Cytokine and nitric oxide responses of monocyte-derived human macrophages to microsporidian spores

Microsporidia are obligate intracellular parasites that emerged as opportunistic pathogens since the onset of the AIDS pandemic. They are capable of disseminating through the body using macrophages as vehicles. We incubated human macrophages with spores of all three Encephalitozoon spp. as well as with Vittaforma corneae, and the number of intracellular spores per cell was determined by fluorescence microscopy. Cell culture supernatants were collected and the content of TNF-alpha, INF-gamma, IL-10, and of nitric oxide was determined. Microsporidian spores did not induce a nitric oxide response in macrophages and there was a negative correlation between the number of intracellular spores and the amount of nitric oxide. TNF-alpha, INF-gamma, and IL-10 increased after simulation of macrophages with microsporidian spores but for TNF-alpha and INF-gamma no clear correlation of cytokine levels with the number of intracellular spores could be observed. A modulation of the nitric oxide response by intracellular microsporidia may contribute to the survival of microsporidia within the macrophage by a mechanism yet unknown.     

Microsporidia as emerging pathogens and the implication for public health: a 10-year study on HIV-positive and -negative patients

Despite recent advances in the understanding and diagnosis of emerging microsporidian pathogens, more research is necessary to elucidate their complex epidemiology. In fact, studies that reflect true human-infecting microsporidian prevalence are still inadequate. The present 10-year study was undertaken to examine the occurrence of Microsporidia in 1989 stools, 69 urine and 200 pulmonary specimens from HIV-positive and HIV-negative patients using PCR and DNA sequencing. In stools, 12.0% were Microsporidia-positive. Prevalences of 13.9% and 8.5% were observed for HIV+ and HIV- samples, respectively. The percentage of children that were Microsporidia-positive (18.8%) was significantly higher than that of adults (10.2%). In stools, Enterocytozoon bieneusi (6.3%) and Vittaforma-like parasites (6.8%) were identified. Based on the internal transcribed spacer (ITS) region of E. bieneusi, Type IV (37.5%), Peru 6 (29.2%), D (12.5%), A (8.3%), C (6.3%) and PtEb II (6.3%) genotypes were identified. Microsporidia were detected in 1.5% and 1.0% of urine and pulmonary specimens, respectively. Encephalitozoonintestinalis was detected in urine. In pulmonary specimens, Encephalitozoon cuniculi and Vittaforma-like parasites were identified. An immunosuppressive condition and youth (children) appear to be risk factors for microsporidian infection. Microsporidia seems to have an important impact on public health in Portugal, highlighting the need to implement routine diagnosis.     

Extraction of microsporidial DNA from modified trichrome-stained clinical slides and subsequent species identification using PCR sequencing

Molecular techniques involving polymerase chain reaction (PCR) and sequencing provide a relatively simple and objective means of identifying microsporidia to species level. We modified previously described methods of DNA extraction and PCR conditions for identification of microsporidia from museum slides, clinical specimens and environmental samples and successfully identified Vittaforma corneae in 11 out of 13 cases of microsporidial infection from used trichrome-stained slides of corneal scrapings from HIV-negative patients with keratoconjunctivitis.     

Heterogeneity among zymodemes of Leishmania infantum from HIV-positive patients with visceral leishmaniasis in south Italy

Abstract Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortaient patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.     

Antibody responses to Cryptosporidium antigen in HIV-positive patients in the Republic of Korea

The diagnosis of cryptosporidiosis has been carried out using coprologic techniques in the Republic of Korea. However, antibody responses to Cryptosporidium have rarely been studied. Serum antibodies from HIV-positive/oocyst-positive Korean patients recognized significantly 31 and 27 kDa antigens, and HIV-negative/oocyst-positive individuals clearly reacted to 15/17 kDa antigens. Compared with oocyst-positive cases, 18.7% and 75.8% of sera from HIV-positive patients reacted to 31 and 27 kDa antigens. Only 11.1% of HIV-negative individuals reacted to 15/17 kDa. Based on these findings, serum antibody responses were different between HIV-positive and HIV-negative individuals infected with Cryptosporidium, and it is suggested that HIV-positive patients are more frequently exposed to C. parvum compared to HIV-negative individuals.     

