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.

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.

Chronic diarrhea and AIDS: insights into studies with non-human primates.

Diarrhea is the pathophysiological reaction of host's gastrointestinal tract to a variety of external stimuli. Classified as a clinical syndrome, diarrhea is the leading cause of mortality and morbidity worldwide. Clinical manifestations can occur in two major forms: A) acute, which usually resolves in less than three weeks and B) chronic, which can last for months. Because of its impact on the host immune system, acquired immune deficiency syndrome (AIDS) is currently the major cause of chronic diarrhea in many parts of the world. It is estimated that up to 90% of HIV-infected individuals with symptoms of AIDS exhibit clinical diarrhea [9, 74, 55]. In SIV-infected rhesus macaques, intense infiltration of intestinal lamina propria with virus-containing lymphocytes and macrophages can be found within days after experimental virus inoculation [25, 57]. In addition to acute enteropathy syndrome, viral infection ultimately leads to other alterations of the gastrointestinal tract including persistent and/or chronic diarrhea, a condition similar to untreated AIDS of human patients. In this short review, the chronic diarrhea is presented from the perspective of the non-human primate or simian model of AIDS (SAIDS), and its most common opportunistic and pathogenic co-infections.

The diagnostic value of electron microscopy in human immunodeficiency virus-positive patients with gastrointestinal disease.

BACKGROUND: Our aim was to determine the diagnostic value of electron microscopy in evaluating the etiology of gastrointestinal disease in patients infected with the human immunodeficiency virus (HIV). METHODS: A retrospective review of electron microscopic and light microscopic results of all HIV-positive patients with gastrointestinal and liver diseases was made during a 3-year period from June 1995 to June 1998. RESULTS: A total of 145 HIV-positive patients had their electron microscopy specimens reviewed. Of these, 136 were investigated for diarrhea, and the other 9 for increased liver enzymes. Twenty-seven of the 145 (18.6%) HIV-positive patients had a pathogen identified by electron microscopy, compared with only 13 of 145 (9%) identified by light microscopy (P < 0.005). The sensitivity of light microscopy for detecting opportunistic pathogens was 68%. Twenty-one of the 27 (77.8%) patients diagnosed by electron microscopy had microsporidiosis, and the most commonly diagnosed species was Enterocytozoon bieneusi. Light microscopy failed to identify 12 cases of microsporidiosis and 2 cases of leishmaniasis. CONCLUSIONS: Electron microscopy contributes substantially to the identification of pathogens in HIV-positive patients. Light microscopy failed to identify one of every two pathogens diagnosed by electron microscopy.

[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.

Differential metabolic response in AIDS-related chronic protozoal diarrhoea.

OBJECTIVE: As the pro-inflammatory cytokine tumour necrosis factor-alpha is greater in microsporidiosis than cryptosporidiosis, there may be a distinct metabolic response between the two organisms. DESIGN: Male HIV seropositive subjects with an untreated AIDS-defining diagnosis of microsporidiosis or cryptosporidiosis had measurement of oxygen consumption and carbon dioxide production by indirect calorimetry and body composition analysis to express resting energy expenditure (REE) and substrate oxidation per kilogram of metabolically active tissue. METHODS: Resting energy expenditure (REE), non-protein respiratory quotient (NPRQ), fat and carbohydrate oxidation were calculated from respiratory gas analysis. Fat, fat-free and appendicular muscle masses were measured by dual-energy X-ray absorptiometry. Subjects with protozoal diarrhoea were compared to other newly diagnosed, active opportunistic infections. Controls were asymptomatic HIV-seropositive men matched by peripheral CD4 count. RESULTS: Seven subjects with microsporidiosis and six with cryptosporidiosis were compared with 24 subjects with other AIDS-defining diagnosis (Pneumocystis carinii pneumonia, cytomegalovirus enteritis and Mycobacterium avium-intracellulare) and 10 controls free from secondary infection. Subjects with cryptosporidiosis had a decreased REE, a significantly increased NPRQ (P< 0.05), decreased fat oxidation (P < 0.05) and increased carbohydrate oxidation compared to microsporidiosis. Subjects with other AIDS diagnoses had an increased REE (P < 0.01) and fat oxidation and decreased carbohydrate oxidation compared to cryptosporidiosis, and a similar metabolic response to microsporidiosis. CONCLUSIONS: The metabolic response to cryptosporidiosis differs from microsporidiosis and associated weight loss may be mediated by different mechanisms. Metabolism in other AIDS diagnoses, including microsporidiosis, is compatible with a cachectic response.

