Cystic echinococcosis in northern Tanzania: a pilot study in Maasai herding communities | Parasites and vectors

Ethical approval was granted by Kilimanjaro Christian Medical University College, Moshi, Tanzania (09/08/2019), National Health Ethics Review Committee, National Institute for Medical Research, Dar es Salaam, Tanzania (NIMR/HQ/R .8c/ Vol.I/732), the College of Medical, Veterinary and Life Sciences Ethics Committee, University of Glasgow (No. 200180193) and the Veterinary Medicine Research Ethics Committee of the School of Veterinary Medicine (EA36/20).

Prevalence of abdominal cystic echinococcosis in men

A cross-sectional US survey was carried out in November 2019 in five predominantly Maasai pastoral villages in Ngorongoro and Longido districts of Arusha region, northern Tanzania (Fig. 1).

Fig. 1

Map showing the locations of five villages where ultrasound surveys were conducted on volunteers and the locations of two slaughterhouses where livestock sampling was carried out, in Ngorongoro and Longido districts, Arusha region, in northern Tanzania. The map was created with QGIS version 3.22.1. Boundary shapefiles are from https://gadm.org/ and http://geoportal.icpac.net. Human population counts for 2020 at 3 arcsecond resolution (about 100m at the equator) from https://hub.worldpop.org/

Villages were selected from those that had participated in a previous zoonotic disease research program with an initial selection based on a generalized random tessellation stratified sampling approach (additional details described in [27]). Villages in Ngorongoro and Longido districts were selected for this study on the basis of their agro-ecological characteristics, their predominantly pastoralist households and their proximity to abattoirs in the district to allow linking of human and animal prevalence data. . The human population size of these communities in 2019 was based on 2012 census projections (Tanzania National Bureau of Statistics, Dodoma, Tanzania; https://www.nbs.go.tz/index.php/en/) , assuming a growth rate of 2.9% [28]. After obtaining consent from community leaders, meetings in each village were held to explain in Kiswahili and Maasai languages ​​the purpose and protocol of the project, provide information on EC and cerebral coenurosis, and invite potential participants to attend. ultrasound screening (US). session, the place and date of which have been communicated in advance. Participants were recruited after spontaneously presenting themselves at the study sites, on a first-come, first-served basis. Participants of both sexes were eligible if they were ≥ 7 years old and lived in the target area. The threshold age was decided based on the practicalities of performing surgery for CE in the context of the project. Written informed consent/fingerprinting/assent was obtained from individuals, as well as parents/guardians of minors, wishing to participate in the survey. Participation was voluntary and refreshments were provided to compensate for travel to study sites.

Five villages were selected based on time and resources available. Given the small number of villages included, our goal was to detect CE cases and derive prevalence estimates at the village level rather than in the region as a whole. A sample of 230 people in each village would have allowed us to exclude a CE prevalence ≥ 1% with 90% confidence if no cases were detected.

Each interviewed participant was recorded on a case report form, uniquely identified by an anonymous alphanumeric code that was used on all study documents and data files. Abdominal ultrasound was performed in a confidential environment (i.e. a room or building separate from the waiting and registration area), using a portable SonoSite M-Turbo device ( Fujifilm, Seattle, WA, USA) fitted with a convex probe, either by a sonographer (FT) with extensive experience in the diagnosis of CE or by two study physicians (EA, ASal, who have been trained in the point-of-care abdominal ultrasound during the study) under the constant direct supervision of the primary sonographer. In participants with abdominal EC, defined as a focal lesion with pathognomonic signs specific to the stage of parasitic etiology in the US, data were recorded on the location, number, size, and stage of the cyst according to the WHO Informal Working Group on Echinococcosis (WHO-IWGE) classification [9]. Briefly, CE1 (double-walled unilocular fluid-filled cyst), CE2 (fluid-filled cysts with daughter cysts), CE3a (unilocular fluid-filled cysts with detached parasitic layers) and CE3b (daughter cysts in a solid matrix with wrinkled hypoechoic cysts). parasitic layers) cysts were classified as active, and CE4 (solid contents with folded hypoechoic parasitic layers) and CE5 (CE4 with obvious eggshell calcifications) cysts were classified as inactive. People with CE were managed under the project in accordance with WHO-IWGE Expert Consensus Recommendations [9]. In case of suspected CE lesions (defined as focal lesions without sonographic pathognomonic signs of parasitic or non-parasitic origin), a commercial immunochromatographic test (VIRapid Hydatidosis; Vircell, Granada, Spain) would be performed. Advanced imaging (eg, contrast-enhanced computed tomography) was also available at Kilimanjaro Christian Medical Center (Moshi, Tanzania) if needed. If lesions other than CE with clear or potential medical relevance were detected, the participant received a written report and was instructed to attend an appropriate healthcare facility at their own expense.

