Viral diarrheas - newer advances in diagnosis and management.

PURPOSE OF REVIEW: Viruses are the most common etiological agents of diarrhea in children. Despite rotavirus vaccine introduction, rotavirus remains as the leading cause of death globally, followed by norovirus, which represents a diagnostic challenge. Here, we describe new advances in the diagnosis and management of viral diarrheas. RECENT FINDINGS: Although immunoassays are widely used for their fast turnaround time and low cost, molecular techniques have become the most reliable diagnostic method due to their high sensitivity and capacity to analyze multiple pathogens in gastrointestinal panels. Isothermal nucleic acid amplification assays (LAMP and RPA) are promising techniques since they do not require sophisticated equipment and can be used as point-of-care testing. CRISPR/Cas nucleic acid detection systems are new diagnostic methods with great potential. Several recent published articles describe the role of human intestinal enteroids to characterize norovirus infection, to test new drugs, and for vaccine development. The interaction between the human gut microbiota and gastrointestinal viral infections has been extensively reviewed and offers some innovative mechanisms for therapeutic and preventive measures. SUMMARY: Although important advances have been made, more research is needed to address remaining challenges and further improve diagnostic capabilities and better management strategies for this critical infectious disease.

Knowledge, attitudes and practices of parents towards antibiotic use in rural communities in Peru: a cross-sectional multicentre study.

BACKGROUND: The inappropriate use of antibiotics significantly contributes to the development of antibiotic resistance. There is limited information about the use of antibiotics among parents from rural areas in Peru. This study aimed to describe the knowledge, attitudes and practices towards antibiotics among parents of children < 5 years of age from rural communities in Peru; to explore the association between knowledge and attitudes towards antibiotics and to explore determinants of low knowledge and self-medicating his/her child with antibiotics. METHODS: Cross-sectional study in six rural primary health centres in Peru using a self-administered survey. Crude and adjusted Prevalence Ratios (PR), and 95% Confidence Intervals (95% CI) were calculated to explore determinants of low knowledge and of having self-medicated his/her child with antibiotics. Linear regression was used to explore the association between knowledge and attitudes. RESULTS: A total of 231 parents were included. The largest gap in knowledge was among 183 parents (79%) who did not know that antibiotics cannot cure viral infections. The largest gap in attitudes was among 185 participants (80%) that did not disagree with "If I want my child to receive antibiotics, I would not be satisfied if the doctor refuses to prescribe them". More than half of parents (n = 120, 52%) reported having self-medicated his/her child with antibiotics. A positive correlation was found between knowledge and attitudes (Coefficient 0.53, 95% CI 0.38-0.68) after adjusting for the age and the education of the parent. Parents who were < 20 years old were more likely to have low knowledge about antibiotics (crude PR 2.39, 95% CI 1.32-4.34) compared to those aged > 40 years. Parents who had self-medicated his/her child with antibiotics (n = 120, 52%) were more likely to have purchased antibiotics without prescription (aPR 2.70, 95% CI 1.74-4.19) and to have received antibiotics after the recommendation of a pharmacist (aPR 1.79, 95% CI 1.13-2.82). CONCLUSIONS: Knowledge about antibiotics among parents from rural settings in Peru is limited and highlights the need for educational interventions. Public health policies to limit the acquisition of antibiotics without prescription should be implemented.

Randomized Controlled Trial of Bovine Lactoferrin for Prevention of Sepsis and Neurodevelopment Impairment in Infants Weighing Less Than 2000 Grams.

