Capillaria Ova and Diagnosis of Trichuris trichiura Infection in Humans by Kato-Katz Smear, Liberia.
Among helminth eggs found in human feces, the barrel-shaped eggs of T. trichiura worms are considered to be characteristic, with a length of 50-55 [micro]m, a width of 22-24 [micro]m, and clearly protruding bipolar plugs (6). Similar eggs of other members of the Trichiuridae family may be differentiated from T. trichiura eggs by size and morphology when observed at high magnification, but these eggs have rarely been found in human fecal samples (7-9). Therefore, the presence of eggs of zoonotic members of the Trichiuridae family is generally not considered a confounder for detecting T. trichiura by Kato-Katz smear.
To assess the effect of mass drug administration using ivermectin and albendazole for the elimination of lymphatic filariasis on STH prevalence and intensity, we collected stool samples over a period of 3 years in 2 different areas in Foya district (Lofa County) in northwestern Liberia and in Harper district (Maryland County) in southeastern Liberia (10). We examined a single stool sample per subject by microscopy (magnification x100) with duplicate Kato-Katz smears (41-mg template). We preserved aliquots of randomly selected specimens on FTA cards (GE Healthcare, Little Chalfont, UK) or in RNAlater (ThermoFisher, Waltham, MA, USA) and shipped them to Washington University School of Medicine (St. Louis, MO, USA) for analysis by qPCR. Two experienced microscopists (L.G., A.T. Momolu) examined the samples by Kato-Katz smear in both study areas. For detection of STH by qPCR, we extracted DNA from [approximately equal to] 100 mg of stool and tested it as described by Pilotte et al. (5) with a Quantstudio 6 Flex Thermocycler (Applied Biosystems, Carlsbad, CA, USA) and TaqMan Fast Advanced Mastermix (Applied Biosystems). We used the following primers and probes to detect Schistosoma mansoni DNA: forward primer 5'-TGTGGGAGTCTTTGGTTGTT-3', reverse primer 5'-CAACATGACTGGGAACAGGA-3', probe 5'-AGGTTCAGGTGG/ZEN/GTGTGTTACGAA-31ABkFQ-3'.
We tested 353 stool samples from Foya district by Kato-Katz smear; 31 (8.8%) were positive for A. lumbricoides eggs, 231 (65.4%) for hookworm eggs, 27 (7.6%) for T. trichiura-like eggs, and 276 (78.2%) for S. mansoni eggs. We tested 225 samples from Harper district by Kato-Katz smear; 163 (72.4%) were positive for A. lumbricoides eggs, 65 (28.9%) for hookworm eggs, and 51 (22.7%) for T. trichiura eggs (Table 1). There was good agreement between the results of the Kato-Katz and qPCR tests for the specimens from Harper (80.5%-91.6%), but generally qPCR had higher sensitivity. Our results were consistent with results previously reported with samples from other areas (3, 11). Agreement between the 2 diagnostic tests for samples from Foya ranged from 77.3% to 92.9%, but the sensitivity of the qPCR was unexpectedly low, a finding that was especially true for Ascaris and Trichuris infection (Table 1). Whereas samples positive for Ascaris by Kato-Katz but negative by qPCR had low egg counts, samples positive for Trichuris by Kato-Katz but negative by qPCR had higher counts; 7 samples contained [greater than or equal to] 1,000 barrel-shaped eggs/g of stool (Table 2). We repeated DNA extraction and qPCR and also used an alternative qPCR for T. trichiura (3), but these tests did not improve the agreement between microscopy and qPCR results.
To check further whether Kato-Katz-positive, qPCR-negative stool samples contained T. trichiura eggs, we examined direct smears of stool samples preserved in RNAlater by microscopy (magnification x100 and x400) (Figure 1). The samples positive by qPCR contained eggs (6 measured) with typical T. trichiura morphology; these eggs had a mean ([+ or -]SD) length of 52 [micro]m ([+ or -]2.4 [micro]m) and width of 25.5 [micro]m ([+ or -]1.3 [micro]m). In contrast, qPCR-negative samples contained eggs (31 measured) with a mean ([+ or -]SD) length of 51.8 pm ([+ or -] 1.5 [micro]m) and width of 32.7 [micro]m ([+ or -] 2.1 [micro]m). The qPCR-negative samples also had less pronounced plugs and a thick, striated shell, features that are consistent with eggs of Capillaria hepatica (syn. Calodium hepaticum) and some other Capillaria species (Trichuridae). Eggs of C. philippinensis or C. aerophila that have been observed in human stool samples previously were either smaller or larger than the Capillaria eggs found in Lofa (12,13). Because polar plugs of these eggs are less prominent than those of T. trichiura, and because their shapes are sometimes more oval or round, they can also be confused with A. lumbricoides eggs by low-power microscopy, especially if only a few eggs were detected (Figure 1).
