Lophomonas as a respiratory pathogen-jumping the gun.

Human infections with the protozoan Lophomonas have been increasingly reported in the medical literature over the past three decades. Initial reports were based on microscopic identification of the purported pathogen in respiratory specimens. Later, a polymerase chain reaction (PCR) was developed to detect Lophomonas blattarum, following which there has been a significant increase in reports. In this minireview, we thoroughly examine the published reports of Lophomonas infection to evaluate its potential role as a human pathogen. We examined the published images and videos of purported Lophomonas, compared its morphology and motility characteristics with host bronchial ciliated epithelial cells and true L. blattarum derived from cockroaches, analyzed the published PCR that is being used for its diagnosis, and reviewed the clinical data of patients reported in the English and Chinese literature. From our analysis, we conclude that the images and videos from human specimens do not represent true Lophomonas and are predominantly misidentified ciliated epithelial cells. Additionally, we note that there is insufficient clinical evidence to attribute the cases to Lophomonas infection, as the clinical manifestations are non-specific, possibly caused by other infections and comorbidities, and there is no associated tissue pathology attributable to Lophomonas. Finally, our analysis reveals that the published PCR is not specific to Lophomonas and can amplify DNA from commensal trichomonads. Based on this thorough review, we emphasize the need for rigorous scientific scrutiny before a microorganism is acknowledged as a novel human pathogen and discuss the potential harms of misdiagnoses for patient care and scientific literature.

Metabolic diversity in commensal protists regulates intestinal immunity and trans-kingdom competition.

The microbiota influences intestinal health and physiology, yet the contributions of commensal protists to the gut environment have been largely overlooked. Here, we discover human- and rodent-associated parabasalid protists, revealing substantial diversity and prevalence in nonindustrialized human populations. Genomic and metabolomic analyses of murine parabasalids from the genus Tritrichomonas revealed species-level differences in excretion of the metabolite succinate, which results in distinct small intestinal immune responses. Metabolic differences between Tritrichomonas species also determine their ecological niche within the microbiota. By manipulating dietary fibers and developing in vitro protist culture, we show that different Tritrichomonas species prefer dietary polysaccharides or mucus glycans. These polysaccharide preferences drive trans-kingdom competition with specific commensal bacteria, which affects intestinal immunity in a diet-dependent manner. Our findings reveal unappreciated diversity in commensal parabasalids, elucidate differences in commensal protist metabolism, and suggest how dietary interventions could regulate their impact on gut health.

New Parabasalia symbionts Snyderella spp. and Daimonympha gen. nov. from South American Rugitermes termites and the parallel evolution of a cell with a rotating "head".

Most Parabasalia are symbionts in the hindgut of "lower" (non-Termitidae) termites, where they widely vary in morphology and degree of morphological complexity. Large and complex cells in the class Cristamonadea evolved by replicating a fundamental unit, the karyomastigont, in various ways. We describe here four new species of Calonymphidae (Cristamonadea) from Rugitermes hosts, assigned to the genus Snyderella based on diagnostic features (including the karyomastigont pattern) and molecular phylogeny. We also report a new genus of Calonymphidae, Daimonympha, from Rugitermes laticollis. Daimonympha's morphology does not match that of any known Parabasalia, and its SSU rRNA gene sequence corroborates this distinction. Daimonympha does however share a puzzling feature with a few previously described, but distantly related, Cristamonadea: a rapid, smooth, and continuous rotation of the anterior end of the cell, including the many karyomastigont nuclei. The function of this rotatory movement, the cellular mechanisms enabling it, and the way the cell deals with the consequent cell membrane shear, are all unknown. "Rotating wheel" structures are famously rare in biology, with prokaryotic flagella being the main exception; these mysterious spinning cells found only among Parabasalia are another, far less understood, example.

Fine structure and molecular characterization of two new parabasalid species that naturally colonize laboratory mice, Tritrichomonas musculus and Tritrichomonas casperi.

Tritrichomonas muris is a common flagellated protist isolated from the cecum of wild rodents. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, referred to as Tritrichomonas musculis and Tritrichomonas rainier, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, Tritrichomonas musculus n. sp., and Tritrichomonas casperi n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the 18S rRNA and trans-ITS genetic loci supported their designation as distinct species, related to T. muris. To assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice bred at the National Institutes of Health (NIH) were screened using pan-parabasalid primers that amplify the trans-ITS region. Forty-four percent of mice were positive for parabasalids, encompassing a total of eight distinct sequence types. Tritrichomonas casperi and Trichomitus-like protists were dominant. T. musculus and T. rainier were also detected, but T. muris was not. Our work establishes a previously underappreciated diversity of commensal trichomonad flagellates that naturally colonize the enteric cavity of laboratory mice.

