Polymerase chain reaction for microsporidian DNA in gastrointestinal biopsy specimens of HIV-infected patients.

OBJECTIVE: To study the accuracy of polymerase chain reaction (PCR) for microsporidian DNA in gastrointestinal biopsy specimens of HIV-infected patients for the diagnosis of intestinal microsporidiosis. SETTING: Infectious disease in- and outpatient clinic of a university hospital in Cologne, Germany. PATIENTS: Forty-six HIV-infected patients with diarrhoea. METHODS: PCR and Southern blot hybridization were performed using DNA extracted from intestinal biopsy specimens with primers and probes from the small subunit rRNA gene of Enterocytozoon bieneusi and Septata intestinalis. Histological examination of intestinal biopsy specimens was performed using a fluorescence technique. Transmission electron microscopy of intestinal biopsy specimens was performed in 13 patients. RESULTS: Amplification and Southern blot hybridization with species-specific primers and probes gave positive results in 10 patients for E. bieneusi, and in 10 patients for S. intestinalis. Overall, five cases of double infection with E. bieneusi and S. intestinalis were seen when both primer pairs and probes were used. Histological examination showed microsporidian spores in all 15 cases, but light microscopy was unable to distinguish between species in almost all cases. CONCLUSIONS: PCR detection of microsporidian DNA in intestinal biopsy specimens can be used reliably for the diagnosis of intestinal microsporidiosis in HIV-infected patients and is also useful for species differentiation between microsporidia. Infections with S. intestinalis and double infections with two types of microsporidia appear to be more common than previously described.

Virological treatment failure of protease inhibitor therapy in an unselected cohort of HIV-infected patients.

OBJECTIVE: To determine the rate of virological treatment failure with protease inhibitor therapy in unselected patients and to assess underlying risk factors. DESIGN AND SETTING: Retrospective study in two German tertiary care treatment centres. PATIENTS: A total of 198 HIV-infected patients treated with protease inhibitors in 1996. MAIN OUTCOME MEASURES: Levels of HIV RNA 1-6 months after start of treatment; definition of treatment failure of < 1 log10 reduction in plasma HIV RNA within 6 months after starting protease inhibitor therapy; multivariate analysis of risk factors for treatment failures. RESULTS: A total of 226 treatment episodes with protease inhibitors were evaluable (saquinavir, 83; ritonavir, 47; indinavir, 96). The rate of virological treatment failure was 44% (saquinavir, 64%; ritonavir, 38%; indinavir, 30%). In a multivariate analysis, the following independent risk factors for virological failure were found: CD4 cell count, pretreatment with antiretroviral drugs (number), and protease inhibitor (compound). The relative risk reduction for each CD4 cell count increase was 0.997 (P = 0.012), 2.64 for pretreatment with one or two drugs versus no drug (P = 0.05), 2.97 for pretreatment with more than two drugs versus no drug (P = 0.05), and 4.62 for treatment with saquinavir versus indinavir (P = 0.001). CONCLUSION: An unexpectedly high rate of virological treatment failure of protease inhibitor therapy was found in an unselected cohort of HIV-infected patients. Response to antiretroviral combination therapy in normal clinical practice may considerably differ from results of randomized clinical trials. Further studies are warranted to find optimal treatment strategies for both initial and salvage therapy.

Asymptomatic pneumatosis intestinalis and free abdominal air in a patient with AIDS.

Detection of free abdominal air requires in most cases immediate surgical intervention. However, there may be situations, where invasive procedures are not indicated. We present a case of asymptomatic pneumatosis intestinalis and free abdominal air in a patient with Aids. Pneumatosis intestinalis is a rare entity with accumulation of subserosal or submucosal gas occurring in the small or large bowel. It has been reported in a variety of gastrointestinal disorders, in Aids, after transplantation, with steroid use, and in association with leukemia, lymphoma, vasculitis, collagen vascular disease and chronic obstructive pulmonary disease.

Clinical efficacy of protease inhibitor based antiretroviral combination therapy--a prospective cohort study.

OBJECTIVES: To analyze virological and clinical efficacy of protease inhibitor based antiretroviral regimens in a cohort of unselected HIV-infected patients. METHODS: Prospective analysis of all HIV-infected patients started on protease inhibitor therapy until August 31, 1997 in two outpatient clinics. Partial viral suppression was defined as reduction of HIV-RNA at least 1log(10) below baseline and complete viral suppression as reduction below the limit of detection. Risk factors for clinical and virological failure were analyzed by a Cox proportional hazard model. RESULTS: 387 patients (median observation time 381 days) were analyzed. In 312 patients (81%) partial and in 265 (68%) complete viral suppression was observed. Secondary failure occurred in 75 patients and could be reversed in 11/75. The probability of virological failure at one year was 51% for complete and 47% for partial suppression. CD4-cells increased by a median of 101/microl overall and 39/microl for patients without partial virologic suppression. 57 clinical events or deaths occurred in 44 pts. Risk factors for virological failure were AIDS at baseline (RR 1.6) and use of Saquinavir vs. Indinavir or Ritonavir (RR 1.7), for clinical failure AIDS at baseline (RR 4. 9), CD4-cell count (0.74 for increase of 50/microl), degree of viral suppression (RR 0.1 for complete suppression) and PI used (Saquinavir vs. Indinavir or Ritonavir, RR 2.7). CONCLUSIONS: Virological failure of PI based combination therapy is common and associated with advanced HIV-infection. Clinical failure is associated with advanced HIV-infection and failure to suppress viral replication.

Polymerase chain reaction for diagnosis and species differentiation of microsporidia.

