CaFÉ: A Sensitive, Low-Cost Filtration Method for Detecting Polioviruses and Other Enteroviruses in Residual Waters

Abstract

Acute flaccid paralysis (AFP) surveillance has been used to identify polio cases and target vaccination campaigns since the inception of the Global Poliovirus Eradication Initiative (GPEI) in 1988. To date, only Afghanistan and Pakistan have failed to interrupt wild poliovirus transmission. Circulation of vaccine-derived polioviruses (VDPV) continues to be a problem in high-risk areas of the Eastern Mediterranean, African, and Southeast Asian regions. Environmental surveillance (ES) is an important adjunct to AFP surveillance, helping to identify circulating polioviruses in problematic areas. Stools from AFP cases and contacts (>200,000 specimens/year) and ES samples (>642 sites) are referred to 146 laboratories in the Global Polio Laboratory Network (GPLN) for testing. Although most World Health Organization supported laboratories use the two-phase separation method due to its simplicity and effectiveness, alternative simple, widely available, and cost-effective methods are needed. The CAFÉ (Concentration and Filtration Elution) method was developed from existing filtration methods to handle any type of sewage or residual waters. At $10-20 US per sample for consumable materials, CAFÉ is cost effective, and all equipment and reagents are readily available from markets and suppliers globally. The report describes the results from a parallel study of CAFÉ method with the standard two-phase separation method. The study was performed with samples collected from five countries (Guatemala, Haïti, Thailand, Papua New Guinea, and the Philippines), run in three laboratories-(United States, Thailand and in the Philippines) to account for regional and sample-to-sample variability. Samples from each site were divided into two 500 ml aliquots and processed by both methods, with no other additional concentration or manipulation. The results of 338 parallel-tested samples show that the CAFÉ method is more sensitive than the two-phase separation method for detection of non-polio enteroviruses (p-value < 0.0001) and performed as well as the two-phase separation method for polioviruses detection with no significant difference (p-value > 0.05). The CAFÉ method is a robust, sensitive, and cost-effective method for isolating enteroviruses from residual waters.

Keywords: enterovirus; environmental surveillance; filtration; poliovirus; two-phase separation; vaccine-derived poliovirus; wastewater.

FIGURE 1 |

Geographical location of sample collection cities during the parallel and pilot studies (shown in red circles) (A) Haïti 1) Cap-Haïtien. 2) Gonaïves, 3) Saint Marc, 4) Port au prince (B) Guatemala: 1) San Juan Sacatepéquez, 2) Villa Nueva (C) Thailand 1) Udonthani, 2) Tak, 3) Bangkok. (D) The Philippines: 1) Baguio, 2) Manila, 3) Quezon City. (E) Papua New Guinea: 1) Port Moresby.

FIGURE 2 |

Schematic assembly of a 1 L stainless-steel coffee press, used to press wastewater samples.

FIGURE 3 |

Concentration and Elution Filtration (CaFÉ) method workflow chart. 1) Sample is pressed using coffee press with 11 µm filter. 2) Viral particles are extracted from Sediment—11 µm filter with beef extract and chloroforrn-dithizone. 3) Supernatant is added to the main pressed sample. 4) MgCl₂ is added to the pressed sample-supernatant mixture and pH is adjusted to 3.5. 5) Sample is filtered using 5 µm filter. 6) Filtrate is then, filtered again with 0.45 µm filter and filter pieces from the two filtration stages are combined and eluted with beef extract. 7) The resulted eluate is chloroform treated. 8) Concentrate is inoculated into RD and L20B cell lines after antibiotics treatment. 9) Inoculated samples are observed for 5 days post-inoculation, following WHO ENVS Virus Isolation Algorithm.

FIGURE 4 |

Poliovirus screening results forthe parallel and pilot studies between two-phase separation method and CaFÉ method by poliovirus genotype (n = 345), negative for poliovirus or mixture of multiple polioviruses. NPEV = non-poliovirus enterovirus, Sabin = Sabin-like type 1 or 3, PV2 = Poliovirus type 2, VDPV = Vaccine Derived poliovirustype 1, 2 or 3.