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

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.

Performance and safety of the induced sputum procedure in young children in Malawi: a prospective study.

BACKGROUND: Collecting sputum specimens is a challenge in infants and young children. We assessed the performance and safety of induced sputum (IS) collection in this population, embedded in a prospective study evaluating respiratory cryptosporidiosis in Malawian children with diarrheal disease. METHODS: We assessed the sputum quality and correlation with detection of Cryptosporidium spp. and evaluated safety and adverse events in 162 children. RESULTS: Among 159 stool specimens tested, 34 (21%, 95% CI 15.0 to 28%) were positive for Cryptosporidium spp. There were 160 IS and 161 nasopharyngeal (NP) specimens collected. IS and NP specimen collection was performed for each patient. The majority of IS specimens (122/147; 83%) were clear in appearance and 132/147 (90%) were of good quality. Among the respiratory specimens tested, 10 (6.3%, 95% CI 2.5 to 10%) IS and 4 (3%, 95% CI 0 to 5%) NP were positive for Cryptosporidium spp. When stool cryptosporidium PCR was the gold standard, IS PCR sensitivity was higher (29%, 95% CI 22 to 37%) compared with NP PCR (12%, 95% CI 7 to 17%) for detection of Cryptosporidium spp. One (0.4%) adverse event occurred, consisting of a drop in oxygen saturations at the 30-min postprocedure evaluation. Consciousness level, median respiratory rate and oxygen saturations were unchanged, before or after IS. CONCLUSIONS: IS provides good quality specimens, is more sensitive than NP specimens for diagnosis of respiratory cryptosporidiosis, and collection can be performed safely in children hospitalized with diarrheal disease.