in 2%, and Trichuris trichiura 0%. All pregnant women with intestinal parasitic infection were anemic. Multiplex real-time PCR showed a high detection rate as compared to microscopy (100% vs. 14.7%). Polyparasitism was detected in 11% of cases using multiplex real-time PCR. Conclusion: Multiplex real-time PCR enabled the detection of intestinal parasites in pregnant anemic women with distinct advantage over microscopy. It can potentially be used in an epidemiological surveillance study and can potentially influence the treatment in practice for intestinal parasitic infection causing anemia in pregnant women.

Keywords: Anemia, intestinal parasitic infections, multiple parasite detection, multiplex real-time polymerase chain reaction, pregnant women

How to cite this article:
Gupta V, Zahiruddin QS, Gaidhane A, Deshpande P, Tambekar A. Application of multiplex real-time polymerase chain reaction in diagnosis of intestinal parasitic infection among anemic pregnant women. J Datta Meghe Inst Med Sci Univ 2022;17:881-6

How to cite this URL:
Gupta V, Zahiruddin QS, Gaidhane A, Deshpande P, Tambekar A. Application of multiplex real-time polymerase chain reaction in diagnosis of intestinal parasitic infection among anemic pregnant women. J Datta Meghe Inst Med Sci Univ [serial online] 2022 [cited 2023 Apr 1];17:881-6. Available from: http://www.journaldmims.com/text.asp?2022/17/4/881/369489

Introduction

Intestinal parasitic infection is a serious public health problem throughout the world, particularly in developing countries. Amebiasis caused by Entamoeba histolytica, ascariasis caused by Ascaris lumbricoides, hookworm infestation caused by Ancylostoma duodenale, giardiasis caused by Giardia duodenalis, and trichuriasis caused by Trichuris trichiura are the common infections found globally.^[1]

In India, intestinal parasites are major public health problems.^[2] Anemia is defined as medical condition in which there are less than normal hemoglobin (Hb) levels in body.^[3] Reduced Hb would lead to reduced oxygen-carrying capacity of red blood cells to the tissues. It is more prevalent in developing countries because of multiple factors related to nutrition and high prevalence of parasitic infections.

Intestinal parasitic infections, especially helminths and protozoans, can cause anemia in the pregnancy. Helminths and protozoal infections can lead to chronic blood loss from intestine and predispose women to develop anemia. The result of this is low pregnancy weight and intrauterine growth retardation followed by low birth weight, which is associated with greater risk of infections and high perinatal morbidity rates.^[4]

Studies conducted worldwide have reported the varying magnitude of intestinal parasitic infections and anemia among pregnant women, but there is a paucity of published data, particularly on intestinal helminths, parasites causing anemia in India.

Several microscopic-based methods are available and widely used to identify the intestinal helminth eggs and E. histolytica cysts. However, they have their limitation with regard to sensitivity.^[5]

Polymerase chain reaction (PCR)-based techniques are assuming a dominant place in modern diagnostic infectious pathology. They are more sensitive than microscopy for helminths and protozoal infections.^[6] Furthermore, adapting PCR assay to multiplex real-time platforms enrolls simultaneous detection of multiple parasites.

The aim of the current study was to detect intestinal parasitic infections in pregnant anemic women using two multiplex real-time PCR.

Methods

Study population and anemia

Pregnant women taking anti-helminthic/anti-protozoan drugs within the past 2 weeks and very sicking with confirmed acute and/or chronic disease-causing anemia were excluded from the study. Pregnant women were labelled as anemic if Hb < 11.0 g/dl. They were further categorized as mild (Hb levels between 10 and 10.9 g/dl), moderate (Hb 7.0–9.9 g/dl), and severe (Hb <7.0 m/dl).^[7]

Study place

The study was carried out in the Molecular Epidemiology Laboratory, Research and Development, Datta Meghe Institute of Medical Sciences, Wardha, and Department of Pathology of Government Institute of Medical Sciences, Greater Noida, India.

Sample collection

Fecal samples were collected from a total of 234 pregnant women, originating from rural communities in Central India. About 10–20-ml stool was collected in a clean, dry, leak-proof primary container, having 2.5% potassium dichromate w/v (1:1 dilution). The collected container(s) was labeled with the patient's name, ID number, date of birth, and date. Samples were immediately stored at 4°C.

Transportation

The primary container was placed in a durable, watertight container referred to as the secondary container. Several primary containers were placed in one secondary container. Cold chain was maintained for primary and secondary containers.

Microscopy

All fecal samples were examined microscopically and enumerated for Ascaris, hookworms, and Trichuris eggs using a simple sodium nitrate flotation as previously described,^[8] and for the presence of protozoan cysts and oocysts using zinc sulfate centrifugal flotation^[9] Microscopy and concentration method Supplemental File S1.

