Clinical validation of loop-mediated isothermal amplification for the detection of Escherichia coli sequence type complex 131.

The dissemination of Escherichia coli multidrug-resistant (MDR) STc131 is related to its persistence in the human gastrointestinal tract as efficient gut colonizers. Infection and prevention measures are the cornerstones for preventing STc131 spread. Oral decolonization therapies that target ST131 are being developed. There are no rapid methods available to identify STc131 in human specimens. A loop-mediated isothermal amplification (LAMP) assay (named LAMP-ST131) was developed for the detection of STc131 on well-characterized E. coli isolates and then compared to culture and PCR for urines and stool swabs. With E. coli isolates (n = 720), LAMP-ST131 had a sensitivity (sens) of 100% [95% confidence interval (C.I.) = 98.1-100%)] and a specificity (spec) of 98.9% (95% C.I. = 97.5-99.5%). On urines (n = 550), LAMP-ST131 had a sens of 97.6% (95% C.I. = 89.68-94.33%) and a spec of 92.3% (95% C.I. = 87.68-99.88%), while on stool swabs (n = 278), LAMP-ST131 had a sens of 100% (95% C.I. = 88.7-100%) and a spec of 83.9% (95% C.I. = 78.8-87.9%). LAMP-ST131 detected 10 (urines) and 100 (stool swabs) gene copies/µL. LAMP-ST131 accurately identified STc131 within E. coli isolates and human specimens. The implementation of LAMP-ST131 will aid genomic surveys, enable the rapid implementation of effective infection prevention measures, and identify patients suitable for ST131 decolonization therapies. Such approaches will curb the spread of STc131 and decrease incidence rates of global MDR E. coli infections. IMPORTANCE: We developed an accurate non-culture-based loop-mediated isothermal amplification (LAMP) methodology for the detection of (sequence type) STc131 among Escherichia coli isolates and human specimens. The use of LAMP-ST131 for global genomic surveillance studies and to identify patients that are suitable for ST131 decolonization therapies will be important for decreasing multidrug-resistant E. coli infections across the globe.

Clostridioides difficile Near-Patient Testing Versus Centralized Testing: A Pragmatic Cluster Randomized Crossover Trial.

BACKGROUND: Management of suspected Clostridioides difficile infection (CDI) in the hospital setting typically results in patient isolation, laboratory testing, infection control, and presumptive treatment. We investigated whether implementation of rapid near-patient testing (NPT) reduced patient isolation time, hospital length of stay (LOS), antibiotic usage, and cost. METHODS: A 2-period pragmatic cluster randomized crossover trial was conducted. Thirty-nine wards were randomized into 2 study arms. The primary outcome measure was effect of NPT on patient isolation time using a mixed-effects generalized linear regression model. Secondary outcomes examined were hospital LOS and antibiotic therapy based on a negative binomial regression model. Natural experiment (NE), intention-to-treat (ITT), and per-protocol (PP) analyses were conducted. RESULTS: During the entire study period, a total of 656 patients received NPT for CDI and 1667 received standard-of-care testing. For the primary outcome, a significant decrease of patient isolation time with NPT was observed (NE, 9.4 hours [P < .01]; ITT, 2.3 hours [P < .05]; PP, 6.7 hours [P < .1]). A significant reduction in hospital LOS was observed with NPT for short stay (NE, 47.4% [P < .01]; ITT, 18.4% [P < .01]; PP, 34.2% [P < .01]). Each additional hour delay for a negative result increased metronidazole use (24 defined daily doses per 1000 patients; P < .05) and non-CDI-treating antibiotics by 70.13 mg (P < .01). NPT was found to save 25.48 US dollars per patient when including test cost to the laboratory and patient isolation in the hospital. CONCLUSIONS: This pragmatic cluster randomized crossover trial demonstrated that implementation of CDI NPT can contribute to significant reductions in isolation time, hospital LOS, antibiotic usage, and healthcare cost. Clinical Trials Registration. NCT03857464.

Amplicon Deep Sequencing Reveals Multiple Genetic Events Lead to Treatment Failure with Atovaquone-Proguanil in Plasmodium falciparum.