Polymerase chain reaction (PCR) techniques have been developed for the detection of microsporidian DNA in different biological samples. We used sequence data of the rRNA gene for the identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. cuniculi, and E. hellem in different biological samples of HIV-infected patients by PCR, Southern blot hybridization, restriction endonuclease digestion analysis, cloning, and comparative genetic sequencing. One primer pair was used for amplification of the entire small subunit (SSU)-rRNA gene of E. bieneusi, E. intestinalis, and E. hellem from samples with electron microscopy confirmed infection. The amplified 1.2 kb SSU-rRNA gene fragments were ligated into a pMOSBlue T-vector, transfected into pMOSBlue competent cells, and were used as positive controls. Several primer pairs and hybridization probes were used to amplify and identify microsporidian DNA from different samples. Light microscopical examination of samples was performed in all patients and transmission electron microscopy was done on a subset of patient samples. DNA products were obtained from all samples with confirmed microsporidial infections. The identity of the DNA fragments was determined by Southern blot hybridization or by restriction endonuclease digestion analysis or by DNA sequencing. The results show that PCR is a reliable and sensitive indicator for the presence of microsporidian DNA in different biological samples of HIV-infected patients. PCR can be used further for species differentiation of microsporidia, even between species which cannot be differentiated by light and/or electron microscopy.

Guillain-Barré syndrome in a patient with non-Hodgkin's lymphoma.

We describe a case of Guillain-Barré syndrome (GBS) in a patient with non-Hodgkin's lymphoma (NHL). A 21-year-old woman with a newly diagnosed stage IV high-grade lymphoma (precursor T-cell NHL according to the R.E.A.L. Classification) developed flaccid quadriparesis and bilateral facial diplegia after three weeks of treatment with vincristine, daunorubicin, L-asparaginase and prednisolone. The clinical course and neurological examination were consistent with GBS. Despite treatment with intravenous immunoglobulins her neurological symptoms progressed. Plasmapheresis was therefore initiated followed by intravenous immunoglobulins. After partial remission of neurologic symptoms, induction chemotherapy with cyclophosphamide and cytarabine was continued without any further complication. Three months later, the lymphoma was in complete remission. GBS has been described in Hodgkin's disease and after bone marrow transplantation but is rare in NHL. In patients with NHL who develop neurological symptoms, drug toxicity and nervous system infiltration are the leading cause of neuropathology, but GBS should be considered in the differential diagnosis.

Detection of microsporidia (Enterocytozoon bieneusi) in intestinal biopsy specimens from human immunodeficiency virus-infected patients by PCR.

Intestinal microsporidiosis has been implicated as a major cause of chronic diarrhea in human immunodeficiency virus (HIV)-infected patients. So far diagnosis depends on direct visualization of the parasites by light and transmission electron microscopy. We evaluated the diagnostic value of microsporidian DNA amplification by PCR on duodenal biopsy specimens obtained from patients with and without intestinal microsporidiosis caused by Enterocytozoon bieneusi. Thirteen HIV-infected patients (all CDC stage C3) were studied. Eight patients had intestinal microsporidiosis caused by E. bieneusi (n = 6), Septata intestinalis (n = 1), and Encephalitozoon cuniculi (n = 1); microsporidioses were diagnosed by light microscopy of stool samples and confirmed by light and electron microscopy of intestinal biopsy specimens. Five patients had no microsporidia in their stool samples or in their intestinal biopsy specimens, as examined by light and electron microscopy. Additionally, DNA prepared from Toxoplasma gondii derived from mouse ascites was used as a further control. A 353-bp DNA fragment of the small-subunit rRNA gene could be amplified from all six biopsy specimens infected with E. bieneusi, and the nature of the PCR products was confirmed by Southern blot hybridization. No amplification of DNA fragments was seen by using DNA extracted from biopsy specimens with S. intestinalis or E. cuniculi infection or without microsporidian infection and with template DNA extracted from T. gondii. The results suggest that PCR testing of intestinal biopsy specimens may be a useful approach to diagnosing microsporidiosis in HIV-infected patients.

Limited value of PCR for detection of Toxoplasma gondii in blood from human immunodeficiency virus-infected patients.

Cerebral toxoplasmosis is a common, opportunistic, and often life-threatening disease in HIV-infected patients. Diagnosis is supported mainly by clinical evidence and computerized tomography or magnetic resonance imaging scans, but brain images may share features with other brain diseases occurring in HIV-infected patients. To determine the diagnostic value of PCR for the detection of Toxoplasma gondii in blood from HIV-infected patients, we examined 89 blood samples from 59 HIV-infected patients. PCR and Southern blot hybridization were done with DNA extracted from blood samples from 20 patients with confirmed cerebral toxoplasmosis and from 10 patients with suspected but not confirmed cerebral toxoplasmosis. The samples were taken before and 7 to 10 days after the beginning of antiparasitic therapy. For 9 patients who suffered from cerebral toxoplasmosis more than 6 months prior to the study and for 20 patients without any evidence for toxoplasmosis only one blood sample per patient was examined. PCR gave positive results with 5 of the 20 blood samples from patients who suffered from cerebral toxoplasmosis. After 7 to 10 days of therapy PCR results became negative in all these five cases. No amplification was seen with DNA from blood samples from the other 54 patients as the target. The results presented here show that PCR testing of blood samples from HIV-infected patients is of limited value for the diagnosis of cerebral toxoplasmosis. The sensitivity was only 25%, but the specificity was very high (100%), so this technique may be useful for discriminating between cerebral toxoplasmosis and other brain diseases which may be mistaken for toxoplasmosis.