OBJECTIVES: To determine the effect of bovine lactoferrin on prevention of late-onset sepsis (LOS) and neurodevelopment delay. STUDY DESIGN: Randomized, double-blind, controlled trial in neonates with a birth weight of 500-2000 g in 3 neonatal units in Lima, Peru, comparing bovine lactoferrin 200 mg/kg/day with placebo administered for 8 weeks. The primary outcome was the first episode of culture-proven LOS or sepsis-associated death. Neurodevelopment delay was assessed by the Mullen Scales at 24 months corrected age. RESULTS: Of the 414 infants enrolled, 209 received bovine lactoferrin and 205 received placebo. LOS or sepsis-associated death occurred in 22 infants (10.5%) in the bovine lactoferrin group vs 30 (14.6%) in the placebo group; there was no difference after adjusting for hospital and birth weight; hazard ratio 0.73 (95% CI, 0.42-1.26). For infants with birth weights of <1500 g the hazard ratio was 0.69 (95% CI, 0.39-1.25). The mean age-adjusted normalized Mullen composite score at 24 months was 83.3 ± 13.6 in the bovine lactoferrin group vs 82.6 ± 13.1 in the placebo group. Growth outcomes and rehospitalization rates during the 2-year follow-up were similar in both groups, except for significantly less bronchiolitis in the bovine lactoferrin group (rate ratio, 0.34; 95% CI, 0.14-0.86). CONCLUSIONS: Supplementation with bovine lactoferrin did not decrease the incidence of sepsis in infants with birth weights of <2000 g. Growth and neurodevelopment outcomes at 24 months of age were similar. Neonatal bovine lactoferrin supplementation had no adverse effects. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01525316.

Pneumocystis primary infection in infancy: Additional French data and review of the literature.

Data on features of Pneumocystis primary infection in infancy are still fragmented. To study Pneumocystis primary infection, 192 infants who were monitored for acute pulmonary disease or fever over a 40-month period were retrospectively investigated. P. jirovecii detection on archival nasopharyngeal aspirates was performed using a qPCR assay. Factors associated with P. jirovecii were assessed using univariate and multivariate analyses. P. jirovecii genotypes in infants and a control group of adults contemporaneously diagnosed with Pneumocystis pneumonia were identified using unilocus, bilocus, and multilocus sequence typing (MLST). P. jirovecii was detected in 35 infants (18.2%). The univariate analysis pointed out four factors: viral infection (P = .035, OR [IC 95], 2.2 [1.1-4.7]), lower respiratory tract infection (P = .032, OR [IC 95], 2.5 [1.1-5.9]), absence of hospital discharge after birth (P = .003, OR (IC 95), 0.1 (0.02-0.5]), and the 63-189-day group (P < .001, OR [IC 95], 42.2 [5.4-332]). The multivariate analysis confirmed these two latter factors (P = .02, OR [IC 95], 0.1 [0.02-0.72]; P = .005, OR [IC 95], 11.5 [2.1-63.5]). Thus, P. jirovecii acquisition mostly takes place in the community. A comparison of these data with those of previously published studies showed that median and interquartile range of positive-infant ages were close to those observed in Chile, Denmark, and Peru, highlighting similar characteristics. Common unilocus or bilocus genotypes were identified in infants and adults, whereas no MLST genotypes were shared. Therefore, a common reservoir made up of infected infants and adults is still hypothetical. Finally, primary infection is a worldwide phenomenon occurring at the same time in childhood regardless of geographical location, rather than an incidental event.

International prospective observational cohort study of Zika in infants and pregnancy (ZIP study): study protocol.