Atovaquone-proguanil (AP) is used as treatment for uncomplicated malaria, and as a chemoprophylactic agent against Plasmodium falciparum. Imported malaria remains one of the top causes of fever in Canadian returning travelers. Twelve sequential whole-blood samples before and after AP treatment failure were obtained from a patient diagnosed with P. falciparum malaria upon their return from Uganda and Sudan. Ultradeep sequencing was performed on the cytb, dhfr, and dhps markers of treatment resistance before and during the episode of recrudescence. Haplotyping profiles were generated using three different approaches: msp2-3D7 agarose and capillary electrophoresis, and cpmp using amplicon deep sequencing (ADS). A complexity of infection (COI) analysis was conducted. De novo cytb Y268C mutants strains were observed during an episode of recrudescence 17 days and 16 h after the initial malaria diagnosis and AP treatment initiation. No Y268C mutant reads were observed in any of the samples prior to the recrudescence. SNPs in the dhfr and dhps genes were observed upon initial presentation. The haplotyping profiles suggest multiple clones mutating under AP selection pressure (COI > 3). Significant differences in COI were observed by capillary electrophoresis and ADS compared to the agarose gel results. ADS using cpmp revealed the lowest haplotype variation across the longitudinal analysis. Our findings highlight the value of ultra-deep sequencing methods in the understanding of P. falciparum haplotype infection dynamics. Longitudinal samples should be analyzed in genotyping studies to increase the analytical sensitivity.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: An Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with >2 million genomes sequenced at this writing. The rise of more transmissible variants of concern that affect vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, clinical reporting for laboratories, and emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretell a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: an Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with more than 2 million genomes sequenced at the time of writing. The rise of more transmissible variants of concern that impact vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, and clinical reporting for laboratories, as well as emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretells a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

The Role of Ultrasensitive Molecular Methods for Detecting Malaria-The Broader Perspective.

Ultrasensitive molecular diagnostics are lowering the limit of detection for malaria parasites in the blood and providing insights not captured by conventional tools such as microscopy and rapid antigen tests. Low-level malaria infections identified by molecular tools may influence clinical outcomes, transmission events, and elimination efforts. While many ultrasensitive molecular methods require well-equipped laboratories, technologies such as loop-mediated isothermal amplification and recombinase polymerase amplification provide more portable and analytically sensitive solutions. These tools may benefit asymptomatic patient screening, antenatal care, and elimination campaigns. We review the recent evidence, offer our perspective on the impact of these new tests, and identify future research priorities.

Latent Class Analysis for the Diagnosis of Clostridioides difficile Infection.

BACKGROUND: Clostridioides difficile infection (CDI) is an opportunistic disease that lacks a gold-standard test. Nucleic acid amplification tests such as real-time polymerase chain reaction (PCR) demonstrate an excellent limit of detection (LOD), whereas antigenic methods are able to detect protein toxin. Latent class analysis (LCA) provides an unbiased statistical approach to resolving true disease. METHODS: A cross-sectional study was conducted in patients with suspected CDI (N = 96). Four commercial real-time PCR tests, toxin antigen detection by enzyme immunoassay (EIA), toxigenic culture, and fecal calprotectin were performed. CDI clinical diagnosis was determined by consensus majority of 3 experts. LCA was performed using laboratory and clinical variables independent of any gold standard. RESULTS: Six LCA models were generated to determine CDI probability using 4 variables including toxin EIA, toxigenic culture, clinical diagnosis, and fecal calprotectin levels. Three defined zones as a function of real-time PCR cycle threshold (Ct) were identified using LCA: CDI likely (>90% probability), CDI equivocal (<90% and >10%), CDI unlikely (<10%). A single model comprising toxigenic culture, clinical diagnosis, and toxin EIA showed the best fitness. The following Ct cutoffs for 4 commercial test platforms were obtained using this model to delineate 3 CDI probability zones: GeneXpert®: 24.00, 33.61; Simplexa®: 28.97, 36.85; Elite MGB®: 30.18, 37.43; and BD Max™: 27.60, 34.26. CONCLUSIONS: The clinical implication of applying LCA to CDI is to report Ct values assigned to probability zones based on the commercial real-time PCR platform. A broad range of equivocation suggests clinical judgment is essential to the confirmation of CDI.

Post-mortem molecular investigations of SARS-CoV-2 in an unexpected death of a recent kidney transplant recipient.

