INTRODUCTION
Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheagenic pathogen associated with diarrheal illnesses among patients in developed and developing countries. Recent studies have identified EAEC in patients with persistent diarrhea infected with human immunodeficiency virus (HIV).1,2 EAEC, which were first described in 1987 and recognized by its distinctive adherence to HEp-2 cells in an aggregative, stacked, brick-like pattern, is a member of the di-arrheagenic E. coli family, which is a diverse group of organisms.3 Diarrheagenic E. coli are divided into at least six categories based on clinical associations, serotyping, and phenotypic assays of different virulence characteristics. These include enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and more recently, diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC).4 EAEC has been increasingly isolated and characterized around the world from human clinical, animal, and environmental samples.5–7 However, frequencies of EAEC virulence genes among patients with diarrhea in the Limpopo Province, South Africa, are not known.
EAEC disease is typically characterized by a mucoid, watery diarrhea, often accompanied by fever, nausea, and vomiting.8 This pathogenicity may be caused by the ability of EAEC to adhere to the intestinal mucosa by means of aggregative adherence fimbriae AAF/I, AAF/II, and AAF/III, which are encoded by the aggA, aafA, and agg-3 genes, respectively, on a large 60-megadalton plasmid (pAA). This adherence is followed by damage of the mucosal epithelium and exfoliation of epithelial cells.9 The production of a cyto-toxin and several enterotoxins including plasmid-encoded toxin, heat-stable enterotoxin, Shigella enterotoxins 1 and 2, cryptic secreted protein AspU, flagellin (fliC), and a protein involved in intestinal colonization may contribute to secretory diarrhea.10 The aggR gene controls the expression of adherence factors as well as a dispersin protein, and a large cluster of genes encoded on the EAEC chromosome and has been successfully used to identify E. coli strains with aggregative characteristics.6
Infection with EAEC is diagnosed by the isolation of E. coli from the stools of patients and the analysis of such isolates by the HEp-2 assay or with DNA probes. Different molecular methods have been described for the detection and characterization of EAEC by amplification of specific pathogenic genes. These include a recently described multiplex polymerase chain reaction (PCR) assay using a DNA probe from the pAA plasmid of EAEC (previously known as CVD432 or AA probe, representing the aatA gene), the transcriptional activator aggR, and a novel antiaggregation protein (dispersin) encoded by the aap gene (formerly known as aspU).11 In the present study, we used the multiplex PCR assay, as well as a quantitative real-time PCR assay that detects the aggR gene, to determine the distribution of EAEC virulence genes in stool samples collected from patients attending local public hospitals with known HIV status and school children in the Vhembe District of South Africa in relation to different markers such as lactoferrin, diarrhea, and occult blood.
MATERIALS AND METHODS
Ethical clearance.
The study was reviewed and approved by the research and ethical committees of the University of Venda, the Department of Health and Welfare, and the Department of Education in Polokwane, Limpopo Province South Africa, before the initiation of the study. The objectives of the study were explained to all subjects and/or their legal guardian and written informed consent was obtained before sample collection.
Study site and sample collection.
The stool samples were randomly collected from patients with diarrhea or intestinal complains during their first visit to the laboratories of the major hospitals including Tshilidzini, Elim, Donald Frazer and Siloam. Stool samples were also collected randomly from apparently healthy students whose parents had consented to the study in a meeting prior to the beginning of the study at two primary schools serving the local population of the Venda region. The samples were aliquoted in 1.5-mL tubes (Eppendorf, Hamburg, Germany) without dilution for diarrheic (unformed) samples or diluted in sterile saline for non-diarrheic (formed) stools and were kept in −80°C until being sent to the University of Virginia for molecular analysis. A total of 322 stool samples were collected of which 255 were from hospital patients and 67 were from primary school children.
Genomic DNA purification
Three different stool pretreatment methods were used. These were 1) six cycles of freezing in liquid nitrogen for one minute and thawing in boiling water for one minute, 2) alkaline treatment of the stool with KOH and dithiothreitol (DTT), or 3) no treatment. Quantitative real-time PCR and conventional PCR using the aggR gene indicated that alkaline treatment yielded more DNA, as indicated by smaller threshold cycle (Ct) values (Figure 1). Thus, for the rest of the study, 250 μL of stool sample homogenates were thoroughly mixed with 66 μL of 1M KOH and 18 μL of 1M DTT and incubated at 65°C for 30 minutes. Genomic DNA was purified from the stool samples using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. DNA was purified from EAEC 042 and 17-2 cultures by the phenol-chloroform method as previously described.12
Multiplex PCR detection of EAEC virulence genes from stool samples.