Management of protozoal diarrhoea in HIV disease.

Since the first reported case of HIV infection in 1981, many HIV-seropositive patients have died as a result of diarrhoea induced by opportunistic protozoal infections: pathogens that would normally cause only a transient illness in immunocompetent individuals. The introduction of highly active antiretroviral therapy (HAART) in 1996 has been associated with a significant decline in incidence and mortality arising from infections such as cryptosporidia and microsporidia. Previously, there were no chemotherapeutic agents known to be effective in eradicating these parasites, but since the availability of HAART, the memory of the emaciated terminally ill patient with advanced AIDS suffering from refractory diarrhoea will hopefully be a thing of the past. Significant advances in the knowledge of the pathogenesis of HIV disease, earlier detection and thus treatment of the virus, and availability of improved diagnostic techniques and HAART have transformed the way HIV-associated diarrhoea is managed. In this review, we look specifically at the management of protozoa-induced diarrhoea.

Prevalence of intestinal pathogens in HIV patients with diarrhea: implications for treatment.

Patients infected with the human immunodeficiency virus (HIV) commonly experience diarrhea at some time during their illness. A variety of enteric pathogens are identified in 50-80% of these patients, depending on the intensity of the diagnostic work-up that is done. In addition to the common enteric pathogens, several unusual enteric pathogens are recognized to cause diarrhea especially in HIV patients. These include protozoan parasites such as Cryptosporidia, Isospora belli, Cyclospora cayatenensis and Microsporidium species bacteria such as enteropathogenic Escherichia coli and Mycobacterium avium-intracellulare, fungi including Candida albicans and Histoplasma capsulatum, and viruses such as astroviruses and caliciviruses. Diagnosis of these infections sometimes involves special procedures not readily available every where, and empiric therapy based on knowledge of the likely pathogens has been advocated for developing countries. This article reviews the currently available data on geographic variation of enteric pathogens in HIV patients with diarrhea and outlines a rational strategy for empiric therapy of these patients.

Small intestine pathogens in AIDS: conventional and opportunistic.

The small intestine, coming in direct contact with ingested potential pathogens, depends on active mucosal immunity to withstand invasion and damage. In patients with AIDS and severe impairment of immunoregulatory lymphocytes, proliferation of protozoal, viral, bacterial, and fungal pathogens produces diarrhea and malabsorption. When noninvasive tests of stool and blood fail to identify responsible organisms, endoscopy can reveal mucosal lesions which are suggestive if not diagnostic. Cryptosporidium, cf2E. intestinalis, cf1CMV, MAC, and other infections can be identified by intestinal biopsy quicker and often at lower overall cost than they can be by culture.

A 3-year follow-up of a brazilian AIDS patient with protracted diarrhea caused by Enterocytozoon bieneusi

Enterocytozoon bieneusi e o mais comum microsporidio agente de infeccoes gastrointestinais que ocorre predominantemente em pessoas com AIDS. Em todo o mundo os microsporidios sao reconhecidos como importantes patogenos oportunistas, entretanto poucos casos ja foram diagnosticados no Brasil, provavelmente devido ao pouco conhecimento do quadro clinico que os agentes produzem ou a dificuldades no diagnostico laboratorial. No presente trabalho relatamos o caso de um paciente brasileiro HIV-positivo acompanhado durante 3 anos, em que foram detectados esporos de microsporidios nas fezes, identificados como Enterocytozoon bieneusi por microscopia eletronica e PCR. O paciente apresentava diarreia cronica, contagem de linfocitos CD4 abaixo de 100/mm3 e fez uso de albendazol em diferentes ocasioes com melhora transitoria da diarreia, que reaparecia logo que a droga era suspensa...

Diagnostic yield of duodenal biopsy and aspirate in AIDS-associated diarrhea.

