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.

HIV-Associated Complications: A Systems-Based Approach.

Persons with human immunodeficiency virus (HIV) infection often develop complications related directly to the infection, as well as to treatment. Aging, lifestyle factors, and comorbidities increase the risk of developing chronic conditions such as diabetes mellitus and chronic kidney disease. HIV-associated neurologic complications encompass a wide spectrum of pathophysiology and symptomatology. Cardiovascular and pulmonary conditions are common among persons with HIV infection. Although some specific antiretroviral medications have been linked to disease development, traditional risk factors (e.g., smoking) have major roles. Prevention and management of viral hepatitis coinfection are important to reduce morbidity and mortality, and new anti-hepatitis C agents produce high rates of sustained virologic response. Antiretroviral-associated metabolic complications include dyslipidemia, hyperglycemia, and loss of bone mineral density. Newer options generally pose less risk of significant systemic toxicity and are better tolerated. Family physicians who care for patients with HIV infection have a key role in identifying and managing many of these chronic complications.

Crofelemer, a novel agent for treatment of non-infectious diarrhea in HIV-infected persons.

Crofelemer is the first US FDA-approved drug for symptomatic relief in HIV-infected persons on antiretroviral therapy (ART) who have non-infectious diarrhea. With the availability of ART, there is increased survival and decrease in gastrointestinal opportunistic infections. However, diarrhea secondary to ART and HIV enteropathy is common in HIV-infected persons. Crofelemer is manufactured from the red latex sap of the Croton lechleri tree in South America. It has a unique mechanism leading to inhibition of chloride ion secretion by blocking chloride channels in the gastrointestinal lumen. This reduces efflux of sodium and water, which in turn reduces the frequency and consistency of diarrhea. Crofelemer is well tolerated due to minimal systemic absorption and has a good safety profile. The availability of crofelemer will likely have a positive impact on the quality of life in HIV-infected persons and also increase compliance to ART.

Enteropathies in the developing world: neglected effects on global health.

A spectrum of enteropathies, characterized by small intestinal inflammation, reduced absorptive capacity, and increased intestinal permeability, commonly affect people in developing countries. This subclinical intestinal pathology facilitates microbial translocation across the compromised intestinal barrier, leading to chronic systemic inflammation that may adversely impact health. Environmental enteropathy (EE), ubiquitous among people living in unhygienic conditions, likely mediates two interlinked public health problems of childhood, stunting and anemia, and underlies poor oral vaccine efficacy in developing countries. Human immunodeficiency virus (HIV) enteropathy, which frequently overlaps with EE, may contribute to immune activation and modulate HIV disease progression. The interacting effects of infection and enteropathy drive a vicious cycle that can propagate severe acute malnutrition, which underlies almost half of under-5-y deaths. Enteropathies are therefore highly prevalent, interacting causes of morbidity and mortality in developing countries. Interventions to prevent or ameliorate enteropathies have potential to improve the health of millions of people in developing countries.

Detection of Dientamoeba fragilis in patients with HIV/AIDS by using a simplified iron hematoxylin technique.

INTRODUCTION: Studies strongly indicate Dientamoeba fragilis as one of the causes of diarrhea in human immunodeficiency virus (HIV) patients. METHODS: The objective of the present study was to evaluate the prevalence of D. fragilis associated with the causes of diarrhea in 82 HIV/ AIDS patients hospitalized at the Instituto de Infectologia Emílio Ribas from September 2006 to November 2008. RESULTS: In total, 105 samples were collected from 82 patients. Unprotected sex was the most frequent cause of HIV infection (46.3%), followed by the use of injectable or non-injectable drugs (14.6%). Patients presented with viral loads of 49-750,000 copies/ mL (average: 73,849 ± 124,850 copies/mL) and CD4 counts ranging of 2-1,306 cells/mm³ (average: 159 ± 250 cells/mm³). On an average, the odds of obtaining a positive result by using the other techniques (Hoffman, Pons and Janer or Lutz; Ritchie) were 2.7 times higher than the chance of obtaining a positive result by using the simplified iron hematoxylin method. Significant differences were found between the methods (p = 0.003). CONCLUSIONS: The other techniques can detect a significantly greater amount of parasites than the simplified iron hematoxylin method, especially with respect to Isospora belli, Cryptosporidium sp., Schistosoma mansoni, and Strongyloides stercoralis, which were not detected using hematoxylin. Endolimax nana and D. fragilis were detected more frequently on using hematoxylin, and the only parasite not found by the other methods was D. fragilis.

