Performance of skimmed milk flocculation and real-time polymerase chain reaction for the concentration, detection, and quantification of Taenia solium in urban wastewater from an endemic region of the Peruvian central highlands

Abstract

The taeniasis/cysticercosis complex, caused by Taenia solium, is a significant zoonotic disease in low- and middle-income countries. Although this complex has been extensively studied in rural communities, information on its status in urban areas remains limited due to the financial and logistical challenges of conducting large-scale assessments in cities. This study aimed to demonstrate the feasibility of using wastewater monitoring for the epidemiological surveillance of T. solium in cities. We evaluated four extraction methods (magnetic beads, bead-beating, spin column, and thermal shock) on serial dilutions of T. solium eggs. Statistical analysis demonstrated that the magnetic bead method achieved the highest quantifications of genomic copies (GC). These values were used to develop a simple linear regression to establish an equivalence between the GC of the internal transcribed spacer 1 (ITS-1) region and eggs. Subsequently, we assessed the performance of skimmed milk flocculation, along with the impact of inhibitory substances, in two distilled water samples and two wastewater samples (250 mL each) seeded with low (5000) and high (25000) eggs numbers. Recovery percentages in distilled water were 98 ± 6 % (high concentration) and 26 ± 11 % (low concentration), while in wastewater, they were 76 ± 11 % and 12 ± 3 %, respectively. Over 16 months, wastewater monitoring in a highly endemic city of the Peruvian Central Highlands detected T. solium in 17 % (12/70) of samples using a real-time polymerase chain reaction targeting the T. solium ITS-1 region, with concentrations ranging from 8.92E+02 to 8.07E+06 GC/100 mL, equivalent to 0.7 and 4510 eggs/100 mL, respectively. Sanger sequencing of the cytochrome c oxidase subunit 1 gene confirmed the presence of T. solium in 4 of the 12 positive samples for the ITS-1 region. In conclusion, T. solium can be detected and quantified in wastewater, supporting its use for urban human taeniasis surveillance.

Keywords: Peruvian central highlands; Recovery efficacy; Skimmed milk flocculation; Taenia solium; Urban wastewater.