C. Franzen

Molecular Techniques for Detection, Species Differentiation, and Phylogenetic Analysis of Microsporidia

SUMMARY Microsporidia are obligate intracellular protozoan parasites that infect a broad range of vertebrates and invertebrates. These parasites are now recognized as one of the most common pathogens in human immunodeficiency virus-infected patients. For most patients with infectious diseases, microbiological isolation and identification techniques offer the most rapid and specific determination of the etiologic agent. This is not a suitable procedure for microsporidia, which are obligate intracellular parasites requiring cell culture systems for growth. Therefore, the diagnosis of microsporidiosis currently depends on morphological demonstration of the organisms themselves. Although the diagnosis of microsporidiosis and identification of microsporidia by light microscopy have greatly improved during the last few years, species differentiation by these techniques is usually impossible and transmission electron microscopy may be necessary. Immunfluorescent-staining techniques have been developed for species differentiation of microsporidia, but the antibodies used in these procedures are available only at research laboratories at present. During the last 10 years, the detection of infectious disease agents has begun to include the use of nucleic acid-based technologies. Diagnosis of infection caused by parasitic organisms is the last field of clinical microbiology to incorporate these techniques and molecular techniques (e.g., PCR and hybridization assays) have recently been developed for the detection, species differentiation, and phylogenetic analysis of microsporidia. In this paper we review human microsporidial infections and describe and discuss these newly developed molecular techniques.

Microsporidiosis: human diseases and diagnosis

Microsporidia are considered opportunistic pathogens in humans because they are most likely to cause diseases if the immune status of a host is such that the infection cannot be controlled. A wide spectrum of diseases has been reported among persons infected with microsporidia and different diagnostic techniques have been developed during the last decade.

Cryptosporidia and microsporidia--waterborne diseases in the immunocompromised host.

Cryptosporidia and microsporidia are emerging parasitic pathogens in immunocompetent and immunocompromised individuals. Cryptosporidium infects several wild and domestic animals that excrete oocysts into the environment and contaminated water represents the major source of infection for humans. Waterborne transmission of Cryptosporidium is a major risk for humans and appropriate measures have to be taken to protect immunocompetent and immunocompromised individuals to become infected. For microsporidia, the sources and ways of transmission are not well documented. Although several animal hosts have been identified recently, the relevant reservoirs of human microsporidia are still unknown. Also, the routes of spreading are unknown. Is microsporidiosis a zoonotic disease that will be transmitted through close contact with infected animals or is contaminated surface water responsible for transmission and represents a relevant reservoir? This review is designed to give information on these two emerging intestinal parasites in a format that will be useful to clinical microbiologists, physicians interested in infectious diseases, and public health personnel.

Ribosomal RNA of Nosema algerae and phylogenetic relationship to other microsporidia

Abstract Microsporidia are intracellular parasites that are common in invertebrates. Taxonomic classification is mostly restricted to morphologic and physiologic data. Limited data are available about taxonomic classification using DNA-sequence data for analysis. We examined the small-subunit (SSU) rDNA, the intergenic spacer (ITS) region, and a part of the large-subunit (LSU) rDNA of Nosema algerae, a parasite of mosquitoes, taken from a laboratory colony of Anopheles stephensi. Target gene amplifications were done by polymerase chain reaction (PCR) and, after cloning, DNA fragments were sequenced. The SSU-rDNA sequence obtained was aligned with several other microsporidian SSU-rDNA sequences available from the GenBank or EMBL data bases and was analyzed by different methods. On the basis of the results of our phylogenetic analysis, we suggest that our N. algerae isolate is not closely related to other microsporidia belonging to the genus Nosema.

Detection of Isospora belli by polymerase chain reaction using primers based on small-subunit ribosomal RNA sequences.

Abstract The aim of the present study was to use small-subunit (SSU)-rRNA sequences of Isospora belli to design specific primer pairs and a hybridization probe for the detection of Isospora belli in human samples by PCR and Southern blot hybridization. PCR amplification with the primer pairs produced correct DNA fragments with target DNA from samples of Isospora belli-infected patients and from cloned SSU-rRNA of Isospora belli. The nature of the PCR products was confirmed by Southern blot hybridization. No amplification was seen with template DNA extracted from other parasites. Although Isospora belli infections can be easily diagnosed using light microscopy, molecular-based techniques may prove useful as an additional diagnostic tool.

Taxonomic position of the human intestinal protozoan parasite Isospora belli as based on ribosomal RNA sequences

Abstract The taxonomic positions of Isospora belli and other members of the genus Isospora are controversial. We determined the small-subunit ribosomal RNA of I. belli and used this sequence in combination with other coccidian RNA sequences for analysis of the taxonomic position of I. belli. The phylogenetic trees we obtained provide molecular evidence for three clades within a monophyletic group that represents the suborder Eimeriina. The clade containing I. belli consists of tissue-cyst-forming coccidia (Toxoplasma and Neospora) and members of the genus Isospora (I. ohioensis, I. suis, I. belli). The second clade, representing a sister clade of that containing the Isospora species, contains members of the genus Sarcocystis. The third one consists of members of the family Eimeriidae, including Eimeria and Cyclospora species. This shows that although I. belli as well as other members of the genus Isospora belong to the suborder Eimeriina, the family to which they belong is not Eimeriidae but rather Sarcocystidae. We suggest that the genus Isospora should be removed from the family Eimeriidae and placed into the family Sarcocystidae within the suborder Eimeriina.