Solid organ transplant recipients are vulnerable to severe infection during induction therapy. We report a case of a 67-year-old male who died unexpectedly 10 days after receiving a kidney transplant on February 10, 2020. There was no clear cause of death, but COVID-19 was considered retrospectively, as the death occurred shortly after the first confirmed case of COVID-19 in Canada. We confirmed the presence of SARS-CoV-2 components in the renal allograft and native lung tissue using immunohistochemistry for SARS-CoV-2 spike protein and RNA scope in situ hybridization for SARS-CoV-2 RNA. Results were reaffirmed with the Food and Drug Administration Emergency Use Authorization approved Bio-Rad SARS-CoV-2 digital droplet PCR for the kidney specimen. Our case highlights the importance of patient autopsies in an unfolding global pandemic and demonstrates the utility of molecular assays to diagnose SARS-CoV-2 post-mortem. SARS-CoV-2 infection during induction therapy may portend a fatal clinical outcome. We also suggest COVID-19 may be transmittable via renal transplant.

Role of Hsp90 in Plasmodium falciparum Malaria.

Studies in mammalian and yeast chaperone systems have significantly advanced our understanding of the biochemistry of heat shock protein 90 (Hsp90), particularly as it pertains to structural details, interaction regulation, and function. Harnessing this body of knowledge, parasitologists have made tremendous steps in understanding the role of the Hsp90 chaperone machine in protozoal parasites, especially in malaria. Heat shock proteins are a crucial part of the signaling events involved in malaria parasite acclimatization in the host. Even though studies of the role of Hsp90 in malaria have been thorough and discovered some of the most intricate details of the utility of the Hsp90 interactome for pathogen-host interactions, the full and elaborate extent of the role of Hsp90 homologs in malaria remains to be uncovered. This chapter discusses the current knowledge about the biochemistry and cellular role of Hsp90 in Plasmodium falciparum malaria. In addition, the involvement of both parasitic and host Hsp90s by the parasite in the infected cell is discussed.

Active case detection of malaria in pregnancy using loop-mediated amplification (LAMP): a pilot outcomes study in South West Ethiopia.

BACKGROUND: 125 million women are pregnant each year in malaria endemic areas and are, therefore, at risk of Malaria in Pregnancy (MiP). MiP is the direct consequence of Plasmodium infection during pregnancy. The sequestration of Plasmodium falciparum parasites in the placenta adversely affects fetal development and impacts newborn birth weight. Importantly, women presenting with MiP commonly develop anaemia. In Ethiopia, the Ministry of Health recommends screening symptomatic women only at antenatal care visits with no formal intermittent preventive therapy. Since MiP can display low-level parasitaemia, current tests which include microscopy and RDT are challenged to detect these cases. Loop mediated isothermal Amplification (LAMP) technology is a highly sensitive technique for DNA detection and is field compatible. This study aims to evaluate the impact of active malaria case detection during pregnancy using LAMP technology in terms of birth outcomes. METHODS: A longitudinal study was conducted in two health centres of the Kafa zone, South West Ethiopia. Both symptomatic and asymptomatic pregnant women were enrolled in the first or second trimester and allocated to either Standard of Care (SOC-microscopy and RDT) or LAMP (LAMP, microscopy and RDT). Women completed at least three visits prior to delivery, and the patient was referred for treatment if Plasmodium infection was detected by any of the testing methods. The primary outcome was to measure absolute birth weight, proportion of low birth weight, and maternal/neonatal haemoglobin in each arm. Secondary outcomes were to assess the performance of microscopy and RDT versus LAMP conducted in the field. RESULTS: One hundred and ninety-nine women were included and assigned to either LAMP or SOC. Six were lost to follow up. In this cohort, 66.8% of women did not display any clinical symptoms and 70.9% were multi-parous. A reduced proportion of low birth weight newborns was observed in the LAMP group (0%) compared to standard of care (14%) (p <0.001). Improved neonatal haemoglobin was observed in the LAMP (13.1 g/dL) versus the SOC (12.8 g/dL) (p = 0.024) arm. RDT and microscopy had an analytical sensitivity of 66.7% and 55.6% compared to LAMP as a reference standard. CONCLUSIONS: These results support the use of highly sensitive tools for rapid on-site active case detection of MiP which may improve birth outcomes in the absence of IPT. However, further large-scale studies are required to confirm this finding.