Members of the subfamily Capillaridae are animal parasites with somewhat divergent life cycles, and most do not infect humans. Pseudoinfections with C. hepatica occur; eggs found in stool are present because they were consumed in infected animal liver. However, actual infections with C. hepatica do not lead to the passing of eggs in stool (9). Other species such as C. philippinensis cause true infections (and autoinfection) with eggs found in stool; the infection is linked to consumption of raw fish. Human capillariasis has not been reported from Liberia, and only isolated case reports have been published from sub-Saharan Africa (7-9). We performed DNA sequencing to better characterize the Capillaria species found in Foya. Using the primers Kt875351.1 (5'-CCCTAGTTGCGACTTTAAACGA-3') and Capillaria 18S1R (5'- TCCACCAACTAAGAACGGCC-3'), we were able to amplify and sequence a 288-bp portion of the 18S rDNA from T. trichiura qPCR-negative samples that contained only eggs morphologically identified as Capillaria spp. (GenBank accession no. MG859285). The DNA fragment was 100% identical to orthologs of C. hepatica (accession no. MF287972.1), Aonchothecaputorii (C. putorii) (accession no. LC052356.2), and Pearsonema plica (C. plica) (accession no. MF621034.1), Capillaria worm species that have varying life cycles and host species but that are only 95% identical to the ortholog of T. trichiura.
The life cycle and the medical importance of the Capillaria species found in humans in northwestern Liberia remain to be elucidated. In our study some subjects showed high Capillaria egg loads that may indicate a true infection rather than pseudoinfection. However, transient high egg counts have been reported in persons with pseudoinfections (7). Whereas consumption of bush meat in Foya is common, consumption of raw or undercooked fish, which is necessary for transmission of C. philippinesis, is rare.
This study shows that Capillaria eggs similar to those of C. hepatica are not uncommon in stool samples collected in Liberia. These eggs can be misidentified by Kato-Katz smear as T. trichiura or as A. lumbricoides, which can confound results of STH surveys. The misidentification can also lead to an incorrect assumption that antihelminthic treatment was ineffective. Our results also illustrate the value of qPCR for validating Kato-Katz test results and for explaining unexpected findings.
DOI: https://doi.org/ 10.3201/eid2408.180184
We thank Emanuel B. Gray and Aaron T. Momolu for their expert help during field work, as well as Laura Rinaldi and Guiseppe Cringoli for their advice concerning animal Capillaria species.
The study was funded in part by grant GH5341 from the Bill & Melinda Gates Foundation.
About the Author
Ms. Fischer is a medical technician and staff scientist at the Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. For the last 20 years, her research has focused on medical helminthology and tropical medicine.
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Kerstin Fischer, Abakar Gankpala, Lincoln Gankpala, Fatorma K. Bolay, Kurt C. Curtis, Gary J. Weil, Peter U. Fischer
Author affiliations: Washington University School of Medicine, St. Louis, Missouri, USA (K. Fischer, K.C. Curtis, G.J. Weil, P.U. Fischer); National Public Health Institute of Liberia, Charlesville, Liberia (A. Gankpala, L. Gankpala, F.K. Bolay)
Address for correspondence: Peter U. Fischer, Washington University School of Medicine-Infectious Diseases Division, Department of Medicine, 4444 Forest Park Blvd, St. Louis, MO 63108, USA; email: Pufischer@wustl.edu
Caption: Figure. Helminth eggs found in stool samples from persons in Lofa County, Liberia. A, B) Eggs of Trichuris trichiura in samples positive for T. trichiura by Kato-Katz smear and by qPCR. C-F) Eggs of Capillaria spp. in samples positive for T. trichiura by Kato-Katz smear but negative for T. trichiura by qPCR. G) Egg of Capillaria spp. in sample positive for Ascari lumbricoides by Kato-Katz smear but negative for A. lumbricoides by qPCR. H) Egg of A. lumbricoides in sample positive for A. lumbricoides by Kato-Katz smear and qPCR. Scale bars indicate 20 [micro]m. qPCR, quantitative PCR.
