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Objectives In the elderly population, chronic diseases are common determinants of mobility limitations and comorbidity consistently shows a strong association with functional status. This study was designed to evaluate the role of single chronic diseases and of their combination on functional recovery after rehabilitative treatment in disabled elderly patients. Design With respect to the difference in magnitude of their disabling effect, diseases were classified into 2 groups: “more disabling” diseases (COPD, heart failure, peripheral artery diseases, diabetes, and not life-threatening cancer) and “less disabling” diseases (anemia, kidney, gastrointestinal, and liver diseases). Setting 35-bed Geriatric Evaluation and Rehabilitation Unit. Participants We studied 710 patients (age 77.8 ± 7.4 years, 76.2% females), consecutively admitted for stroke, Parkinson’s disease, and osteoarthritis. Measurements A multidimensional evaluation for mobility (Tinetti-score), cognitive status (MMSE), and somatic health (Greenfield’s Individual Disease Severity Index-IDS, Burden of diseases-BoD) was performed. Functional recovery was decided based on the Delta-Tinetti, which is the difference of the values between admission and discharge. Results We tested, in a multivariate regression model, the predictive role of single chronic conditions and of their combinations on functional recovery, after having adjusted for which diseases are direct causes of disability (stroke, Parkinson’s disease, and osteoarthritis) and other potential predictors (age, sex, cognitive function, depressive symptoms, albumin, and c-reactive protein). A negative prediction of functional recovery was expressed by the “more disabling” diseases group. The determinants of poor recovery were characterized by the combination of “more disabling diseases” rather than single condition effects, independently by age, cognitive, and functional status on admission. Conclusion Our study adds a new perspective about the role of COPD, heart failure, peripheral artery diseases, diabetes and not life-threatening cancer on functional recovery, emphasizing their combined impact in elderly people.

An epidemiologic field study was conducted in the village of Borbòn in Esmeraldas province in northern Ecuador to compare different parasitologic methods in the diagnosis of infection with the Entamoeba histolytica/Entamoeba dispar complex. The results of two stool antigen detection assays (the Prospect Entamoeba histolytica microplate assay and the E. histolytica II assay) were compared with isoenzyme characterization of the amebic isolates. Nearly all (176 of 178, 98.9%) subjects were positive for intestinal parasites on direct microscopic examination, and cysts and/or vegetative forms morphologically consistent with the E. histolytica/E. dispar complex were recorded in 48 of 178 cases (27%). Culture in Robinson's medium was positive for amebic stocks in 89 (50%) of the 178 samples tested. Of the 37 isolates successfully stabilized, cloned, and characterized by zymodeme analysis, seven (18.9%) showed isoenzyme patterns of E. histolytica, whereas 26 (70.3%) showed patterns of E. dispar. The remaining four strains were identified as Entamoeba coli (three isolates; 8.1%) and Dientamoeba fragilis (one strain; 2.7%).The immunochromatographic tests showed different degrees of sensitivity and specificity when compared with isoenzyme characterization as the reference technique. The microplate assay, which does not discriminate between E. histolytica and E.dispar, showed a sensitivity of 54.5% and a specificity of 94% for both these amebic species. In contrast, the second-generation E. histolytica II test had a sensitivity of 14.3% and a specificity of 98.4% for E. histolytica sensu stricto. Our survey clearly demonstrated that more specific and sensitive diagnostic tests, such as stool antigen detection assays and isoenzyme analysis, are needed to establish the actual worldwide distribution of E. histolytica and E. dispar.

Encephalitozoon hellem is a microsporidian species responsible for opportunistic infections in AIDS patients. Use of a novel chitinase-based method allowed unsheared chromosomal DNA to be recovered from eleven E. hellem isolates derived from three geographic regions. All isolates were typed by 18S rDNA sequencing, which showed that they belonged to internal transcribed spacer type 1. After ethidium bromide staining, pulsed-field gel electrophoresis (PFGE) analysis discriminated two new karyotypes comprising 7 and 8 chromosomal bands respectively, ranging in size from 205- to 272-kb pairs. Genomic size was estimated to be 2.39 Mb. Our data indicate PFGE is useful for typing E. hellem and confirms genetic diversity among E. hellem genotypes.

