• View in gallery
    Figure 1.

    Lateral flow test for the detection of Cryptosporidium in stool specimens. The lateral flow on the left is an example of a negative test result where only the control line (upper) is positive. A positive test result is shown on the right with both the control and test lines visible.

  • 1.

    Fayer R, Ungar BLP, 1987. Cryptosporidium spp. and cryptosporidiosis. Pediatr Infect Dis J 6: 879.

  • 2.

    Liu L, Johnson HL, Cousens S, Perin J, Scott S, Lawn JE, Rudan I, Campbell H, Cibulskis R, Li M, Mathers C, Black RE, 2012. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379: 21512161.

    • Search Google Scholar
    • Export Citation
  • 3.

    Fayer R, Morgan U, Upton SJ, 2000. Epidemiology of Cryptosporidium: transmission, detection and identification. Int J Parasitol 30: 13051322.

    • Search Google Scholar
    • Export Citation
  • 4.

    Chappell CL, Okhuysen PC, Langer-Curry R, Widmer G, Akiyoshi DE, Tanriverdi S, Tzipori S, 2006. Cryptosporidium hominis: experimental challenge of healthy adults. Am J Trop Med Hyg 75: 851857.

    • Search Google Scholar
    • Export Citation
  • 5.

    DuPont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski W, 1995. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 332: 855859.

    • Search Google Scholar
    • Export Citation
  • 6.

    Guerrant DI, Moore SR, Lima AA, Patrick PD, Schorling JB, Guerrant RL, 1999. Association of early childhood diarrhea and cryptosporidiosis with impaired physical fitness and cognitive function four–seven years later in a poor urban community in northeast Brazil. Am J Trop Med Hyg 61: 707713.

    • Search Google Scholar
    • Export Citation
  • 7.

    Mondal D, Haque R, Sack RB, Kirkpatrick BD, Petri WA Jr, 2009. Attribution of malnutrition to cause-specific diarrheal illness: evidence from a prospective study of preschool children in Mirpur, Dhaka, Bangladesh. Am J Trop Med Hyg 80: 824826.

    • Search Google Scholar
    • Export Citation
  • 8.

    Haque R, Roy S, Siddique A, Mondal U, Rahman SMM, Mondal D, Houpt E, Petri WA Jr, 2007. Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. Am J Trop Med Hyg 76: 713717.

    • Search Google Scholar
    • Export Citation
  • 9.

    Liu J, Kabir F, Manneh J, Lertsethtakarn P, Begum S, Gratz J, Becker SM, Operario DJ, Taniuchi M, Janaki L, Platts-Mills JA, Haverstick DM, Kabir M, Sobuz SU, Nakjarung K, Sakpaisal P, Silapong S, Bodhidatta L, Qureshi S, Kalam A, Saidi Q, Swai N, Mujaga B, Maro A, Kwambana B, Dione M, Antonio M, Kibiki G, Mason CJ, Haque R, Iqbal N, Zaidi AK, Houpt ER, 2014. Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study. Lancet Infect Dis 14: 716724.

    • Search Google Scholar
    • Export Citation
  • 10.

    Llorente M, Clavel A, Varea M, Olivera S, Castillo F, Sahagún J, Rubio M, Gómez-Lus R, 2002. Evaluation of an immunochromatographic dip-strip test for the detection of Cryptosporidium oocysts in stool specimens. Eur J Clin Microbiol Infect Dis 21: 624625.

    • Search Google Scholar
    • Export Citation
  • 11.

    Coris BioConcept Inc., 2012. Crypto-Strip (package insert). Gembloux, Belgium: Coris BioConcept, Inc.

  • 12.

    Hawash Y, 2014. Evaluation of an immunoassay-based algorithm for screening and identification of Giardia and Cryptosporidium antigens in human faecal specimens from Saudi Arabia. J Parasitol Res 2014: 213745.

    • Search Google Scholar
    • Export Citation
  • 13.