Detection of Dientamoeba fragilis in patients with HIV/AIDS by using a simplified iron hematoxylin technique / Detecção de Dientamoeba fragilis em pacientes com HIV/AIDS utilizando a técnica de hematoxilina férrica simplificada

INTRODUCTION: Studies strongly indicate Dientamoeba fragilis as one of the causes of diarrhea in human immunodeficiency virus (HIV) patients. METHODS: The objective of the present study was to evaluate the prevalence of D. fragilis associated with the causes of diarrhea in 82 HIV/ AIDS patients hospitalized at the Instituto de Infectologia Emílio Ribas from September 2006 to November 2008. RESULTS: In total, 105 samples were collected from 82 patients. Unprotected sex was the most frequent cause of HIV infection (46.3%), followed by the use of injectable or non-injectable drugs (14.6%). Patients presented with viral loads of 49-750,000 copies/ mL (average: 73,849 ± 124,850 copies/mL) and CD4 counts ranging of 2-1,306 cells/mm³ (average: 159 ± 250 cells/mm³). On an average, the odds of obtaining a positive result by using the other techniques (Hoffman, Pons and Janer or Lutz; Ritchie) were 2.7 times higher than the chance of obtaining a positive result by using the simplified iron hematoxylin method. Significant differences were found between the methods (p = 0.003). CONCLUSIONS: The other techniques can detect a significantly greater amount of parasites than the simplified iron hematoxylin method, especially with respect to Isospora belli, Cryptosporidium sp., Schistosoma mansoni, and Strongyloides stercoralis, which were not detected using hematoxylin. Endolimax nana and D. fragilis were detected more frequently on using hematoxylin, and the only parasite not found by the other methods was D. fragilis.

INTRODUÇÃO: Estudos indicam a Dientamoeba fragilis como uma das causas de diarréia em pacientes com HIV/AIDS. MÉTODOS: Os objetivos deste estudo foram avaliar a prevalência de D. fragilis associadas com as causas de diarréia em pacientes com HIV/AIDS internados no Instituto de Infectologia Emílio Ribas (IIER). Oitenta e dois pacientes internados no IIER fizeram parte deste estudo de setembro de 2006 a novembro de 2008. RESULTADOS: No total, 105 amostras foram coletadas a partir de 82 pacientes neste estudo. Sexo desprotegido foi à causa mais frequente para a aquisição do HIV (46,3%), seguido pelo uso de drogas injetáveis ou não injetáveis (14,6%). Relações heterossexuais foram os mais citados (19,5%). Pacientes apresentaram carga viral entre 49 e 750.000 (média de 7.849 ± 124.850) e CD4 variando de 2 a 1.306 (média de 159 ± 250). Em média, as chances de um resultado ser positivo com outras técnicas foram 2,7 vezes maiores do que a chance de um resultado positivo com hematoxilina férrica simplificada. Foram encontradas diferenças significativas entre os métodos (p=0,003). CONCLUSÕES: As outras técnicas são capazes de detectar uma quantidade significativa maior de parasitas em comparação com a hematoxilina férrica simplificada, especialmente em relação à Isospora belli, Cryptosporidium sp., Schistossoma mansoni e Strongyloides stercoralis que não foram encontrados utilizando a hematoxilina e a Endolimax nana e D. fragilis foram mais detectados pela hematoxilina férrica simplificada, principalmente a D. fragilis que não foi detectada pelos outros métodos.

Occurrence of gastrointestinal opportunistic disorders in AIDS despite the use of highly active antiretroviral therapy.

Since the introduction of highly active antiretroviral therapy (HAART), the frequency of opportunistic disorders (ODs) of the gastrointestinal (GI) tract in human immunodeficiency virus (HIV)-infected patients has fallen dramatically. We have found, however, that despite the use of HAART, HIV-infected patients can still present with GI ODs. To evaluate the prevalence of GI ODs in HIV-infected patients on HAART who were undergoing endoscopic evaluation for GI-related symptoms. From January 1996 through February 2002, all HIV-infected patients undergoing GI endoscopy were prospectively identified; mucosal biopsies were obtained in a standardized fashion and histologic specimens were examined by a single GI pathologist. All the patients on HAART presenting with a GI OD are described. Results showed that 294 patients (88% men; mean age, 36.5 +/- 10 years; median CD4 lymphocyte count, 64 cells/microl; range, 1-884; median viral RNA level, 40,357 copies/ml; range, 0-7,721,715) underwent 401 upper and/or lower endoscopies during the study period. The use of HAART increased from 10% in early 1996 to 85% in 2002. Nevertheless we found that 27 patients (9%) presented with a GI OD despite HAART. Forty percent of the patients with a GI OD were noncompliant with HAART. We conclude that GI ODs can occur despite HAART and normal CD4 counts. The prevalence of GI ODs in HIV-infected patients taking HAART is 9%. The reasons for this are multifactorial and likely include noncompliance with medications, viral resistance to the drugs, and decreased drug bioavailability. Although the use of HAART has led to a decreased incidence of GI ODs in AIDS, the gastroenterologist evaluating these patients should not discard the possibility that the GI symptoms in HIV-infected patients taking HAART may be secondary to an OD, even when the CD4 count is normal and the viral load is low.