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi,Encephalitozoon hellem,and Vittaforma corneae

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.     

Prevalence and Exoenzyme Secretion by Candida Albicans Isolates from Oral and Vaginal Mucosas of HIV-Infected Women

A cross-sectional study was performed to evaluate the prevalence and the aetiology of forms of mucosal fungal infections of HIV-negative and HIV-positive women. Candida albicans was the predominate specie isolated from both groups of patients, with remarkable proportion of isolation from symptomatic women. All 239 C. albicans isolates, regardless of their source, showed activity of proteinase and phospholipase. It was verified that isolates with particularly higher levels of exoenzymes production were significantly more common in HIV-positive patients. However, isolates obtained from the HIV-positive patients in use of HAART, with protease inhibitor, presented lower levels of these exoenzymes, similar to the levels observed in the isolates from HIV-negative patients.     

Myospora metanephrops (n. g., n. sp.) from marine lobsters and a proposal for erection of a new order and family (Crustaceacida; Myosporidae) in the Class Marinosporidia (Phylum Microsporidia)

In this study we describe, the first microsporidian parasite from nephropid lobsters. Metanephrops challengeri were captured from an important marine fishery situated off the south coast of New Zealand. Infected lobsters displayed an unusual external appearance and were lethargic. Histology was used to demonstrate replacement of skeletal and other muscles by merogonic and sporogonic stages of the parasite, while transmission electron microscopy revealed the presence of diplokaryotic meronts, sporonts, sporoblasts and spore stages, all in direct contact with the host sarcoplasm. Analysis of the ssrDNA gene sequence from the lobster microsporidian suggested a close affinity with Thelohania butleri, a morphologically dissimilar microsporidian from marine shrimps. Whilst morphological features of the lobster parasite are consistent with members of the family Nosematidae, molecular data place the parasite closer to members of the family Thelohanidae. Due to the contradiction between morphological and molecular taxonomic data, we propose the erection of a new genus in which the lobster parasite is the type species (Myospora metanephrops). Furthermore, we recommend the erection of a new family (Myosporidae) and a new order (Crustaceacida) to contain this genus. The taxonomic framework presented could be further applied to the re-classification of existing members of the Phylum Microsporidia.     

隐球菌性脑膜炎治疗的更新

隐球菌性脑膜炎(cryptococcal meningitis,CM)是一种由新型隐球菌和格特隐球菌引起的机会性真菌感染性疾病。尽管近些年来,随着CM诊断方法与治疗手段的重大进展,患者整体病死率明显下降,但其发病率在世界范围内仍呈升高趋势,在我国尤为明显。虽然CM常发生在有免疫缺陷的人群如HIV患者,但HIV阴性的人群患病率却逐年升高,且二者的治疗方案不尽相同。本文总结了国内外关于HIV阳性与HIV阴性隐球菌性脑膜炎患者治疗的新进展,旨在对HIV阳性和HIV阴性隐球菌性脑膜炎患者的治疗提供依据。     

Comparison between human immunodeficiency virus positive and negative patients with tuberculosis in Southern Brazil.

The objective of this study is to determine the different characteristics of human immunodeficiency virus (HIV) positive and negative patients treated for tuberculosis (TBC) in a tertiary hospital in Southern Brazil. We conducted a retrospective cohort study over a 5-year period, from January 1992 through December 1996. We reviewed medical charts of patients from our institution who received TBC treatment. We reviewed 167 medical charts of patients with confirmed TBC. HIV positivity was detected in 74 patients. There were statistically significant difference between HIV positive and negative patients in sex and age. HIV-infected patients showed significantly more signs of bacteremia than HIV-negative patients. Extra-pulmonary TBC was present respectively in 13 (17.6%) and 21 (22.6%) HIV positive and negative patients. There was a significant difference between chest radiograph presentation in HIV positive and negative patients. There were significantly lower hematocrit, hemoglobin, leukocyte and lymphocyte levels in HIV-positive compared to HIV-negative patients. Outcome was significantly different in the two groups with a death rate of 36.5% and 10.8% in HIV-positive and in HIV-negative patients. The difference between HIV positive and negative patients may have importance in the diagnosis, management and prognosis of patients with TBC.     