Cryptosporidium parvum is a coccidian protozoon that causes diarrhoeal enteritis in immunocompetent and immunocompromised humans and other mammals. Sometimes, chiefly in HIV-infected subjects, anatomical sites other than gastro-intestinal tract, such as the biliary and respiratory tree, are involved. We performed an experimental respiratory infection in immunosuppressed albino rats with a C. parvum human-derived isolate, to confirm the possibility of a primary infection at this site and to evaluate the protozoan damages by light and also by transmission electron microscopy (TEM). The animals were infected intratracheally with 1 x 10(6) C. parvum oocysts/ml and, from the 7th day post-infection, biological specimens of trachea, bronchi, lung and ileum were zoopsied. A sole cryptosporidial colonization of the respiratory tract, from the trachea to the median bronchi, without lung parenchyma infection, was observed. Moreover 13/33 (39.4%) rats also developed intestinal infection. TEM study of the respiratory tree specimens demonstrated that cryptosporidia infect either ciliated or goblet cells, and confirmed the role of microvilli in the parasite cell adhesion. The most relevant alterations involved the ciliated cells, with loss of cilia and nuclear and cytoplasmic damages.

The microsporidian Encephalitozoon hellem is being reported with increasing frequency in HIV-positive subjects, as an agent of disseminated microsporidiosis without involving the gastrointestinal tract. We describe a case of pulmonary microsporidiosis in a 27-year-old Italian man with AIDS who developed fever, cough, and dyspnea. A chest X-ray showed multiple bilateral pulmonary opacities and mediastinal lymph-node enlargement. Stained smears of bronchoalveolar lavage sediment showed oval structures consistent with microsporidian spores. Viral, bacterial and fungal cultures were repeatedly negative, whereas microsporidia were successfully cultured in human and bovine fibroblast cell lines. Analysis of electron micrographs indicated that the isolate belonged to the genus Encephalitozoon. Based on further immunological, biochemical and molecular studies it was characterized as E. hellem. Even though a temporary improvement with albendazole therapy was noticed, the patient deteriorated clinically and died of severe respiratory distress.

A total of 77 mentally retarded male inpatients residing in a psychiatric institution in northern Italy were screened for the presence of stool parasites, Entamoeba histolytica particularly. Parasitological stool examination showed Entamoeba spp. (E. histolytica and/or E. dispar) in 26 cases (33.7%). In vitro culture on Robinson's medium was positive in 16 cases (61.1%); in 11 cases we could stabilize and clone the isolates and proceed to electrophoretic assays. In all cases, patterns of pathogenic zymodemes were found (zymodeme II, 3 isolates; zymodeme XII, 4 isolates; zymodeme XIV, 4 isolates). All isolates were therefore identified as E. histolytica.

Malaria is the most diffused parasitic disease that affects humans.A rapid and accurate diagnosis is a prerequisite for an effective treatment. The development of a new ready-to-use test for the screening and the typing of malaria solve the temperature storage requests of common molecular tests and PCR reagents. The performances of STAT-NAT Malaria Screening and STAT-NAT Malaria Typing kits were tested on samples obtained from patients suspected to be infected by malaria’s parasites, Immigrants (I) and International Travellers (IT), and were compared with the gold standard method (microscopy). Samples of peripheral blood of 35 patients (25 IT and 10 I) obtained from San Matteo Hospital in Pavia were analysed.The microscopy identified 18 of them as positive samples: 11 samples were positive for Plasmodium falciparum (5 IT, 6 I), 3 for Plasmodium vivax (3 IT), 2 for Plasmodium ovale (1I, 1 IT) and 2 for Plasmodium malariae (2 I). No co-infections were found. STAT-NAT Malaria kits confirmed the results of 18 positive samples as well as of 17 negative samples (100% sensitivity). In a case STAT-NAT Malaria Typing kit identified the presence of Plasmodium ovale in a sample instead of Plasmodium vivax, as previously diagnosed with the microscopic analysis.The epidemiological analysis and a new microscopic diagnosis confirmed the infection as caused by Plasmodium ovale species (100% specificity). We conclude that the association of the freeze-dried STAT-NAT Malaria tests could be determinant for the correct detection and typing of malaria, mainly where the temperature controlled storage could be problematic.

Microsporidia are primitive mitochondria-lacking spore-forming eukaryotic protozoa that infect a wide variety of animals and also humans. Of the five genera (Encephalitozoon, Enterocytozoon, Septata, Nosema and Pleistophora) that cause infections in humans, Enterocytozoon bieneusi, Septata intestinalis, and Encephalitozoon hellem are being increasingly identified in patients with acquired immunodeficiency syndrome (AIDS). E. bieneusi causes gastrointestinal disease, S. intestinalis causes gastrointestinal and disseminated disease, and E. hellem causes ocular as well as disseminated disease. We have established in continuous culture a strain of microsporidia isolated from the urine and throat washings of an Italian AIDS patient and identified it as Encephalitozoon hellem, based on its ultrastructural morphology, antigenic pattern, and polymerase chain reaction-amplified small subunit ribosomal RNA. We believe that this is the first time that a strain of microsporidia has been isolated from the throat washings of a patient with microsporidiosis.