Antimalarial activity of tetrahydro-ß-carbolines targeting the ATP binding pocket of the Plasmodium falciparum heat shock 90 protein.

A series of tetrahydro-ß-carboline derivatives of a lead compound known to target the heat shock 90 protein of Plasmodium falciparum were synthesized and assayed for both potency against the parasite and toxicity against a human cell line. Using a rationalized structure based design strategy, a new lead compound with a potency two orders of magnitude greater than the original lead compound was found. Additional modeling of this new lead compound suggests multiple avenues to further increase potency against this target, potentially paving the path for a therapeutic with a mode of action different than any current clinical treatment.

Practical Guidance for Clinical Microbiology Laboratories: Laboratory Diagnosis of Parasites from the Gastrointestinal Tract

This Practical Guidance for Clinical Microbiology document on the laboratory diagnosis of parasites from the gastrointestinal tract provides practical information for the recovery and identification of relevant human parasites. The document is based on a comprehensive literature review and expert consensus on relevant diagnostic methods. However, it does not include didactic information on human parasite life cycles, organism morphology, clinical disease, pathogenesis, treatment, or epidemiology and prevention. As greater emphasis is placed on neglected tropical diseases, it becomes highly probable that patients with gastrointestinal parasitic infections will become more widely recognized in areas where parasites are endemic and not endemic. Generally, these methods are nonautomated and require extensive bench experience for accurate performance and interpretation.

Prevalence and Epidemiological Characteristics of Asymptomatic Malaria Based on Ultrasensitive Diagnostics: A Cross-sectional Study.

BACKGROUND: As the global public-health objectives for malaria evolve from malaria control towards malaria elimination, there is increasing interest in the significance of asymptomatic infections and the optimal diagnostic test to identify them. METHOD: We conducted a cross-sectional study of asymptomatic individuals (N = 562) to determine the epidemiological characteristics associated with asymptomatic malaria. Participants were tested by rapid diagnostic tests (CareStart, Standard Diagnostics [SD] Bioline, and Alere ultrasensitive RDT [uRDT]), loop-mediated isothermal amplification (LAMP), and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine malaria positivity. Hemoglobin values were recorded, and anemia was defined as a binary variable, according to World Health Organization guidelines. RESULTS: Compared to reference qRT-PCR, LAMP had the highest sensitivity (92.6%, 95% confidence interval [CI] 86.4-96.5), followed by uRDT Alere Malaria (33.9%, 95% CI 25.5-43.1), CareStart Malaria (14.1%, 95% CI 8.4-21.5), microscopy (5.0%, 95% CI 1.8-10.5), and SD Bioline (5.0%, 95% CI 1.8-10.5). For Plasmodium falciparum specimens only, the sensitivity for uRDT Alere Malaria was 50.0% (95% CI 38.8-61.3) and SD Bioline was 7.3% (95% CI 2.7-15.3). Based on multivariate regression analysis with qRT-PCR as the gold standard, for every 3.2% increase in the prevalence of asymptomatic malaria, hemoglobin decreased by 1 gram per deciliter (prevalence ratio 0.968, 95% CI 0.940-0.997; P = .032). Deletions (4.8%) in hrp2 were noted. CONCLUSIONS: While uRDT Alere Malaria has superior sensitivity to rapid diagnostic tests and microscopy in detecting asymptomatic malaria, LAMP is superior still. Ultrasensitive diagnostics provide the accurate prevalence estimates of asymptomatic malaria required for elimination.

Multicenter Clinical Validation of the Molecular BD Max Enteric Viral Panel for Detection of Enteric Pathogens.

