Caspar Franzen

Reply: 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 log 10 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.

eradication of Cryptosporidia and Microsporidia Following Successful Antiretroviral Therapy

Objectives: Incidence of opportunistic protozoal infections causing diarrheal illnesses in patients with HIV has decreased since the introduction of highly active antiretroviral therapy (HAART). The objective of this study was to determine whether the parasites, cryptosporidia, and microsporidia were effectively eradicated or only suppressed following treatment. Design: Six HIV‐positive patients with diarrheal symptoms caused by cryptosporidia or microsporidia were prospectively followed up with stool samples and duodenal biopsies. Samples were taken before HAART, between 1 to 3 months, and 6 months post‐HAART. Methods: Duodenal samples were analyzed using routine histology and transmission electron microscopy. Stool samples were analyzed by both light microscopy and polymerase chain reaction (PCR) techniques. Results: Patients who responded successfully to HAART eradicated both cryptosporidial and microsporidial organisms. Symptoms improved within 1 month of therapy but complete eradication of the organisms was only observed after 6 months of treatment. Conclusions: AIDs‐related cryptosporidiosis and microsporidiosis can be cured following successful antiretroviral therapy.

Detection of Microsporidia in Travelers with Diarrhea

ABSTRACT We examined stool specimens of 148 returning travelers from an outpatient department for tropical diseases for the appearence of microsporidia using light microscopy and PCR. Intestinal microsporidiosis was diagnosed for five patients by light microscopy and for nine patients by PCR. Some cases were diagnosed only by PCR, indicating that the true prevalence has to be determined by highly sensitive techniques, such as PCR.

Transfer of the Members of the Genus Brachiola (Microsporidia) to the Genus Anncaliia Based on Ultrastructural and Molecular Data

ABSTRACT. Two microsporidian genera, Anncaliia Issi, Krylova, & Nicolaeva 1993 and Brachiola Cali et al. 1998 , possess a Nosema‐type life cycle and unique cell surface ornamentations, which include precocious electron‐dense coating of the plasmalemma and a variety of secretory structures deposited on the parasite surface and scattered in the host cell cytoplasm. Comparative analysis of ultrastructure of Anncaliia meligethi (the type species of the genus Anncaliia) and of B. vesicularum and B. algerae (the best‐studied members of the genus Brachiola) clearly demonstrated that these microsporidia share many distinctive morphological features. The comparison of small subunit ribosomal DNA sequences showed high sequence identity of A. meligethi and B. algerae. Phylogenetic analyses indicated that the rDNA sequences of A. meligethi clustered with those of B. algerae suggesting a close relatedness of these microsporidia. The combination of molecular and morphological data provided clear evidence that these microsporidia belong to the same genus and therefore, warranted emendation of the genus Anncaliia and establishments of the following new combinations: Anncaliia vesicularum nov. comb., Anncaliia algerae nov. comb., Anncaliia connori nov. comb., and Anncaliia gambiae nov. comb. The generic name Brachiola is submerged according to the rule of priority.

Morphological and Molecular Investigations of Tubulinosema ratisbonensis gen. nov., sp. nov. (Microsporidia: Tubulinosematidae fam. nov.), a Parasite Infecting a Laboratory Colony of Drosophila melanogaster (Diptera: Drosophilidae)

Abstract. A new species of microsporidia from Drosophila melanogaster was investigated by light and electron microscopy and by ribosomal RNA (rRNA) sequencing. This microsporidium and the previously described Nosema kingi and Nosema acridophagus have been transferred to the new genus Tubulinosema gen. nov. with the following characters: nuclei are in diplokaryotic arrangement during the life cycle. All stages are in direct contact with the host cell cytoplasm, slightly anisofilar polar tube with the last coils being smaller in diameter arranged in one or two rows on both sides of the diplokaryon and small tubuli on the surface of late meronts. Spores are oval or slightly pyriform. Thick endospore wall, thinner over anchoring disc. This new genus and the genus Brachiola have been placed in a new family Tubulinosematidae fam. nov. Phylogenetic analysis of small subunit rRNA sequences by different methods placed Tubulinosema spp. in one clade with the genus Brachiola forming its sister clade, which is distant from the clade containing the true Nosema spp. including Nosema bombycis.

Cell invasion and intracellular fate of Encephalitozoon cuniculi (Microsporidia)

Microsporidia are obligate intracellular parasites that utilize a unique mechanism to infect host cells, which is one of the most sophisticated infection mechanisms in biology. Microsporidian spores contain a long coiled polar tube that extrudes from the spores and penetrates the membranes of new host cells. We have initiated a study to investigate the invasive process and intracellular fate of the microsporidium Encephalitozoon cuniculi. Here we show that relatively few cells were infected through the traditional penetration of the polar tube from outside. Rather, phagocytosis of spores occurred at least 10 times more frequently than injection of sporoplasms. Some spores extruded their polar tube inside the cells following phagocytosis. Membranes of the vacuoles surrounding the internalized spores were positive for late endosomal and lysosomal markers. Spores that remained inside these compartments disappeared within 3 days. Thus, our studies demonstrate that in addition to the unique way in which microsporidia infect host cells, E. cuniculi spores can also gain access to host cells by phagocytosis. The presence of intracellular spores that have extruded their polar tube shows that some spores germinate after phagocytosis, thus escaping from the phagosomes that mature into lysosomes.

