A pilot comparative study between serological and genetic investigations in relationship to clinical outcomes on patients with cystic echinococcosis

Cystic echinococcosis (CE) is a parasitic disease spread worldwide. It establishes a major public health problem in the regions where sheep and cattle are bred and used for the consumption. The CE is endemic and occurs frequently in Mediterranean regions, Middle East, Asia, North and East Africa, Australia and South America, and some European countries (McManus et al., 2003). In Turkey, the CE has a negative impact on the national economy as well as the health, and still remains one of the most important and serious helminthic diseases. It is quite common due to the wide prevalence of stray dogs and the lack of necessary sanitary precautions (Altintas, 2003; Šnábel, 2009).

In humans, CE may be represented by wide spectrum of clinical manifestations. Clinical signs and symptoms of the disease are not specific and depend on the location of the cyst. The growth rates of cysts may vary between cysts in the same organ or within the same individual and between individuals in various regions. Early diagnosis and prompt treatment are essential to treat disease efficiently. Both, imaging techniques (US, CT) and serology provide useful and complementary information about the character of the cyst that may be relevant for therapeutic intervention. Serological tests provide an extremely important information related to the diagnosis and prognosis of disease (Yolasigmaz et al., 2006; Manzano-Roman et al., 2015).

The CE genotype could affect the biological outcome and disease management In human G1 genotype is the most common (88.44 %) while the G6/G7 genotypes which are closely related strains are less frequent it (11.07 %). On the other hand the G5, G8, and G10 genotypes occur in human rarely (Alvarez Rojas et al., 2014). So far only one human case with G4 genotype was identified (Kim et al., 2017). However, for many genotypes of E. granulosus sensu lato we still have insufficient information regarding e.g. geographical distribution, host specificity, morphology and infectivity. In a study which compared the cysts locations, their size and other features isolated from various strains (E.granulosus G1, E.ortleppi and E.canadensis G6) was noted that G6 genotype showed accelerated growth rate (Guarnera et al. 2004, Alvarez Rojas et al., 2014).

Studies on the genotyping of E. granulosus isolates obtained from different geographic regions of Turkey and intermediate hosts (sheep, cattle, goat, camel, buffalo, horses, mules, and mouflon) confirmed the occurrence of G1-G3, G4 genotypes. Meanwhile in another group of intermediate hosts (sheep, cattle, goat, camel, buffalo, horses, mules) and human regarding the E. granulosus G1,G3 and G7 genotypes were confirmed (Simsek & Eroksuz 2009; Šnábel et al., 2009; Eryıldız et al., 2012; Altintas et al., 2013; Utuk et al., 2013; Gokpinar et al., 2017).

These reports also showed that both, the early CE diagnosis and appropriate hospitalization are very important for lessening the number of fatal cases and serious health outcome (Khachatryan, 2017). In addition, a good symptoms distinction and adequate information about the disease play an important role in death cases prevention (Belhassen-García, M, 2014). The, molecular identification of human CE cases should be suggested for better understanding of pathology, the disease outcome and epidemiology. The essential question which needs to be addressed is whether the link between clinical outcomes and distinct CE genotypes exists.

The aim of this study was to investigate the association between antigenic presentation and antibody response in CE genotype defined patients where the clinical outcomes based CE genotype were compared and analyzed.

Informed written consent was obtained from each participant. The study was approved by the local Clinical Research Ethical Committee.

Human CE caused by the tapeworms Echinococcus granulosus s.s is among the most pathogenic helminthic zoonoses. The larval stages of the tapeworm Echinococcus granulosus sensu lato are the causative agent of CE, one of the most important cestodes infections responsible for the morbidity and mortality in humans and significant economic losses in livestock (Salamatin et al., 2017). Around one million or more people are currently suffering from CE globally. The financial burden with up to $2 billion lost annually on the livestock industry is substantial (Torgerson & Macpherson, 2011). In Turkey, as determined by DNA sequence analysis of COX1 region, the G1 is the dominant genotype in human and the other intermediate hosts (Utuk & Simsek, 2008). The current study which supported this interpretation examined the CE agents responsible for causing 29 cases of liver CE and all were found to be G1. It would also be interesting to see and compare results from different localities and CE patients with different genotypes and with clinical outcomes from various Turkey regions.