For standardization purposes, we used two previously described EAEC reference strains (042 and 17-2) as positive controls and strain K-12 as a negative control. A multiplex PCR protocol previously described was used with modifications.11 Briefly, each PCR tube contained 25 μL of reaction mixture composed of (final concentrations) 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 100 mg/mL of gelatin, 5% (v/v) glycerol, 200 mM each of dATP, dCTP, dGTP, and dTTP (Qiagen), and 2.5 units/25 μL of AmpliTaq polymerase (Gibco-BRL, Gaithersburg, MD). Also included were a mixture of the six primers and a 5-μL portion of the DNA extract. The cycling conditions were 1 cycle for 2 minutes at 50°C; 1 cycle for 5 minutes at 95°C; 40 cycles for 45 seconds at 95°C, 45 seconds at 55°C, and 45 seconds at 72°C; and a final extension step for 10 minutes at 72°C in a thermal cycler (Bio-Rad Laboratories, Hercules, CA). After amplification, 10 μL of the PCR mixture was visualized after electrophoresis on a 2% agarose gel in Tris-acetate-EDTA buffer (Gibco-BRL) by staining with ethidium bromide. Only the presence of the correctly sized gene PCR product(s) was interpreted as a positive result. The sequences of the primers and the expected amplicon sizes are shown in Table 1.
Quantitative real-time PCR detection of the aggR gene.
A quantitative real-time PCR using SYBR-Green -490 (Bio-Rad Laboratories) and based on the protocol described above was established and used to confirm the detection of EAEC in the stool samples. The reaction was performed in a Bio-Rad iCycler iQ (Bio-Rad Laboratories) using iCycler Version 3.0 software, and the results were analyzed with a user-defined threshold of 200 PCR baseline-subtracted curve-fit relative fluorescence units. Melt curve data collection and analysis was enabled at cycles 3 and 4 with increase setpoint temperatures after cycle 2 by 0.5°C. Standard cultures with known numbers of EAEC cells were used as reference and positive controls; water and E. coli K-12 were used as negative controls in each reaction. The level of positivity of the samples was indicated by the Ct values.
Intestinal inflammation: detection of fecal lactoferrin.
Stool supernatants were tested for the presence of lactoferrin using the Leuko-Test (TechLab, Blacksburg, VA) according to the manufacturer’s specifications, including appropriate kit controls. The level of lactoferrin in the stool samples was quantified using the inflammatory bowel disease (IBD) check and the IBD scan kits from TechLab following the manufacturer’s instructions.
Test for occult blood.
Occult blood in the stool samples was detected with the Hemoccult test kit (Beckman Coulter Inc., Fullerton, CA) following manufacturer’s instructions.
Statistical analysis.
Results were analyzed using SPSS software version 10.1 (SPSS Inc., Chicago, IL). The chi-square test was used to compare the proportions of patients who provided the stool samples based on parameters such as di-arrheal symptoms, sex, age, origin, and lactoferrin or occult blood tests results. Differences were considered significant when the P value was < 0.05.
RESULTS
Demographic information on the sample population.
The age of the hospital patients varied between 2 weeks and 88 years, with most patients between 10 and 39 years of age and school children between 3 and 15 years of age. Table 2 shows the demographic information of the sample population. A total of 148 (58%) persons in the hospitals were female and 34 (51%) persons in the schools were female. Diarrhea was common among hospital patients (65%), as well as intestinal inflammation indicated by increased lactoferrin levels (56%), and occult blood in the stools (43%). The HIV status of the hospital patients was known and 44 were HIV positive, among whom 27 (61.4%) were female. The HIV-positive individuals were found at all age groups, but the highest percentage was among those ≥ 20 years of age.
Prevalence of EAEC virulence-related genes in the study-population.
Agarose gel electrophoresis of the PCR products showed bands of different sizes expected for each of the genes. Figure 2 shows a representative agarose gel of the PCR products positive of samples for one, two, or all three genes detected. Figure 3 shows results obtained from the quantitative real-time PCR that included different standards and controls. There was complete agreement between the standard PCR and the quantitative real-time PCR when the Ct valued considered positive was ≤ 37 for the quantitative real-time PCR. At least one of the three pathogenic genes detected in this study was found in 52 (16.1%) samples, with higher prevalence (χ2 = 7.876, P = 0.005) in the hospitals with 50 (19.6%) positive cases than in the schools where only 2 (3%) individuals were infected (Table 2). Of the three genes, aap was the most common in 49 (94%) cases, followed by aggR in 41 (79%) cases and the AA probe in 36 (69%) cases. Three different patterns of infections were observed: those positive only for one gene, some positive for two genes, and some positive for all three genes (Figure 2). However, most individuals were infected with strains carrying all three genes, accounting for 34 (65.4%) cases, compared with 12 (23%) with one gene and 6 (11.5%) with two genes. EAEC was more common among individuals less than five years of age. The prevalence of infection among HIV-positive individuals was significantly higher (χ2 = 5.360, P = 0.021) with 13 (29.5%) infections than the rest of the study population with 39 (14%) infections.