OBJECTIVES: To evaluate the diagnostic yield of performing duodenal biopsies and aspirates in AIDS patients with chronic diarrhea. METHODS: Retrospective review of esophagogastroduodenoscopy (EGD) records from January 1993 to March 1995 to identify those patients who underwent EGD for evaluation of AIDS associated diarrhea and had a duodenal biopsy and/or aspirate. Biopsies were examined for pathogens using routine histology and special stains, viral culture, and electron microscopy. Duodenal aspirates were evaluated for ova and parasites. All patients had previous negative stool studies. Pathology laboratory charges (hospital and professional fees) for each test and charges per positive test were determined. RESULTS: Of the 57 patients included in this study, 56 had a duodenal biopsy and 42 had a duodenal aspirate. An established pathogen was identified in only 15 (26%) patients. One patient had both Mycobacterium avium complex and microsporidia. Pathogens were identified in seven patients by hematoxylin and eosin stain, in three patients by acid-fast bacillus stain, and in six patients by electron microscopy. No pathogens were identified with Gomori's methenamine silver stain (44 patients), duodenal aspirate for ova and parasites (46 patients), immunoperoxidase stains (4 patients), or viral culture (4 patients). Cryptosporidia were identified in six, microsporidia in five, Mycobacterium avium complex in three, and Giardia lamblia and adenovirus each in one patient. CONCLUSIONS: In this series, the diagnostic yield of EGD with duodenal biopsy and aspirate in AIDS associated diarrhea was low. Pathogens were identified in 26% of patients; predominantly Cryptosporidium organisms and microsporidia. The routine performance of aspiration of duodenal contents for parasite examination and staining of duodenal tissue with Gomori's methenamine silver stain for fungal identification are not recommended. One should consider obtaining tissue for electron microscopy whenever duodenal biopsies are performed. The utility of EGD in AIDS associated diarrhea may improve as more effective therapies become available.

Uncertain clinical significance of duodenal mucosal abnormalities in HIV-infected individuals. Results of a case-control study.

Previous research has described abnormalities of duodenal mucosal morphology in human immunodeficiency virus (HIV)-infected individuals. We wanted to determine the frequency of disturbed villus architecture and investigate its relationship to HIV-related chronic diarrhea. We conducted a case-control study of 120 HIV-infected men, 63 with and 57 without chronic diarrhea. Stools were cultured for bacteria and examined for ova and parasites; esophagogastroduodenoscopy and flexible sigmoidoscopy with mucosal biopsies were performed. Biopsy tissue was examined using light and electron microscopy to detect enteric pathogens and to evaluate mucosal morphology. The mean CD4+ cell count was 143/min3, and enteric pathogens were detected in 56 of 120 men (47%). In approximately half the study sample (57%), duodenal villus architecture was normal; complete villus flattening was not observed. We detected no association between chronic diarrhea and altered villus architecture. Although further study is needed to clarify the pathogenesis of altered duodenal mucosal morphology, our results suggest that the clinical significance of the abnormalities may be small.

Prevalence of intestinal parasites in adult patients with enteropathic AIDS in north-eastern Tanzania.

A six month study was conducted in north-eastern Tanzania to determine the prevalence of pathogenic intestinal parasites among adult patients with enteropathic AIDS. A total of 352 patients were recruited of whom 158 (45%) had chronic diarrhoea. Of the 352 patients, 123 (35%) had intestinal parasites. Of the 123, 77 (62.6%) patients had chronic diarrhoea. The types of parasites detected were Cryptosporidium, Isospora belli, Strongyloides stercoralis, Schistosoma mansoni, Trichuris, trichiura, Ascaris lumbricoides, hookworm and Entamoeba histolytica. The prevalence of intestinal parasites was significantly higher in patients with chronic diarrhoea than in those without (P < 0.05). Cryptosporidium and Isospora belli were only detected in patients with chronic diarrhoea and were thus the most likely cause of the diarrhoea. This study has established that coccidian parasites are the most important gut opportunistic infections in Tanzanian patients with enteropathic AIDS. The fact that a high proportion of patients with chronic diarrhoea (51.3%) had no identifiable parasitic agents, suggests that other infectious agents or alternative mechanisms other than infections are responsible for the diarrhoea.

