Etiological Spectrum of Infective Diarrhea in Renal Transplant Patient by Stool PCR: An Indian Perspective

Multiplex PCR

The FilmArray GI panel test is FDA, Conformité Européene–In Vitro Diagnostics (CE-IVD) and Therapeutic Goods Administration (TGA) certified nested PCR assay. Two hundred microliters of the specimen in Cary-Blair transport medium was subject to FilmArray GI Panel testing, according to the manufacturer's instructions. The FilmArray GI Panel test consists of automated nucleic acid extraction, reverse transcription, amplification, and analysis, with results available in 1 h per run per specimen. Each FilmArray GI Panel pouch contains an internal nucleic acid extraction control and PCR control. The FilmArray GI Panel runs were considered valid if the run completed normally and internal controls passed. The FilmArray GI Panel software performs automated result analysis with each target in a valid run reported as detected or not detected. If either internal control fails, the software automatically provides a result of invalid for all panel analytes. All specimens having invalid results were retested.

The panel targets include:-

CMV

All transplant patients with diarrhea but negative stool PCR, CMV RT-PCR, and colonoscopy were done. Both the tests were also sought when the patient presented with >2 organ involvement along with diarrhea. Test to diagnose CMV infections were not done in control group

Real-time PCR for CMV DNA quantification

QPCR for CMV was performed in both biopsy and plasma samples in all the patients using the artus CMV RG PCR kit (Qiagen, Hilden, Germany), as per the manufacturer's instructions. The assay targets a 105 bp region of the glycoprotein gene of CMV genome. Internal control was added to each sample before extraction, and each batch of PCR included four positive and one negative control. CMV viral load levels were then expressed as the number of CMV DNA copies/mg of tissue for intestinal biopsy samples and in copies/mL for plasma samples. The dynamic range of the assay is 102–106 copies/mL with a lower limit of detection of 57.1 copies/mL.

Extraction of DNA from intestinal biopsy and blood

All the biopsy samples were collected in a sterile plain vial and transported to the laboratory on ice. Extraction of DNA from biopsy tissue sample was done using the automated DNA extraction system (QIAsymphony, Qiagen, Germany) using the DSP virus/pathogen kit, with slight modifications. Tissue was weighed, and 5 mg of the tissue was digested by proteinase K before processing. The volume was adjusted to 750 μl with AVE buffer (Qiagen, Germany). Plasma (750 μl) samples were directly processed in the automated Qiasymphony, as per manufacturer instructions.

Statistical analysis

Quantitative values for variables were given as medians ± extreme values or means ± standard deviations, and qualitative data were given as numbers and percentages. Pearson's Chi-square test or the Fisher exact test was used to compare qualitative variables. Between-group comparison of numeric parametric data was done by unpaired t-test. Results were considered to be statistically significant for two-sided P values < 0.05.

Microbiological yield

A total of 86.3% of samples yielded one or more organisms in stool PCR. A total of 181 organisms were isolated from 110 diarrheal event. Bacterial infection was the most common etiology, with almost 50% of all infections being bacterial in origin [Figure 1]. The majority of infections were coinfections, with 68% of positive PCR had two or more organisms [Figure 2]. Overall, Norovirus G1/G2 (20%) was the most common organism isolated, followed by G. lamblia (17%), and Enteropathogenic Escherichia coli (EPEC) (16%) [Table 2]. The frequency of Norovirus infection was significantly higher as overall infection as well as isolated infection in the transplant group than the control group [Table 3]. Frequency of Cryptosporidium spp. and EPEC was also significantly higher in the transplant group than the control group. PCR was positive in 87.4% of samples in the control group. 270 organisms were isolated from the 194 diarrheal events. GI Infection was dominated by bacteria 64.4%, followed by an equal incidence of parasite and viral infection [Figure 1]. Overall, G. lamblia, Enteroaggregative E. coli (EAEC), Shigella/Enterotoxigenic E. coli (ETEC) were present in equal frequency (13%) [Tables 2 and 3]. Clostridium difficile, Vibrio cholerae, Salmonella, and Rotavirus were significantly higher in the control group than the transplant group.

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Figure 1

Graph showing the frequency of various categories of gastrointestinal pathogens detected by stool PCR in each group

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Figure 2

Graph showing the frequency of the number of organisms per person in coinfection detected by stool PCR in each group

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The spectrum of diarrhea in the non-transplant population

The spectrum of etiological agents was much more diverse among the control population. A comparison of various studies regarding the frequency of the infective organisms has been shown in Table 5. Most of the studies were conducted in west and tested only few organisms.[58910] Findings in our study closely matches with the study from Turkey by Arslan et al., although they had a small sample size (n = 33).[8] More than 85% of cases were positive for an infectious agent; Giardia lamblia was the most common organism isolated in both studies. In other study from south India by Ramakrishna B et al., using stool PCR, evaluated 106 patients with acute and chronic diarrhea.[11] 50% of the sample yielded one or more organisms. As compared to our findings of 53% coinfection among controls, in their study, 48% had coinfections. Similar to our results, EAEC was the most organisms, EAEC and EPEC and EAEC and ETEC were the most common coinfections. This reinforces the fact that the spectrum of infective gastrointestinal illness largely depends upon the region, food habits, and socio-economic state of the population.

