Prevalence of Protozoan Pathogens Among Diarrheic Children Under 5 Years in Public Hospital of Ethiopia During the Global COVID 19 Pandemic.

Acute childhood diarrhea is one of the leading causes of childhood morbidity and mortality in sub-Saharan African countries. Entamoeba histolytica and Giardia lamblia are the common cause of childhood diarrhea in the region. However, there are only few studies on protozoa causing diarrhea in sub-Saharan African countries. This study was conducted to investigate the relative prevalence and explore risk factors of E. histolytica and G. lamblia among diarrheic children of under 5 years in a public hospital of Ethiopia. A retrospective study was conducted among diarrheic children at Hiwot Fana hospital, Ethiopia. Records of all diarrheic children less than 5 years who had sought medical treatment in the hospital from September 1, 2020 to December 31, 2022 were included. Data were collected from 1257 medical records of the children using a structured data-collection format. Data were entered into an Excel sheet and exported into SPSS version 22 for data processing and analysis. Descriptive statistical tests, Chi-square, and logistic region analysis were applied to determine predictors of protozoa infections. Of the 1257 cases, 962 (76.5%) had watery diarrhea and the remaining 239 (19.0%) had dysentery. The combined prevalence of E. histolytica and G. lamblia among diarrheic children was 11.8% (95% CI: 9.6-13.4). As the age of children increased, the frequency of these two protozoan infections was significantly increased compared to children with other causes. There were more diarrhea cases during the summer season including those associated with E. histolytica and G. lamblia. This study revealed that 1 in 10 causes of diarhhea among young children in the study area was likely caused by E. histolytica and G. lamblia. These findings call for community-based safe water and food safety interventions in order to reduce childhood diarrhea caused by protozoan infections in resource-poor settings.

Omega-3 and omega-6 polyunsaturated fatty acids and their potential therapeutic role in protozoan infections.

Protozoa exert a serious global threat of growing concern to human, and animal, and there is a need for the advancement of novel therapeutic strategies to effectively treat or mitigate the impact of associated diseases. Omega polyunsaturated fatty acids (ω-PUFAs), including Omega-3 (ω-3) and omega-6 (ω-6), are constituents derived from various natural sources, have gained significant attention for their therapeutic role in parasitic infections and a variety of essential structural and regulatory functions in animals and humans. Both ω-3 and ω-6 decrease the growth and survival rate of parasites through metabolized anti-inflammatory mediators, such as lipoxins, resolvins, and protectins, and have both in vivo and in vitro protective effects against various protozoan infections. The ω-PUFAs have been shown to modulate the host immune response by a commonly known mechanism such as (inhibition of arachidonic acid (AA) metabolic process, production of anti-inflammatory mediators, modification of intracellular lipids, and activation of the nuclear receptor), and promotion of a shift towards a more effective immune defense against parasitic invaders by regulation the inflammation like prostaglandins, leukotrienes, thromboxane, are involved in controlling the inflammatory reaction. The immune modulation may involve reducing inflammation, enhancing phagocytosis, and suppressing parasitic virulence factors. The unique properties of ω-PUFAs could prevent protozoan infections, representing an important area of study. This review explores the clinical impact of ω-PUFAs against some protozoan infections, elucidating possible mechanisms of action and supportive therapy for preventing various parasitic infections in humans and animals, such as toxoplasmosis, malaria, coccidiosis, and chagas disease. ω-PUFAs show promise as a therapeutic approach for parasitic infections due to their direct anti-parasitic effects and their ability to modulate the host immune response. Additionally, we discuss current treatment options and suggest perspectives for future studies. This could potentially provide an alternative or supplementary treatment option for these complex global health problems.

Quercetin as a Promising Antiprotozoan Phytochemical: Current Knowledge and Future Research Avenues.

Despite tremendous advances in the prevention and treatment of infectious diseases, only few antiparasitic drugs have been developed to date. Protozoan infections such as malaria, leishmaniasis, and trypanosomiasis continue to exact an enormous toll on public health worldwide, underscoring the need to discover novel antiprotozoan drugs. Recently, there has been an explosion of research into the antiprotozoan properties of quercetin, one of the most abundant flavonoids in the human diet. In this review, we tried to consolidate the current knowledge on the antiprotozoal effects of quercetin and to provide the most fruitful avenues for future research. Quercetin exerts potent antiprotozoan activity against a broad spectrum of pathogens such as Leishmania spp., Trypanosoma spp., Plasmodium spp., Cryptosporidium spp., Trichomonas spp., and Toxoplasma gondii. In addition to its immunomodulatory roles, quercetin disrupts mitochondrial function, induces apoptotic/necrotic cell death, impairs iron uptake, inhibits multiple enzymes involved in fatty acid synthesis and the glycolytic pathways, suppresses the activity of DNA topoisomerases, and downregulates the expression of various heat shock proteins in these pathogens. In vivo studies also show that quercetin is effective in reducing parasitic loads, histopathological damage, and mortality in animals. Future research should focus on designing effective drug delivery systems to increase the oral bioavailability of quercetin. Incorporating quercetin into various nanocarrier systems would be a promising approach to manage localized cutaneous infections. Nevertheless, clinical trials are needed to validate the efficacy of quercetin in treating various protozoan infections.

