An Epidemiological, Molecular Characterization and Risk Assessment of Enchinococcus spp in Punjab Province, Pakistan

Abstract

An Epidemiological, Molecular Characterization and Risk Assessment of Echinococcus spp. in Punjab Province, Pakistan Echinococcosis is a zoonotic parasitic disease-causing serious health problem in both humans and animals in different endemic regions across the world. There are two different forms of human echinococcosis: Cystic echinococcosis (CE) and alveolar echinococcosis (AE). CE is caused by the larval stage of Echinococcus granulosus sensu lato and AE by the larval stage of Echinococcus multilocularis. The present study was aimed to determine the prevalence of human cystic echinococcosis in Punjab Province, impact of epidemiological factors on the intensity of CE, molecular characterization, haplotype analysis, knowledge, attitude, and practices (KAPs), risk factor analysis, ELISA assay and to determine the mobility assay for anti-cestodal compounds effectiveness against disease causing agents. The results of this study based on all available clinical records from 8 hospitals revealed a total of 488 patients with echinococcosis were diagnosed. Higher number of histopathologically diagnosed cases were reported for Lahore (n=263), followed by Rawalpindi (n=189), Gujrat (n=12), Bahawalpur (n=10), Multan (n=5), Jhelum (n=3), Attock (n=3) and Kharian (n=3), respectively. Among the 488 cases, Majority of the cases were from rural areas while low CE cases were from urban areas. Within the gender wise distribution, of the 448 patients, 52.66% were females and 47.34% were males, showing higher incidence rate in females. Different age groups showed differential number of cases. It was found that the highest number of individuals infected with CE were under the age group >40-50 with 24.18%. The age group above 70 was least infected with 2.05% of the total patients. Surprisingly, total 37 cases (7.58%) were children with age 0-10, indicating children were also vulnerable to hydatid cyst infection. The echinococcosis cases identified in present study were mostly Pakistani (n=483, 98.98%) and rest were Afghani (n=5, 1.02%). These belong to two major ethnicities of Pakistan. Among studied cases, 240 individuals (49.18%) were Pathan and 248 (50.82%) were Punjabi. The infected individuals were associated with different occupations. Out of 448, the first two most xii vulnerable occupations were farmer (n=99, 20.29%) and butcher (n=37, 7.58%). Other 352 individuals (72.13%) had different professions. In the current study, 488 human hydatid cysts samples were collected from surgically confirmed patients with echinococcosis, from different areas of Punjab, Pakistan. We succeeded to get DNA from only 404 samples, males were 210 (52%) and females were 194 (48%). Among the 404 human echinococcosis samples analyzed, males were 210 (52%) and females were 194 (48%) and this difference was substantially. A larger proportion (76.5%) of echinococcosis cases was reported from rural areas, which have closer contact or association with dogs compared with that in urban areas (23.68%). The mean age of the echinococcosis-positive patients was 32.72 years old, with the oldest patient being 75 years old and the youngest being 5 years old. The liver (53%) was most affected organ, followed by the lung (22.5%), spleen, and others. The cox1 gene was amplified by PCR and yielded a amplification product of 446 bp, while cytb yielded a 580 bp fragment, and nad1 yielded a 900 bp product in all samples. The nucleotide sequences of all sequenced Pakistani samples were aligned with the reference sequences of each genotype within E. granulosus, which were retrieved from GenBank. According to the sequence analysis of the cox1, cytb, and nad1 genes, E. granulosus s.s. (n = 399), E. canadensis (G6/7), (n = 2), and E. multilocularis (n=1) were detected. The findings showed that majority of the patients (399/404) were infected with E. granulosus s.s. In the sequence comparison, the cytb gene from the Pakistan samples of E. granulosus s.s. showed 100% similarity with the other selected reference sequences. All the other compared G6, G7, and G10 samples, including the samples from Pakistan, showed the same sequences. For the nad1 gene, PUN-131- Pakistan was conserved when compared with the selected genotypes, whereas point mutations and substitutions were found in some of the other compared sequences. PUN 116-Pakistan sample had point mutations reported from France and Turkey, while other sequences of E. granulosus s.s were conserved. For KAPs analysis, a total of 793 questionnaires were filled from three categories i.e. rural, urban population and butchers. The percentage of questionnaires filled from villagers was 34.3% and from urban populations who have dogs was 29.4% while percentage from the sites of butcher and abattoirs was 36.3%. Participants involved in this study were dog owners and peoples who keep animals. By combining the data from xiii three different sites we concluded that most of the participants were male with 60.02% (476/793). The percentage of female participants was low as 39.97% (317/793). The interviewed participants were adults of age group of 15-35 years old with highest percentage of 68.47% (543/793) while other age groups are of 36-56 years old with 26.61% (211/793) and from 57-77 years old with 4.92% (39/793). Diagnostically significant titers are present in a serum dilution of 1:32 to 1:64 or higher. Titers between 1:512 and 1:2048 are regarded as mean serum titers in patients with cystic echinococcosis. Low positive titers (1:32 to 1:128) should only be assessed together with the positive result of a second serological method such as CFR, and ELISA etc. The seroprevalence was 13.28% in male and 12.8% in female. Age wise infection was found to be higher in age between 15-36 as 19.38%, as compared to other age groups. Ethnic attribute also showed differences in seroprevalence rate of CE infection as pathans have high CE infection rate as found 6 infected people out of 32 with seroprevalence 18.75% as compared to Punjabis(11.84%) and others (1.25%). Findings showed that the seroprevalence of CE is much more in uneducated people as compared to people having primary, secondary and post-secondary education. Butchers were found to be infected much more as compared to the people with other occupations. The seroprevalence was 23.07%, in butchers while it was 5.88% in farmers and livestock keepers and others with multiple professions it was 2.43%. The anti-cestodal activity showed a significant effect against Echinococcus spp. These findings suggested that E. granulosus s.s. is the dominant species in humans in Pakistan. In addition, E. canadensis (G6/G7) and E. multilocularis are circulating in the country. Further studies are required to explore the genetic diversity in both humans and livestock.