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Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis [1]

['Mahboubeh Hadipour', 'Department Of Parasitology', 'Mycology', 'School Of Medicine', 'Isfahan University Of Medical Sciences', 'Isfahan', 'Hossein Yousofi Darani', 'Hamid Talebzadeh', 'Department Of Surgery', 'Mohammad Eslamian']

Date: 2023-11

Abstract Background Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated. Methods To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 μl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I. Results Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases. Conclusions Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.

Author summary Cystic echinococcosis (CE) is a disease caused by the larvae of the worm Echinococcus granulosus. Ingesting the eggs of these worms which are excreted in the feces of infected dogs, can result in the formation of cysts in the liver or lungs of humans or herbivorous animals. Although ultrasound is commonly employed for diagnosing these cysts, hydatid cysts can frequently be misdiagnosed as cystic lesions of different origins. Hence, the necessity for faster, more accurate, and non-invasive diagnostic methods is evident. In this study, serum samples were collected from 35 sheep with hydatid cysts, and DNA was extracted and purified. Specific primers were then utilized to amplify the free DNA of the sera via polymerase chain reaction (PCR). Our findings indicate that the diagnostic sensitivity of this disease can be enhanced up to 95% by increasing the volume of serum, employing larger amounts of DNA template in the PCR reaction, and utilizing nested PCR assay. If applied to human samples, this method holds great promise for significantly improving the sensitivity and accuracy of CE diagnosis.

Citation: Hadipour M, Darani HY, Talebzadeh H, Eslamian M, Aboutalebian S, Harandi MF, et al. (2023) Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis. PLoS Negl Trop Dis 17(10): e0011715. https://doi.org/10.1371/journal.pntd.0011715 Editor: Susan Madison-Antenucci, Wadsworth Center, UNITED STATES Received: June 11, 2023; Accepted: October 10, 2023; Published: October 19, 2023 Copyright: © 2023 Hadipour et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All data are included in the paper. Funding: This work was supported by the deputy of research and technology, Isfahan University of Medical Sciences, Isfahan, Iran, Grant No. 3991123, to HY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Cystic echinococcosis (CE) is a neglected tropical zoonosis caused by larvae of the tapeworms Echinococcus granulosus sensu lato. In the life cycle of the parasite, canines are known as the definitive hosts in which the adult tapeworms develop in the small intestine after ingesting livestock offal infected by hydatid cysts. The dogs excrete the infective eggs into the environment through defecation. The eggs are ingested by livestock as intermediate hosts, followed by developing the eggs into the larvae known as hydatid cysts which are located in different organs, especially in the liver and lungs [1,2]. Human indeed is an accidental host that is not directly involved in the parasite life cycle. Cystic echinococcosis is a cosmopolitan disease with an annual incidence ranging from <1 to 200 per 100,000 in humans and a mortality rate of 2% which may be increased if sufficient care management is not provided [3]. Every year, CE causes the loss of about 1 million disability-adjusted life years (DALYs) and USD 3 billion in expenses, including treatment and livestock losses [4]. Elimination or control of CE is difficult, thus active surveillance of the disease should be considered