Cystic echinococcosis in slaughtered cattle

A survey was carried out at district authority slaughterhouses, located in Wasso village in Ngorongoro district and Eworendeke village in Longido district, to determine the prevalence of E. granulosus sl infection in cattle, sheep and goats. These abattoirs serve the herding communities of Ngorongoro and Longido districts and receive livestock from each district, including areas where US human screening has been conducted.

Assuming that approximately 50% of animals would have detectable parasitic cysts and that 50% of these cysts would be due to E. granulosuis sl [14, 21]our target sample size in each slaughterhouse to estimate a prevalence of 25% of E. granulosus sl among livestock with 5% error and 95% confidence interval was 290 animals, with a target of about 100 animals of each species.

All consecutive slaughtered ruminants accessing slaughterhouses between October and December 2020 were assigned a unique code and were inspected by local slaughterhouse inspectors for the presence of visible cysts in the abdomen and lungs. If present, a random cyst was removed from each of three sites, i.e. liver parenchima, liver surface/peritoneum, and lungs (Fig. 2), and preserved in 70% ethanol . Species, age group based on adult incisor pairs, and location of visible cysts were reported on a case report form.

Figure 2
Figure 2

Flow chart showing livestock inspection in slaughterhouses and sampling strategy for parasitic cysts

Prior to the start of the sampling period, training was provided to inspectors on the procedures for studying and identifying parasitic cysts in the form of online sessions due to COVID-19 restrictions. However, no attempt to identify parasitic cysts to the genus level (Echinococcus Where Cysticercus) was requested from inspectors as the training that could be provided was not considered sufficient to ensure correct identification. Due to travel disruptions related to COVID-19, in-person morphological characterization of the cysts by experienced study personnel was not possible. Attempts have been made to use videoconferencing to differentiate between specimens of E. granulosus sl and Taenia hydatigena/Cysticercus tenuicollis, another tapeworm transmitted between canids and cattle forming cyst-like metacestodes usually hanging from the surface of the viscera. However, this was not successful and so identification relied on molecular analysis of a subset of removed cysts, selected as detailed in the Molecular Analysis of Metacestodes section.

Molecular analysis of metacestodes

DNA was extracted from one parasitic cyst per animal. In animals with cysts removed from more than one location, DNA extraction was prioritized from the cyst removed from the liver parenchyma, followed by the lungs, and finally from the liver surface/peritoneum. This diet has been used for E. granulosus sl. cysts more frequently affect the hepatic parenchyma, then the lungs, while C. tenuicollis is most often localized on serous surfaces. DNA was extracted in the Kilimanjaro Clinical Research Institute (KCRI) laboratory from a 1.5-2 cm fragment of each cyst wall using the DNeasy Blood and Tissue Kit ( Qiagen, Hilden, Germany) following the manufacturer’s instructions. Nuclease-free water was included as a negative control in each work session to verify the absence of contamination. The DNA was sent to the WHO Collaborating Center on Epidemiology, Detection and Control of Cystic and Alveolar Echinococcosis (Istituto Superiore di Sanità, Rome, Italy). Molecular identification of metacestode species was performed according to the protocol published by Santolamazza et al. [29]. Briefly, a fragment of the mitochondrial cytochrome oxidase I gene (COX1) was amplified by PCR and the 444 bp fragment digested with AluminumI. Restriction fragment length polymorphism (RFLP) identifies G1–G3 genotypes (Echinococcus granulosus in the strict sense [E.granulosus s.s.]) from G4 to G10 E. granulosus genotypes; the latter are further identified in G4, G5, G6/7 and G8/10 by banding patterns after multiplex PCR. Nope-E. granulosus sl samples were submitted for bi-directional Sanger sequencing of the COX1 PCR product for species identification. The resulting forward and reverse sequences were aligned to generate unique consensus sequences, which were compared to nucleotide sequences deposited in GenBank using BLASTn (nucleotide Basic Local Alignment Search Tool).

Data analysis

Continuous variables were described as mean and standard deviation or median and interquartile range, as appropriate; categorical variables were described as numbers and percentages. The prevalence of human CE at the village level was reported with exact binomial 95% confidence intervals (CI). Given the small number of villages in each district, we did not calculate a 95% CI at the district level or directly compare the prevalence at the district level. We derived 95% CIs for abattoir-level prevalence (since they receive animals from a large area) and compared abattoir-level prevalence using a chi-square (χ) test. The exact binomial confidence intervals were derived in R version 4.1.1. using the pair package (R Foundation for Statistical Computing, Vienna, Austria). The Chi-square test was performed using basis functions in R.

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