True molecular phylogenetic position of the cockroach gut commensal Lophomonas blattarum (Lophomonadida, Parabasalia).

Lophomonas blattarum is a facultative commensal gut dweller of common pest cockroaches. Its cells are roughly spherical in shape with an apical tuft of ~50 flagella. Controversially, it has been implicated in human respiratory infections based on light microscopic observations of similarly shaped cells in sputum or bronchoalveolar lavage fluid. Here, we have sequenced the 18S rRNA gene of L. blattarum and its sole congener, Lophomonas striata, isolated from cockroaches. Both species branch in a fully supported clade with Trichonymphida, consistent with a previous study of L. striata, but not consistent with sequences from human samples attributed to L. blattarum.

Lophomonas blattarum-like organism in bronchoalveolar lavage from a pneumonia patient: current diagnostic scheme and polymerase chain reaction can lead to false-positive results.

Lophomonas blattarum is an anaerobic protozoan living in the intestine of cockroaches and house dust mites, with ultramicroscopic characteristics such as the presence of a parabasal body, axial filament, and absence of mitochondria. More than 200 cases of Lophomonas infection of the respiratory tract have been reported worldwide. However, the current diagnosis of such infection depends only on light microscopic morphological findings from respiratory secretions. In this study, we attempted to provide more robust evidence of protozoal infection in an immunocompromised patient with atypical pneumonia, positive for Lophomonas-like protozoal cell forms. A direct search of bronchoalveolar lavage fluid via polymerase chain reaction (PCR), transmission electron microscopy (TEM), and metagenomic next-generation sequencing did not prove the presence of protozoal infection. PCR results were not validated with sufficient rigor, while de novo assembly and taxonomic classification results did not confirm the presence of an unidentified pathogen. The TEM results implied that such protozoal forms in light microscopy are actually non-detached ciliated epithelial cells. After ruling out infectious causes, the patient's final diagnosis was drug-induced pneumonitis. These findings underscore the lack of validation in the previously utilized diagnostic methods, and more evidence in the presence of L. blattarum is required to further prove its pathogenicity.

Ancient and pervasive expansion of adaptin-related vesicle coat machinery across Parabasalia.

The eukaryotic phylum Parabasalia is composed primarily of anaerobic, endobiotic organisms such as the veterinary parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis, the latter causing the most prevalent, non-viral, sexually transmitted disease world-wide. Although a parasitic lifestyle is generally associated with a reduction in cell biology, T. vaginalis provides a striking counter-example. The 2007 T. vaginalis genome paper reported a massive and selective expansion of encoded proteins involved in vesicle trafficking, particularly those implicated in the late secretory and endocytic systems. Chief amongst these were the hetero-tetrameric adaptor proteins or 'adaptins', with T. vaginalis encoding ∼3.5 times more such proteins than do humans. The provenance of such a complement, and how it relates to the transition from a free-living or endobiotic state to parasitism, remains unclear. In this study, we performed a comprehensive bioinformatic and molecular evolutionary investigation of the heterotetrameric cargo adaptor-derived coats, comparing the molecular complement and evolution of these proteins between T. vaginalis, T. foetus and the available diversity of endobiotic parabasalids. Notably, with the recent discovery of Anaeramoeba spp. as the free-living sister lineage to all parabasalids, we were able to delve back to time points earlier in the lineage's history than ever before. We found that, although T. vaginalis still encodes the most HTAC subunits amongst parabasalids, the duplications giving rise to the complement took place more deeply and at various stages across the lineage. While some duplications appear to have convergently shaped the parasitic lineages, the largest jump is in the transition from free-living to endobiotic lifestyle with both gains and losses shaping the encoded complement. This work details the evolution of a cellular system across an important lineage of parasites and provides insight into the evolutionary dynamics of an example of expansion of protein machinery, counter to the more common trends observed in many parasitic systems.

Molecular phylogeny of Spirotrichonymphea (Parabasalia) with emphasis on Spironympha, Spirotrichonympha, and three new genera Pseudospironympha, Nanospironympha, and Brugerollina.

