We found a surprisingly low prevalence of all parasites, including the protozoa of interest. A retrospective analysis published in 2009 reported rates of parasitic infection based on stool microscopy at this same clinic of 13.3% and 18.1% of children for G. lamblia
and E. histolytica
That study compiled data from an entire year and all of the 10 communities that visit the clinic, whereas our study was more limited. Seasonal variability or the individual communities tested may have played a role in the difference in rates of infection between our study and theirs.
As expected, there was only fair agreement between microscopy and ELISA, represented by the low Kappa coefficient of 0.49. We had anticipated that microscopy would miss cases of Giardia
spp. detected by ELISA, but we also found samples were reported positive by microscopy and not by ELISA. Because microscopy has traditionally been the gold standard for diagnosis of Giardia
spp., its specificity is unclear from the literature, but is in part related to the skill and experience of the microscopist. However the specificity of stool ELISA is reported to be 100%, and this testing modality is less user dependent.8
Unfortunately, we had no facilities to perform the polymerase chain reaction, but given the test parameters of stool ELISA for detecting Giardia
spp., results by this method are likely more reliable.
Use of ELISA enabled detection of an additional 29 cases of Giardia
infection, more than doubling the true positive case detection by microscopy among 620 children. Increasing the sensitivity of diagnostics for enteric infections may be important for preventing morbidity. Studies in Brazil have shown reduced physical fitness associated with early childhood diarrhea years afterwards, growth deficits of 3.6 cm at seven years of age, and cognitive deficits.14–16
Although mortality from diarrheal illness has greatly decreased since 1955, rates of illness have not changed, suggesting that morbidity remains severe.17
In performing this study we hoped to determine the feasibility of using ELISA stool detection routinely in a small rural clinic. The laboratory available consisted of a small sink, two desks, small waste basket, and a window. Equipment included a light microscope and a centrifuge. There was no available refrigeration for laboratory use. In this setting, it was possible to perform ELISAs. Because the lack of refrigeration meant that samples had to be tested the day they arrived, batching samples over multiple days was not possible. The samples were collected at home 1–2 days before being brought to the laboratory and rarely were tested the next business day. This limitation may have adversely affected the ELISA results. However, it more likely affected the detection of protozoa by microscopy, which might explain the low sensitivity of microscopy in our study. Conducting four ELISAs required significant space and produced more solid waste than was typical for the laboratory. Limitations affecting the ELISA performance included the type of water used for mixing the buffer solution, lack of an optical density reader, and lack of refrigeration. However, positive and negative controls were consistently within manufacturer's specifications, reassuring us that this did not affect our results.
The TRI-COMBO showed a good correlation with individual assays. In locations with high numbers of all three parasites, this correlation would provide a simplified method of screening stools for a variety of infections after which positive samples could be tested further for individual pathogens. The time required for a single laboratory technician to perform three separate ELISAs is substantial and using the TRI-COMBO could decrease this time considerably.
The prevalence of Giardia spp., E. histolytica, and Cryptosporidium spp. among school children in the Palajunoj Valley measured by stool antigen ELISA was lower than previously reported at 8.4%, 0.3% and 0.5%, respectively. Use of a stool antigen ELISA can greatly augment case detection of these pathogens and is feasible in a rural laboratory, but does require additional equipment and laboratory space, and produces more solid waste than standard microscopy. Use of a triple-screening ELISA such as the TRI-COMBO is accurate and can be used to simplify the ELISA screening process where giardiasis, cryptosporidiosis, and/or amebiasis are suspected.