Occurrence of EAEC pathogenic genes and pathologic conditions.
In the present study, three variables including diarrhea on the basis of type of stool samples, intestinal inflammation on the basis of the level of lactoferrin, and occult blood were used to characterize the pathogenicity of EAEC strains present in the stools. The pathogenicity index, defined as the ratio of the prevalence of EAEC genes in diarrheal, lactoferrin-positive or occult blood–positive samples divided by the prevalence in nondiarrheal, lactoferrin-negative or occult blood–negative samples, was calculated for all three variables depending on the characteristics of the patients such as age, HIV status, and infection characteristics.
EAEC infections were significantly associated with diarrhea (χ2 = 8.387, P = 0.004). Thirty seven (71.2%) of all EAEC-positive samples were diarrheal compared with 49.3% for samples negative for EAEC. Table 3 shows the infection level (based on the prevalence of any of the three genes) and diarrheal pathogenicity index in the population depending on sample origin, age, HIV status, and infection characteristics such as number of genes involved, multiple or single infection, and the number of cells in the stool samples. The diarrheal pathogenicity index was higher for individuals less than five years of age and in HIV-positive individuals. The diarrheal pathogenicity index was also higher when all three genes were present in the same stool sample and when the number of cells carrying the aggR gene was more than 106 per gram of stool. When other infections were excluded, the diarrheal pathogenicity index was higher in samples with all three genes. Similar results were obtained for the inflammatory pathogenicity index (Table 4) based on the detection of increased lactoferrin levels in stool samples (> 7.5 μg of lactoferrin per gram of stool). EAEC infections were significantly associated with intestinal inflammation (χ2 = 6.565, P = 0.010) and 61.5% of stools that were positive for EAEC genes had increased lactoferrin levels compared with 42.2% for samples negative for EAEC genes.
The stool samples were also tested for occult blood and samples positive for EAEC genes were more likely to have occult blood (odds ratio = 5.069, 95% confidence interval = 2.665–9.644) even when the number of cells carrying the aggR gene was lower in the stool (Table 5). Of the samples positive for at least one EAEC gene, 69.2% had occult blood compared with only 30.7% for samples negative for EAEC genes (χ2 = 27.725, P < 0.00001). The occult blood pathogenicity index was higher for samples containing aggR than in samples with the other two genes.
Multiple infections.
We previously described the detection of different organisms in these samples, including Cryptosporidium spp., Entamoeba histolytica, E. bieneusi, Campylobacter jejuni, and Clostridium difficile.12 Table 6 shows cases of mixed infections involving EAEC with different pathogenic organisms detected in previous studies. Of the 52 samples that tested positive for at least one virulence-related EAEC gene, 31 were mixed with other organisms and 21 were single EAEC infections. Of the 21 single EAEC positive samples, 6 were negative for both lactoferrin and occult blood. Seven were positive for both occult blood and lactoferrin, five were positive only for occult blood, and three were positive only for lactoferrin.
DISCUSSION
Over the past few years, EAEC have been increasingly characterized in developing countries, and recent data have suggested that EAEC are emerging as diarrheal agents in developed nations.2 However, the true distribution of these organisms as well as their pathogenicity has not been well studied in South Africa, particularly in the Venda region. In the present study, we detected three EAEC pathogenic genes by using a recently developed multiplex PCR. We evaluated these genes in relation to HIV status, diarrheal symptoms, and intestinal inflammation determined by elevated lactoferrin and occult blood in a sample population composed of hospital patients with known HIV status and school children in the Vhembe district of South Africa.
Different methods have been described for the detection of EAEC and have suggested the existence of two different categories of EAEC (typical and atypical).13 Typical EAEC carry the pAA plasmid were originally detected with an AA probe. The two other genes studied here (aap and aggR) are more frequently detected in PCR-positive isolates. Atypical EAEC isolates that do not carry the AA plasmid or carry a plasmid (pAA) with some deletions or rearrangements can still cause diarrheal disease. Alternatively, they harbor other plasmids that contain some of these genes through horizontal gene transfer.14–16 Typical EAEC has been described as the most common and most virulent strains in other studies.13,17 We found the AA probe in 66% of all EAEC-positive samples. It can be hypothesized that the other 34% of the cases might be caused by atypical strains. However, more studies need to be conducted to confirm this possibility.
