How a discerning cytological examination can aid in the diagnosis of infectious diseases: case reports.

Infections caused by uncommon and resistant pathogens in unusual sites have been increasingly reported in medical literature. We describe four cases of rare cytological findings and clinical impact for patients. In the first case, Aspergillus sp and Pneumocystis jirovecii were observed in the bronchoalveolar lavage of a patient with severe systemic lupus. In the second and third cases, we describe the presence of Trichomonas sp and Strongyloides sp larvae in samples of pleural and peritoneal fluid, respectively. The fourth report is about a patient with a wrist subcutaneous nodule whose synovial aspiration and cytology revealed the presence of brown septate hyphae. The early identification of the infectious agent in the cytological examination was essential for the introduction and/or re-adaptation of therapy in the four cases described. Patients in this report were immunocompromised with severe comorbidities, conditions often associated with unfavorable clinical outcomes.

HEALTH SCREENING OF THE EUROPEAN ENDANGERED SPECIES PROGRAM CAPTIVE POPULATION OF THE PINK PIGEON (NESOENAS MAYERI).

The population of the Mauritian pink pigeon (Nesoenas mayeri) fell to fewer than 20 individuals in the 1970s. Following intensive conservation efforts, the free-living population is now estimated to be 470 individuals. However, because of the population bottleneck the species remains at risk of extinction because of genetic loss and inbreeding depression. A European captive population was established in 1977 and a European Endangered Species Program (EEP) was formalized in 1992. As birds in the EEP captive population possess unique alleles not observed in the surviving free-living birds, the EEP management plan recommends transferring EEP birds to Mauritius to improve genetic diversity. Health screening of the current EEP population to identify circulating pathogens was performed. Forty-two birds from three collections in the United Kingdom and one in Jersey were screened for a wide range of pathogens, present clinically or subclinically, including important viruses, bacteria, protozoa, and helminths. Eleven birds tested positive for at least one pathogen: Trichomonas spp. (5), Yersinia kristensenii (2), Yersinia aleksiciae (1), coccidial oocysts (3), and strongyle ova (3). None of the positive birds showed overt signs of clinical disease, although two birds with Trichomonas spp. had suboptimal body condition. Genotyping of one Trichomonas gallinae sample revealed a type-C strain (low pathogenicity). The results from this screening will contribute towards a disease risk assessment, to create a pre-export protocol for translocation of captive EEP birds to Mauritius.

How a discerning cytological examination can aid in the diagnosis of infectious diseases: case reports

Infections caused by uncommon and resistant pathogens in unusual sites have been increasingly reported in medical literature. We describe four cases of rare cytological findings and clinical impact for patients. In the first case, Aspergillus sp and Pneumocystis jirovecii were observed in the bronchoalveolar lavage of a patient with severe systemic lupus. In the second and third cases, we describe the presence of Trichomonas sp and Strongyloides sp larvae in samples of pleural and peritoneal fluid, respectively. The fourth report is about a patient with a wrist subcutaneous nodule whose synovial aspiration and cytology revealed the presence of brown septate hyphae. The early identification of the infectious agent in the cytological examination was essential for the introduction and/or re-adaptation of therapy in the four cases described. Patients in this report were immunocompromised with severe comorbidities, conditions often associated with unfavorable clinical outcomes.

Diverse Trichomonas lineages in Australasian pigeons and doves support a columbid origin for the genus Trichomonas.

Trichomonas is a significant protist genus, and includes T. vaginalis, the most prevalent sexually transmitted non-viral infection of humans, and T. gallinae of rock doves (Columba livia), one of the earliest known avian pathogens. New Trichomonas genotypes, including T. vaginalis-like isolates, have been discovered in American columbid hosts, suggesting geographically widespread cryptic diversity of Trichomonas in pigeons and doves. We sampled 319 birds from 22 columbid species in Australia, Papua New Guinea, New Zealand and southern Africa and uncovered 15 novel lineages of Trichomonas, more than doubling the known diversity of this parasite genus and providing evidence for frequent host-switching that eventually gave rise to T. vaginalis in humans. We show that Trichomonas has a columbid origin and likely underwent Miocene expansion out of Australasia. Our chronological topology for Trichomonas is calibrated on the evolution of a host phenotypic trait associated with ecological entrapment of the most basal extant lineage of Trichomonas in Ptilinopus fruit-doves.

Incidence and Predictors of Chlamydia, Gonorrhea and Trichomonas Among a Prospective Cohort of Cisgender Female Sex Workers in Baltimore, Maryland.