    R-Biopharm Inc., 2010. RIDA QUICK Cryptosporidium (package insert). Darmstadt, Germany: R-Biopharm Inc.

  • 14.

    CLONIT, 2012. CRYPTO + GIARDIA dipstick (package insert). Milano, Italy: CLONIT Inc.

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Evaluation of a Rapid Lateral Flow Point-of-Care Test for Detection of Cryptosporidium

Molly E. FleeceDepartment of Medicine, University of Virginia, Charlottesville, Virginia.

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Jack HeptinstallTechLab, Inc., Blacksburg, Virginia.

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Shaila S. KhanInternational Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh.

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Mamum KabirInternational Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh.

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Joel HerbeinTechLab, Inc., Blacksburg, Virginia.

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Rashidul HaqueInternational Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh.

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William A. Petri Jr.Department of Medicine, University of Virginia, Charlottesville, Virginia.

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A new rapid lateral flow fecal antigen detection test for Cryptosporidium was evaluated using diarrheal stool samples from a cohort of children in Bangladesh. The test had a sensitivity of 100% and a specificity of 94% when compared with enzyme-linked immunosorbent assay antigen detection.

Diarrheal diseases are a major cause of morbidity and mortality in the world.1,2 Cryptosporidium is an enteric protozoan parasite that is transmitted through the fecal-oral route, typically by consumption of contaminated food and water.3,4 The parasite has a low infectious dose resulting in diarrhea and abdominal pain.4,5 Cryptosporidium is a common cause of waterborne diarrheal disease worldwide, both in developing and developed countries as well as urban and rural areas; however, due to poor sanitation and urban crowding, Cryptosporidium infections are more prevalent in underdeveloped areas.1,4 Although infections do occur in immunocompetent hosts, immunocompromised hosts and children tend to have a more severe and prolonged disease course.46 There is need for a practical point-of-care diagnostic test that is rapid, reliable, and feasible for use in the field.

Cryptosporidium lateral flow (TechLab, Inc., Blacksburg, VA) is a newly developed immunochromatographic assay that qualitatively detects Cryptosporidium antigen in fecal specimens. It is a dipstick that uses a monoclonal antibody sandwich design to detect Cryptosporidium oocyst wall antigen. The assay flow begins with a diluted specimen that is drawn up via capillary action, the liquid fraction of which liberates membrane-embedded gold particles conjugated with anti-Cryptosporidium antigens. This mixture then flows to the visible reaction window where additional anti-Cryptosporidium antibodies are immobilized and capture antigen–gold complexes for a visual positive result.

The data presented here are of the first field test of the Cryptosporidium lateral flow focusing on the sensitivity and specificity of this rapid dipstick test. All diarrheal stool samples were collected from a cohort of children living in an urban slum in Bangladesh where Cryptosporidium is prevalent.7 The specimens were tested at the International Centre for Diarrhoeal Disease Research, Bangladesh. The samples were stored on average for 2 years at −20°C until testing in batches. Real-time polymerase chain reaction (PCR) testing had been performed on all diarrheal stool samples before this study.8 As a comparison of measurement of the presence/absence of Cryptosporidium antigen, enzyme-linked immunosorbent assays (ELISAs) were performed using the Cryptosporidium II test (TechLab, Inc.). The lateral flow was tested on 50 diarrheal stool samples known to be Cryptosporidium positive by PCR and 50 negative diarrheal stool samples. In addition, 100 randomly selected diarrheal stool specimens from children 6–12 months of age were tested using Cryptosporidium lateral flow and compared with the results of PCR testing.

Fecal samples were brought to room temperature and mixed thoroughly before beginning the test. Fifty microliters of specimen were transferred via pipette into the specimen dilution tube containing diluent (buffered protein solution). The sample end of a test strip was inserted into the specimen dilution tube. Results were read visually after 10 minutes. A sample was interpreted as positive if both test and control lines were present (Figure 1). The color of the lines ranged from dark red to light pink, recognizing that color intensity did not correlate with strength of positivity. A sample was interpreted as negative if only the control line was visible. The test was considered invalid if the control line was absent.