We found a genetic distribution of typical EAEC pathogenic genes similar to that obtained by other investigators.11,13,18 However, the prevalence of the samples with all three genes was lower than that previously described where a rate of 82% was obtained compared with 65.4% in our study.11 This could be explained by the heterogeneity of EAEC strains and the possible infection of the patients by multiple strains presenting different genotypes. The genetic characterization of isolates from the region is needed and will help to verify this hypothesis.
We found EAEC pathogenic genes in 15.5% of all the samples used. This new rate is higher than previously described (5.8%), a finding that confirms the emergence of EAEC strains in the region.19 This finding highlights the need for the design of surveillance strategies to control EAEC diarrhea and to prevent the transmission of EAEC in the region through water or food contamination. Similar rates have been described in other regions of the world such as in Hanoi, Vietnam, where 11.5% of children less than five years of age were infected with EAEC detected by a multiplex PCR.20 In our study, the diarrheal pathogenicity index was higher in children less than five years of age, which indicated the increased susceptibility of this population to EAEC infections. These results are consistent with studies in Rio de Janeiro, Brazil, where EAEC was the most frequent diarrhoeagenic E. coli, accounting for 14.6% of isolates in children with diarrhea and 11.1% in children without diarrhea,21 and in Nigeria, where EAEC was found in 10.3% of the persons in a study carried out in small-town and rural primary health care centers.22 A similar prevalence was found in a developed country (Switzerland) where EAEC isolates were found in specimens of 19 (10.2%) of 187 children with diarrhea and in 3 (2.2%) of 137 children without diarrhea (P = 0.006) and were the most frequently detected bacteria associated with diarrhea.23
EAEC have also been associated with a weakened immune system such as in patients infected with HIV and in patients with acquired immunodeficiency syndrome (AIDS). They have also been described as the most common pathogen among HIV-positive patients in many countries even though the rates of infection vary from country to country. In this study, we found a higher rate of EAEC infection among HIV-positive patients (29.5%) compared with Senegal, where EAEC was found in 19.6% of HIV-positive patients and was the most common pathogen in these individuals.24 In Switzerland, EAEC genes were detected in 22% of HIV-positive patients with diarrhea; in Zambia, EAEC was found in both HIV-positive patients and controls even though cytotoxic phenotypes were only isolated from the AIDS patients with no evidence of seasonality in the frequency of isolation and no evidence of long-term carriage.25–27
Different markers of pathogenesis have been described in EAEC infections including fecal cytokines such as interleikin-8 (IL-8) and IL-1R, lactoferrin, and occult blood.28,29 Volunteer challenge studies have demonstrated heterogeneity in the ability of EAEC isolates to cause disease, and several studies have been unable to make clear associations with EAEC and diarrhea. In this study, more EAEC-positive samples had increased levels of lactoferrin and diarrhea, and EAEC in the stools was significantly associated with occult blood (P < 0.001). Although EAEC have been associated with bloody stool samples, the relationship with occult blood has not been clearly described.25 A study in the Central African Republic indicated that EAEC were most frequently identified in HIV-positive patients with persistent diarrhea, and 42.8% of the patients with EAEC as the only pathogen had bloody diarrhea.27 Occult blood in stools of individuals infected with EAEC was tested in a study that did not find a significant association between EAEC infection and occult blood in the stools; only 4 (31.1%) of EAEC-positive stool samples had occult blood and 27 (60.0%) of EAEC-positive stool samples had lactoferrin.30
Our study found a significant association between EAEC infections and occult blood in the stool, which may indicate a different pathogenic manifestation of these organisms in this part of the world. Studies elsewhere have indicated that the best characterized E. coli pathotypes require multiple genes to be fully/highly virulent. For example, ETEC with heat-labile toxin, heat-stable toxin, and colonization factor antigens; EPEC with bundle forming pili and the eae gene encoding the adhesin intimin that is responsible for intimate attachment of bacteria to epithelial cells; and Shiga toxin–producing E. coli (STEC) with Shiga-like toxin and eaeA that encode intimin, which is also involved in attachment of bacteria to enterocytes are highly virulent.31–35 However, the presence of multiple genes has not been associated with pathogenesis in EAEC. This study has shown that strains with all three genes were more pathogenic in terms of diarrhea production, intestinal inflammation indicated by lactoferrin levels in stools, and occult blood.
The results obtained in this study have demonstrated that EAEC is an important etiologic agent of diarrhea in the Venda region of South Africa, as indicated by its high prevalence among hospital patients. Furthermore, EAEC may be a treatable cause of diarrhea in patients with AIDS.36 This study also confirms the usefulness of molecular methods such as PCR and quantitative real-time PCR in detection and possible quantification of EAEC in stool samples in regions such as Venda that are endemic for these bacteria. Quantitative real-time PCR indicated that a certain threshold related to the number of cells was needed for the EAEC to cause pathologic symptoms such as diarrhea and inflammation.