BACKGROUND: Cisgender female sex workers (CFSWs) have elevated rates of sexually transmitted infections (STI) yet are underrepresented in targeted programming and research in the United States. We examined the prevalence, incidence and predictors of chlamydia, gonorrhea, and trichomonas infection among CFSW. METHODS: Two hundred fifty street-based CFSWs were recruited into a prospective observational cohort in Baltimore, Maryland using targeted sampling in 2016 to 2017 and completed surveys and STI testing at baseline, 3, 6, 9, and 12 months. Cox proportional hazards regression was used to model the predictors of STI. RESULTS: Mean age was 36 years, and 66.5% of respondents were white. Baseline prevalence of chlamydia, gonorrhea, trichomonas was 10.5%, 12.6%, and 48.5%, respectively. The incidence of chlamydia, gonorrhea, and trichomonas was 14.3, 19.3, 69.1 per 100 person-years. Over one year of observation, past year sex work initiation predicted both chlamydia incidence (adjusted hazard ratio [aHR], 2.7; 95% confidence interval [CI], 1.3-6.0) and gonorrhea incidence (aHR, 1.7; 95% CI, 1.0-2.8). Client sexual violence predicted gonorrhea incidence (aHR, 2.9; 95% CI, 1.2-7.1) and having female sexual partners predicted trichomonas incidence (aHR, 3.4; 95% CI, 1.3-8.5). Having a usual health care provider (aHR, 0.6; 95% CI, 0.5-0.7) was inversely associated with trichomonas. CONCLUSIONS: In this study of urban US street-based CFSW, interpersonal and structural factors differentially predicted STIs, and infection rates remained elevated through follow-up despite regular testing, notification, and treatment referral. Focused and multifaceted interventions for sex workers and their sexual partners are urgently needed.

Observational cross-sectional study of Trichomonas tenax in patients with periodontal disease attending a Chilean university dental clinic.

BACKGROUND: The oral flagellated protozoan Trichomonas tenax has been associated with patients with periodontal disease. However, no recent studies have been conducted on the prevalence of T. tenax in Chile. The aim of this study was to determine the presence of T. tenax in patients with periodontal disease, admitted to the Dental Clinic of the University of Antofagasta, Chile, through Polymerase Chain Reaction (PCR) amplification of the beta-tubulin gene. METHODS: An observational, cross-sectional study was conducted on 50 patients diagnosed with periodontal disease, 20 of them with gingivitis and 30 with periodontitis. T. tenax was identified by PCR amplification of the beta-tubulin gene. Associations between the protozoan and periodontal disease or the presence of risk factors to establish T. tenax infection were determined using the chi-square test and binary logistic regression analysis. RESULTS: T. tenax was present in 28 out of 50 (56%) of patients with periodontal disease, and was more prevalent when associated with periodontitis (21 out of 30; 70%) than dental plaque-induced gingivitis (7 out of 20; 35%). Non-statistically-significant associations were observed between the presence of T. tenax and age, gender, smoking habit or diabetes. Statistically significant associations were observed between the presence of T. tenax and periodontal disease, and between T. tenax and the Periodontal Screening and Recording (PSR) index. CONCLUSION: T. tenax showed a high presence in patients with progressive states of periodontal diseases. Consequently, T. tenax detection is strongly recommended in patients with periodontal disease diagnosis and with a PSR index greater than 3.

Multilocus Analysis Resolves the European Finch Epidemic Strain of Trichomonas gallinae and Suggests Introgression from Divergent Trichomonads.

In Europe, Trichomonas gallinae recently emerged as a cause of epidemic disease in songbirds. A clonal strain of the parasite, first found in the United Kingdom, has become the predominant strain there and spread to continental Europe. Discriminating this epidemic strain of T. gallinae from other strains necessitated development of multilocus sequence typing (MLST). Development of the MLST was facilitated by the assembly and annotation of a 54.7 Mb draft genome of a cloned stabilate of the A1 European finch epidemic strain (isolated from Greenfinch, Chloris chloris, XT-1081/07 in 2007) containing 21,924 protein coding genes. This enabled construction of a robust 19 locus MLST based on existing typing loci for Trichomonas vaginalis and T. gallinae. Our MLST has the sensitivity to discriminate strains within existing genotypes confidently, and resolves the American finch A1 genotype from the European finch epidemic A1 genotype. Interestingly, one isolate we obtained from a captive black-naped fruit dove Ptilinopsus melanospilus, was not truly T. gallinae but a hybrid of T. gallinae with a distant trichomonad lineage. Phylogenetic analysis of the individual loci in this fruit dove provides evidence of gene flow between distant trichomonad lineages at 2 of the 19 loci examined and may provide precedence for the emergence of other hybrid trichomonad genomes including T. vaginalis.