a realistic goal by detecting the parasite in livestock particularly in sheep herds [5]. Carcass inspection is the primary method for identifying sheep with cystic echinococcosis, which has a great limitation. Serological diagnosis of infection in animals has received less attention despite the potential value of such tests for hydatid control programs. The tests have limitations including low specificity (cross-reaction with other taeniid cestode species) and the complexity and unstablity of natural antigen sources [6]. Diagnosing hydatid cysts in humans is based on clinical findings, imaging, and immunodiagnostic tests [7,8]. Clinical manifestations of the disease are not specific and often appear in the later stages of the disease [9,10]. Ultrasound (Us) imaging is considered a safe, low-cost, and rapid technique for diagnosing, following up, and screening of CE [11]. However, hydatid cysts can frequently be misdiagnosed as cystic lesions of different origins [12,13]. Serologic tests based on detecting a parasite antigen or host antibody can be helpful [14,15]; however, they have limitations including cross-reactions, variation in sensitivity and specificity in different conditions, and a lack of differentiating the present and past infections [16–18]. DNA-based assays such as conventional polymerase chain reaction (PCR), real-time PCR, and loop-mediated isothermal amplification (LAMP) have been used for accurate assessment of E. granulosus prevalence in definitive host feces [19–22] or identification of different genotypes in intermediate host [23,24]. Cell-free DNAs (cfDNAs) are extracellular nucleic acid fragments that can be released into body fluids from any exotic cell or organ in various tissue due to apoptosis, cell necrosis, and secretions. cfDNAs can be detected in body fluids including whole blood, serum, plasma, urine, and saliva. The typical features of cfDNAs are small length, low richness, and fast degradation. It is estimated that the size of cfDNA varies from ~40–200 base pairs (bp), with a dominant peak of 166 bp. The concentration of cfDNA in 1 mL of human plasma in normal conditions is estimated to be around 1~10 ng; however, under certain circumstances (more cell death and/or deficient removal of the dead cells) or after exercise, the concentration increases to hundreds of nanograms [25]. They have a half-life of about 10–15 min and are typically removed by the liver. Recently, several studies have demonstrated that specific circulating cfDNAs are promising diagnostic targets for some human diseases, such as autoimmune diseases, metabolic disorders, and cancers [26–28]. Given the current limitations in the laboratory diagnosis of echinococcosis by immunological and imaging approaches, new diagnostic approaches based on the detection of parasite cfDNA should be considered. Recent studies have reported the presence of Echinococcus-derived cfDNA in plasma, serum, and urine samples of infected patients, offering a promising approach toward non-invasive diagnosis. However, PCR or qPCR methods for detecting Echinococcus cfDNA in patients’ sera have shown limited sensitivity, with reported rates of 20–25%. Thus, these methods have yet to be improved and established as reliable diagnostic tools for echinococcosis [25]. To address this issue, our study aimed to enhance molecular techniques’ sensitivity, specificity, and diagnostic validity for detecting E. granulosus sensu lato cfDNA. In the present study we present a semi-nested PCR assay for detecting cfDNA in sheep sera with a potential application for diagnosing CE in human samples. We hope that this study opens up a new avenue for the diagnosis of human echinococcosis in the future.