[Gastrointestinal manifestations of HIV infection]. / Gastrointestinale Manifestationen der HIV-Infektion.

BACKGROUND: HIV-related immunodeficiency particularly affects the mucosal immune system. Therefore, the gastrointestinal tract is target to numerous HIV-associated diseases. Since the introduction of highly active antiretroviral therapy (HAART) the prevalence of HIV-related secondary diseases has significantly declined. Their clinical appearance, however, remained unchanged. RESULTS: This review summarizes the most important gastrointestinal complications of HIV disease and their treatment focusing on key symptoms and signs. In addition, appropriate diagnostic strategies are proposed. CONCLUSIONS: Given the high number of secondary gastrointestinal diseases and their potential impact on prognosis and quality of life of HIV-infected patients, it is important to employ an effective diagnostic and therapeutic approach to these patients. Control of HIV replication and immune deficiency by HAART offers new therapeutic options in cryptosporidiosis and microsporidiosis and is crucial for the long-term prevention of opportunistic enteric infections or HIV-related malignancies.

[Chronic diarrhea: value of microbiology in diagnosis]. / Chronische Diarrhoe: Stellenwert der Mikrobiologie in der Diagnostik.

Chronic diarrhoea of the adult is defined as diarrhea during 30 days or longer. Frequent causes of chronic diarrhea in the immunocompetent adult without recent travel to developing countries are noninfectious processes, including laxatives misuse, diseases causing chronic maldigestion, osmotically active artificial sweeteners (i.e. sorbitol), hormonal disorders or drugs with intestinal side effects. Infectious agents as the cause of chronic diarrhea are important in two populations, namely in travelers returning from tropical countries bearing a significant risk of intestinal parasitic infections and in immunocompromised patients, especially AIDS patients with CD4 cell counts below 50 per microliter. Intestinal parasites and C. difficile, Y. enterocolitica, Shigellae and Cytomegalovirus are the most important causative agents of chronic diarrhea. Intestinal pathogens were identified in 46% of chronic, but only in 16.5% of acute diarrhea episodes of HIV-infected patients. An extensive medical history including recent travel as well as the detailed characteristics of onset of symptoms and of their time course is essential for the diagnosis. All patients should have a complete differential blood count, ESR, determination of electrolytes, liver enzymes, creatinine, blood glucose, and serum albumin. Tests to exclude hyperthyriodism, or pancreatic insufficiency as well as a d-xylose absorption test can be included, if appropriate. Microbiological-parasitological investigations are obligatory in patients with chronic diarrhea returning from countries with increased risk of traveler diarrhea, in cases of suspected immunodeficiency, if sudden onset of symptoms with fever is reported, after antibiotic treatment, and in children below six years of age. As a rule, stool specimens are appropriate, for the detection of cytomegalovirus colonic biopsies are necessary. In the latter case colonosigmoidoscopy has no diagnostic advantage. One single stool specimen is sufficient for the detection of bacteria or toxins, in contrast to parasitological investigations, where only three consecutive specimens provide sufficient diagnostic sensitivity.

Special histologic stains are rarely beneficial for the evaluation of HIV-related gastrointestinal infections.