Early Detection of Histomoniasis in Blood Samples by PCR and Sequencing.

Histomoniasis is a deadly disease of turkeys causing devastating economic losses to the poultry industry. In field outbreaks, a presumptive diagnosis is made based on gross pathology lesions and confirmed by histopathology. An early detection tool with quick turnaround time is needed to prevent the spread of histomoniasis. With this objective, two studies were conducted in turkeys. In Study 1, 40 poults were housed in two pens (20 poults/pen) and challenged at 14 days of age with Histomonas meleagridis by intracloacal route. Blood samples were collected 4 days postchallenge. Fifty-five percent (22/40) of the blood samples tested positive for H. meleagridis based on PCR using primers targeted against the 18S rRNA gene and confirmed by sequencing. In Study 2, 40 poults were housed in two groups and raised in floor pens. Groups 1 and 2 served as negative and challenge controls, respectively. At 14 days of age, the birds in Group 2 were challenged with H. meleagridis by intracloacal route. Blood samples were collected 2 days postchallenge. Five percent (1/20) of the blood samples tested positive for H. meleagridis, based on PCR and confirmed by sequencing. The results from both studies indicate that H. meleagridis DNA can be detected in the blood samples by PCR and confirmed by sequencing as early as 4 days postchallenge. This early detection method could be applied in field outbreaks to detect and confirm histomoniasis as early as possible.

Detección temprana de histomoniasis en muestras de sangre mediante PCR y secuenciación La histomoniasis es una enfermedad mortal de los pavos que causa pérdidas económicas devastadoras a la industria avícola. En los brotes de campo, se realiza un diagnóstico presuntivo basado en lesiones patológicas macroscópicas y se confirma mediante histopatología. Se necesita una herramienta de detección temprana con un tiempo de respuesta rápido para prevenir la propagación de la histomoniasis. Con este objetivo, se realizaron dos estudios en pavos. En el Estudio 1, se alojaron 40 pavipollos en dos corrales (20 pavipollos/corral) y se desafiaron a los 14 días de edad con Histomonas meleagridis por vía intracloacal. Se recolectaron muestras de sangre a los cuatro días después del desafío. El cincuenta y cinco por ciento (22/40) de las muestras de sangre resultaron positivas para H. meleagridis según el método de PCR utilizando iniciadores dirigidos contra el gene 18S rRNA y confirmado mediante secuenciación. En el Estudio 2, se alojaron 40 pavipollos en dos grupos y se criaron en corrales en piso. Los grupos 1 y 2 sirvieron como controles negativos y de desafío, respectivamente. A los 14 días de edad, las aves del Grupo 2 fueron expuestas a H. meleagridis por vía intracloacal. Se recolectaron muestras de sangre dos días después del desafío. El cinco por ciento (1/20) de las muestras de sangre dieron positivo para H. meleagridis, según el método de PCR y confirmado mediante secuenciación. Los resultados de ambos estudios indican que el ADN de H. meleagridis puede detectarse en las muestras de sangre mediante PCR y confirmarse mediante secuenciación tan pronto como cuatro días después de la exposición. Este método de detección temprana podría aplicarse en brotes de campo para detectar y confirmar la histomoniasis lo antes posible.

Severe lophomoniasis in a patient with diabetes and past history of COVID-19 in Central Iran: case report.

In this case report, we address the diagnostic challenges and clinical implications of severe infection with Lophomonas blattarum in a patient initially suspected of experiencing long COVID symptoms. We describe the patient's medical history, initial symptoms, diagnostic tests, and treatment. A female patient with diabetes in her early 60s presented with severe shortness of breath and was initially diagnosed with diabetic ketoacidosis (DKA). After resolution of her DKA symptoms, persistent respiratory issues led to a COVID-19 test, which was negative. A chest computed tomography scan revealed abnormalities, prompting bronchoscopy and bronchoalveolar lavage fluid analysis, which confirmed the presence of L. blattarum. Notably, the protozoan remained mobile and viable even after a 4-day transport at ambient temperature. This case emphasizes the importance of considering alternative diagnoses and improving awareness about L. blattarum infection in patients with respiratory symptoms, for timely and accurate management.

