Diagnosis of protozoa diarrhoea in Campylobacter patients increases markedly with molecular techniques

BackgroundCryptosporidium and Giardia are major food and water-borne causes of diarrhoea globally, and two of the most notified infectious diseases in New Zealand. Diagnosis requires laboratory confirmation carried out mostly via antigen or microscopy-based techniques. However, these methods are increasingly being superseded by molecular techniques for diagnostics. Here we investigate the level of protozoa coinfection identified by molecular methods in Campylobacter positive samples missed through use of antigen-based assays and then investigated different molecular testing protocols. MethodsWe report the findings of two observational studies; the first among 111 people with diarrhoea during a large Campylobacter outbreak in Havelock North, and the second a study during normal surveillance activities among 158 people presenting with diarrhoea and a positive Campylobacter test, but negative Cryptosporidium and/or Giardia antigen-based diagnostic test result. The molecular methods used for comparison with the antigen-based tests were in-house end-point PCR tests targeting the gp60 gene for Cryptosporidium and gdh gene for Giardia. DNA extraction was performed with and without bead-beating and comparisons with commercial real-time quantitative (qPCR) were made using clinical samples diluted down to 10-5 for Cryptosporidium positive samples. ResultsThe coinfection prevalence was 9% (n= 10, 3-15% 95%CI) for Cryptosporidium and 21% (n=23, 12- 29% 95%CI) for Giardia in the 111 Campylobacter patients of the Havelock North outbreak. The coinfection prevalence was 40% (n=62, 32-48% 95%CI) for Cryptosporidium and 1.3% (n=2, 0.2-4.5% 95%CI) for Giardia in the 158 routine surveillance samples. Sequencing identified Cryptosporidium hominis, C. parvum, and Giardia intestinalis assemblages A and B among patients. We found no statistical difference in positive test results between samples using end-point PCR with or without bead-beating prior to DNA extraction, or between the in-house end-point PCR and qPCR. The qPCR Ct value was 36 (35-37 95%CI) for 1 oocyst, suggesting a high limit of detection. DiscussionIn surveillance and outbreak situations we found diagnostic serology testing substantially underdiagnoses Cryptosporidium and Giardia coinfections in Campylobacter patients. These findings suggest that the impact of protozoa infections may be underestimated, through underdiagnosis, but molecular techniques likely improve detection capabilities. Laboratories need to understand clinical, rather than analytical, test sensitivity, to allow clinicians to better understand the disease aetiologies of patients that enable better health advice.