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6%) of the HIV-positive subjects and in 13/26 (50%) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.     

A comparison of two microsporidian parasites in enterocytes of AIDS patients with chronic diarrhea.

Enterocytozoon bieneusi was first described by electron microscopy in 1985 in intestinal biopsies from an AIDS patient. It has subsequently been observed in many AIDS patients with chronic diarrhea from the U.S.A., Africa, and Europe. Morphologically, this parasite meets the criteria for being a microsporidian but has unique features justifying the creation of a a new genus and family. It has organelles not seen in microsporida before, i.e. elongated nuclei, electron-lucent inclusions, electron-dense discs, and development of multiple polar tubules in a single cell prior to the final cytokinetic process producing many sporoblasts. However, it produces typical microsporidian spores. Recently, a second type of microsporidian has been observed in similar biopsies from an AIDS patient which resembles an Encephalitozoon except that it secretes a fine network of material in which the developing organisms become embedded. During sporogony, each cell appears to be in a separate chamber. These two parasites are morphologically and pathologically compared.     

Shorter Telomere Length - A Potential Susceptibility Factor for HIV-Associated Neurocognitive Impairments in South African Woman

The neuropathogenesis of the human immunodeficiency virus (HIV) may manifest as various neurocognitive impairments (NCI). HIV-positive individuals also have significantly shorter telomere length (TL) in peripheral blood mononuclear cells (PBMCs) and CD8+ T cells compared to HIV-negative individuals. Additionally, reduced TL has been found to be associated with chronic psychological stress. This study focused on the effects of HIV-infection and chronic stress associated with childhood trauma on telomere length, and investigated whether leukocyte TL (LTL), in particular, represents a risk factor for NCI. Eighty-three HIV-positive and 45 HIV-negative women were assessed for childhood trauma and were subjected to detailed neurocognitive testing. Blood from each participant was used to extract Deoxyribonucleic acid (DNA). Relative LTL were determined by performing real time quantitative PCR reactions as described by Cawthon et al. (2002). As expected, relative LTL in the HIV-positive individuals was significantly shorter than that of HIV-negative individuals (F = 51.56, p = <0.01). Notably, a significant positive correlation was evident between relative LTL and learning performance in the HIV-positive group. In addition, a significant negative correlation was observed between relative LTL and verbal fluency, but this association was only evident in HIV-positive individuals who had experienced trauma. Our results suggest that reduced LTL is associated with worse learning performance in HIV-positive individuals, indicating that TL could act as a susceptibility factor in increasing neurocognitive decline in HIV-infected individuals.     

An autopsy study describing causes of death and comparing clinico-pathological findings among hospitalized patients in Kampala, Uganda

Background

Information on causes of death in HIV-infected patients in Sub-Saharan Africa is mainly derived from observational cohort and verbal autopsy studies. Autopsy is the gold standard to ascertain cause of death. We conducted an autopsy study to describe and compare the clinical and autopsy causes of death and contributory findings in hospitalized HIV-infected and HIV-uninfected patients in Uganda.

Methods

Between May and September 2009 a complete autopsy was performed on patients that died on a combined infectious diseases gastroenterology ward in Mulago Hospital in Kampala, Uganda. Autopsy cause of death and contributing findings were based on the macro- and microscopic post-mortem findings combined with clinical information. Clinical diagnoses were reported by the ward doctor and classified as confirmed, highly suspected, considered or not considered, based on information derived from the medical chart. Results are reported according to HIV serostatus.