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.

Differential diagnosis of lophomaniasis in patients with COVID-19 in northern Mexico: a case report / Diagnóstico diferencial de lofomaniasis en pacientes con COVID-19 en el norte de México: reporte de un caso

Pulmonary lophomoniasis is a rare infection produced by a multiflagellated and anaerobic pyriform or oval protozoan belonging to the family of Lophomonadidae. The study aimed learn the differential diagnosis of lophomoniasis in patients with COVID-19 in northern Mexico. Clinical case of a 37-years-old male patient diagnosed with pneumonia, respiratory syndrome, hemoptysis, and fever, which suggested pulmonary tuberculosis. Bronchial lavage was performed, and laboratory tests were requested, an RT-PCR test to search for SARS-CoV-2, which was positive. The results for TB and KOH for fungi were negative. In addition to the protocol, a fresh examination was performed by placing a drop from the sample on a glass slide and observing it with a 10X objective, then 40X searching for clinically structural elements. As a result, multiflagellated cellular elements in the continuous movement were observed that morphologically correspond to the genus Lophomonas spp concluding the bacteriological protocol of bronchial secretions should consider fresh examination to search for trophozoites of Lophomonas spp. Medical and laboratory personnel are unaware of the protozoa Lophomonas spp, since the fresh examination in the analysis protocol is not considered. This paper reports the first case of Lophomonas infection in a patient caused by chronic lung disease. (AU)

La lofomoniasis pulmonar es una infección rara producida por un protozoo piriforme u ovalado multiflagelado y anaeróbico perteneciente a la familia de los Lophomonadidae. El estudio tuvo como objetivo conocer el diagnóstico diferencial de lofomoniasis en pacientes con COVID-19 en el norte de México. Caso clínico de un paciente masculino de 37 años con diagnóstico de neumonía, síndrome respiratorio, hemoptisis y fiebre, que sugería tuberculosis pulmonar. Se realizó lavado bronquial y se solicitaron pruebas de laboratorio, prueba RT-PCR para búsqueda de SARS-CoV-2, la cual resultó positiva. Los resultados de TB y KOH para hongos fueron negativos. Además del protocolo, se realizó un nuevo examen colocando una gota de la muestra en un portaobjetos de vidrio y observándola con un objetivo de 10X, luego 40X en busca de elementos clínicamente estructurales. Como resultado se observaron elementos celulares multiflagelados en movimiento continuo que morfológicamente corresponden al género Lophomonas spp, por lo que el protocolo bacteriológico de secreciones bronquiales debe considerar examen en fresco para búsqueda de trofozoítos de Lophomonas spp. El personal médico y de laboratorio desconoce la presencia del protozoo Lophomonas spp, ya que en el protocolo de análisis no se considera el examen en fresco. Este artículo reporta el primer caso de infección por Lophomonas en un paciente causado por una enfermedad pulmonar crónica. (AU)

Evaluation of a rapid immunochromatographic test for the diagnosis of intestinal protozoan infections among patients attending a rural outreach outpatient department in Northern India.

Despite great efforts, intestinal protozoan infections remain a significant healthcare concern worldwide. Although many point-of-care (POC) tests are increasingly being used, microscopic examination of stool specimens remains the mainstay for their diagnosis, especially in resource-limited settings. We assessed the utility of rapid POC tests based on immunochromatography among patients from rural Northern India. A total of 78 patients were enrolled in the study. Out of nine specimens that tested positive for Giardia duodenalis on microscopy, an immunochromatographic test (ICT) could detect only five (55.55%). Entamoeba histolytica/dispar was demonstrated in two specimens on microscopy, both of which were missed by ICT. Its overall sensitivity, specificity, and positive and negative predictive value were 50%, 98.5%, 83.3%, and 93%, respectively. Its performance was considered unsatisfactory. Although ICT-based tests provide a relatively rapid and less labor-intensive alternative, they should be used to supplement and not replace stool microscopy.

[Unconventional Laveran]. / Laveran décalé.