BACKGROUND: Until recently, Zika virus (ZIKV) infections were considered mild and self-limiting. Since 2015, they have been associated with an increase in microcephaly and other birth defects in newborns. While this association has been observed in case reports and epidemiological studies, the nature and extent of the relationship between ZIKV and adverse pregnancy and pediatric health outcomes is not well understood. With the unique opportunity to prospectively explore the full spectrum of issues related to ZIKV exposure during pregnancy, we undertook a multi-country, prospective cohort study to evaluate the association between ZIKV and pregnancy, neonatal, and infant outcomes. METHODS: At research sites in ZIKV endemic regions of Brazil (4 sites), Colombia, Guatemala, Nicaragua, Puerto Rico (2 sites), and Peru, up to 10,000 pregnant women will be recruited and consented in the first and early second trimesters of pregnancy and then followed through delivery up to 6 weeks post-partum; their infants will be followed until at least 1 year of age. Pregnant women with symptomatic ZIKV infection confirmed by presence of ZIKV RNA and/or IgM for ZIKV will also be enrolled, regardless of gestational age. Participants will be tested monthly for ZIKV infection; additional demographic, physical, laboratory and environmental data will be collected to assess the potential interaction of these variables with ZIKV infection. Delivery outcomes and detailed infant assessments, including physical and neurological outcomes, will be obtained. DISCUSSION: With the emergence of ZIKV in the Americas and its association with adverse pregnancy outcomes in this region, a much better understanding of the spectrum of clinical outcomes associated with exposure to ZIKV during pregnancy is needed. This cohort study will provide information about maternal, fetal, and infant outcomes related to ZIKV infection, including congenital ZIKV syndrome, and manifestations that are not detectable at birth but may appear during the first year of life. In addition, the flexibility of the study design has provided an opportunity to modify study parameters in real time to provide rigorous research data to answer the most critical questions about the impact of congenital ZIKV exposure. TRIAL REGISTRATION: NCT02856984 . Registered August 5, 2016. Retrospectively registered.

Correction to: International prospective observational cohort study of Zika in infants and pregnancy (ZIP study): study protocol.

Following publication of the original article [1], the author mentioned that two additional NIH staff were involved in the development of the protocol who did not receive recognition in the Acknowledgments section in their published article.

Lactoferrin and prematurity: a promising milk protein?

Lactoferrin (Lf) is the major whey protein in milk, with multiple beneficial health effects including direct antimicrobial activities, anti-inflammatory effects, and iron homeostasis. Oral Lf supplementation in human preterm infants has been shown to reduce the incidence of sepsis and necrotizing enterocolitis. In preclinical models of antenatal stress and perinatal brain injury, bovine Lf protected the developing brain from neuronal loss, improved connectivity, increased neurotrophic factors, and decreased inflammation. It also supported brain development and cognition. Further, Lf can prevent preterm delivery by reducing proinflammatory factors and inhibiting premature cervix maturation. We review here the latest research on Lf in the field of neonatology.

Factors affecting lactoferrin concentration in human milk: how much do we know?

Lactoferrin (LF) is a breast milk glycoprotein with antimicrobial and anti-inflammatory effects. Its beneficial properties in infants, especially in those born preterm, are currently being studied in clinical trials. However, the maternal and nursing infant factors that may affect LF concentration in breast milk are still not clear. We conducted a systematic review to investigate the factors that may affect the concentration of LF in breast milk. We used a 2-step approach to identify the eligible studies according to inclusion/exclusion criteria, and to determine which studies would be considered. We included 70 qualified articles from 29 countries with publication dates ranging from 1976 to 2015. We described the correlation between LF concentration in breast milk and lactation stage; 10 maternal factors, such as race, parity, among others; and 2 infant factors: infections and prematurity. Colostrum has the highest LF levels, but they decrease with days postpartum. No other factor has been consistently associated with LF concentration. A major limitation of the majority of the published studies is the small sample size and the different methods used to measure LF concentration. Therefore, there is a need for large, multicenter studies with standardized study design, sample collection, and LF measurement methods to identify clinically significant factors associated with LF expression in breast milk, which will help promote exclusive breastfeeding in preterm infants.

Key features of invasive pneumococcal isolates recovered in Lima, Peru determined through whole genome sequencing.