Results

Fifty-three complete autopsies were performed in 66% HIV-positive, 21% HIV-negative and 13% patients with an unknown HIV serological status. Infectious diseases caused death in 83% of HIV-positive patients, with disseminated TB as the main diagnosis causing 37% of deaths. The spectrum of illness and causes of death were substantially different between HIV-positive and HIV-negative patients. In HIV-positive patients 12% of postmortem diagnoses were clinically confirmed, 27% highly suspected, 16% considered and 45% not considered. In HIV-negative patients 17% of postmortem diagnoses were clinically highly suspected, 42% considered and 42% not considered.

Conclusion

Autopsy examination remains an important tool to ascertain causes of death particularly in settings with limited access to diagnostic testing during life. HIV-positive patients continue to die from treatable and clinically undiagnosed infectious diseases. Until rapid-point of care testing is available to confirm common infections, empiric treatment should be further investigated.     

Antisperm antibodies in human immunodeficiency virus infection: effects on fertilization and embryonic development

Sera from human immunodeficiency virus (HIV)-infected males (n = 10) and females (n = 5) were analyzed for the presence of antisperm antibodies reacting against sperm-specific antigens. Of the HIV-positive males tested, sera of 40% were positive for human sperm extract (HSE), 70% for protamine, and 70% for fertilization antigen (FA-1) for at least one class of antibodies, compared to sera from HIV-negative males. Of the HIV-positive females tested, sera of 40% were positive for HSE, 30% for protamine, and 30% for FA-1 compared to sera from HIV-negative females. The majority of the sperm antigen-reactive antibodies belonged to the IgG class. The reactions observed with FA-1 were weaker than those with other antigens. Ninety percent of HIV-positive male sera and 80% of the HIV-negative female sera were found to contain immune complexes, 20% of which showed the presence of FA-1. HIV-positive male or female sera did not bind to any specific protein on the Western blot of HSE. The minimal amount of free anti-FA-1 antibodies present in sera did not bind to live sperm in the sperm immobilization technique, sperm agglutination technique, or immunobead binding technique and thus were incapable of affecting human sperm penetration of zona-free hamster ova (SPA). Nor did HIV-positive sera induce any apparent abnormality in the development of 2-cell embryos to blastocysts in vitro in murine bioassay. In conclusion, these results indicate that HIV-infected patients have sperm-specific antibodies in their sera that do not adversely affect SPA and murine embryo bioassay. There was a high incidence of immune complex formation after HIV infection. These data will provide the basis for exploring further the role of sperm antigens in altering the immunoregulatory mechanisms after HIV infection.     

Septata Intestinalis N. G., N. Sp., an Intestinal Microsporidian Associated With Chronic Diarrhea and Dissemination In Aids Patients

ABSTRACT. Intestinal microsporidiosis in patients diagnosed with acquired immunodeficiency syndrome (AIDS) and having chronic diarrhea was first reported in 1985 and the associated microsporidian was named Enterocytozoon bieneusi. the intracellular developmental cycle of E. bieneusi in enterocytes has been demonstrated and many cases have been reported worldwide. This report presents the life cycle of a second intestinal microsporidian, associated with the same symptoms, in five AIDS patients. This new microsporidian also infects enterocytes but its pathology and morphology differ from that of E. bieneusi. It involves lamina propria macrophages, fibroblasts, and endothelial cells and can disseminate to infect other parts of the body, e.g. the kidney and gall bladder. the parasite cycle includes development of rounded uninucleate and elongated bi- or tetranucleate cells without the formation of plasmodial stages. Sporogony is similar to the more typical development of microsporidia with sporoblast morphogenesis occurring after the last cell division. the development of cells within chambers of a septate, honeycomb-like, parasite-secreted fibrillar network and surrounded by a parasitophorous vacuole, however, is unique to this microsporidian, justifying the establishment of a new genus and species, Septata intestinalis n. g., n. sp.