Parasitic Disease Surveillance, Mississippi, USA

Richard S. Bradbury; Meredith Lane; Irene Arguello; Sukwan Handali; Gretchen Cooley; Nils Pilotte; John M. Williams; Sam Jameson; Susan P. Montgomery; Kathryn Hellmann; Michelle Tharp; Lisa Haynie; Regina Galloway; Bruce Brackin; Brian Kirmse; Lisa Stempak; Paul Byers; Steven Williams; Fazlay Faruque; Charlotte V. Hobbs

Disclosures

Emerging Infectious Diseases. 2021;27(8):2201-2204. 

In This Article

The Study

To investigate the current prevalence of these infections, we conducted a pilot study to identify STH and other potentially endemic parasitic infections in convenience samples of specimens collected from patients in Mississippi. We deidentified fresh fecal samples submitted for diagnostic testing from patients at the University of Mississippi Medical Center (UMMC; Jackson, Mississippi, USA) during March 30, 2017–February 22, 2018, and serum samples submitted during October 28, 2017–March 29, 2018. This study was approved by the UMMC Institutional Review Board; the Centers for Disease Control and Prevention (CDC) was determined to be nonengaged and therefore did not undertake a separate institutional review board review.

We froze two 250-mg aliquots of feces for later DNA extraction. Where sample volume allowed, we performed microscopic examination using the saturated salt (specific gravity 1.2) passive flotation method as previously described.[4] We extracted DNA by using the SurePrep Soil DNA isolation kit (ThermoFisher, https://www.thermofisher.com) after conducting initial bead beating for 3 minutes using zirconium beads. We stored DNA extracts at −80°C and sent them to the CDC for real-time PCR analysis. At CDC, each sample was initially tested for inhibition and poor DNA extraction by a real-time PCR assay targeting the human cytochrome B gene.[5] Samples positive by this inhibition and extraction control were then tested by multiparallel real-time PCR for STH.[6] A cycle threshold (Ct) ≤35 was considered to represent a positive result. Any positive PCR results were confirmed by duplicate testing.

We froze the deidentified serum samples at –80°C and sent them to CDC, where they were tested for antibodies to Toxocara spp., S. stercoralis, Cryptosporidium spp., and G. duodenalis using MAGPIX multiplex serology (ThermoFisher) (Appendix, https://wwwnc.cdc.gov/EID/article/27/8/20-4318-App1.pdf) to detect evidence of prior exposure. For statistical calculations, we used Excel (Microsoft, https://www.microsoft.com) and R version 3.3.1 (https://www.r-project.org).

A total of 650 fecal samples were obtained from UMMC patients. The median age of patients providing fecal samples for this analysis was 56 years (range 2–95 years). We obtained samples sufficient to perform saturated salt centrifugal flotation on 507 samples (80%). We found no samples to contain helminth eggs or larvae. Sufficient sample for DNA extraction was available for 631 (99.5%) samples. Of these fecal DNA extracts, a negative inhibition and extraction control excluded 37 samples. We tested 224 DNA extracts for Ancylostoma spp., N. americanus, S. stercoralis, A. lumbricoides, and T. trichiura by real-time PCR. (Table 1)

Because prior work in Alabama[3] detected only N. americanus and S. stercoralis infections, we screened an additional 370 DNA extracts for these helminths only. Of these 370 samples, 2 DNA extracts yielded positive amplicons for S. stercoralis (Ct 29.57 and 30.48). The first of these samples (Ct 29.57) yielded no amplification curve on repeat testing and was interpreted as representing an initial false-positive result. The second sample (Ct 30.48) was positive upon confirmatory retesting (Ct 28.52 and 30.49). (Table 1)

A total of 1,960 postdiagnostic serum samples from Mississippi residents were available for multiplex serologic testing. The median age of patients providing serum samples for this analysis was 38 years (range 0–94 years). Of the 1,960 samples, 646 (33.0%) reacted with the Cp17 antigen of C. parvum (range 87–48,448 mean fluorescence intensity [MFI]), and 1,076 (54.9%) reacted with Cp23 (range 377–56,727 MFI). Of those samples, 538 (27.4%) reacted with both C. parvum antigens (Figure 1, panel A), suggesting prior Cryptosporidium species infection. A total of 111 samples (5.7%) reacted with the G. duodenalis VSP3 antigen (range 84–48,547 MFI) (Figure 1, panel B). A total of 38 (1.9%) samples contained antibodies to the Cp17, Cp23 and VSP3 antigens (Figure 1, panel C), demonstrating prior exposure to both Cryptosporidium and G. duodenalis infections. A total of 172 (8.8%) samples contained antibodies to Toxocara spp. Tc-CTL-1 antigen (range 23.2–33,814 MFI) (Table 2; Figure 2, panel A). When Toxocara-seropositive participants ≤6 years of age were excluded, 167/1,814 (9.2%) of UMMC patient samples were seropositive. A total of 9 (0.4%) samples contained antibodies reacting with the recombinant S. stercoralis NIE-1 antigen (range 16.2–11248 MFI) in MAGPIX serologic testing, of which 4 (0.2%) were positive in the confirmatory S. stercoralis CrAg-ELISA (range 9.94–57.7 IU/mL) (Figure 2, panel B).

Figure 1.

Places of residence of participants with antibody levels suggesting prior exposure to Cryptosporidium spp. Cp17 and Cp23 (n = 538) (A), Giardia duodenalis VSP3 (n = 111) (B), and Cryptosporidium spp. Cp17 and Cp23 and Giardia duodenalis VSP3 (combined) (n = 38) (C), Mississippi, USA. All serologic assays were performed using MAGPIX multiplex recombinant antigen beads (ThermoFisher, https://www.thermofisher.com) on convenience serum samples collected at the University of Mississippi Medical Center (Jackson, MS, USA) during October 28, 2017–March 29, 2018.

Figure 2.

Places of residence of participants with antibody levels suggesting prior exposure to Toxocara spp. Tc-CTL-1 (n = 172) (A) and Strongyloides stercoralis Ss-NIE-1 (n = 4) (B), Mississippi, USA. All serologic assays were performed using MAGPIX multiplex recombinant antigen beads (ThermoFisher, https://www.thermofisher.com) on convenience serum samples collected at the University of Mississippi Medical Center (Jackson, MS, USA) during October 28, 2017–March 29, 2018. Only those samples confirmed by a subsequent S. stercoralis crude L3 larval antigen (CrAg) ELISA are included.

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