Acanthamoeba keratitis (AK) is a corneal disease caused by members of a genus of free-living amoebae and is associated predominantly with contact lens (CL) use. This study reports 16 cases of culture-proven AK diagnosed in northern Italy. Genotype identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. A 405 bp region of the 18S rRNA gene (ASA.S1) including diagnostic fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the nuclear small-subunit rRNA gene sequence excluding the highly variable DF3 region. Phylogenetic analysis was also performed on the sequences obtained. All patients complained of monolateral infection; 11 (68.75%) admitted improper CL disinfection. In 14/16 (87.5 %) subjects, corneal scrapings were stained with calcofluor white and haematoxylin and eosin and, in ten cases (62.5 %), microscopy was positive for Acanthamoeba cysts. In vitro culture on 3 % non-nutrient agar plates was obtained in all cases (100 %), whereas cloning and axenic growth were positive for 14 amoebic stocks (87.5 %). PCR analysis had 100 % sensitivity and specificity compared with in vitro axenic culture, showing positive amplification from 15 isolates. All Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK on biological samples. Genotyping allowed inclusion of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in northern Italy.

A cohort of 51 homosexuals who were either HIV-positive or had AIDS was followed prospectively with parasitologic stool examination and in vitro culture in order to determine the incidence of E. histolytica infection. Amoebic isolates were further characterized by electrophoretic isoenzyme study. Five subjects (9.8%) were found to be infected with E. histolytica. None of the amoebic isolates were found to be pathogenic by isoenzyme analysis.

SUMMARYEntamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0·27 μm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0·74 and 0·22 mm, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

Evaluation of the ability to detect symptoms of osteoarthritis and prescription of analgesics in older adults with different degrees of cognitive impairment.Patients consecutively admitted to a Geriatric Evaluation and Rehabilitation Unit (GERU) for disability due to orthopedic, neurological, or cardiopulmonary diseases, after a surgical intervention, or for behavioral disturbances of dementia. Subjects with a recent orthopedic intervention were excluded. Eight hundred eighty-eight patients were analyzed (mean age 78.4 +/- 7.2 years, 69.5% female) and divided into 4 groups according to cognitive impairment: severe (Mini-Mental State Examination, MMSE 0-12), moderate (MMSE 13-18), mild (MMSE 19-24), or absent (MMSE 25-30).Patients underwent a geriatric multidimensional assessment; the diagnosis of musculoskeletal pain was based on patients' direct report, objective signs (inflammatory signs around the joints), and "pain behaviors" (reductions in activity, social withdrawal, self-protective maneuvers, increased alterations in facial expressions or body postures, observable displays of distress). Analgesic drugs were registered on discharge in subjects with symptomatic osteoarthritis.Of the patients admitted to GERU, 16.8% (n = 149) had osteoarthritis; this percentage is different according to cognitive impairment (MMSE 0-12: 1%, MMSE 13-18: 8%, MMSE 19-24: 17.4%, MMSE 24-30: 34.1%, P.001). If patients admitted for osteoarthritis are excluded, the new diagnoses of pain due to osteoarthritis are 306 (41.4%), without differences between cognitive status groups. On discharge, analgesic drugs are prescribed to 28.8% of patients with symptomatic arthritis (n = 131); the percentage is lower in patients with severe cognitive impairment in comparison with subjects with better cognitive functions.There is a poor attention to chronic pain due to osteoarthritis in community-dwelling elders with cognitive impairment. In a rehabilitative setting, an accurate evaluation of objective signs allows the diagnosis of symptomatic osteoarthritis. However, the link between the diagnosis and the treatment is not direct, since the prescription of analgesics is lower in patients with a more severe cognitive impairment.

Leishmaniases are endemic in 88 countries of five continents (only Australia is safe); 72 of these are distributed in tropical and subtropical areas, whereas 16 are included in the Temperate Zone [1].

To develop an alternative genotyping tool, the genetic diversity of Encephalitozoon hellem was examined at the polar tube protein (PTP) locus. Nucleotide sequence analysis of the PTP gene divided 24 E. hellem isolates into four genotypes, compared to two genotypes identified by analysis of the internal transcribed spacer of the rRNA gene. The four PTP genotypes differed from each other by the copy number of the 60-bp central repeat as well as by point mutations. A simple PCR test was developed to differentiate E. hellem genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping of E. hellem in clinical samples.