Figure 1.
Figure 1.

Lateral flow test for the detection of Cryptosporidium in stool specimens. The lateral flow on the left is an example of a negative test result where only the control line (upper) is positive. A positive test result is shown on the right with both the control and test lines visible.

Citation: The American Society of Tropical Medicine and Hygiene 95, 4; 10.4269/ajtmh.16-0132

We first tested 50 diarrheal stool samples known to contain Cryptosporidium DNA by PCR and 50 negative controls. Using ELISA as the reference standard for antigen detection, the Cryptosporidium lateral flow had a sensitivity of 100%, 94% specificity, 89% positive predictive value, and 100% negative predictive value (Table 1). Three of the four discrepant specimens (i.e., that were positive by Cryptosporidium lateral flow and negative by Cryptosporidium II test) were confirmed negative via PCR (with the fourth PCR positive).

Table 1

Comparison of the Cryptosporidium lateral flow to the Cryptosporidium II ELISA for diarrheal stool samples

Assay type Cryptosporidium II ELISA (+) Cryptosporidium II ELISA (−)
Lateral flow (+) 34 4
Lateral flow (−) 0 62

ELISA = enzyme-linked immunosorbent assay.

We also evaluated the field adaptability of the lateral flow by testing 100 randomly selected diarrheal stool samples from the same cohort in Bangladesh, the vast majority of which did not have Cryptosporidium. There were no false positives: none of the 96 Cryptosporidium-negative samples had a positive lateral flow result. Of the four diarrhea samples with detectable Cryptosporidium DNA by PCR, the Cryptosporidium lateral flow detected one true positive sample with a Ct value 31.5. The three PCR (+) samples that were not detected by the lateral flow were most likely true negatives (i.e., Cryptosporidium was not the cause of diarrhea), as they had substantially lower amounts of Cryptosporidium DNA (Ct values of 35.2, 36.2, and 38.0). It has previously been shown that the strength of association of PCR (+) samples with diarrhea increases at higher pathogen loads.9

Available alternative rapid antigen detection dipstick tests include the Crypto Uni-Strip (Coris BioConcept, Gembloux, Belgium), RIDA QUICK Cryptosporidium (R-Biopharm, Darmstadt, Germany), and Crypto + Giardia dipstick (CLONIT, Milano, Italy).1014 All these tests have comparable time to results and easy visual result interpretation; however, the other available rapid antigen detection tests above involve at least one additional step in comparison to the Cryptosporidium lateral flow test. We concluded that the Cryptosporidium lateral flow has a comparable sensitivity and specificity to the Cryptosporidium II ELISA and is rapid, reliable, and easy to use in the field.

  • 1.

    Fayer R, Ungar BLP, 1987. Cryptosporidium spp. and cryptosporidiosis. Pediatr Infect Dis J 6: 879.

  • 2.

    Liu L, Johnson HL, Cousens S, Perin J, Scott S, Lawn JE, Rudan I, Campbell H, Cibulskis R, Li M, Mathers C, Black RE, 2012. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379: 21512161.

    • Search Google Scholar
    • Export Citation
  • 3.

    Fayer R, Morgan U, Upton SJ, 2000. Epidemiology of Cryptosporidium: transmission, detection and identification. Int J Parasitol 30: 13051322.

    • Search Google Scholar
    • Export Citation
  • 4.

    Chappell CL, Okhuysen PC, Langer-Curry R, Widmer G, Akiyoshi DE, Tanriverdi S, Tzipori S, 2006. Cryptosporidium hominis: experimental challenge of healthy adults. Am J Trop Med Hyg 75: 851857.

    • Search Google Scholar
    • Export Citation
  • 5.