DNA extraction^[10]

DNA extraction from the stool samples was done using kits and reagents from by HiMedia Cat. No. MB544 Lot No. 0000316458, 0000362802. Steps were followed according to the instruction manual. Briefly, 1-ml TE buffer was added to the collected stool sample and vortexed vigorously followed by centrifugation at ×8000 g (10,000 rpm) for 3 min. Supernatant was discarded. The pellet was resuspended thoroughly in 500 μl of lysis solution. To 200 μl of resuspended solution, 20 μl of the proteinase K solution (20 mg/ml) was added and vortexed and incubated for 30 min at 55°C. A 25 μl of RNase solution was added and incubated for 5 min at room temperature (15°C–25°C). Thereafter, 200 μl of stool lysis buffer was added, vortexed thoroughly, and incubated at 70°C for 10 min. For removal of inhibitors, 250 μl of inhibitor removal solution was added, vortexed, and incubated at 4°C for 5 min. Following this, centrifugation of the tube was done for 1 min at ×10,000 g (12,000 rpm) at room temperature. Supernatant was transferred to a clean collection tube (2.0 ml), and 200 μl of binding solution was added and vortexed for few seconds. Lysate was loaded on the HiElute Miniprep Spin Column and centrifuged for 1 min at ×10,000 g (12,000 rpm) at room temperature. The flow-through was discarded and 500 μl of diluted Wash Solution was added and centrifuged at × 10,000 g (12,000 rpm) at room temperature for 1 min. The flow-through was again discarded and the step was repeated one more time. The flow-through was discarded and centrifuged the HiElute Miniprep Spin column at ×10,000 g (12,000 rpm) at room temperature for an additional 1 min to remove any residual ethanol. The column was transferred to a fresh uncapped collection tube 2.0 ml and 200 μL of elution buffer was added directly onto the center of the column membrane. The tube was centrifuged for 1 min at ×10,000 g (12,000 rpm) at room temperature. The eluate was transferred to a fresh 2-ml collection tube for longer DNA storage.

Primer design

Five major parasites and helminths were selected for this study: A. lumbricoides, E. histolytica, G. duodenalis, A. duodenale, and T. trichiura. The target genes of parasite were selected from GenBank and selected as follows [Table 1] and [Table 2]. The set of primers was designed using GenScript Real-Time PCR (TaqMan) primer design software (https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool). Each primer for amplification was selected from size 18–27 base pairs (optimally 20 base pairs), GC content of 36%–50%, melting temperature ranging from 52°C to 60°C, and probe melting temperature ranging from 62°C to 70°C.

Table 1: Multiplex (triplex) real-time polymerase chain reaction setup overview

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Table 2: Multiplex (duplex) real-time polymerase chain reaction setup overview

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Multiplex polymerase chain reaction

Extracted DNA was run in triplex and duplex real-time PCR reaction. The triplex included A. lumbricoides, E. histolytica, and G. duodenalis [Table 1], and the duplex included A. duodenale and T. trichiura [Table 2]. The PikoReal™ reverse transcription-PCR system (Thermo Fisher Scientific, TCR0096, Foster City, CA, USA) was used. Master mix was prepared using TaqMan mixture 2 × 5 μl; 0.36 μl each forward and reverse primers 25 μM; 0.25 μl probe each 10 μM; DNA template 2.5 μl, and nuclease-free water. Final reaction volume was set at 10 μl, with UNG incubation at 50°C: 2 min, polymerase activation at 95°C: 10 min, followed by 45 cycles of denaturation (95°C: 15 s), and annealing and extension (60°C: 1 min). Reaction profile was selected as FAM (6-carboxyfluorescein [6-FAM]) for positive signals of A. lumbricoides, HEX (hexachlorofluorescein) for the positive signals of G. duodenalis, Cy5 (cyanine) for positive signals of E. histolytica, and ROX (6-carboxy-X-rhodamine) to minimize the noise of unwanted signals for multiplex (triplex) real-time PCR. FAM for positive signals of A. duodenale, HEX for positive signals of T. trichiura, and ROX to minimize the noise of unwanted signals were selected for the reaction profile for other multiplex (duplex) real-time PCR. A signal detected in the fluorescence channels of FAM, Cy5, and HEX before 40 cycles was considered being positive. Threshold and Cq values were obtained and interpreted.

Ethical consideration

Ethical permission for conducting the study was obtained by the Institutional Ethics Committee vide letter number: Ref. No. DMIMS (DU)/IEC/2014-15/1202 dated March 31, 2015. Informed consent was taken from all the patients.

