Application of a multiplex PCR assay for the detection of gastrointestinal pathogens in a rural African setting

The present study revealed a large proportion (96.6 %) of Luminex positive stool samples among children with diarrhoeal disease, with co-infections detected in more than 75 % of symptomatic cases. The samples analysed in the current study are a subset of samples collected for a larger study on diarrhoeal disease in Ghanaian children. In that study a mix of culture and PCR methods was used to diagnose isolates and a positive result was found in 79.5 % of the analysed samples [22]. A previous multicentre study conducted in sub-Sahara Africa and South Asia detected pathogens in 83 % of diarrhoea samples, using a mixture of conventional bacterial culture methods, immunoassays as well as molecular detection methods for viruses [1]. As expected, lower positivity rates are reported with routine detection methods (i.e. bacterial culture, ELISA, latex agglutination tests, singleplex real-time PCRs, stool microscopy, viral culture) by laboratories from industrialised countries, such as the Netherlands (6.4 %), the United States (8.3 %) and New Zealand (18 %) [9, 17, 19]. The same three laboratories increased the detection frequency of gastrointestinal pathogens 2–4-fold when using a multiplex real-time PCR assay. Compared to the present study, similarly high detection rates (78 %) have been reported from asymptomatic children attending day care facilities in the Netherlands using a quantitative real-time multiplex PCR [26].

Parallel to the high detection rate, co-infections occur in most of the diarrhoeal samples in the present study. Co-infections seem to be less frequent in the United States (14.1 %) and rather exceptional in the Netherlands (0.9 %), although PCR methods were applied in both settings [17, 19], however mainly on adult patients. This difference is probably explained by the high number of asymptomatically circulating pathogens among children living in rural Ghana under low hygiene and sanitary conditions [27]. Those circumstances raise the question, whether highly sensitive multiplex PCR methods are able to provide valuable information for the diagnosis of gastrointestinal infection in sub-Saharan Africa.

Multiplex molecular diagnostic tests might have an important role in epidemiological surveillance, research projects and outbreak investigations [6]. They might also contribute to a better understanding of complex and severe clinical cases as well as to the application of efficient treatment options [21]. However the reduction of antibiotic prescriptions due to multi-parametric molecular diagnostic tools is still debated. Previous studies showed increases as well as decreases of antibiotic consumption, when multiplex PCRs for the diagnosis of respiratory pathogens were investigated [28, 29].

Studies from resource-rich settings demonstrated that the use of stool multiplex PCR panels might be cheaper than using different individual methods for the same number of pathogens when all costs of reagents and technicians’ time are taken into account [9, 30]. However those costs might still be too high for rural laboratories in sub-Saharan Africa. Furthermore, the lack of technical expertise on molecular diagnostic techniques might be a constraint.

It might be worth to investigate whether for this study setting a sequential or stepwise diagnostic algorithm might be more suitable and cost-effective. Within such an algorithm, all children with diarrhoea could be tested for pathogens with a high AF in a first screening procedure. Only those children tested negative would undergo further screening for other pathogens. Another approach would be the development of quantitative or semi-quantitative PCR assays, which distinguish between causative and colonizing pathogens [31, 32]. In general, the decision on the most appropriate diagnostic tests for resource-poor settings must not rely on test evaluations in industrialized countries.

The study has several limitations. Sensitivities and specificities of the Luminex PCR assay cannot be assessed since results have not been compared to a reference method or confirmed by another assay. A previous study, which analysed a different sub-population within the same stool sample collection using a mixture of conventional and PCR detection methods, revealed similar AFs, but lower positivity rates [22]. Indeed, the Luminex assay has been evaluated with high sensitivities and specificities in previous studies [18, 19, 21, 30, 33‐36]. However the Luminex PCR requires open manipulation of an amplified product, which carries the potential for carryover contamination, resulting in false positive results. We recruited cases as well as controls at the hospital OPD, hence the control group consists of children being sick with conditions other than diarrhoea. Occasionally gastrointestinal pathogens might lead to vomiting without diarrhoeal symptoms, e.g. as described for Cryptosporidium parvum [37], which may result in an underestimation of the association between gastrointestinal pathogens and diarrhoea in this study.