Prevalence and Spectrum of infective diarrhea in the transplant population

The prevalence of infective diarrhea among the transplant recipient has been varied from 38-77% depending upon the region and method of diagnosis.[8121314] In the landmark trial, the DIDACT study, 69 of 108 (64%) patients with diarrhea had infective origin.[15] In a study from turkey, renal and liver transplant recipients were compared with the immunocompetent population for the cause of diarrhea.[8] Infective cause for diarrhea was elucidated in 76% of transplant patients and 89% of the immunocompetent population. In a study from America, liver and kidney transplant recipients were compared with community-acquired and hospital-acquired diarrhea.[16] The majority of episodes had no identifiable cause (60-75%) and were self-limited (90%). The infective cause was found in 30%, and 19% of community-acquired and hospital-acquired diarrhea, respectively.

The etiological spectrum of infective agents in acute gastroenteritis in transplant patients is mainly region centered. Table 6 reviews and compares the various pathogenic organisms found in recent studies in the transplant population. In the present study, bacterial were the most common among all organisms in both cases and control, although individually Norovirus and G. lamblia were the most common among cases and controls, respectively.

Co-infections and its implication in the transplant population

Co-infection is a common finding in an acute diarrheal illness.[1718192021] Stool PCR is superior in detecting the multiple organisms than the conventional methods.[45] In the study by Coste et al., conventional methods failed to detect any co-infection in the transplant population, whereas the stool PCR found that 23% infections were actually co-infections.[4] In the study, Amar et al., after the application of PCR, the detection rate of multiple infections significantly increased by 73%.[5] Many of the studies have looked upon a specific association between viral and bacterial infections.[172223] In this study, co-infections were more in the transplant group (67% vs. 57%), but it did not reach the statistically significant level (p = 0.06). Existence of gut flora as commensal is a known fact but do the gut pathogens have a symbiotic, independent or suppresses each other's growth is largely unknown. According to a study by Byoyofo et al., parasites have specifically lower prevalence with viral infections. This was hypothesized due to antiviral cytokines like interferon are upregulated locally by the parasite, the viral infection is suppressed in the presence of parasitic infection.[22] Paradoxically, in this study, the most common combination was found to be with Norovirus with G. lamblia (19%) in the transplant group. This may be attributed to the immunosuppressed state of the transplant recipients. In the controls, we did get fewer patients who have co-infections that include viruses with the parasites. Many studies have correlated the severity of diarrhea in terms of duration and number of stools/days with the co-infections.[2425262728] In our study, no such pattern was observed, and clinical features were inadequate to predict the multiplicity of organisms and vice-versa. One of the potential benefits of diagnosing coinfections apart from more targeted therapy is the prevention of incompletely diagnosed case who could be either termed as resistant infection or attributing it to immunosuppression and subsequently modification of the drugs.

Norovirus was the most common infective pathology in 20% of transplant patients. Norovirus is the leading cause of diarrhea in the united states, even among non-transplant patient.[29] Norovirus is notorious for causing chronic infection among immunocompromised patients.[30313233] In a study by Roos et al., they demonstrated Sapo/Norovirus was present in 90% of patients with chronic diarrhea.[34] Chronic Norovirus is a known complication in renal transplant patients.[35] The incidence of Norovirus from India in the transplant population is mostly lacking. The prevalence of Norovirus in India is 5–15% in children presenting with acute gastroenteritis.[363738] In our control population, 9% of patients were harboring Norovirus at the time of presentation. Although the incidence is almost half of the transplant population, nevertheless, Norovirus thus contributes to a chunk of cases among the general population, which mostly go undiagnosed as conservative methods do not detect the virus.

G. lamblia is endemic in India, and its prevalence range from 5.5% to 70%, especially in children from northern India in community-based surveys.[39] Intestinal parasite infestation is generally rare among developed countries, attributing it to better socio-economic conditions. It is the second most common intestinal pathogens detected in our study (17%). Giardiasis also has frequent occurrence along with other pathogens in transplant patients.

The incidence of diarrhea due to CMV disease is estimated to be 1-20%.[81215164041] In the present study, we did not find any CMV cases in patients presented over two years. Since only 18% of patients underwent CMV PCR, and only 4% underwent colonoscopy, there could be a chance of under-diagnosis of the infection. Further studies that are specifically designed to detect CMV are needed to get the actual incidence of the disease.

There are several limitations to the study. First, due to its retrospective nature, complete elimination of selection and information bias was not possible. Second, rather than including all consecutive patients of diarrhea, only those patients were included who were admitted and underwent stool PCR, this have resulted in missing a considerable number of cases (29%). As such the results in this study provide a glimpse of problem of post-transplant diarrhea and further studies are needed to provide a complete picture of this issue.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.