Simonetta Gatti

Fatal Naegleria fowleri meningoencephalitis, Italy.

We report the first case of primary amebic meningoencephalitis in Italy, in a 9-year-old boy. Clinical course was fulminant, and diagnosis was made by identifying amebas in stained brain sections and by indirect immunofluorescence analysis. Naegleria fowleri was characterized as genotype I on the basis of polymerase chain reaction test results.

Microsporidian Spore Wall: Ultrastructural Findings on Encephalitozoon hellem Exospore

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.

Pneumocystis carinii pneumonia and tuberculosis in Tanzanian patients infected with HIV

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.

Genotyping Encephalitozoon hellem isolates by analysis of the polar tube protein gene.

ABSTRACT To develop an alternative genotyping tool, the genetic diversity ofEncephalitozoon 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 differentiateE. hellem genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping ofE. hellem in clinical samples.

Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica

Since 1993, strains of Entamoeba histolytica sensu lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba 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 for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. 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 both E. 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 polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Asymptomatic Respiratory Tract Microsporidiosis Due to Encephalitozoon hellem in Three Patients with AIDS

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.

Pulmonary microsporidiosis due to Encephalitozoon hellem in a patient with AIDS

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.

Transmission of Entamoeba histolytica within a family complex

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.

Isolation and identification of Encephalitozoon hellem from an Italian AIDS patient with disseminated microsporidiosis.

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, Septula, Nosema and Pleistophora) that cause infections in humans, Enterocytozoon bieneusi. Septula intestinulis, and Encephulitozoon hellem are being increasingly identified in patients with acquired immunodeficiency syndrome (AIDS). E. bieneusi causes gastrointestinal disease, S. intestinulis 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.

Isolation and genotyping of Acanthamoeba strains from corneal infections in Italy

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.