PIP: A 6-month study was conducted in northeastern Tanzania to assess the prevalence of pathogenic intestinal parasites among adult patients with enteropathic AIDS. Of the 352 patients recruited, 158 had chronic diarrhea and 123 had intestinal parasites. 77 of the 123 patients with intestinal parasites had chronic diarrhea. Cryptosporidium, Isospora belli, Strongyloides stercoralis, Schistosoma mansoni, Trichuris trichiura, Ascaris lumbricoides, hookworm, and Entamoeba histolytica were detected. The prevalence of intestinal parasites was significantly higher in patients with chronic diarrhea than in those without. Cryptosporidium and Isospora belli were only detected in patients with chronic diarrhea and were therefore the most likely cause of the diarrhea. Coccidian parasites have thus been identified as the most important gut opportunistic infections in Tanzanian patients with enteropathic AIDS. However, that 51.3% of patients with chronic diarrhea had no identifiable parasitic agents suggests that other infectious agents or alternative mechanisms are responsible for the condition.

Isosporíase como causa frequente de diarréia crônica em pacientes com AIDS em nosso meio / Isosporiasis and chronic diarrhea in AIDS

Säo apresentadas algumas consideraçöes sobre a diarréia crônica em pacientes com AIDS em nosso meio, chamando a atençäo para a alta prevalência da infecçäo pela Isospora belli.

Enteropatia pelo HIV / HIV enteropathy

A diarréia crônica é a manifestaçäo gastrointestinal mais comum em pacientes infectados pelo HIV. Deve-se a um conjunto de múltiplos fatores, entre eles o próprio HIV, que causa alteraçöes estruturais da mucosa do intestino delgado. A atrofia vilosa foi a alteraçäo mais freqüentemente observada, provavelmente devido a um desarranjo da funçäo regulatória do sistema imune sobre a estrutura da mucosa. É encontrada já na fase inicial da infecçäo pelo HIV, mesmo sem alteraçöes nos níveis sangüíneos de células CD4+ e sem infecçöes por microrganismos oportunistas. Conhecida como enteropatia pelo HIV, seu diagnóstico obriga que todas as outras possíveis causas de alteraçöes estruturais da mucosa tenham sido excluídas, após cuidadosa investigaçäo diagnóstica. Os autores fazem uma revisäo bibliográfica e discutem os mecanismos propostos para a participaçäo do HIV nessas alteraçöes.

[Acute and chronic diarrhea in AIDS: study of 435 (HIV+) patients, in Buenos Aires]. / Diarrea aguda y crónica en SIDA: estudio de 435 pacientes HIV(+), en Buenos Aires.

OBJECTIVE: Assessment of the rate of Diarrhoea in an infected HIV population of our medium. Assessment of its etiology, risk factors and response to treatment in both presentation of disease (Acute and chronic). DESIGN: This is a retrospective analysis of HIV and/or AIDS patients and diarrhea. The analytic points were: the clinical pattern of diarrhea (acute or chronic), risk factors, age, sex, etiology, stage of the disease, and response rate to treatment. PLACE: Hospital Prof. A. Posadas, that is situated in the greater Buenos Aires, and that functions as a referral centre for AIDS patients. PATIENTS AND METHODS: 435 case records that were followed up through June 1987/ March 1994 were reviewed; 109 of the total number suffered from diarrhea. RESULTS: The rate of diarrhoea in the studied population was 25% (109/435). This represented the first symptom of HIV infection in 18.3% of the patients (20/109), 49% had acute diarrhea and 51% had chronic diarrhea. An etiologic agent was established in 52.3% of the chronic diarrhoeas and in the 17.7% of the acute ones, with a 35.4% of a global isolation when basic stool test were made. Sixty three percent of the patients with chronic diarrhoea were in stage IV of HIV infection. The 78.3% of the acute diarrhoeas and 46% of the chronic ones were responsive to specific or symptomatic treatment. CONCLUSIONS: HIV antibodies determinations should be included in the study protocol of diarrhea, especially in young patients. Chronic diarrhea could be related to final stages of the disease, with lower response to treatment. Endoscopy studies should be useful when the stool tests are negative, specially in the Cytomegalovirus (CMV) and Mycobacterium complex avium intracellulare (MAI) search.