Charles Louis Alphonse Laveran - 18 June 1845 - 18 May 1922: first French Nobel Prize in Medicine, "in recognition of his work on the role played by protozoa in causing diseases". One hundred years after his death, only written records remain of his work and life. The witnesses to this period are no more. Alphonse Laveran has become an "object" of history.He was deeply involved in a turbulent historical period, marked by crises of regime change (Monarchy/Empire/Republic), military events (French colonial expansion in North Africa from 1830, the wars of 1870 and 1914-1918) and their consequences (the medical impact of infections in the colonial empire and during armed conflicts, the Dreyfus affair, among others), the advent of Pasteurian "microbiology" and the deciphering of the causes and modes of transmission of infectious diseases. A player on the edge of the military and civilian worlds, with their own, sometimes incompatible, visions of the aims and objectives to be pursued, Alphonse Laveran lived through these upheavals in a society in the throes of change, in his family and scientific environment.Paradoxically, the primary sources available to us for learning about this scientist and man are both abundant and "scarce" for us in the 21st century. His scientific publications and many of his speeches at various academies, committees and meetings are for the most part public and accessible, giving us a vision of a professional in scientific and medical research in action, presenting and convincing people of his ideas and theoretical and practical insights. The writings of his contemporaries, both public and private, shed light on - distort? - the man's many facets. On the other hand, there are few surviving sources on the man and his vision of life, his life and that of his family and friends.We will rely on the archives that have been preserved, in particular by the organisations that welcomed him during his military and civilian career, as well as by his wife Marie Laveran and his colleague Marie Phisalix, one of the first doctors of medicine in France and a renowned herpetologist. These two female figures have preserved and contributed to his memory. Let's take a closer look at the man behind the scientist, as we can imagine him through the traces that remain.

Teleost innate immunity, an intricate game between immune cells and parasites of fish organs: who wins, who loses.

Fish, comprising over 27,000 species, represent the oldest vertebrate group and possess both innate and adaptive immune systems. The susceptibility of most wild fish to parasitic infections and related diseases is well-established. Among all vertebrates, the digestive tract creates a remarkably favorable and nutrient-rich environment, which, in turn, renders it susceptible to microparasites and macroparasites. Consequently, metazoan parasites emerge as important disease agents, impacting both wild and farmed fish and resulting in substantial economic losses. Given their status as pathogenic organisms, these parasites warrant considerable attention. Helminths, a general term encompassing worms, constitute one of the most important groups of metazoan parasites in fish. This group includes various species of platyhelminthes (digeneans, cestodes), nematodes, and acanthocephalans. In addition, myxozoans, microscopic metazoan endoparasites, are found in water-dwelling invertebrates and vertebrate hosts. It is worth noting that several innate immune cells within the fish alimentary canal and certain visceral organs (e.g., liver, spleen, and gonads) play active roles in the immune response against parasites. These immune cells include macrophages, neutrophils, rodlet cells, and mast cells also known as eosinophilic granular cells. At the site of intestinal infection, helminths often impact mucous cells number and alter mucus composition. This paper presents an overview of the state of the art on the occurrence and characteristics of innate immune cells in the digestive tract and other visceral organs in different fish-parasite systems. The data, coming especially from studies employed immunohistochemical, histopathological, and ultrastructural analyses, provide evidence supporting the involvement of teleost innate immune cells in modulating inflammatory responses to metazoan and protozoan parasitic infections.

A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

How colonization bottlenecks, tissue niches, and transmission strategies shape protozoan infections.

Protozoan pathogens such as Plasmodium spp., Leishmania spp., Toxoplasma gondii, and Trypanosoma spp. are often associated with high-mortality, acute and chronic diseases of global health concern. For transmission and immune evasion, protozoans have evolved diverse strategies to interact with a range of host tissue environments. These interactions are linked to disease pathology, yet our understanding of the association between parasite colonization and host homeostatic disruption is limited. Recently developed techniques for cellular barcoding have the potential to uncover the biology regulating parasite transmission, dissemination, and the stability of infection. Understanding bottlenecks to infection and the in vivo tissue niches that facilitate chronic infection and spread has the potential to reveal new aspects of parasite biology.

The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

Mapping the poultry insectome in and around broiler breeder pullet farms identifies new potential Dipteran vectors of Histomonas meleagridis.

