Detection of intestinal parasites was overall higher using multiplex PCR compared to microscopy in our study. While Cryptosporidium spp. detection was similar, multiplex PCR yielded more G. lamblia, B. hominis, and D. fragilis but less E. histolytica and C. cayetanensis. Notably, microscopy is unable to distinguish E. histolytica (pathogen) from E. dispar (non-pathogen) whereas molecular detection is specific for E. histolytica. Multiplex PCR is known to be more sensitive than conventional techniques for parasite detection therefore it is not clear why C. cayetanensis was not detected using multiplex PCR in our study . Shigella spp./enteroinvasive enteroinvasive E. coli was the most commonly detected organism (s) in our study at 80.2%, followed by ETEC (73.3%), Aeromonas spp. (73.3%), EAEC (59.3%) and EPEC (54.7%). This is similar to a study among symptomatic Peruvians in which Shigella spp./enteroinvasive E. coli was the most commonly detected organism at 93% followed by G. lamblia (70%), EAEC (60%), ETEC (48%) and EPEC (41%). G.lamblia was of lower prevalence in our study.
In contrast, detection of diarrheagenic E. coli strains are much lower when examining stool from patients in developed countries, highlighting their common frequency in sanitation poor settings . Even in asymptomatic individuals, prevalence of diarrheagenic strains of E. coli are high in developing countries. For instance, one study detected an enteropathogen in 61% of asymptomatic travelers, with EPEC, EAEC and ETEC being most commonly detected at 42, 28, and 9%, respectively .
Our study reported a 24.5% prevalence of enteric viruses with the most common being norovirus GI/GII at 16.3%. This finding is higher than that reported in other studies examining diarrheal stool from adults with HIV/AIDS, which showed an enteric virus prevalence of 15.9–17%. However, those studies were done using less sensitive conventional techniques [21, 22]. A study examining microbiological etiologies of diarrhea in adult Peruvians detected enteric viruses, with multiplex PCR, in 18% of samples, the majority being norovirus GI/GII at 12% . To our knowledge, no study has yet examined diarrheic stool on HIV/AIDS patients using multiplex PCR for comparison.
Debate exists in the literature as to whether B. hominis, D. fragilis and Aeromonas spp. are true enteropathogens with virulence potential [23, 24]. Among several studies examining HIV populations, there has been no statistically significant difference between B. hominis found in HIV patients with or without diarrhea  and in D. fragilis found in HIV-negative or HIV-positive individuals . Several studies on HIV-positive patients did report higher detection of Aeromonas spp. in those with diarrhea compared to those without [27, 28]. The lack of a control group, unfortunately, makes our study unable to draw any conclusions with respect to the aforementioned organisms and their intrinsic virulence.
Corresponding to known literature, lower CD4 cell counts in our study were associated with a longer duration of diarrhea . Lower CD4 cell counts were not associated with known opportunistic enteropathogens (Cryptosporidium and Isospora) but our sample size was small (N = 9). We detected an association between lower CD4 cell counts and the presence of enteric viruses (norovirus GI/II, rotavirus A, adenovirus 40/41) in this study. Only one virus was detected when CD4 cell count was > 500/μL. Several studies have associated enteric viruses with more advanced HIV infection, suggesting that CD4 deficiency helps mediate enteric viral replication and infection [22, 30, 31]. Astrovirus has been implicated as a potential opportunistic pathogen but none was detected in this study . A study examining stool from symptomatic HIV patients with diarrhea in London, UK found that adenovirus and rotavirus were associated with significantly lower CD4 cell counts compared to coronavirus or norovirus . Our study found that rotavirus and norovirus were independently associated with lower CD4 cell counts. Adenovirus occurred more frequently at lower CD4 cell counts but was not statistically significant, likely due to small sample size in our study (N = 5).
Ninety-four percent of specimens in our study contained a mixture of enteropathogens, with an average of 5 organisms per sample. When excluding B. hominis, D. fragilis and Aeromonas spp., prevalence of mixed infection was still high at 90% with a mean of 4 organisms per sample. High prevalence of mixed enteropathogens has been demonstrated in stool from residents and travelers in developing countries. For instance, 25% and 53% of stool specimens from asymptomatic travelers and travelers with diarrhea, respectively, had two or more bacterial pathogens present when using molecular techniques . All stool samples from symptomatic Peruvians had mixed enteropathogens present with a mean of 4.4 pathogens per specimen when tested using the FilmArray® panel . In a longitudinal cohort study of 147 infants in Dhaka, Bangladesh, multiple enteropathogens were also observed in both asymptomatic surveillance and diarrheal stools with the average number of organisms being 4.3 and 5.6, respectively . In contrast, no enteropathogens were detected in non-diarrheal stool samples taken from infants within their first year of life in Virginia, US . Our study has several limitations. Notably, there was no control group for comparison and our overall sample size was not large enough to provide any reliable conclusions on less commonly detected enteropathogens (e.g. Cryptosporidium spp., Campylobacter spp.).
Conventional approaches, including culture, microscopy, and antigen-based tests have significant limitations such as the limit of detection and the need for labour-intensive procedures. Molecular diagnostics, especially PCR based tests, are rapidly changing research and practice in infectious diseases. A syndromic approach to the diagnosis of diarrheal disease, with its broad range of potential infectious etiologies, may benefit from the use of multiplex molecular formats. However, their applicability due to cost and capital equipment requirements in developing countries is unclear.
In our study, we found a high number of mixed enteropathogens in diarrheal stools taken from symptomatic HIV patients from Ethiopia when using multiplex PCR. To our knowledge, this is the first study to demonstrate this finding in people living with HIV/AIDS in a developing setting using multiplex molecular methods. Most specimens were mixed with diarrheagenic E. coli strains which were similar in prevalence to those seen among diarrheal stools in symptomatic Peruvians . This finding highlights the difficulties in determining colonization versus infection when using extremely sensitive diagnostic methods such as multiplex PCR, and the specific etiological role of each organism in an individual patient. The use of quantitative cutoffs for enteropathogen detection using multiplex PCR has been explored but further clinical studies are needed, especially in developing countries, where high rates of colonization with diarrheagenic strains of E. coli exists [33, 34]. One concern is that inappropriate antimicrobial use may escalate with this technology without a fuller understanding of infection versus colonization. Nevertheless, simplification of these technologies, reduction in cost, and better clinical understanding will undoubtedly enhance patient management.