The conventional methodology for gastrointestinal pathogen detection remains time-consuming, expensive, and of limited sensitivity. The objective of this study was to evaluate the performance of the BD Max enteric viral panel (Max EVP) assay for identification of viral pathogens in stool specimens from individuals with symptoms of acute gastroenteritis, enteritis, or colitis. Prospective and archival stool specimens from adult and pediatric patients with diarrhea were collected in Cary-Blair medium or unpreserved containers. The results for specimens tested by the Max EVP (on the BD Max platform) were compared to those obtained by the reference method (alternate PCR assays, followed by bidirectional sequencing). Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. A total of 2,239 specimens were collected, with 2,148 being included for analysis. In this population, 39.6% of specimens were from outpatients, 42.1% were from patients <21 years old, and 49.7% were from females. Prevalence rates for prospective specimens were 7.3%, 4.5%, 3.5%, 2.4%, and 1.2% for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. PPA was 92.8%, 84.9%, 93.0%, 100%, and 95.6%, for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. NPA was ≥99.4% for all targets. In conjunction with the clinical presentation, laboratory findings, and epidemiological information, the Max EVP assay is effective for the differential diagnosis of enteric disease caused by norovirus, sapovirus, astrovirus, rotavirus, and adenovirus. This assay can be used individually for patients at high risk for a viral enteropathogen (e.g., in outbreak settings) or as an adjunct to other enteric bacterial panels.

Ultrasensitive loop mediated isothermal amplification (US-LAMP) to detect malaria for elimination.

BACKGROUND: Malaria elimination requires diagnostic methods able to detect parasite levels well below what is currently possible with microscopy and rapid diagnostic tests. This is particularly true in surveillance of malaria at the population level that includes so-called "asymptomatic" individuals. METHODS: The development of the first ultrasensitive loop mediated amplification method capable of detecting malaria from both whole blood and dried blood spots (DBS) is described. The 18S rRNA and corresponding genes that remain stable on DBS for up to 5 months are targeted. RESULTS: In the case of Plasmodium falciparum, lower limits of detection of 25 parasite/mL and 50-100 parasite/mL from whole blood and DBS were obtained, respectively. A sensitivity of 97.0% (95% CI 82.5-99.8) and specificity of 99.1% (95% CI 97.6-99.7) was obtained for the detection of all species in asymptomatic individuals from Africa and Asia (n = 494). CONCLUSION: This tool is ideally suited for low middle-income countries where malaria is endemic and ultrasensitive surveillance of malaria is highly desirable for elimination.

Risk prediction for severe disease and better diagnostic accuracy in early dengue infection; the Colombo dengue study.

BACKGROUND: A major challenge in dengue management in resource limited settings is the confirmation of diagnosis. Clinical features of dengue often overlap with other infections and molecular diagnostic tools are not readily accessible to clinicians at hospitals. In addition, the prediction of plasma leakage in dengue is also difficult. Hematocrit level and ultrasound scans (combined with clinical parameters) are helpful to detect plasma leakage once it has happened, not before. METHODS: Colombo Dengue Study (CDS) is a prospective cohort study of clinically suspected adult dengue patients recruited from the National hospital of Sri Lanka (within the first 3 days of fever) that aimed to a) identify clinical and basic laboratory test parameters to differentiate dengue from non-dengue fever, b) evaluate the comparative efficacy of loop-mediated isothermal amplification (LAMP) for dengue diagnosis (vs. NS1 antigen test and RT-qPCR) and c) identify early associations that are predictive of plasma leakage or severe dengue. The basic laboratory tests considered here included hematological parameters, serum biochemistry and inflammatory markers. RESULTS: Only 70% of clinically suspected patients were confirmed as having dengue by either the NS1 antigen test or RT-qPCR. On a Bayesian latent class model which assumes no "gold standard", LAMP performed equally or better than RT-qPCR and NS1 antigen test respectively. When confirmed dengue patients were compared with others, the earlier group had significantly lower lymphocyte counts and higher aspartate aminotransferase levels (AST) within the first 3 days of fever. Confirmed dengue patients with plasma leakage had a lower mean age and a higher median baseline AST level compared to those without plasma leakage (p < 0.05). CONCLUSION: Clinical suspicion overestimates the true number of dengue patients. RT-LAMP is a potentially useful low-cost diagnostic tool for dengue diagnosis. Confirmed dengue patients had significantly higher AST levels and lower lymphocyte counts in early disease compared to others. In confirmed dengue patients, younger age and a higher AST level in early infection were associated with subsequent plasma leakage.

Susceptibility Testing of Medically Important Parasites.