    DuPont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski W, 1995. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 332: 855859.

    • Search Google Scholar
    • Export Citation
  • 6.

    Guerrant DI, Moore SR, Lima AA, Patrick PD, Schorling JB, Guerrant RL, 1999. Association of early childhood diarrhea and cryptosporidiosis with impaired physical fitness and cognitive function four–seven years later in a poor urban community in northeast Brazil. Am J Trop Med Hyg 61: 707713.

    • Search Google Scholar
    • Export Citation
  • 7.

    Mondal D, Haque R, Sack RB, Kirkpatrick BD, Petri WA Jr, 2009. Attribution of malnutrition to cause-specific diarrheal illness: evidence from a prospective study of preschool children in Mirpur, Dhaka, Bangladesh. Am J Trop Med Hyg 80: 824826.

    • Search Google Scholar
    • Export Citation
  • 8.

    Haque R, Roy S, Siddique A, Mondal U, Rahman SMM, Mondal D, Houpt E, Petri WA Jr, 2007. Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. Am J Trop Med Hyg 76: 713717.

    • Search Google Scholar
    • Export Citation
  • 9.

    Liu J, Kabir F, Manneh J, Lertsethtakarn P, Begum S, Gratz J, Becker SM, Operario DJ, Taniuchi M, Janaki L, Platts-Mills JA, Haverstick DM, Kabir M, Sobuz SU, Nakjarung K, Sakpaisal P, Silapong S, Bodhidatta L, Qureshi S, Kalam A, Saidi Q, Swai N, Mujaga B, Maro A, Kwambana B, Dione M, Antonio M, Kibiki G, Mason CJ, Haque R, Iqbal N, Zaidi AK, Houpt ER, 2014. Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study. Lancet Infect Dis 14: 716724.

    • Search Google Scholar
    • Export Citation
  • 10.

    Llorente M, Clavel A, Varea M, Olivera S, Castillo F, Sahagún J, Rubio M, Gómez-Lus R, 2002. Evaluation of an immunochromatographic dip-strip test for the detection of Cryptosporidium oocysts in stool specimens. Eur J Clin Microbiol Infect Dis 21: 624625.

    • Search Google Scholar
    • Export Citation
  • 11.

    Coris BioConcept Inc., 2012. Crypto-Strip (package insert). Gembloux, Belgium: Coris BioConcept, Inc.

  • 12.

    Hawash Y, 2014. Evaluation of an immunoassay-based algorithm for screening and identification of Giardia and Cryptosporidium antigens in human faecal specimens from Saudi Arabia. J Parasitol Res 2014: 213745.

    • Search Google Scholar
    • Export Citation
  • 13.

    R-Biopharm Inc., 2010. RIDA QUICK Cryptosporidium (package insert). Darmstadt, Germany: R-Biopharm Inc.

  • 14.

    CLONIT, 2012. CRYPTO + GIARDIA dipstick (package insert). Milano, Italy: CLONIT Inc.

Author Notes

* Address correspondence to William A. Petri Jr., Infectious Diseases and International Health, 1709A Carter-Harrison Bldg., University of Virginia, Charlottesville, VA 22908-1340. E-mail: wap3g@virginia.edu

Financial support: This work was supported by NIH grant 5R01 AI043596 to William A. Petri Jr. William A. Petri Jr. is a consultant for TechLab, Inc.

Authors' addresses: Molly E. Fleece and William A. Petri Jr., Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, E-mails: mef8w@hscmail.mcc.virginia.edu and wap3g@virginia.edu. Jack Heptinstall and Joel Herbein, Research and Development, Techlab, Inc., Blacksburg, VA, E-mails: jheptinstall@techlab.com and jherbein@techlab.com. Shaila S. Khan and Rashidul Haque, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh, E-mails: skhan@icddrb.org and rhaque@icddrb.org. Mamun Kabir, Parasitology, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh, E-mail: mamunk@icddrb.org.

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