Before PCV7 introduction, invasive pneumococcal disease (IPD) was responsible for approximately 12,000-18,000 deaths annually among children <5years in Latin America. In Peru, PCV7 was introduced in 2009. We used whole genome sequencing to deduce key features of invasive strains collected in Lima, Peru from 2006 to 2011. We sequenced 212 IPD isolates from 16 hospitals in Lima pre (2006-2009; n=133) and post (2010-2011; n=79) PCV7 introduction; 130 (61.3%) isolates were from children≤5years old. CDC's Streptococcus lab bioinformatics pipeline revealed serotypes, sequence types (STs), pilus genes, PBP types and other resistance determinants. During the pre-PCV7 period, serotype 14 was the most common serotype (24.8%), followed by 6B (20.3%), 19F (10.5%), and 23F (6.8%). Post-PCV7, the proportion of PCV7 serotype 6B decreased significantly (to 6.3%), while 19F (16.3%), 14 (15.0%), 23F (7.5%), and 19A (7.5%) were the most common serotypes; only serotypes 3 and 10A increased significantly. Overall, 82% (n=173) of all isolates carried at least one resistance determinant, including 72 (34%) isolates that carried resistance determinants against 3 or more antimicrobial classes; of these 72 isolates, 56 (78%) belonged to a PCV7 serotype. Eighty-two STs were identified, with 53 of them organized in 14 clonal complexes. ST frequencies were distributed differently pre and post-PCV7 introduction, with only 18 of the 57 STs identified in years 2006-2009 isolates also observed in years 2010-2011 isolates. The apparent expansion of a 19F/ST1421 lineage with predicted ß-lactam resistance (PBP type 13:16:20) and carrying resistance determinants against four additional antimicrobial classes was observed.

Evaluation of the efficacy, safety and acceptability of a fish protein isolate in the nutrition of children under 36 months of age.

OBJECTIVE: To determine the effect of a fish protein isolate (FPi), administered over 6 months, on the growth of children aged 6-36 months, measured by Z-scores of height-for-age (HAZ) and weight-for-height (WHZ), compared with the standard meal without FPi; and to determine the safety and acceptability of FPi daily consumption. DESIGN: Cluster-randomized community-based controlled trial. For 6 months, the centres received either FPi replacing 50 % of total proteins in the diet or standard protein. HAZ and WHZ were used to determine the effect on growth. Acceptability was determined by daily consumption, measured by weighing the servings before and after consumption. SETTING: Day care centres and community nutritional centres in northern Lima, Peru. SUBJECTS: Children (n 441) aged 6-36 months. RESULTS: Four centres were randomized to the intervention with FPi, five centres were randomized to the standard control diet. More than 36 900 meals were prepared and administered in a supervised manner. Both groups received the same amounts of energy and proteins daily (proteins about 12-15 % of total energy). Growth of children who received the FPi diet was similar to that of children with the standard diet. Consumption was similar in the FPi and control groups (70 v. 80 % of amount offered, respectively). The protein was safe and well tolerated. No adverse events were reported. However, the cost of the intervention with FPi was 20-40 % lower v. the standard diet with animal protein derived from beef, chicken, eggs or liver. CONCLUSIONS: The FPi was well accepted and there was no significant difference in growth between both groups. FPi is a potential source of animal protein at lower cost.

Pathogenicity Island O-122 in enteropathogenic Escherichia coli strains is associated with diarrhea severity in children from Lima Peru.

EPEC is an attaching and effacing diarrheal pathogen that carries a large pathogenicity island, locus for enterocyte effacement (LEE). Recently, the pathogenicity island PAI O-122 was described among non-LEE effectors and found to be associated with diarrhea among atypical EPEC strains. It is unknown if incomplete PAI O-122 could be associated with diarrhea duration and severity. To identify these virulence determinants we analyzed 379 EPEC strains isolated from Peruvian children. EPEC was diagnosed by PCR(eae+, stx-) and classified as typical(t-EPEC) or atypical(a-EPEC). To characterize PAI O-122 we amplified three modules by PCR: Module 1(pagC), Module 2(senA, nleB and nleE) and Module 3(lifA/efa-1). To characterize the large ORF lifA/efa-1 we amplified the regions known as efa-N, efa-M and efa-C. Clinical information was obtained from the cohort study. A total of 379 EPEC strains were able to analyze PAI O-122 genes, 128 (10.4%) EPEC strains were isolated from 1235 diarrhea episodes and 251(9.2%) from 2734 healthy controls. t-EPEC strains were isolated from 14.8% (19/128) of children with diarrhea and 25/251(10.0%) from healthy controls. The most frequent PAI O-122 genes were nleE(37.7%), senA(34.6%) and nleB(37.5%), with similar prevalence among diarrhea and control samples. However, lifA/efa-1 was more common among diarrhea cases than healthy control cases (30.5% vs. 21.1%, p<0.05). The presence of complete PAI O-122 was associated with diarrhea episodes of higher severity among single pathogen infection (33.3% vs. 1.8%, p<0.05) mainly due to the presence of a complete lifA/efa-1 gene. In summary, the gene lifA/efa-1 is significantly associated with diarrheal episodes of higher severity, suggesting to be an important virulent factor.