Empirical Liposomal Amphotericin-B Therapy in a Neutropenic Patient: Breakthrough of Disseminated Blastoschizomyces capitatus Infection*

Blastoschizomyces capitatus (Trichosporon capitatum) is an uncommon fungal pathogen. Infections have mostly been seen in immunocompromised patients and use of broad spectrum antibiotics was identified as a risk factor. Treatment has been extremely difficult. A report is presented about a case of fatal B. capitatum infection with clinical septicemia and multiorgan failure during intravenous liposomal amphotericin B therapy.

Randomized controlled monocentric comparison of once daily ceftriaxone with tobramycin and cefotaxime three times daily with tobramycin in neutropenic fever.

Abstract A prospective, randomized, controlled monocentric trial was performed to evaluate the efficacy and safety of once daily ceftriaxone 2 g plus tobramycin 5 mg/kg in comparison to cefotaxime 2 g t.i.d. plus tobramycin 5 mg/kg qd in the treatment of neutropenic fever. In cases of fever ≥38.5  °C and a neutrophil count below 1000/μl, patients with hematological malignancies were assigned to ceftriaxone or cefotaxime, each with tobramycin. The primary endpoint was defined as defervescence <37.5  °C on day 4–6 followed by at least 7 afebrile days. Secondary endpoints were overall response, defined as defervescence on day 25 and toxicity. There were 160 episodes of 114 patients included. Fever of unknown origin accounted for 79 episodes (51%), microbiologically defined infection for 36 (23%), clinically defined infection for 27 (17%), and both clinically and microbiologically defined infection for 14 episodes (9%). On an intent-to-treat basis 156 episodes could be evaluated for the primary endpoint. Ceftriaxone plus tobramycin and cefotaxime plus tobramycin resulted in a primary response in 46.9% and 45.3%, respectively. Overall response was achieved on study day 25 in 87.7% and 80%, respectively. No significant difference in toxicity was observed. Once-daily ceftriaxone plus tobramycin was not inferior to cefotaxime t.i.d. plus tobramycin qd in the empirical treatment of neutropenic fever.

Intestinal microsporidiosis with Septata intestinalis in a patient with AIDS—response to albendazole

Microsporidiosis is a common finding in HIV-infected patients who have diarrhoea. The species most commonly causing gastrointestinal disease is Enterocytozoon bieneusi. Recently Septata intestinalis has been described as a cause of diarrhoea and disseminated infection in patients with AIDS. A 44-year-old homosexual man with severe immunodeficiency (CD4 cell count 40/microliters) had a history of watery diarrhoea for 2 weeks. Microsporidian spores measuring 1.2 to 1.5 x 2.5 to 3.0 microns were found in stool samples. Electron microscopy of duodenal biopsies confirmed the diagnosis of intestinal microsporidiosis and showed parasitophorous vacuoles with the typical ultrastructure of S. intestinalis. The patient was treated with albendazole (400 mg twice daily) and became asymptomatic within 4 days. No spores could be detected in stool samples after a treatment period of 14 days. About 25 infections with S. intestinalis have been reported to date, and the case presented here is the first in a German patient.

Intestinal Microsporidiosis in Patients with Acquired Immunodeficiency Syndrome - Report of Three More German Cases

SummaryIntestinal microsporidiosis withEnterocytozoon bieneusi was diagnosed in three of 18 HIV-infected patients with chronic diarrhoea. In two cases all known stages of the life cycle ofE. bieneusi (merogonial plasmodia, sporogonial plasmodia, sporoblasts, spores) were found in duodenal biopsies by electron microscopical examination, whereas in the third case only merogonial and sporogonial stages were seen. Spores were also visible by light microscopy in semithin sections. Two patients were treated with albendazole (2×400 mg/day for 4 weeks) but showed no response. These findings underline the concept of the worldwide distribution of this parasite and verify that it is also frequent in Germany.ZusammenfassungEine intestinale Microsporidiose mitEnterocytozoon bieneusi konnte bei drei von 18 HIV-infizierten Patienten mit chronischem Durchfall durch elektronenmikroskopische Untersuchungen diagnostiziert werden. In zwei Fällen wurden alle bekannten Stadien des Entwicklungszyklus vonE. bieneusi in duodenalen Biopsien gefunden (Meronten, Sporonten, Sporoblasten, Sporen) wohingegen in einem Fall nur Meronten und Sporonten gesehen wurden. Die Sporen waren auch lichtmikroskopisch in Semidünnschnitten sichtbar. Zwei Patienten wurden mit Albendazol behandelt (2×400 mg/Tag für 4 Wochen), besserten sich jedoch nicht in ihrer Symptomatik. Diese drei Fälle einer intestinalen Microsporidiose unterstreichen das Konzept der weltweiten Verbreitung dieser Parasiten und belegen, daß sie auch in Deutschland häufiger vorkommen.