Imported tropical infections have become a serious health problem in industrialized countries, and have greatly contributed to the birth of a new branch of medicine called travel medicine. Several specific factors play an important role in this pathology, including the progressive and constant increase of subjects who travel to tropical and subtropical countries for various reasons (tourism, job, missionary institutions, etc.), the extracommunitary, often underground, flow of immigration, and the adoption of children from the same areas. A high percentage of imported tropical pathology is constituted by intestinal helminthic and protozoan infections, in particular those due to the Entamoeba histolytica / Entamoeba dispar complex. The aim of our study was to evaluate E. histolytica / E. dispar incidence in a casuistry composed of international travelers (IT), adult immigrants (AI), and adopted children (AC) collected from 1994–1999.

Since 1985 microsporidia have been recognized as a cause of emerging infections in humans, mainly in immunocompromised human immunodeficiency virus-positive subjects. As chitin is a basic component of the microsporidian infective stage, the spore, we evaluated in vitro the susceptibility of a human-derived strain of Encephalitozoon hellem to nikkomycin Z, a peptide-nucleoside antibiotic known as a competitive inhibitor of chitin synthase enzymes. Transmission electron microscopy showed that this drug, at 25 μg/ml, reduced the number of parasitic foci by about 35% ± standard deviation after 7 days of culture ( P < 0.0001) and induced cell damage of both mature and immature spores and also other sporogonic and merogonic stages. In particular, an irregular outline of the cell shape and an abnormally condensed cytoplasm in meronts and sporonts were documented. Also, the polar tubule and the polaroplast membranes appeared disarrayed in the sporoblast stage. The spore wall showed an enlarged endospore and delaminated exospore. Mature spores had a complete cytoplasmic disorganization and a swollen and delaminated cell wall. No ultrastructural cell damage was observed in uninfected control cultures treated with the drug.

The mitotic process in microsporidian Encephalitozoon hellem, a known human pathogen, has been studied with the aim of elucidating some ultrastructural aspects of its nuclear division. The presence of a nuclear spindle, of "electrondense spindle plaques" associated with the nuclear envelope and of cytoplasmic double walled vesicles are reported. We suggest that these "electrondense spindle plaques" serve as foci for intranuclear and cytoplasmic microtubule arrangements, similar to the microtubule organizing centers within the centrosomes of animal cells. The extent to which the microsporidial division process is comparable with that of more familiar eukaryotes such as yeast cells is discussed.

Microsporidia of the genera Enterocytozoon and Encephalitozoon have been identified as frequent causes of intestinal and disseminated infections, respectively, in patients with AIDS. Even though most subjects infected with these protozoa develop overt disease, simple colonization without illness may occur, as we observed in three severely immunosuppressed patients with AIDS. The parasites, recognized in and isolated from bronchoalveolar lavage sediment specimens, were characterized as Encephalitozoon hellem. Colonization of the bronchial tree was temporary, and treatment with albendazole was not needed to clear the infection.

Isoenzyme analysis by starch-gel electrophoresis has proved to be a useful method for the biochemical differentiation of pathogenic Entamoeba histolytica and non-pathogenic E. dispar isolates. Of the 24 known zymodemes, 3 are laboratory-made and have not previously been identified in humans. Parasitology screening was carried out in a psychiatric institution. Two amebic stocks were isolated and characterized that had never previously been found in humans and that have protein patterns identical to that of the laboratory-made zymodeme XX.

A study of the spore wall of Encephalitozoon hellem was performed on thin sections, freeze-fracture, and deep-etched samples to obtain information on spore wall organization and composition. Our observations demonstrate that the spore wall is formed by an inner 30-35 nm electron-lucent endospore and an outer 25-30 nm electron-dense exospore. The exospore is a complex of three layers: an outer spiny layer, an electron-lucent intermediate lamina and an inner fibrous layer. Freeze-fracture and deep-etching techniques reveal that the intermediate lamina and the inner fibrous layer result from the different spatial disposition of the same 4-nm thick fibrils. In thin sections the endospore reveals a scattered electron-dense material that appears in the form of trabecular structures when analyzed in deep-etched samples. The presence of chitin in the exospore is discussed.

SUMMARYSince 1993, strains ofEntamoeba histolytica sensn latohave been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains toE. histolytica sensu stricto, the non-pathogenic strains toEntamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases forE. histolytica, only a partial sequence has been published forE. dispar. Here we report a SSU rDNA sequence forE. dispar. Compared to those ofE. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from bothE. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphicDdeI restriction site which allows, after cleavage of the fragment,E. histolyticaandE. disparto be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

A limited outbreak of symptomatic intestinal and extraintestinal amoebiasis within a family complex is described. The infection was almost certainly transmitted by a Philippino housemaid, who was an asymptomatic carrier of Entamoeba histolytica infection acquired in her native country. Starch-gel electrophoresis showed isoenzyme patterns characteristic of pathogenic zymodeme XIX in all the amoebic isolates.