During a 28-month period, endoscopic mucosal biopsy specimens from all HIV-infected patients were submitted for routine histologic evaluation. Immunoperoxidase staining for cytomegalovirus and herpesvirus antigens (esophagus), mycobacterial and fungal staining, and Gram staining of mucosal biopsy specimens were done. Special fungal and acid-fast stains were selectively performed in patients with absolute CD4 cell counts of less than 200 cells per microliter (200 x 10(6)/L) and/or with diarrhea and or wasting syndrome. Treatment was based on the endoscopic and histologic findings, and long-term follow-up was performed. The 121 symptomatic HIV-infected patients underwent 221 upper and/or lower endoscopies with 285 biopsy sites. The sensitivity and specificity of H&E staining for the diagnosis of gastrointestinal cytomegalovirus were 97% and 100%, respectively. The results of fungal and mycobacterial stains neither altered therapy nor identified previously undiagnosed infections in any patient. Long-term follow-up revealed no patient in whom an infection was missed on routine H&E, which affected outcome. Routine H&E staining is accurate for the diagnosis of gastrointestinal opportunistic infections in HIV-infected patients. Special histologic stains for fungal, mycobacterial, and viral infections did not increase the diagnostic yield or alter medical therapy but doubled the costs.

Accuracy of endoscopic databases for assessing patient symptoms: comparison with self-reported questionnaires in patients infected with the human immunodeficiency virus.

BACKGROUND: Endoscopic databases are increasingly used for clinical research, but their validity as research instruments has not been assessed. We compared the accuracy of endoscopic indications recorded in an endoscopic database with patient symptom questionnaires. METHODS: All patients infected with the human immunodeficiency virus referred to the outpatient gastroenterology practice were prospectively evaluated using recognized symptom questionnaires. For patients undergoing esophagogastroduodenoscopy, the procedure indications recorded in the endoscopic database and the patient's self-reported symptom scores were compared. RESULTS: Ninety-three patients were evaluated. The symptoms of nausea/vomiting, diarrhea, and anorexia were highly predictive for the presence of these symptoms on the patient questionnaires. The symptoms of dyspepsia/abdominal pain did not predict well the presence of these symptoms on the questionnaire. Patients reported frequent and severe symptoms that were not recorded as indications for the procedures. The overall agreement (kappa statistic) was highly variable, from slight (kappa = 0.07 for anorexia) to moderate (kappa = 0.44 for diarrhea). CONCLUSIONS: Endoscopic indications are variably associated with self-reported symptom scores. These findings raise concerns about using some endoscopic database indications as accurate representations of patients' symptoms. Until performance characteristics of a given database are known, symptom-oriented research should use validated questionnaires whenever possible.

[Endoscopic evaluation of chronic human immunodeficiency virus-related diarrhea]. / La place de l'endoscopie dans le diagnostic etiologique des diarrhées chroniques des sidéens.

Chronic diarrhea is an important clinical problem in patients infected with HIV. Data assessing the diagnostic yield of upper and lower endoscopy are limited. We reported 10 cases of HIV-infected patient referred to our hospital for chronic diarrhea from March 1995 to June 1999. 60% of the pathogens were identified obviously by stool studies. Cryptosporidium and Mycobacterium avium intracellulare (MAI) were the most common organisms. In this study, endoscopy identified 2 additional cases of MAI and one of 5 cryptosporidia detected in stool. Immunologic test identified a CMV infection in one case. Stool tests and endoscopy identified obviously 80% of the pathogens. Most investigators and us agree that stool studies should be the first diagnostic test. In patients with negative stool studies, lower endoscopy is more cost-effective than upper endoscopy and indicated as an initial exam.

Evaluation of upper gastrointestinal tract symptoms in patients infected with HIV.

OBJECTIVE: Upper gastrointestinal tract (UGI) symptoms are frequent in patients infected with the human immunodeficiency virus (HIV), but little published information exists about their characteristics or methods of evaluation. We evaluated the prevalence of nonesophageal UGI symptoms in a referral population, the utility of esophagogastroduodenoscopy (EGD) for diagnosis, and clinical predictors of abnormal endoscopic findings in patients infected with HIV. METHODS: All HIV-infected patients referred to.the outpatient gastroenterology clinics were prospectively evaluated using recognized symptom questionnaires. EGD indications, results, and the patients' self-reported symptom scores were compared. HIV-infected patients undergoing EGD were compared with HIV-infected patients not receiving an EGD and with symptomatic non-HIV-infected patients undergoing EGD. RESULTS: A total of 201 patients completed 280 questionnaires. Among 93 patients who underwent endoscopy, severe symptoms occurring at least several times per week included: anorexia (70%), upper abdominal pain (34%), vomiting (32%), or a recent weight loss of approximately 15 lb (31%). Patients undergoing EGD had more frequent/severe symptoms, but did not have differences in overall well-being or mean GI symptom score. The frequency of substantial and treatable endoscopic findings among patients infected with HIV was comparable to that found in the non-HIV-infected control group. There were no independent symptoms predicting substantial or treatable disease on EGD. CONCLUSIONS: We conclude that: 1) upper gastrointestinal symptoms are common in HIV-infected patients referred for GI consultation; 2) symptomatic HIV patients have a high prevalence of both treatable and untreatable upper GI pathologies; 3) and physicians use symptom frequency and severity to select patients for EGD, but these factors correlate poorly with abnormalities on EGD. Given this discrepancy, longitudinal study is needed to determine whether treating endoscopic abnormalities improves UGI symptoms.

