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

Background Cryptosporidium 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 However, these methods are increasingly being superseded by molecular techniques for diagnostics.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. Methods We 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. Results The 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. Discussion In 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by Massey University (AP), Royal Society Te Aparangi Grant RDF-MAU170 (DTSH), New Zealand Ministry of Health Contract Number 355766-02 (DTSH), and The Percival Carmine Chair in Epidemiology and Public Health (DTSH). The Ministry of Health supported the investigation into the Havelock North outbreak and were informed of this work through annual reports, but had no specific role in this investigation. The other funders had no role in the work. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Havelock North outbreak data were collected through routine public health investigation activities in which the 2006 guidelines from the National Ethics Advisory Committee, Ministry of Health, Wellington, New Zealand, state that these activities do not require ethics committee review and ethical oversight was waived by Massey University Human Ethics Committee. The surveillance data study was approved by Massey University Human Ethics Committee number SOA 17/09. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data are available in the manuscript or publicly available via GenBank and accession numbers are provided.