A direct method for detection of Pneumocystis carinii was evaluated in 14 patients with impaired immune function (3 seropositive for HIV, 8 with AIDS and 3 with heart transplants) and signs and symptoms suggestive of Pneumocystis carinii pneumonia. Direct examination by phase-contrast and interference-contrast microscopy of fresh clinical specimens obtained by sputum induction, bronchoalveolar lavage or transbronchial lung biopsy was found to be a simple and rapid method for detection of Pneumocystis carinii, the sensitivity of the method being comparable to that of the classical toluidine blue O and Diff-Quik staining methods. These findings suggest that this direct microscopy technique could be considered for routine clinical application in patients with suspected Pneumocystis carinii pneumonia.

Between August and December 1991 in Tanzania, a study to determine the prevalence of Pneumocystis carinii and of tuberculosis occurred among 83 18-38 year old HIV seropositive people living in the rural area of Malenga Makali in Iringa district. The adults had difficulty breathing, cough, fever of at least 2 weeks duration, or overt pneumonia. 3.6% of the sputum samples were confirmed positive for P. carinii. 38.5% of preparations and 13.2% of cultures tested positive for Mycobacterium tuberculosis. All these isolates were completely sensitive to standard antibiotics. 2 of the 3 patients testing positive for P. carinii also had pulmonary tuberculosis. These findings showed that sputum contains many mycobacteria. They also confirmed that TB is associated with HIV infection in several African countries and that P. carinii infection occurs less frequently than it does in Europe and the US, but occurs nevertheless. A possible explanation for the low prevalence of P. carinii infection in Africa is that more virulent infections kill AIDS patients before P. carinii pneumonia has a chance to develop. The researchers admit that their inability to use more suitable specimens obtained by bronchoalveolar lavage or transbronchial biopsy could have resulted in considerable underdiagnosis. They recommended further clinical research to determine the real importance of P. carinii in developing countries.

Ultrastructural studies were carried out to describe the nuclear division cycle of a strain of Encephalitozoon hellem isolated from an Italian AIDS patient. The nuclear division occurs during the proliferative vegetative phase and it is characterized by the intranuclear mitosis and by the lack of centrioles. The spindle termini are electron dense spindle plaques (ESPs), resembling to the spindle pole bodies (SPBs) of Saccharomycetes. The ESPs are bifacial organella forming microtubules on both nucleoplasic and cytoplasmic faces. In the outer layer of the spindle plaque are present vesicular elements lined by a double membrane of unknown function. The peculiar morphological features of E. hellem ESPs indicate that both intranuclear spindle and cytoplasmic microtubules are involved in the nuclear division.

A la suite d'une recherche parasitologique conduite sur differents echantillons d'eau et de boue d'egouts de la ville de Pavie (Italie) a ete possible l'isolement de 2 nouvelles souches d'E. m. La caracterisation morpho-biologique des souches a ete effectuee a l'aide de 2 methodes, tels que la culture en milieux hypotoniques et le test de thermoresistance in vitro, qui d'apres les travaux d'autres auteurs s'etaient montres deja de bons marqueurs mene pour la differenciation entre E. histolytica et les souches laredo thermoadaptables

Samples from therapeutic swimming pools and mud basins were cultured for free-living amoebae. Seven strains of pathogenic Naegleria species were isolated. Although some of the strains were as virulent as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis, they were identified as Naegleria australiensis with the indirect fluorescent-antibody technique. The virulence of the isolates for mice corresponded with the cytopathic effect for Vero cells. The N. australiensis strains were isolated from swimming pools with water temperatures ranging from 32 to 35 degrees C and from mud with temperatures from 25 to 43 degrees C. The presence of pathogenic N. australiensis in the swimming pools did not correlate with bacterial indicators.

A subspecies of Naegleria australiensis, N. australiensis italica, pathogenic for mice, was recently isolated and identified from an Italian thermal spa. We describe the histopathological changes of the central nervous system with experimental infection of albino mice. The histopathological patterns are intermediate to those seen with infection caused by N. fowleri and N. australiensis or Acanthamoeba spp. An acute inflammatory reaction was present within the choroid plexus, ependyma, midbrain, cerebellum, and basal ganglia. Occasional single amebic trophozoites were found within some microabscesses. Cysts were not identified. Involvement of the olfactory neuroepithelium and of the nasal mucosa was not detected.