Table 1. Comparison of sensitivity of Kato-Katz smear and quantitative PCR results for 778 stool samples tested for soil-transmitted helminths, Foya and Harper districts, Liberia Site and species No. positive * Kato-Katz smear sensitivity, % Foya district, n = 353 Ascaris lumbricoides 34 91.2 Hookworm ([dagger]) 247 93.5 Trichuris trichiura 27 100 Schistosoma mansoni 307 89.9 Harper district, n = 225 A. lumbricoides 180 90.6 Hookworm f 99 65.7 T. trichiura 86 59.3 Site and species qPCR sensitivity, % McNemar p value Foya district, n = 353 Ascaris lumbricoides 17.6 <0.0001 Hookworm ([dagger]) 83.4 <0.0001 Trichuris trichiura 7.4 <0.0001 Schistosoma mansoni 84.0 0.0573 Harper district, n = 225 A. lumbricoides 98.9 0.0013 Hookworm f 89.9 0.0005 T. trichiura 94.2 0.0001 * Samples that tested positive by either method. ([dagger]) Hookworm was Necator americanus. No Ancylostoma duodenale was detected. Table 2. Demographics and Kato-Katz and qPCR results for patients positive for Trichuris trichiura infection by microscopy, Liberia * Demographics Microscopy, epg Year Patient Age, Village Tt Al Hk Sm no. y/sex 2014 P320529 45/F Yallahun 576 0 360 24 P320683 35/F Kpombu 12 0 0 0 P320695 16/M Kpombu 24 0 0 72 P320620 15/M Foya-Dundu 12 120 0 288 P320746 9/F Bandenin 24 0 0 0 P320452 7/F Felaloe 12 0 0 120 P320596 6/F Foya-Dundu 12 0 0 90 P320656 6/F Kpombu 120 0 0 504 2016 P331772 36/M Kpormbu 3,048 0 12 24 P331921 35/M Felaloe 60 0 0 12 P331783 34/F Kpormbu 420 0 0 0 P330724 26/M Keyabendu 4,224 0 0 456 P331791 6/F Kpormbu 12 0 156 12 P331962 6/F Bandenin 12 0 0 168 P331983 6/F Bandenin 36 0 0 5,304 2017 P341287 61/M Mendikorma 1,464 0 0 0 P341282 56/M Mendikorma 540 0 216 0 P341284 50/M Mendikorma 60 0 0 132 P342148 45/M Keyabendu 1,368 0 0 192 P340246 39/M Kamatahun 120 0 0 216 P340307 19/F Bambuloe 2,028 0 0 1,188 P340133 12/M Fokolahun 1,020 16,392 0 0 P340183 9/F Kpelloe 72 0 0 0 Ndama P341308 9/F Mendikorma 36 0 108 0 P341326 9/M Mendikorma 456 0 0 0 P341327 6/M Mendikorma 2,076 0 0 0 P340147 5/M Fokolahun 48 0 0 0 qPCR, cycle threshold Year Tt Al Na Sm 2014 Neg Neg 31.7 30.5 Neg Neg Neg 28.4 Neg Neg Neg 23.8 Neg Neg 32.2 23.5 Neg Neg Neg 26.51 Neg Neg Neg 23.9 Neg Neg Neg 27.6 Neg Neg Neg 21.3 2016 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 30.4 Neg Neg 33.1 33.4 Neg Neg Neg 29.6 Neg Neg Neg 28.1 2017 Neg Neg Neg 33.1 Neg Neg 28.3 Neg Neg Neg Neg Neg Neg Neg 34.5 Neg Neg Neg Neg 30.0 Neg Neg Neg 24.1 25.3 16.7 Neg Neg Neg Neg Neg 36.0 Neg Neg Neg 28.5 Neg Neg 26.5 30.4 Neg Neg Neg Neg 30.94 26.93 Neg Neg * T. trichiura infection was confirmed by qPCR in only 2 patients, but 25 had Capillaria eggs in their stool. Al, Ascaris lumbricoides; epg, eggs per gram of stool; Hk, hookworm; Na, Necator americanus; Neg, negative; Sm, Schistosoma mansoni; Tt, T. trichiura.
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|Author:||Fischer, Kerstin; Gankpala, Abakar; Gankpala, Lincoln; Bolay, Fatorma K.; Curtis, Kurt C.; Weil, Gar|
|Publication:||Emerging Infectious Diseases|
|Date:||Aug 1, 2018|
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