Discussion Cystic echinococcosis has been registered by the World Health Organization as one of the 20 neglected diseases targeted for control or elimination until 2030. Limitations in diagnostic methods, the low efficacy and side effects of the available drugs, and the problems in the implementation of preventive measures are among the challenges of CE management and control [29]. There are several approaches for CE control and prevention, including an effective livestock vaccine, dog deworming, tailored educational programs, more effective antiparasitic treatments, and the development of better diagnostics for humans as well as the definitive and intermediate hosts humans [5]. Sheep as a domestic herbivorous animals is the key intermediate host of E. granulosus in most endemic regions of the world. The disease causes much economic damage due to increased mortality, decreased productivity, loss of body weight, and high costs of sanitary, highlighting the need for a sensitive and accurate diagnosis technique [30]. The most common method for diagnosis of cystic echinococcosis in domestic livestock is the post-mortem examining of the organs and tissuses at abattoirs. Imaging has been used effectively on a small scale but is not suitable for large-scale epidemiological surveys. The potential of ultrasound investigation for CE diagnosis has shown promising sensitivity (>70%), however, there is variability in specificity caused by the common ovine cysticercosis caused by Taenia hydatigena in many endemic regions [31]. Serological tests for CE in sheep have been generally inconsistent and contradictory, attributed to main challenges in serological diagnosis such as cross-reactivity with other cestodes, false positive results and low sensitivity due to limited immune responses to parasite antigens [32]. cfDNA has been largely applied for detecting parasites such as Plasmodium, Trypanosoma, Leishmania, Schistosoma, and Wuchereria spp. with high accuracy [33]. The limited studies have detected Echinococcus cfDNA in human patients, using PCR-based methods, next-generation sequencing (NGS), and DNA-deep sequencing [25]. NGS and DNA-deep sequencing has demonstrated high sensitivity, but due to costs and inaccessibility in most endemic areas, they are not appropriate for routine clinical examination. PCR-based methods had a sensitivity of 20 to 25% for detecting Echinococcus cfDNA in human patients’ sera. This low sensitivity of the assays might be due to the low concentration of Echinococcus cfDNA in serum and the highly fragmented nature of cfDNA [34]. Moradi et al. reported that the low sensitivity of their PCR method in the detection of Echinococcus cfDNA might be because of the primers detecting long cfDNA fragments (400 or 450 bp); therefore, amplification of the shorter fragment was recommended [35]. Baraquin and colleagues detected parasite-derived cfDNA in the serum of humans infected with alveolar echinococcosis (AE). They also detected parasite-derived cfDNA in sera of experimentally infected animals using real time PCR and droplet digital PCR (dPCR). They used two primers selected from two genomic regions, including a nuclear-repeated region (U1 snRNA) and a mitochondrial region (Nad5). All animals infected with AE and only 25% of human samples were positive. They concluded that a very low concentration of cfDNA in samples is the reason for the low sensitivity of their PCR assay [36]. Toribio et al. detected E. granulosus cfDNA in urine samples of 9 out of 12 patients, by using EgG1Hae III primer to amplify a 133 bp fragment. This primer is present with ~7000 copies arranged in tandem in groups of 2–6 repeats in the E. granulosus genome and possibly is one of the most highly represented nucleic acid species in the Echinococcus-derived cfDNA. In their study, patients that based on imaging methods harbored the active cyst in the liver were positive with the PCR; however, patients harboring calcified liver cysts gave variable results possibly because of the period of calcification and the level of inflammation in the cyst-proximal microenvironment. The sensitivity of the method for identifying the cysts situated outside of the liver, either in inactive or active stages, was less than the sensitivity of the test for liver cysts [37]. In this study, we investigated specific cfDNA detection in the serum samples of sheep infected with E. granulosus as a diagnostic method. Given the low concentration of cfDNA in serum samples, two different volumes of serum samples and different amounts of extracted DNA were compared. Furthermore, to improve the sensitivity of the detection, standard PCR was compared with semi-nested PCR. After evaluation of the results, we found that a combination of some factors, including the application of a larger volume of clinical specimens, a more amount of template DNA, and a two-step PCR amplification, created a higher level of sensitivity (94.2%) in the detection of a low amount of specific cfDNA. The study proves that parasitic cfDNA can be detected with high sensitivity in intermediate hosts infected with echinococcosis. Due to the lack of obvious or specific signs/symptoms and the existence of small cysts during the early stages, timely diagnosis of the disease in humans is difficult. Presently, a combination of imaging and serological techniques is recommended for the diagnosis of CE in humans, which is not straightforward and has some limitations. Therefore, developing a sensitive, accurate, and non-invasive diagnosis method is essential. In this study, we demonstrated that E. granulosus cfDNA present in the serum samples of naturally infected sheep, can be detected with high sensitivity. Hence, it is possible to apply this assay for diagnosing human echinococcosis. Since one of the biggest diagnostic problems is small cysts, thus more investigations are needed using this method on the serum samples of patients with cysts of various stages and sizes.

Conclusion We report a sensitivity of 94.2% by an improved PCR method in detecting E. granulosus sensu lato cfDNA in serum samples of the sheep naturally infected with a hydatid cyst. This high level of sensitivity was related to establishing a new practical protocol based on using a higher volume of sera for DNA extraction, applying a higher amount of DNA in the PCR reaction, and running a semi-nested PCR method. This study provides insights into the diagnosis of human echinococcosis with a higher specificity and sensitivity.

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