Use of nested PCR for the detection of trichomonads in bronchoalveolar lavage fluid.

BACKGROUND: The methods routinely used to detect trichomonads in the lungs are not sensitive enough, and an effective method is urgently needed. METHOD: Primers were first designed to match the conserved area of the 18S rRNA gene of trichomonads. Then, nested PCR was carried out to detect trichomonads in bronchoalveolar lavage fluid (BALF). Finally, all positive specimens were subjected to DNA sequencing and phylogenetic analysis. RESULTS: Among 115 bronchoalveolar lavage fluid samples, ten samples tested positive in nested PCR (10/115), while no samples were positive in wet mount microscopy (0/115) (P < 0.01). Among the ten positive specimens, two were identified as Tetratrichomonas spp. and the other eight as Trichomonas tenax in phylogenetic analysis. CONCLUSIONS: Nested PCR is an effective way to detect trichomonads in bronchoalveolar lavage fluid.

An outbreak of trichomonosis in European greenfinches Chloris chloris and European goldfinches Carduelis carduelis wintering in Northern France.

Avian trichomonosis is a common and widespread disease, traditionally affecting columbids and raptors, and recently emerging among finch populations mainly in Europe. Across Europe, finch trichomonosis is caused by a single clonal strain of Trichomonas gallinae and negatively impacts finch populations. Here, we report an outbreak of finch trichomonosis in the wintering populations of Chloris chloris (European greenfinch) and Carduelis carduelis (European goldfinch) from the Boulonnais, in northern France. The outbreak was detected and monitored by bird ringers during their wintering bird ringing protocols. A total of 105 records from 12 sites were collected during the first quarter of 2017, with 46 and 59 concerning dead and diseased birds, respectively. Fourteen carcasses from two locations were necropsied and screened for multiple pathogens; the only causative agent identified was T. gallinae. Genetic characterization was performed by four markers (small subunit ribosomal RNA, hydrogenosomal iron-hydrogenase, and RNA polymerase II subunit 1 genes, and the internal transcribed spacers (ITS) region) and confirmed the T. gallinae strain to be A1, which affects the finch populations of Europe. This was also confirmed by an ITS-based phylogenetic analysis which further illustrated the diversity of the Trichomonas infecting birds. Preliminary data on the survival and dispersion of infected birds were obtained from ring-returns of diseased individuals. The anthropogenic spread of diseases through bird feeding practices is highlighted and some suggestions to prevent pathogen transmission via backyard supplementary feeders for garden birds are given.

Captive Agamid lizards in Germany: Prevalence, pathogenicity and therapy of gastrointestinal protozoan and helminth infections.

Reptiles are becoming popular pets in many parts of the world. They are also known to harbor numerous gastrointestinal parasites. We used faecal smears to examine 748 stool samples from 14 different agamid lizard species. In addition, we used coproantigen ELISA tests (11 samples) and immunofluorescence assays (IFA) (19 samples) to detect reptile Cryptosporidium infections. In 28 cases, veterinarians requested therapy to treat oxyurid- and/or Isospora amphiboluri-infections and resent fecal samples after proposed therapy and anti-parasitic treatments had been applied. We also performed complete dissections of 24 deceased agamas in order to specify protozoan and helminth parasite infections. Overall, the examined fecal samples contained 6 different taxa. Oxyurids (Pharyngodonidae) were the most prevalent nematodes (41.2%), followed by I. amphiboluri (17.0%), Entamoeba spp. (0.8%), Choleoeimeria spp. (0.5%), Trichomonas spp. (0.3%), Cryptosporidium spp. (0.3%) and Strongyloides-like nematodes (0.1%). I. amphiboluri infections were significantly more prevalent (Chi-square test: χ2 = 21,5, df = 1, P < 0.001) in juvenile agamid lizards (31.9%) than in adults (14.2%). One of 11 (9.1%) coproantigen ELISA-examined samples was positive for Cryptosporidium. In 10.5% of the samples we found oocysts of Cryptosporidium. Thirteen (54.2%) of necropsied agamid lizards were infected with endoparasites and it is likely that three (12.5%) of them died due to severe parasitic infections. 74.0% of the samples that were submitted after therapy had been applied were negative. The high prevalences and pathological findings of several clinical parasitoses observed in these exotic reptiles calls for more detailed investigations on agamid gastrointestinal parasite fauna.