Spirotrichonymphea, one of the six classes of phylum Parabasalia, are characterized by bearing many flagella in spiral rows, and they occur exclusively in the guts of termites. Phylogenetic relationships among the 13 described genera are not well understood due to complex morphological evolution and a paucity of molecular data. One such understudied genus is Spironympha. It has been variously considered a valid genus, a subgenus of Spirotrichonympha, or an "immature" life cycle stage of Spirotrichonympha. To clarify this, we sequenced the small subunit rRNA gene sequences of Spironympha and Spirotrichonympha cells isolated from the hindguts of Reticulitermes species and Hodotermopsis sjostedti and confirmed the molecular identity of H. sjostedti symbionts using fluorescence in situ hybridization. Spironympha as currently circumscribed is polyphyletic, with both H. sjostedti symbiont species branching separately from the "true" Spironympha from Reticulitermes. Similarly, the Spirotrichonympha symbiont of H. sjostedti branches separately from the "true" Spirotrichonympha found in Reticulitermes. Our data support Spironympha from Reticulitermes as a valid genus most closely related to Spirotrichonympha, though its monophyly and interspecific relationships are not resolved in our molecular phylogenetic analysis. We propose three new genera to accommodate the H. sjostedti symbionts and two new species of Spirotrichonympha from Reticulitermes.

Free-living Trichomonads are Unexpectedly Diverse.

The vast majority of the more than 450 described species of Parabasalia are intestinal symbionts or parasites of animals. This endobiotic life-history is presumably ancestral although the root of Parabasalia still needs to be robustly established. The half-dozen putatively free-living species thus far described are likely independently derived from endobiotic ancestors and represent the most neglected ecological group of parabasalids. Thus, we isolated and cultivated 45 free-living strains of Parabasalia obtained from a wide variety of anoxic sediments to conduct detailed morphological and SSU rRNA gene phylogenetic analyses. Sixteen species of trichomonads were recovered. Among them, we described seven new species, three new genera, two new families, and one new order. Most of the newly described species were more or less closely related to members of already described genera. However, we uncovered a new deep-branching lineage without affinity to any currently known group of Parabasalia. The newly discovered free-living parabasalids will be key taxa in comparative analyses aimed at rooting the entire lineage and deciphering the evolutionary innovations involved in transitioning between endobiotic and free-living habitats.

First Co-morbidity of Lophomonas blattarum and COVID-19 Infections: Confirmed Using Molecular Approach.

INTRODUCTION: Lophomoniasis is caused by Lophomonas spp., a new emerging protozoan, which commonly affects the human lower respiratory tract. The Lophomonas parasite mostly lives commensally in the hindgut of cockroaches. CASE PRESENTATION: We present the case of a 33-year-old woman, 30 weeks pregnant, who had severe COVID-19. She was intubated upon admission and began the routine COVID-19 treatment. To rule out possible super infection dual with COVID-19, microscopic examination of the patient's mini-bronchoalveolar lavage (mini-BAL) specimen, revealed L. blattarum, which was identified by the SSU rRNA-PCR and sequencing approaches (accession number: MZ093069). According to that, the patient was treated successfully with metronidazole. CONCLUSION: To prevent serious complications, lophomoniasis should be listed in co-morbidity cases of COVID-19 infection during the COVID-19 pandemic worldwide. To the best of our knowledge, this is the first co-infection of Lophomonas blattarum and COVID-19 in the world which has been confirmed using a molecular approach.

Molecular Phylogenetic Position of Microjoenia (Parabasalia: Spirotrichonymphea) from Reticulitermes and Hodotermopsis Termite Hosts.

Microjoenia are obligate symbionts of termites. The genus was erected in 1892 for small cells with many flagella that insert near, but not directly from, the cell apex, and an axostyle that can protrude from the cell posterior. Although ultrastructural studies have been carried out on three Microjoenia species to date, no molecular data have been directly attributed to any species. Microjoenia are classified within the parabasalian class Spirotrichonymphea, which is characterized by flagellar bands that emerge near the cell apex and proceed posteriorly in a right-handed helix. In Microjoenia, however, the flagellar bands are very short and proceed longitudinally or with a weakly observable helix. In this study, we have amplified and sequenced the 18S ribosomal RNA gene from individually isolated Microjoenia cells from Reticulitermes and Hodotermopsis hosts as part of an ongoing effort to understand the phylogeny of Spirotrichonymphea and their coevolution with termites. In our 18S rRNA gene phylogeny, Microjoenia forms the sister lineage to Spirotrichonympha, though many other evolutionary relationships within Spirotrichonymphea remain unresolved.