HIV-positive individuals are at a higher risk of infection by EAEC and had higher levels of lactoferrin than HIV-negative individuals. This is the first study to significantly associate EAEC with occult blood in the stools. Occult blood after infection with EAEC in these patients might be caused by pathogenic factors such the plasmid-encoded toxin, which is highly homologous to the EspP protease of EHEC and to EspC of EPEC and the protein involved in colonization.37,38 This study has also confirmed heterogeneity among infecting EAEC isolates that indicates the presence of multiple strains in the population, which may lead to recurrent infections and outbreaks in Venda, South Africa. It thus underscores the importance of regular monitoring and characterization of E. coli strains and clinical symptoms in infected patients, which are crucial for epidemiologic control. Regular monitoring will also be helpful in surveys of emergence of genetic rearrangements of virulence factors and in tracking emergence of new pathogenic strains. Thus, the pathogenicity of EAEC in Venda may involve diarrhea and intestinal inflammation as determined by the lactoferrin test. Occult blood or fecal lactoferrin testing may also provide potential information about EAEC infections in this region.
Sequences of primers used in the study*
| Gene target | Orientation | Sequence | Amplicon size (bp) |
|---|---|---|---|
| * bp = basepairs. | |||
| aap | Forward Reverse | 5′-CTT GGG TAT CAG CCT GAA TG-3′ 5′-AAC CCA TTC GGT TAG AGC AC-3′ | 310 |
| aggR | Forward Reverse | 5′-CTA ATT GTA CAA TCG ATG TA-3′ 5′-AGA GTC CAT CTC TTT GAT AAG-3′ | 457 |
| AA probe | Forward Reverse | 5′-CTG GCG AAA GAC TGT ATC AT-3′ 5′-CAA TGT ATA GAA ATC CGC TGT T-3′ | 629 |
Demographic information on the study population*
| Characteristic | Hospitals | Schools | Total |
|---|---|---|---|
| * HIV = human immunodeficiency virus; EAEC = enteroaggregative Escherichia coli. | |||
| Total no. | 255 | 67 | 322 |
| Age range (years) | 0–88 | 3–15 | 0–88 |
| Population by age groups (years) | |||
| 0–5 | 35 (13.7%) | 5 (7.5%) | 40 (12.4%) |
| 6–19 | 85 (33.3%) | 62 (92.5%) | 147 (45.7%) |
| ≥ 20 | 135 (52.9%) | 0 | 135 (41.9%) |
| Sex | |||
| Female | 148 (58%) | 34 (51%) | 182 (57%) |
| Male | 107 (42%) | 33 (49%) | 140 (43%) |
| Sample characteristics | |||
| Bloody diarrhea | 8 (3.1%) | 0 | 8 (2.5%) |
| Diarrheal | 159 (62.4%) | 9 (13.4%) | 168 (52.2%) |
| Nondiarrheal | 88 (34.5%) | 58 (86.6%) | 146 (45.3%) |
| HIV seropositive | 44 (18.4%) | – | 44 |
| Elevated lactoferrin level | 143 (56%) | 7 (10.4%) | 150 (46.6%) |
| Occult blood positive | 110 (43%) | 9 (13%) | 119 (37%) |
| Presence of any of the 3 EAEC genes | 50 (19.6%) | 2 (3%) | 52 (16.1%) |
EAEC infections in stool samples of the study population*
| Characteristics | Diarrheal stools (n = 170) | Non-diarrheal stools (n = 152) | Total | Risk estimate: OR (95% CI) | χ2, P | PI |
|---|---|---|---|---|---|---|
| * Differences are significant if P < 0.05. EAEC = enteroaggregative Escherichia coli; OR = odds ratio; CI = confidence interval; PI = pathogenicity index (ratio of occurrence of EAEC virulence-related genes in diarrheal samples to that in non-diarrheal samples)2; HIV = human immunodeficiency virus; qPCR = quantitative polymerase chain reaction. | ||||||
| Origin | ||||||
| Hospitals | 35 (20.5% | 15 (9.8%) | 50 (19.6%) | 1.433 (0.735–2.793) | χ2 = 1.124; P = 0.289 | 2.1 |
| Schools | 1 (0.6%) | 1 (0.7%) | 2 (3%) | 8.286 (0.465–147.694) | χ2 = 2.840; P = 0.092 | 1 |
| Age group, years | χ2 = 3.401; P = 0.183 | |||||
| 0–5 | 7 (4.1%) | 1 (0.7%) | 8 (20%) | 1.615 (0.166–15.723) | 5.9 | |
| 6–19 | 8 (4.7%) | 7 (4.6%) | 15 (10.2%) | 2.537 (0.862–7.464) | 1.02 | |
| > 20 | 21 (12.4%) | 8 (10.5%) | 29 (21.5%) | 1.528 (0.618–3.777) | 1.2 | |
| HIV status | χ2 = 6.754; P = 0.009 | |||||
| Positive (n = 44) | 12 (27.3%) | 1 (2.3%) | 13 (29.5%) | 1.941 (0.970–3.883) | 11.9 | |
| Negative (n = 278) | 24 (8.6%) | 15 (5.4%) | 39 (14%) | 2.308 (0.242–21.968) | 1.6 | |