Statistical analysis

The data were cleaned, coded, and double entered in Excel. Then, data were exported to SPSS version 22 (SPSS Inc., Chicago, IL, USA) software for analysis. Results were presented using tables and figures, and conclusion and recommendations were derived accordingly.

Results

Stool samples were obtained from total 234 antenatal women enrolled in this study. A total of 216 out of 234 antenatal women were aged ≤29 years and 18/234 were >29 years of age.

The distribution of species-specific intestinal parasites detected by multiplex real-time PCR among pregnant women is depicted in [Figure 1].

Figure 1: Distribution of species-specific intestinal parasites detected by multiplex real-time polymerase chain reaction among pregnant woman

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The overall prevalence of anemia was 193/234 (82.5%). Mean Hb levels were 9.81 g/dl (standard deviation ± 1.39). Predominant type of anemia was of moderate type. The distribution of status of anemia by species-specific intestinal parasites among pregnant women is depicted in [Figure 2]. Pregnant women infected with A. lumbricoides, A. duodenale, E. histolytica, and multiple parasites had more prevalence of anemia.

Figure 2: Distribution of severity of anemia by species-specific intestinal parasites detected by multiplex real-time polymerase chain reaction among pregnant women

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[Figure 3] depicts the comparison of parasite detection by multiplex real-time PCR and microscopy. Only 6.4% (15/234) were positive overall on microscopy, while 40.1% were detected positive by multiplex real-time PCR. [Table 3] shows the comparison of microscopic examination and multiplex real-time PCR in terms of detection of parasites in pregnant anemic women. The multiplex real-time PCR showed a higher correct identification rate than did the microscopic examination (100% vs. 14.7%). Moreover, incorrect results were obtained more frequently with microscopic examination than with multiplex real-time PCR (85.3% vs. 0%).

Figure 3: Comparison of parasite detection by multiplex real-time polymerase chain reaction and microscopy. Data presented combined for all 234 study participants

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Table 3: Comparison of microscopic examination and multiplex real-time polymerase chain reaction in terms of detection of parasites in pregnant anemic women

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One case was positive for Entamoeba on microscopy but negative for E. histolytica on multiplex real-time PCR. This could be due to detection of other nonpathogenic species of Entamoeba such as Entamoeba dispar on microscopy but could not be detected by real-time PCR as the primers were specific for pathogenic species E. histolytica.

[Table 4] depicts the results of kappa agreement statistics in multiplex real-time PCR and microscopy parasite in anemic pregnant women. The highest value of kappa agreement was found in E. histolytica, i.e., 0.804.

Table 4: Multiplex real-time polymerase chain reaction and microscopy parasite prevalence agreement statistics

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Detection of multiple infections

Along with higher detection rates of all individual target organisms, the multiplex PCR was also superior in terms of sensitivity in detecting samples with multiple infections. E. histolytica and A. lumbricoides were simultaneously detected in 6.34% and A. duodenale and A. lumbricoides in 4% of total positive cases. Polyparasitism was detected by multiplex real-time PCR in 11% of cases, while it was not detected on microscopy [Figure 4].

Figure 4: Polyparasitism observed in the 10 out of 96 samples on multiplex real-time polymerase chain reaction

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Discussion

Our study offers a detailed picture of detection of intestinal parasitic infection among anemic pregnant women using multiplex real-time PCR. We found that among the intestinal helminthes infections causing anemia, A. lumbricoides and A. duodenale were common among pregnant women with anemia.

The overall detection of A. lumbricoides in this present study was 81% comparable with other studies from different parts of the world; Egwunyenga et al.^[11] reported 86% in Southeast Nigeria; Wekesa et al.^[12] reported 47.6% in Kenya; Mengist et al.^[13] reported 28% in Ethiopia; Dwedar et al.^[14] reported 69.6% in Egypt. In India, Segal et al.^[15] reported only 3.4%.

Our study used real-time PCR and reported a higher prevalence as it is a more sensitive and more accurate method of diagnosis.^[16] The other studies utilized microscopic methods that are less reliable and often miss the diagnosis.

The higher prevalence of ascariasis may also be attributed to poor personal hygiene and low economic status. The eggs of the parasite are known to adhere to dust fruits and vegetables, and pregnant women may inadvertently get infected by eating contaminated food items.^[12] Human ascariasis is the most common infection because of spread through fecal pollution of soil. The infection can be acquired accidentally by ingesting embryonated eggs in contaminated food, drink, or soil. Ascariasis ova are also spread by coprophagous animals and thus can be transported to locations far from the defecation site. Well-protected eggs can withstand drying and can survive very lengthy periods in soil.