In the last 2 decades, renewed attention to neglected tropical diseases (NTDs) has spurred the development of antiparasitic agents, especially in light of emerging drug resistance. The need for new drugs has required in vitro screening methods using parasite culture. Furthermore, clinical laboratories sought to correlate in vitro susceptibility methods with treatment outcomes, most notably with malaria. Parasites with their various life cycles present greater complexity than bacteria, for which standardized susceptibility methods exist. This review catalogs the state-of-the-art methodologies used to evaluate the effects of drugs on key human parasites from the point of view of drug discovery as well as the need for laboratory methods that correlate with clinical outcomes.

Epidemiological and genomic characterization of community-acquired Clostridium difficile infections.

BACKGROUND: Clostridium difficile infection (CDI) is a major cause of morbidity and mortality in North America and Europe. The aim of this study was to identify epidemiologically-confirmed cases of community-acquired (CA)-CDI in a large North American urban center and analyze isolates using multiple genetic and phenotypic methods. METHODS: Seventy-eight patients testing positive for C. difficile from outpatient clinics were further investigated by telephone questionnaire. CA-CDI isolates were characterized by antibiotic susceptibility, pulsed-field gel electrophoresis and whole genome sequencing. CA-CDI was defined as testing positive greater than 12 weeks following discharge or no previous hospital admission in conjunction with positive toxin stool testing. RESULTS: 51.3% (40/78) of the patients in this study were found to have bona fide CA-CDI. The majority of patients were female (71.8% vs. 28.2%) with 50-59 years of age being most common (21.8%). Common co-morbidities included ulcerative colitis (1/40; 2.5%), Crohn's disease (3/40; 7.5%), celiac disease (2/40; 5.0%) and irritable bowel syndrome (8/40; 20.0%). However, of 40 patients with CA-CDI, 9 (29.0%) had been hospitalized between 3 and 6 months prior and 31 (77.5%) between 6 and 12 months prior. The hypervirulent North American Pulostype (NAP) 1-like (9/40; 22.5%) strain was the most commonly identified pulsotype. Whole genome sequencing of CA-CDI isolates confirmed that NAP 1-like pulsotypes are commonplace in CA-CDI. From a therapeutic perspective, there was universal susceptibility to metronidazole and vancomycin. CONCLUSIONS: All CA-CDI cases had some history of hospitalization if the definition were modified to health care facility exposure in the last 12 months and is supported by the genomic analysis. This raises the possibility that even CA-CDI may have nosocomial origins.

Molecular detection of Enteropathogens from diarrheic stool of HIV positive patients in Gondar, Ethiopia.

BACKGROUND: Infectious diarrhea is a common problem in the developing world, especially among people living with HIV/AIDS. Traditional diagnostic methods such as stool culture and microscopic examination are limited by resources and poor sensitivity. The use of molecular diagnostics for enteropathogen detection in this region of sub-Saharan Africa has not been fully explored. We sought to identify risk factors and characterize enteropathogens from diarrheic stools of HIV-positive patients in Gondar, Ethiopia using multiplex molecular panels targeting key infectious agents. METHODS: A cross-sectional study of 100 stool samples was performed. Samples were collected consecutively from HIV- positive patients presenting with diarrhea at University of Gondar Hospital clinic, a major center in NW Ethiopia. Genomic DNA was extracted from stool and processed using a multiplex molecular panel Allplex™ [Seegene, Canada]. Correlations between patient characteristics, symptoms, public health risk factors, and enteropathogen type (s) were studied. Eighty-six samples were successfully analyzed by molecular methods. RESULTS: The mean age was 35 with 43% male. Eighty percent lived in an urban area, 18% had access to well water only, and 81% practiced proper hand hygiene. The majority of patients (72%) were receiving HAART with a median CD4 cell count of 362/µL. Multiple pathogens were detected in 94% of specimens, with an average of 5 enteropathogens per sample. Common bacteria, viruses, and parasites detected were Shigella spp./enteroinvasive E. coli (80%), enterotoxigenic E. coli (73%), Norovirus (16%) and B. hominis (62%). CD4 cell count < 500/ µL was associated with the presence of viruses (p = 0.004) and the absence of STEC (p = 0.010). The use of HAART or CD4 levels was not associated with the number of enteropathogens detected. CONCLUSIONS: Diarrheic stool from HIV-positive outpatients in Gondar, Ethiopia had on average 5 enteropathogens present in their stool. Shigellaspp./enteroinvasive E. coli and enterotoxigenic E. coli are the major pathogens, not dissimilar to immunocompetent individuals in low income countries.