Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context

Nyirenda, James T., Henrion, Marc, Nyasulu, Vita, Msakwiza, Mike, Nedi, Wilfred, Thole, Herbert, Phulusa, Jacob, Toto, Neema, Jere, Khuzwayo C., Winter, Alex, Sawyer, Leigh A., Conrad, Thomas, Hebert, Donnie, Chen, Crystal, Van Voorhis, Wesley C., Houpt, Eric R., Iroh Tam, Pui-Ying ORCID: https://orcid.org/0000-0002-3682-8892 and Operario, Darwin J. (2023) 'Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context'. PLoS ONE, Vol 18, Issue 9, e0289929.

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Abstract

Background:
Cryptosporidium is a gastrointestinal pathogen that presents a serious opportunistic infection in immunocompromised individuals including those living with human immunodeficiency syndrome. The CRYPTOFAZ trial, previously published, was conducted in Malawi to evaluate the efficacy of clofazimine in response to an unmet need for drugs to treat cryptosporidiosis in HIV populations. A combination of rapid diagnostic tests, ELISA, qPCR, and conventional sequencing were employed to detect Cryptosporidium in 586 individuals during pre-screening and monitor oocyst shedding and identify enteric co-pathogens in 22 enrolled/randomized participants during the in-patient period and follow-up visits.

Methodology:
Oocyst shedding as measured by qPCR was used to determine primary trial outcomes, however pathogen was detected even at trial days 41–55 in individuals randomized to either clofazimine or placebo arms of the study. Therefore, in this work we re-examine the trial outcomes and conclusions in light of data from the other diagnostics, particularly ELISA. ELISA data was normalized between experiments prior to comparison to qPCR. The amount of all identified enteric pathogens was examined to determine if co-pathogens other than Cryptosporidium were major causative agents to a participant’s diarrhea.

Conclusion:
ELISA had higher sample-to-sample variability and proved to be equally or less sensitive than qPCR in detecting Cryptosporidium positive samples. Compared to qPCR, ELISA had equal or greater specificity in detecting Cryptosporidium negative samples. Sequencing identified several Cryptosporidium species including viatorum which has never been identified in Malawi and Southern Africa. In addition to Cryptosporidium, enterotoxigenic E. coli was also identified as a pathogen in diarrheagenic amounts in 4 out of 22 participants.

Item Type:	Article
Subjects:	WC Communicable Diseases > Virus Diseases > Acquired Immunodeficiency Syndrome. HIV Infections > WC 503 Acquired immunodeficiency syndrome. HIV infections
Faculty: Department:	Clinical Sciences & International Health > International Public Health Department Clinical Sciences & International Health > Malawi-Liverpool-Wellcome Programme (MLW)
Digital Object Identifer (DOI):	https://doi.org/10.1371/journal.pone.0289929
SWORD Depositor:	JISC Pubrouter
Depositing User:	JISC Pubrouter
Date Deposited:	15 Sep 2023 12:49
Last Modified:	15 Sep 2023 12:49
URI:	https://archive.lstmed.ac.uk/id/eprint/23135

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