A prospective study of endoscopy in HIV-associated diarrhea.

OBJECTIVE: Diarrhea commonly occurs in persons with human immunodeficiency virus (HIV) infection. The optimal use of endoscopic procedures remains poorly studied for patients with HIV-related diarrhea. The purpose of this study is to compare the diagnostic yield of a complete endoscopic work-up including an esophagogastroduodenoscopy and colonoscopy to a more limited approach of biopsies obtainable by flexible sigmoidoscopy. METHODS: A prospective study of 79 patients with HIV-related diarrhea. Upper endoscopy and colonoscopy were performed with tissue biopsies labelled according to location within the colon or small intestine. RESULTS: A new infection was diagnosed in 22 of 79 patients (28%). Biopsy of the left colon yielded an enteric pathogen in 17 of 22 patients (sensitivity: 77%) and in 15 of 15 patients with cytomegalovirus colitis (sensitivity: 100%). Combined left and right colonic biopsies had a sensitivity of 82%. Combined colonic and terminal ileum biopsies missed no pathogens. Duodenal biopsies yielded no additional pathogens beyond those identified by colonoscopy and terminal ileal biopsy. Patients with a new pathogen diagnosed had significantly lower CD4 lymphocyte counts as compared to patients without a new pathogen (p = 0.001). CONCLUSIONS: For patients with CD4 counts < 100/mm3 and unexplained AIDS-related diarrhea, flexible sigmoidoscopy with biopsy is a sufficiently thorough endoscopic evaluation.

In situ hybridization: a molecular approach for the diagnosis of the microsporidian parasite Enterocytozoon bieneusi.

Microsporidia are emerging as opportunistic pathogens in patients with acquired immunodeficiency syndrome (AIDS). Enterocytozoon bieneusi is the most commonly reported microsporidium that is detected in gastrointestinal specimens. This report describes an in situ hybridization technique with a 30-base specific synthetic DNA probe for detection of E bieneusi by light microscopy. Formalin-fixed paraffin-embedded duodenal biopsy specimens from three patients with AIDS, chronic diarrhea, and E bieneusi infection confirmed by electron microscopy were used in this study. Light microscopic examination after colorimetric detection allowed the identification of different stages of the pathogen's life cycle in the cytoplasm of enterocytes. No cross-reactivity was noted between the probe and human DNA. Our study underscores the applicability of a synthetic-labeled oligonucleotide for the detection and identification of E bieneusi in clinical samples.

Diagnostic yield and cost-effectiveness of endoscopy in chronic human immunodeficiency virus-related diarrhea.

BACKGROUND: Endoscopy is commonly performed in patients with chronic human immunodeficiency virus (HIV)-related diarrhea after negative stool studies. The aim of this study was to determine the diagnostic yield and cost-effectiveness of endoscopy in this setting. METHODS: Consecutive HIV-infected patients with chronic unexplained diarrhea who were referred for diagnostic endoscopy were identified. Patient charts, pathology reports, and endoscopy records were reviewed. RESULTS: A total of 479 endoscopic procedures were performed in 307 patients. A pathogen was identified in 147 patients (47.9%); cytomegalovirus was the most frequent organism found (21.5%). The average cost of identifying a pathogen by endoscopy was $3822.94. Colonoscopy had a greater diagnostic yield than flexible sigmoidoscopy (38.7% vs. 22.4%, p = 0.009) and was more cost-effective. The yield of upper endoscopy was 29.6%. In patients with a CD4 count of less than 100/mm3, endoscopy had a higher diagnostic yield (62.8% vs. 8.3%, p < 0.0001) and a lower cost of identifying a pathogen ($2943.92 vs. $21,583.51) than in those with higher CD4 counts. CONCLUSIONS: Endoscopy frequently identifies a pathogen in HIV-related chronic diarrhea. Colonoscopy is the most cost-effective procedure. Endoscopic evaluation has a significantly higher diagnostic yield and is considerably more cost-effective in patients with a CD4 count of less than 100/mm3 than in those with higher CD4 counts.