Microsporidia are strict intracellular parasites that are known to infect a wide array of eukaryotes (including humans), as well as economically important fish and insects.

Their medical importance is mirrored in the fact that they are emerging as causative agents of infectious diseases in HIV/AIDS patients, organ transplant recipients, and also in contact lens wearers, travelers, children and the elderly.

The condition these small and enigmatic microorganisms cause is recognized under a joint name “microsporidiosis”, albeit there are eight different genera that are known to infect humans (and this number is likely to grow). There are three broad clinical manifestations of microsporidiosis, usually grouped into enteric, ocular and disseminated disease categories. Diagnosis is often hard to establish, and treatment options are scarce.

Microbiology and Epidemiology of Microsporidiosis

In microbiological sciences, microsporidia are considered as protozoan organisms, even though comparative molecular and phylogenetic studies point to their close relationship with the kingdom of Fungi. Species of microsporidia known to infect humans have been found in domestic, wild and food-producing farm animals, but also in various water sources, resulting in concerns for potential foodborne, zoonotic and waterborne disease transmission.

Fifteen microsporidian species identified as human pathogens include (in alphabetical order) Anncaliia algerae, Anncaliia connori, Anncaliia vesicularum, Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Enterocytozoon bieneusi, Microsporidium ceylonensis, Microsporidium africanum, Nosema ocularum, Pleistophora sp., Trachipleistophora hominis, Trachipleistophora anthropophthera, Tubulinosema acridophagusand Vittaforma corneae.

The characteristic stage of all the aforementioned microsporidians is a small, Gram-positive spore (typically oval and 1-40 micrometers in diameter) that contains a coiled filament (also known as polar tube) and an infective element (sporoplasm). The spore represents the infective stage that can enter the host by inhalation, ingestion, transplacentally – or even by direct contact.

The spore forces the infective sporoplasm into the host by using the polar tube. Once inside the cell, two phases occur next in the lifecycle: extensive multiplication (either by merogony or schizogony) and spore formation (sporogony). The latter phase is characterized by a formation of a thick wall around the spore that provides resistance to degradation and hostile environmental conditions.

When the spores significantly increase in number (and completely fill the host cell cytoplasm), the disruption of the cell membrane releases the spores into the environment. These liberated mature spores can readily infect new eukaryotic cells and propagate the disease cycle.

Microscopy as the Mainstay of Diagnostic Endeavors

The diagnostic approach used by the majority of laboratories is microscopic examination of the stained smears from clinical specimens (most notably fecal samples) by light microscopy; nevertheless, this method does not allow differentiation of microsporidial pathogens to the species level.

Staining techniques that are most often employed are the Chromotrope 2R method and its faster variation known as Quick-Hot Gram Chromotrope technique. Chemofluorescent brightening dyes such as Calcofluor white are also quite useful when there is a need for rapid identification of spores in the specimens.

The gold standard to identify microsporidia to the species level is transmission electron microscopy (TEM). However, this method is laborious, time-consuming and expensive, thus not feasible for use in routine diagnostics. Indirect immunofluorescence approaches are also an option by using monoclonal or polyclonal antibodies, but are seldom used.

Some specialized institutions offer molecular identification and characterization of Enterocytozoon bieneusi and three Encephalitozoon species by means of polymerase chain reaction (PCR). Other microsporidia can also be molecularly characterized by employing genera-specific primers and subsequent sequencing analysis on an individual basis.