Morphological and Molecular Identification of Emerged Lophomonas blattarum Infection in Mazandaran Province, Northern Iran: First Registry-Based Study.

BACKGROUND: In the last decade, several cases of bronchopulmonary lophomoniasis (BPL) have been recorded. Little information is available about epidemiological aspects on Lophomonas infection among BPL patients. The present study was aimed to investigate the prevalence of Lophomonas spp. infection in patients who were referred to the Iranian National Registry Center for Lophomoniasis (INRCL), using morphological and molecular tests. SUBJECTS AND METHODS: We examined patients enrolled in the INRCL from 2017 to 2019 at the Mazandaran University of Medical Sciences, northern Iran. All bronchoalveolar lavage fluid (BALF) and two nasal discharges of the patients were examined by both microscopic and small-subunit ribosomal RNA (SSU rRNA) PCR methods. To confirm the species of Lophomonas, two positive samples were sequenced. RESULTS: In this study, 321 specimens (including 319 BALF and 2 nasal discharges) were microscopically examined. Lophomonas spp. was found in 45(14%) (n = 44 BAL; n = 1 nasal discharge). The mean age of infected patients was 54.9 ± 17.1 years. The following morphological characteristics were observed in both fresh and Papanicolaou-stained smears to identify Lophomonas spp. All microscopically positive specimens were confirmed with genus-specific PCR technique. The obtained sequences were deposited in Gen Bank under the accession numbers (MN243135-36). The BLAST analysis of our two sequences with the only available sequence in the Gen Bank of the Thailand strain of L. blattarum, showed identity of 99-100% and 98.51%, respectively. CONCLUSION: To the best of our knowledge, this is the first registry-based study regarding lophomoniasis worldwide. According to our study, the conventional PCR test is an available and reliable tool for confirming the Lophomonas parasite in clinical samples. Moreover, the results confirmed that L. blattarum is circulating at least in our region.

Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha.

Cristamonadea is a large class of parabasalian protists that reside in the hindguts of wood-feeding insects, where they play an essential role in the digestion of lignocellulose. This group of symbionts boasts an impressive array of complex morphological characteristics, many of which have evolved multiple times independently. However, their diversity is understudied and molecular data remain scarce. Here we describe seven new species of cristamonad symbionts from Comatermes, Calcaritermes, and Rugitermes termites from Peru and Ecuador. To classify these new species, we examined cells by light and scanning electron microscopy, sequenced the symbiont small subunit ribosomal RNA (rRNA) genes, and carried out barcoding of the mitochondrial large subunit rRNA gene of the hosts to confirm host identification. Based on these data, five of the symbionts characterized here represent new species within described genera: Devescovina sapara n. sp., Devescovina aymara n. sp., Macrotrichomonas ashaninka n. sp., Macrotrichomonas secoya n. sp., and Macrotrichomonas yanesha n. sp. Additionally, two symbionts with overall morphological characteristics similar to the poorly-studied and probably polyphyletic 'joeniid' Parabasalia are classified in a new genus Runanympha n. gen.: Runanympha illapa n. sp., and Runanympha pacha n. sp.

Spirotrichonymphea (Parabasalia) symbionts of the termite Paraneotermes simplicicornis.

The desert dampwood termite Paraneotermes simplicicornis harbors several species of obligately symbiotic protists that support its nutrition by fermenting lignocellulose. Among them are three morphotypes with the dexiotropic spiraling flagellar bands characteristic of Spirotrichonymphea (Parabasalia). The largest morphotype, characterized by an elongated cell apex with axial columella and internally positioned spiraling flagellar bands, was previously described as Spirotrichonympha polygyra. A smaller morphotype, with similarly internalized flagellar bands but a more rounded posterior without a protruding axostyle, was previously reported but not named. The smallest morphotype has surface flagellar bands and can attach to other protist cells by its apex. In this study, we combine light microscopy of live specimens and 18S rRNA gene sequencing of individually isolated cells to better understand the diversity of symbionts in P. simplicicornis. We found that S. polygyra branches distantly from true Spirotrichonympha, which are associated with Reticulitermes termites. Thus, we propose the new genus Cuppa to accommodate C. polygyra n. comb. (type species) and the similar but smaller morphotype Cuppa taenia n. sp. The undescribed smallest morphotype can be excluded from all previously described Spirotrichonymphea genera by molecular and behavioral evidence, so we propose Fraterculus simplicicornis n. gen., n. sp., to accommodate this organism.