A large disparity of detection rates was noted by two methods for ascariasis. Multiplex real-time PCR detected a large number of positive samples not detected by microscopy. This difference may also be attributed to errors in microscopy leading to false-negative results. Such errors are less likely in multiplex PCR due to rigorous controls, while limited controls can be implemented with microscopy which relies heavily on the technical expertise of users.^[16]

Hookworm was detected in 9% of cases, comparable to a prevalence rate of 11% in Kenya.^[17] Studies from other parts of India reported it to be 0.5% in Chennai^[2] in pregnant women. This difference can be attributed to better diagnosis offered by real-time PCR.

However, other parts of the world have reported a prevalence of 74.9% in Kilifi (10), 56.6% in Tanzania, 44.5% in Uganda, and 8.1% in Venezuela.^[18] This difference may be due to difference in geographical location, tropical nature of the study which favors hookworm transmission, and difference in the habit of walking bare feet.^[18]

Our study did not find any case of trichuriasis. Its prevalence has not been reported in pregnant women from India, whereas other parts of the world report it: 1.3% in Kenya^[12] and 1.7% in Nigeria.^[19] Trichuriasis is specifically prevalent in warm humid tropics where fecal contamination of soil and water is a major factor in transmission of infection in the community. The difference in prevalence may be due to environmental factors and sample size used in various studies.

E. histolytica infection was found to be the second most common intestinal parasitic infection in pregnant women (17%). A study from North India reported it to be 4%–6%.^[15] It is important to correctly diagnose intestinal parasitic infection caused by E. histolytica as it is associated with fatal health consequences in pregnant women.^[20] Microscopy detected only 4.7% of cases and showed a moderate kappa agreement (0.804) with real-time PCR which detected 6.8% of cases. Microscopy is an outmoded technique that should not be used to diagnose amebic colitis, a condition that may have fatal consequences in women. It is not a sensitive method and is incapable of differentiating pathogenic E. histolytica from nonpathogenic E. dispar, and prone to give false-positive results (Haque et al., 1998). In our study, we found that one case was positive for Entamoeba on microscopy but negative for E. histolytica on multiplex real-time PCR. This could be due to detection of a nonpathogenic species of Entamoeba on microscopy that could not be detected by PCR as the primers were specific for the pathogenic species E. histolytica.^[21]

G. duodenalis was detected in 2% of cases but was not detected on microscopy in any case. The overall prevalence varies: 0.8% reported by Segal et al.^[15] and 4.6% reported by Dwedar et al. in Indian pregnant women.^[14]

Multiple parasites were detected in 10% of cases. Polyparasitism has been reported in studies that have used multiplex PCR. Llewellyn et al. also detected multiple parasites in 37.9% of cases.^[22] There is a scarcity of literature in India population that have detected multiple intestinal parasites using multiplex real-time PCR. Detection of multiple parasites is important as it has deleterious consequences and requires complex treatment.

In our study, 50% of the cases had moderate anemia in pregnant women and 10% had severe anemia that may lead to fatal outcome in pregnancy such as loss of pregnancy, low weight, intrauterine growth retardation, and low birth weight babies associated with greater risk of infection and perinatal mortality.

Conclusion

In summary, the use of multiplex real-time PCR enabled the detection of 41% of cases of intestinal parasitic infections among pregnant women. It proves to be more sensitive and enables accurate determination of intestinal parasites compared to microscopy. Multiplex PCR has a distinct advantage of detecting mixed parasitic infection than standard microscopy techniques. Multiplex PCR can be used in an epidemiological surveillance study and can potentially influence antibiotics prescribing in practice for intestinal parasitic infection causing anemia in pregnant women.

Acknowledgment

We would like to thank to Dr. Manoj, Nishant, Shital, Prabhakar, and research staff at Research and Development and to the patients enrolled in this study.

Financial support and sponsorship

This research work was supported by a research grant awarded by Public Health Research Initiative supported by the Public Health Foundation of India with financial support from SERB, Department of Science and Technology, India.

Conflicts of interest

There are no conflicts of interest.

Supplemental File

Supplemental file S1

The microscopic examination

Direct wet mount analysis stool specimen was obtained from all patients selected for the study. A direct saline and iodine wet mount of each sample were used to detect intestinal parasites microscopically. The wet mounts were examined under a light microscope at ×100 and ×400 magnifications.^[8]

Formol-ether concentration method

A portion of each preserved stool specimen was taken and processed following standard procedures to determine intestinal helminths. Briefly, 1 g of stool was placed in a clean conical centrifuge tube containing 7-ml 10% formol water by using applicator stick. The resulting suspension was filtered through a sieve into another conical tube. After adding 3–4 ml of diethyl ether to the formalin solution, the content was centrifuged at 3200 rpm for 1 min. The supernatant was discarded; smear was prepared from the sediment and observed under a light microscope with a magnification of 100× and 400× after air-dried.^[9]

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