Oxacillin disk diffusion testing for the prediction of penicillin resistance in Streptococcus pneumoniae.

Objective To 1) describe the correlation between the zones of inhibition in 1-µg oxacillin disk diffusion (ODD) tests and penicillin and ceftriaxone minimum inhibitory concentrations (MICs) of meningeal and non-meningeal strains of Streptococcus pneumoniae and 2) evaluate the usefulness of the ODD test as a predictor of susceptibility to penicillin in S. pneumoniae and as a quick and cost-effective method easily implemented in a routine clinical laboratory setting. Methods S. pneumoniae isolates from healthy nasopharyngeal carriers less than 2 years old, obtained in a multicentric cross-sectional study conducted in various Peruvian hospitals and health centers from 2007 to 2009, were analyzed. Using Clinical and Laboratory Standards Institute (CLSI) breakpoints, the correlation between the zones of inhibition of the ODD test and the MICs of penicillin and ceftriaxone was determined. Results Of the 571 S. pneumoniae isolates, 314 (55%) showed resistance to penicillin (MIC ≥ 0.12 µg/mL) and 124 (21.7%) showed resistance to ceftriaxone (MIC ≥ 1 µg/mL). Comparison of the ODD test zones of inhibition and the penicillin MICs, using the CLSI meningeal breakpoints, showed good correlation (Cohen's kappa coefficient = 0.8239). Conclusions There was good correlation between ODD zones of inhibition and penicillin meningeal breakpoints but weak correlation between the ODD results and non-meningeal breakpoints for both penicillin and ceftriaxone. Therefore, the ODD test appears to be a useful tool for predicting penicillin resistance in cases of meningeal strains of S. pneumoniae, particularly in low- and middle- income countries, where MIC determination is not routinely available.

Virulence factors and mechanisms of antimicrobial resistance in Shigella strains from periurban areas of Lima (Peru).

The study was aimed to describe the serotype, mechanisms of antimicrobial resistance, and virulence determinants in Shigella spp. isolated from Peruvian children. Eighty three Shigella spp. were serogrouped and serotyped being established the antibiotic susceptibility. The presence of 12 virulence factors (VF) and integrase 1 and 2, along with commonly found antibiotic resistance genes was established by PCR. S. flexneri was the most relevant serogroup (55 isolates, 66%), with serotype 2a most frequently detected (27 of 55, 49%), followed by S. boydii and S. sonnei at 12 isolates each (14%) and S. dysenteriae (four isolates, 5%). Fifty isolates (60%) were multi-drug resistant (MDR) including 100% of S. sonnei and 64% of S. flexneri. Resistance levels were high to trimethoprim-sulfamethoxazole (86%), tetracycline (74%), ampicillin (67%), and chloramphenicol (65%). Six isolates showed decreased azithromycin susceptibility. No isolate was resistant to nalidixic acid, ciprofloxacin, nitrofurantoin, or ceftriaxone. The most frequent resistance genes were sul2 (95%), tet(B) (92%), cat (80%), dfrA1 (47%), blaOXA-1like (40%), with intl1 and intl2 detected in 51 and 52% of the isolates, respectively. Thirty-one different VF profiles were observed, being the ipaH (100%), sen (77%), virA and icsA (75%) genes the most frequently found. Differences in the prevalence of VF were observed between species with S. flexneri isolates, particularly serotype 2a, possessing high numbers of VF. In conclusion, this study highlights the high heterogeneity of Shigella VF and resistance genes, and prevalence of MDR organisms within this geographic region.

Challenges in the diagnosis and management of neonatal sepsis.