| Genes | ||||||
| aap | 35 (21%) | 14 (9%) | 49 (94%) | 2.556 (1.316–4.963) | χ2 = 8.052; P = 0.005 | 2.3 |
| Aa probe | 26 (15.5%) | 10 (6.5%) | 36 (69.2%) | 2.564 (1.193–5.511) | χ2 = 6.138; P = 0.013 | 2.4 |
| AggR | 28 (16.7%) | 13 (8.4%) | 41 (79%) | 2.108 (1.049–4.238) | χ2 = 4.528; P = 0.033 | 2 |
| Gene combinations | ||||||
| Any 1 gene only | 8 (4.8%) | 4 (2.6%) | 12 (23%) | 1.827 (0.539–6.194) | χ2 = 0.962; P = 0.327 | 1.8 |
| Any 2 genes | 3 (1.8%) | 3 (1.9%) | 6 (11.5%) | 0.892 (0.177–4.488) | χ2 = 0.019; P = 0.890 | 0.9 |
| All 3 genes | 25 (14.9%) | 9 (5.8%) | 34 (65.4%) | 2.739 (1.236–6.073) | χ2 = 6.558; P = 0.010 | 2.6 |
| Any EAEC gene+ | 36 (21.4%) | 16 (10.4%) | 52 (16.1%) | 2.284 (1.210–4.311) | χ2 = 6.722; P = 0.010 | 2.05 |
| EAEC single infections (EAEC alone) | 14 (8.2%) | 7 (4.6%) | 21 (6.5%) | 1.615 (0.658–3.964) | χ2 = 1.735; P = 0.188 | 1.8 |
| EAEC single with 1 or 2 genes only | 5 (2.9%) | 3 (1.9%) | 1.5 | |||
| EAEC single with all 3 genes | 9 (5.3%) | 4 (2.6%) | 2.04 | |||
| EAEC with 1 other pathogen | 13 (7.6%) | 7 (4.5%) | 20 (6.2%) | 1.715 (0.666–4.418) | χ2 = 1.275; P = 0.259 | 1.7 |
| EAEC with at least 2 other pathogens | 9 (5.4%) | 1 (0.6%) | 10 (3.1%) | 4.193 (0.891–19.718) | χ2 = 3.849; P = 0.050 | 9 |
| No. of cells (intensity of infection: AggR qPCR) | ||||||
| > 106 | 7 (4.1%) | 1 (0.6%) | 8 (2.4%) | 2.744 (0.545–13.804) | χ2 = 120.83; P = 0.0001 | 6.8 |
| 104–106 | 7 (4.2%) | 2 (1.3%) | 9 (2.8%) | 3.221 (0.659–15.748) | χ2 = 2.319; P = 0.128 | 3.2 |
| < 104 | 15 (8.9%) | 9 (5.8%) | 24 (7.4%) | 1.538 (0.653–3.623) | χ2 = 0.980; P = 0.322 | 1.5 |
EAEC infections and stool lactoferrin in the study population*
EAEC infections and occult blood in stool samples of the study population*
Cases of co-infection with other organisms*
| No. of organisms | Diarrhea | Elevated lactoferrin level | Occult blood | |
|---|---|---|---|---|
| * EAEC = enteroaggregative Escherichia coli. | ||||
| Two | Cryptosporidium hominis + EAEC | 0 | 0 | 1 |
| C. hominis + EAEC | 1 | 4 | 1 | |
| C. hominis + EAEC | 1 | 0 | 1 | |
| C. hominis + EAEC | 0 | 0 | 0 | |
| C. hominis + EAEC | 1 | 1 | 1 | |
| C. hominis + EAEC | 1 | 1 | 1 | |
| C. hominis + EAEC | 0 | 0 | 1 | |
| C. hominis + EAEC | 1 | 1 | 0 | |
| Cryptosporidium parvum + EAEC | 0 | 0 | 1 | |
| Clostridium difficile + EAEC | 1 | 1 | 1 | |
| C. difficile + EAEC | 1 | 1 | 0 | |
| C. difficile + EAEC | 0 | 0 | 1 | |
| C. difficile + EAEC | 1 | 1 | 1 | |
| C. difficile + EAEC | 1 | 1 | 1 | |
| Campylobacter jejuni + EAEC | 1 | 1 | 1 | |
| C. jejuni + EAEC | 1 | 1 | 1 | |
| C. jejuni + EAEC | 1 | 1 | 0 | |
| Campylobacter coli + EAEC | 1 | 1 | 1 | |
| Entamoeba histolytica + EAEC | 0 | 0 | 1 | |
| Arcobacter butzleri + EAEC | 1 | 1 | 0 | |
| Three | C. hominis + C. coli + EAEC | 0 | 0 | 1 |
| C. hominis + E. histolytica + EAEC | 1 | 1 | 1 | |
| Enterocytozoon bieneusi + C. difficile + EAEC | 1 | 1 | 1 | |
| E. histolytica + E. bieneusi + EAEC | 1 | 1 | 1 | |
| C. parvum + C. difficile + EAEC | 1 | 1 | 1 | |
| E. histolytica + C. difficile + EAEC | 1 | 1 | 1 | |
| Four | E. bieneusi + C. hominis + C. difficile + EAEC | 1 | 0 | 1 |
| C. jejuni + C. coli + C. difficile + EAEC | 1 | 1 | 1 | |
| Five | C. jejuni + A. butzleri + C. parvum + C. difficile + EAEC | 1 | 1 | 1 |
| Campylobacter concisus + C. jejuni + E. histolytica + C. difficile + EAEC | 1 | 1 | 1 | |
| Six | C. difficile + E. bieneusi + E. histolytica + C. jejuni + C. coli + A. butzleri + EAEC | 1 | 0 | 1 |
| Total | 31 | 31 | 31 | |