BACKGROUND: Histomonas meleagridis can infect chickens and turkeys. It uses the eggs of the cecal worm Heterakis gallinarum as a vector and reservoir. Litter beetles (Alphitobius diaperinus) and other arthropod species have been implicated as potential vectors, but little information about other arthropod species as potential vectors is known. METHODS: Four broiler breeder pullet farms were sampled every 4 months. On each farm, three types of traps were set inside and outside two houses. Trapped arthropod specimens were morphologically identified at order level and grouped into families/types when possible. Selected specimens from abundant types found both inside and outside barns were screened for H. meleagridis and H. gallinarum by qPCR. RESULTS: A total of 4743 arthropod specimens were trapped. The three most frequently encountered orders were Diptera (38%), Coleoptera (17%), and Hymenoptera (7%). Three hundred seventeen discrete types were differentiated. More arthropods were trapped outside than inside. Alpha diversity was greater outside than inside but not significantly influenced by season. The composition of the arthropod populations, including the insectome, varied significantly between trap location and seasons. Up to 50% of litter beetles tested positive for H. meleagridis DNA 4 months after an observed histomonosis outbreak. Sporadically litter beetles were positive for H. gallinarum DNA. Thirteen further arthropod types were tested, and specimens of four Dipteran families tested positive for either one or both parasites. CONCLUSIONS: This study describes the insectome in and around broiler breeder pullet farms and identifies new potential vectors of H. meleagridis through qPCR. The results show a limited but present potential of arthropods, especially flies, to transmit histomonosis between farms.

The most prominent modulated Annexins during parasitic infections.

Annexins (ANXs) exert different functions in cell biological and pathological processes and are thus known as double or multi-faceted proteins. These sophisticated proteins might express on both parasite structure and secretion and in parasite-infected host cells. In addition to the characterization of these pivotal proteins, describing their mechanism of action can be also fruitful in recognizing their roles in the pathogenesis of parasitic infections. Accordingly, this study presents the most prominent ANXs thus far identified and their relevant functions in parasites and infected host cells during pathogenesis, especially in the most important intracellular protozoan parasitic infections including leishmaniasis, toxoplasmosis, malaria and trypanosomiasis. The data provided in this study demonstrate that the helminth parasites most probably express and secret ANXs to develop pathogenesis while the modulation of the host-ANXs could be employed as a crucial strategy by intracellular protozoan parasites. Moreover, such data highlight that the use of analogs of both parasite and host ANX peptides (which mimic or regulate ANXs physiological functions through various strategies) might suggest novel therapeutic insights into the treatment of parasitic infections. Furthermore, due to the prominent immunoregulatory activities of ANXs during most parasitic infections and the expression levels of these proteins in some parasitic infected tissues, such multifunctional proteins might be also potentially relevant as vaccine and diagnostic biomarkers. We also suggest some prospects and insights that could be useful and applicable to form the basis of future experimental studies.

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.

Enterocytozoon bieneusi and Encephalitozoon intestinalis (microsporidia) in HIV-positive patients in central Spain.

Microsporidia are fungi-related eukaryotic intracellular parasites that opportunistically infect immunocompromised individuals such as those infected by the human immunodeficiency virus (HIV). Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most clinically relevant species. We investigated the occurrence and genetic diversity of microsporidial and protist infections in mostly immunocompetent HIV-positive patients in Madrid, Spain. A structured questionnaire was used to retrieve data on factors potentially associated with an increased risk of infection, including sexual attitudes and sex-risk behaviour. Faecal samples (n = 96) from 81 HIV-positive patients were collected and analysed by molecular (PCR and Sanger sequencing) methods. Two microsporidial pathogens were detected: Ent. bieneusi (2.5%, 95% CI: 0.3-8.6) and Enc.intestinalis (4.9%, 95% CI: 1.4-12.2). The two Ent. bieneusi isolates were identified as zoonotic genotype A. Among protists, Entamoeba dispar was the species most prevalently found (33.3%, 95% CI: 23.2-44.7), followed by Blastocystis spp. (19.8%, 95% CI: 11.7-30.1), Giardia duodenalis (13.6%, 95% CI: 7.0-23.0), and Cryptosporidium spp. and Entamoeba histolytica (2.5%, 95% CI: 0.3-8.6 each). Cyclospora cayetanensis and Cystoisospora belli were not detected. Subtypes ST1 (70.6%, 12/17) and ST3 (29.4%, 5/17) were identified within Blastocystis sp., sub-assemblages AII and BIII (50%, 1/2 each) within G. duodenalis, and Cry. parvum and canine-adapted Cry. canis (50%, 1/2 each) within Cryptosporidium spp. Microsporidial and protist parasites were frequent in well-controlled, mostly immunocompetent HIV-positive patients and should be included in diagnostic algorithms when diarrhoea is present.

Opportunistic microsporidial and protist intestinal infections were relatively common in well-controlled HIV-positive patients in Madrid, Spain. These agents should be suspected and appropriately diagnosed in HIV-positive patients presenting with diarrhoea regardless of their immunological status.