For clinical practice it should be noted that the ELISA utilizing crude hydatid cyst fluid has a high sensitivity (over 95 %) but its specificity is often unsatisfactory. It should be remembered that approximately 10 to 20 % of patients with hepatic cysts and about 40 % with pulmonary cysts do not produce detectable specific serum antibodies (IgG) and therefore give false-negative results (Pawlowski et al., 2001). Cysts in the brain, bone, or eye and calcified cysts often induce no or low antibody responses. In routine laboratory practice, usually at least two different tests should be used to obtain the most accurate results (Eckert & Deplazes, 2004). So, in this study ELISA and WB tests were used together to get reliable results. Three patients which were negative by ELISA were found to be positive by Western Blotting in which the USG and CT examinations were also positive.

Study covering Turkey’s east and southeast Anatolian 179 sheep, 19 cattle and 7 goats were examined by the PCR-RFLP method for 205 ribosomal ITS-1 gene region of the E. granulosus isolate. It has been reported that all isolates were of G1 genotype (Utuk et al., 2008). Another study in the western region of Turkey in which 22 E. granulosus isolates (12 sheep, 10 humans) were analyzed by DNA sequencing methods for mitochondrial COX1 and NAD1, and found that G1 genotype were confirmed in 17 isolates, while G1-G3 strain was found in sheep isolate. In Turkey, for the first time G7 strains were detected in two sheep and one human isolate (Šnábel et al., 2009). Analysis of 58 samples (42 human, 13 cattle, and 3 sheep) in Çukurova region of Adana found that the active genotype is solely G1 strain (Eroglu et al., 2016).

The purpose of our study was to determine the cysts genotype obtained from 29 individuals who were diagnosed with CE and compared them with ELISA and Western Blot results. In our recent study, we found that common genotype in human is E. granulosus s.s. G1-G3. There was no consistent correlation between ELISA and Western Blotting results, the number or size of cysts and CE genotype.

Regarding molecular analysis, the COX1 and NAD1 genes and the ITS-1 gene region were amplified in all isolates obtained. When the amplicons of the COX1 and NAD1 genes (COX1:446 bp and NAD1:378 bp) were electrophoresed on agarose gel, a single band was observed. There was no difference in the size of the amplicon’s bands obtained from cysts taken from the same host. The amplicons of the COX1, NAD1 and ITS-1 genes were similar to those obtained from previous studies (Xue et. al 1993; Mwambete et al., 2004; Utuk et al., 2008; Ergin et al., 2010; Eryildiz & Sakru, 2012; Parsa et al., 2011; Mogoye et al., 2013; Adwan, 2013; Yan et al., 2013; Ahmed et al., 2013; Altintas et al., 2013). According to the ELISA results, antibody titers varied broadly and were found low in some patients or very high in others. Immunoblot analysis of EgAg showed many protein bands of with size 8, 12, 20, 22, 24, 36, 75 and 90 kDa. Among of them, 8 – 12 kDa bands, 20 – 22 kDa and 36 kDa bands presented strong reactivity with human serum specimens. Obviously none of the blood serum samples from healthy individuals reacted with EgAg. All patients in our study were found to be infected with the E. granulosus G1-G3 genotype. In conclusion; this was the first study that investigates the correlation between clinical outcomes with specific species or genotypes and serological results using CE parasitic material derived from Turkish human hosts. Our results showed that the E. granulosus s.s. (G1-G3) is predominant in the Aegean Region of Turkey where G7 strain is also present as it was determined in the study published earlier (Šnábel et al., 2009). In order to investigate whether the specific immunoreactions in CE patients is related to the species by DNA sequence analysis and with ELISA and WB results it is necessary to compare different types or genotypes with infected patients antigen and antibody outcomes. In our study it was not possible because all our patients were infected with E. granulosus s.s. (G1-G3 genotype). Unfortunately, the limitation of this study was that there is limited number of patients to search for different genotypes. As reported by Grubor et al., (2017), future studies will give us opportunity to investigate the role of determined genotypes on immunomodulatory effects on parasite infections in humans. However, it might be difficult to understand the host-parasite relationship because of development of CE takes long-time. These findings are extremely important for the better development and improvement of CE diagnosis and treatment, as well as for control strategies and vaccine development.

For many of the genotypes we still have insufficient information. In particular regarding geographical distribution, host relationships in humans and animals, clinical outcomes and pathology. Therefore, these areas of interest need to be investigated more comprehensively on larger groups of patients and hosts. It will be interesting to compare results from different Turkey regions where E. granulosus is quite common. We understand that further studies and collaborations utilizing larger sample sizes are required to examine the links between different genotypes (if possible), localizations, cyst stages as well as clinical and immune parameters outcomes.