Comparison of three methods of DNA extractions for two positive samples (A and B) of enteroaggregative Escherichia coli from South Africa by agarose gel electrophoresis. Both samples were treated with three different methods including KOH (lanes 1 and 4), freeze-thaw (lanes 2 and 5), and no treatment (lanes 3 and 6). Lanes 9–14, negative samples. The respective threshold cycle (Ct) values obtained with the real-time polymerase chain reaction for sample A were 29.16 for 1, 32.83 for 2, and 31.54 for 3 after treatment using the three methods successively. The Ct values for sample B were 28.81 for 4, 32.29 for 5, and 33.34 for 6. These indicated that the KOH treatment was best for DNA purification. Lane M, DNA molecular mass ladder; lane N = negative controls (water and a nonpathogenic Escherichia coli strain). bp = basepairs.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Comparison of three methods of DNA extractions for two positive samples (A and B) of enteroaggregative Escherichia coli from South Africa by agarose gel electrophoresis. Both samples were treated with three different methods including KOH (lanes 1 and 4), freeze-thaw (lanes 2 and 5), and no treatment (lanes 3 and 6). Lanes 9–14, negative samples. The respective threshold cycle (Ct) values obtained with the real-time polymerase chain reaction for sample A were 29.16 for 1, 32.83 for 2, and 31.54 for 3 after treatment using the three methods successively. The Ct values for sample B were 28.81 for 4, 32.29 for 5, and 33.34 for 6. These indicated that the KOH treatment was best for DNA purification. Lane M, DNA molecular mass ladder; lane N = negative controls (water and a nonpathogenic Escherichia coli strain). bp = basepairs.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Comparison of three methods of DNA extractions for two positive samples (A and B) of enteroaggregative Escherichia coli from South Africa by agarose gel electrophoresis. Both samples were treated with three different methods including KOH (lanes 1 and 4), freeze-thaw (lanes 2 and 5), and no treatment (lanes 3 and 6). Lanes 9–14, negative samples. The respective threshold cycle (Ct) values obtained with the real-time polymerase chain reaction for sample A were 29.16 for 1, 32.83 for 2, and 31.54 for 3 after treatment using the three methods successively. The Ct values for sample B were 28.81 for 4, 32.29 for 5, and 33.34 for 6. These indicated that the KOH treatment was best for DNA purification. Lane M, DNA molecular mass ladder; lane N = negative controls (water and a nonpathogenic Escherichia coli strain). bp = basepairs.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Electrophoresis (2% agarose gel, 100 minutes at 80V) of polymerase chain reaction (PCR) products for three different enteroaggregative Escherichia coli genes. The gel was stained with ethidium bromide (2 μg/mL) and photographed under ultraviolet light. Positive samples showed clear bands (310 basepairs [bp]) for aap, 457 bp for aggR, 629 bp for AA. Lane M = 100-bp DNA molecular mass ladder; lane PC, positive control strain 042, lane NC, E. coli negative control strain K-12; lanes 1, 2, 3, 4, 5, and 6–15, positive samples for one, two, or all three genes; lane Blank, water. Some strains were positive for all three genes.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Electrophoresis (2% agarose gel, 100 minutes at 80V) of polymerase chain reaction (PCR) products for three different enteroaggregative Escherichia coli genes. The gel was stained with ethidium bromide (2 μg/mL) and photographed under ultraviolet light. Positive samples showed clear bands (310 basepairs [bp]) for aap, 457 bp for aggR, 629 bp for AA. Lane M = 100-bp DNA molecular mass ladder; lane PC, positive control strain 042, lane NC, E. coli negative control strain K-12; lanes 1, 2, 3, 4, 5, and 6–15, positive samples for one, two, or all three genes; lane Blank, water. Some strains were positive for all three genes.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Electrophoresis (2% agarose gel, 100 minutes at 80V) of polymerase chain reaction (PCR) products for three different enteroaggregative Escherichia coli genes. The gel was stained with ethidium bromide (2 μg/mL) and photographed under ultraviolet light. Positive samples showed clear bands (310 basepairs [bp]) for aap, 457 bp for aggR, 629 bp for AA. Lane M = 100-bp DNA molecular mass ladder; lane PC, positive control strain 042, lane NC, E. coli negative control strain K-12; lanes 1, 2, 3, 4, 5, and 6–15, positive samples for one, two, or all three genes; lane Blank, water. Some strains were positive for all three genes.