Salvage therapy with regimens containing ritonavir and saquinavir in extensively pretreated HIV-infected patients.

OBJECTIVE To evaluate the efficacy and toxicity of salvage regimens containing ritonavir and saquinavir in patients failing highly active antiretroviral therapy (HAART), and to correlate outcome with plasma concentrations of protease inhibitors. DESIGN Prospective, non-randomized interventional study. SUBJECTS AND METHODS Thirty extensively pretreated HIV-infected patients with virological failure under HAART were treated with ritonavir (400 mg twice daily) and saquinavir (600 mg twice daily) and at least one reverse transcriptase inhibitor. HIV-RNA, CD4 cell counts and plasma concentrations of protease inhibitors were determined, and patients were monitored for toxicity at monthly intervals. RESULTS Six patients showed complete virological success (HIV-RNA <200 copies/ml at week 12) which was sustained for a median follow-up of 6.3 months. Partial virological response (decrease of HIV-RNA of >1 log10 at week 12) was achieved by a further three patients. Patients with a virological response had significantly higher CD4 cell increases than patients without virological response (mean increase at week 12: 66x10(6) cells/l versus 6x10(6) cells/l; P = 0.01). No clinical events were observed during 6 months of follow-up. Neither the use of a non-nucleoside reverse transcriptase inhibitor (NNRTI) nor the number of newly introduced drugs influenced the virological response. Plasma concentrations of protease inhibitors did not statistically differ between patients with and without success. Toxicity included gastrointestinal disturbances, lipid abnormalities and liver dysfunction. CONCLUSIONS In extensively pretreated patients, salvage regimens containing ritonavir and saquinavir had only limited and short-term anti-HIV activity and were associated with substantial toxicity. Plasma concentrations of saquinavir were not predictive for virological response.

Fitness effects and transmission routes of a microsporidian parasite infecting Drosophila and its parasitoids

A microsporidian infection was discovered in laboratory cultures of Drosophila species. Ultrastructural examination suggested it belonged to the poorly characterized species Tubulinosema kingi, and morphological and sequence data are presented. We explored how T. kingi affected the fitness of Drosophila melanogaster and D. subobscura, as well as the fitness of 2 of their parasitoids, Asobara tabida and Pachycrepoideus vindemiae. In Drosophila, infections caused changes in most of the traits we looked at that were associated with fitness, in particular causing a 34-55% reduction in early-life fecundity. Parasitoid fitness was affected more severely by infection than that of their hosts, with pupal mortality in particular increasing by 75-89%. We investigated the most important routes of transmission for T. kingi in a laboratory setting. Letting Drosophila larvae feed on medium contaminated with spores from infected dead flies resulted in 100% infection. Low levels of transmission (<10%) were found between larvae, and vertically between mothers and their offspring. Parasitoids developing in infected hosts all became infected, but infected adults were neither able to transmit the pathogen to their offspring nor to their offsprings Drosophila host, either directly, or via contamination of the ovipositor or other body parts. A field survey of Drosophila and their parasitoids in southern England revealed no natural infections. We discuss the potential importance of Microsporidia in parasitoid-host interactions, and for those working with Drosophila in the laboratory.

Detection of polyomavirus BK and JC in children with kidney diseases and renal transplant recipients

Background: Reactivation of polyomavirus is a known reason for severe renal dysfunction in adult renal transplant recipients. Testing for polyomavirus DNA in plasma has been described as a sensitive and specific method to discover viral nephropathy in adult patients. We were now interested in polyomavirus status in a pediatric patient setting. Methods: Plasma and urine samples were obtained from 80 children including 38 children after renal transplantation (group 1), 7 children with different kidney diseases receiving immunosuppressive treatment (group 2) and 35 children with different kidney diseases not receiving immunosuppressive treatment (group 3). A nested polymerase chain reaction method was used for amplification of polyomavirus DNA fragments. Differentiation between JC and BK virus was done by digestion with restriction endonucleases. Results: Polyomavirus DNA was detected in the urine sample of 19 of 38 (50%) renal transplant recipients (group 1), of 1 of 7 (14%) patients from group 2 and in none of the 35 patients of group 3. Plasma samples from 3 (8%) of group 1 patients and from 1 child each of group 2 (14%) and group 3 (3%) were tested positive for polyomavirus DNA. Conclusion: Urinary polyomavirus excretion seems to be more frequent in pediatric patients with kidney diseases receiving immunosuppressive treatment and after renal transplantation than in children with various kidney diseases without immunosuppressive treatment.

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.