Neonatal sepsis is the third leading cause of neonatal mortality and a major public health problem, especially in developing countries. Although recent medical advances have improved neonatal care, many challenges remain in the diagnosis and management of neonatal infections. The diagnosis of neonatal sepsis is complicated by the frequent presence of noninfectious conditions that resemble sepsis, especially in preterm infants, and by the absence of optimal diagnostic tests. Since neonatal sepsis is a high-risk disease, especially in preterm infants, clinicians are compelled to empirically administer antibiotics to infants with risk factors and/or signs of suspected sepsis. Unfortunately, both broad-spectrum antibiotics and prolonged treatment with empirical antibiotics are associated with adverse outcomes and increase antimicrobial resistance rates. Given the high incidence and mortality of sepsis in preterm infants and its long-term consequences on growth and development, efforts to reduce the rates of infection in this vulnerable population are one of the most important interventions in neonatal care. In this review, we discuss the most common questions and challenges in the diagnosis and management of neonatal sepsis, with a focus on developing countries.

Diarrheagenic Escherichia coli phylogroups are associated with antibiotic resistance and duration of diarrheal episode.

Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D are associated with extraintestinal strains. The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogeny and its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibiotic resistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvian children <1 year of age were determined by a Clermont triplex PCR and Kirby-Bauer method, respectively. The distribution of the 369 E. coli strains among the 4 phylogenetic groups was A (40%), D (31%), B1 (21%), and B2 (8%). DEC-control strains were more associated with group A while DEC-diarrhea strains were more associated with group D (P < 0.05). There was a tendency (P = 0.06) for higher proportion of persistent diarrhea (≥ 14 days) among severe groups (B2 and D) in comparison with nonsevere groups (A and B1). Strains belonging to group D presented significantly higher percentages of multidrug resistance than the rest of the groups (P > 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.

Lactoferrin for prevention of neonatal sepsis.

Preterm neonates are at risk to acquire infections. In addition to the high mortality associated with sepsis, these patients are at risk for long-term disabilities, particularly neurodevelopment impairment. Several interventions have been evaluated to reduce rates of infections in neonates but have not proven efficacy. Lactoferrin (LF), a milk glycoprotein with anti-inflammatory, immunomodulatory and anti-microbial properties, has the potential to prevent infections in young children. We performed a review of current and ongoing clinical trials of LF for prevention of neonatal sepsis, and found eleven registered clinical trials that include more than 6,000 subjects. Few of these trials have finished; despite their small sample size, the preliminary results show a trend towards a positive protective effect of LF on neonatal infections. Larger trials are underway to confirm the findings of these initial studies. This information will help to define LF's role in clinical settings and, if proven effective, would profoundly affect the treatment of low birth weight neonates as a cost-effective intervention worldwide.

Fecal contamination of food, water, hands, and kitchen utensils at the household level in rural areas of Peru.

The study described in this article evaluated sources of contamination of children's food and drinking water in rural households in the highlands of Peru. Samples from children's meals, drinking water, kitchen utensils, and caregivers' and children's hands were analyzed for total coliforms and E. coli counts using Petrifilm EC. Thermotolerant coliforms in water were measured using DelAgua test kits while diarrheagenic E. coli was identified using polymerase chain reaction methods (PCR). Thermotolerant coliforms were found in 48% of all water samples. E. coli was found on 23% of hands, 16% of utensils, and 4% of meals. Kitchen cloths were the item most frequently contaminated with total coliforms (89%) and E. coli (42%). Diarrheagenic E. coli was found in 33% of drinking water, 27% of meals, and on 23% of kitchen utensils. These findings indicate a need to develop hygiene interventions that focus on specific kitchen utensils and hand washing practices, to reduce the contamination of food, water, and the kitchen environment in these rural settings.

Phenotypic diversity of type III secretion system activity in enteropathogenic Escherichia coli clinical isolates.

Introduction. Enteropathogenic Escherichia coli (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins.Gap Statement. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in ler expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation.Aim. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression.Methodology. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity.Results. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression.Conclusion. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.