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Quantitative detection of the AggR gene of enteroaggregative Escherichia coli (EAEC) from stool samples by real-time polymerase chain reaction (PCR). A, PCR Amp/Cycle graph for SYBR-490 Step 2. 17-2 and 042 are the two reference strains; 042a and 042b are standard samples of 109 and 106 colony-forming units, respectively). Other graphs represent stool samples positive for the AggR gene. B, Standard graph for SYBR-490 Step 2 showing standards and the stool samples (unknowns) positive for the gene considered. Graphs were generated by the iCycler software based on the standards (genomic DNA isolated pure EAEC standard cultures [042 and 17-2]). Samples were positive when Ct values were ≤ 37. C, Melt curve graph for SYBR-490 generated by the iCycler software for the standard strain (042) and a positive stool sample.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Quantitative detection of the AggR gene of enteroaggregative Escherichia coli (EAEC) from stool samples by real-time polymerase chain reaction (PCR). A, PCR Amp/Cycle graph for SYBR-490 Step 2. 17-2 and 042 are the two reference strains; 042a and 042b are standard samples of 109 and 106 colony-forming units, respectively). Other graphs represent stool samples positive for the AggR gene. B, Standard graph for SYBR-490 Step 2 showing standards and the stool samples (unknowns) positive for the gene considered. Graphs were generated by the iCycler software based on the standards (genomic DNA isolated pure EAEC standard cultures [042 and 17-2]). Samples were positive when Ct values were ≤ 37. C, Melt curve graph for SYBR-490 generated by the iCycler software for the standard strain (042) and a positive stool sample.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Quantitative detection of the AggR gene of enteroaggregative Escherichia coli (EAEC) from stool samples by real-time polymerase chain reaction (PCR). A, PCR Amp/Cycle graph for SYBR-490 Step 2. 17-2 and 042 are the two reference strains; 042a and 042b are standard samples of 109 and 106 colony-forming units, respectively). Other graphs represent stool samples positive for the AggR gene. B, Standard graph for SYBR-490 Step 2 showing standards and the stool samples (unknowns) positive for the gene considered. Graphs were generated by the iCycler software based on the standards (genomic DNA isolated pure EAEC standard cultures [042 and 17-2]). Samples were positive when Ct values were ≤ 37. C, Melt curve graph for SYBR-490 generated by the iCycler software for the standard strain (042) and a positive stool sample.
Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.142
Address correspondence to Amidou Samie, Center for Global Health, Division of Infectious Diseases and International Health, University of Virginia, MR4, Lane Road, Room 3146, Charlottesville, VA 22908. E-mail: samieamidou@yahoo.com
Authors’ addresses: Amidou Samie, Department of Microbiology, University of Venda, Thohoyandou, 0950, South Africa and Center for Global Health, Division of Infectious Diseases and International Health, University of Virginia, MR4, Lane Road, Room 3146, Charlottesville, VA 22908, Telephone: 434-924-5242, Fax: 434-977-5323, E-mail: samieamidou@yahoo.com. Chikwelu Larry Obi, College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences, University of South Africa, Pretoria, South Africa. Rebecca Dillingham, Relena C. Pinkerton, and Richard L. Guerrant, Center for Global Health, University of Virginia, Charlottesville, VA 22908.
Financial support: This study was supported in part by grants from the Ellison Medical Foundation and the Pfizer Foundation to the Centre for Global Health, University of Virginia.
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