Specific clones of Trichomonas tenax are associated with periodontitis.

Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflammatory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 periodontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from periodontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%°) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p<0.05). More frequently detected in periodontal cases, T. tenax is likely to be related to the onset or/and evolution of periodontal diseases.

Trichomonas Detection in Leucorrhea Based on VIBE Method.

Trichomonas examination is one of the important items in the leucorrhea routine detection. And it cannot be recognized by still images because of the unstable morphology and unfixed focal location caused by motion characteristic. We proposed an improved VIBE algorithm. 6 videos (totally 1414 frames) are collected for testing. In order to compare the effects of the algorithms, we segment each frame artificially as ground truth. Experiments show that percentage of correct classification (PCC) achieves 88%. The proposed improved method can effectively suppress the false detection caused by the formed components such as epithelial cells in the leucorrhea microscopic image and the missed detection caused by the background model update during the movement. At the same time, improvements can effectively suppress smear and ghost areas. The algorithm proposed in this paper can be integrated into the leucorrhea automatic detection system.

Presence of trichomonads in subgingival biofilm of patients with periodontitis: preliminary results.

This study aims to evaluate the prevalence of trichomonads in the subgingival biofilm of patients with periodontitis. Secondarily, the trichomonad presence was related to patient characteristics and periodontal clinical parameters, in order to highlight the factor favoring the development of these protozoans. Subgingival biofilm samples were collected from at least two diseased and one healthy site in 50 patients suffering from periodontitis. Trichomonads were identified using phase contrast microscopy. All patient characteristics and periodontal clinical parameter data were then statistically analyzed. From the 50 patients examined, 195 sites were sampled, including 145 diseased ones. Trichomonads were only observed on 16 of the 145 diseased sites (11%) and none in the other 50 healthy sites. Based on these results, 20% (n = 10) of patients were positive for the presence of trichomonads from at least one of the diseased sites collected. Tooth mobility, substantial supra-gingival dental deposits, and severe clinical attachment loss were statistically associated with trichomonad presence. If the subgingival biofilm of male patients over the age of 50 seemed to be more frequently contaminated with trichomonads, this data was not statistically supported. This preliminary study indicates for the first time that in periodontitis-involved patients, trichomonads are observed in the subgingival biofilm collected from diseased sites with severe bone loss, but not from healthy teeth. Further investigations are needed to fully explore the role of this microorganism in the etiology of periodontal disease.

Molecular identification of Trichomonas tenax in the oral environment of domesticated animals in Poland - potential effects of host diversity for human health.

INTRODUCTION: The protozoan Trichomonas tenax is considered to be a human specific flagellate of the oral cavity, found in humans with poor oral hygiene and advanced periodontal disease. Morphological variability and great similarity between species occurring in humans and animals, complicate the specific identification of trichomonads, using microscopic examination and other standard parasitological techniques. OBJECTIVE: The aim of the study was to search for and identify T. tenax in domesticated animals using molecular methods. The obtained data were assessed in terms of potential effects of a spread of the species deriving from the animals in the human environment. MATERIAL AND METHODS: 301 animals: 142 dogs, 57 cats and 102 horses, were examined in terms of their mouth status and occurrence of trichomonads. ITS1-5.8S rRNA-ITS2 region was amplified and sequenced. RESULTS: Finally, 7 dogs, 3 cats and 1 horse were diagnosed positive for T. tenax by PCR. In the oral cavity of 9 /11 animals, gingivitis and dental plaque accumulation were diagnosed. 9 /11 sequences of trichomonad isolates showed 100% identity with T. tenax sequence derived from the GenBank. The sequences of 2 isolates differed by substitutions. CONCLUSIONS: It was proved that T. tenax, considered so far as a human specific parasite, can also inhabit the oral cavity of dog, cat and horse. To summarize, T. tenax was detected in the mouths of different domesticated animals, indicating that in Poland it can colonize a wider range of hosts than previously known. The owners of 3 dogs showed oral tissue inflammation of different intensity and were also positive for T. tenax; therefore, oral trichomonosis spread from humans to domestic animals and conversely should be taken into consideration.

Prevalence of Haemoproteus columbae (Apicomplexa: Haemoproteidae) and Trichomonas gallinae (Metamonada: Trichomonadidae) Infections Among Pigeons (Columba livia) in West Azerbaijan Province, Iran.

A cross-sectional survey was performed on domestic pigeons (Columba liviadomestica) to evaluate the prevalence of Haemoproteus columbae and Trichomonas gallinae in West Azerbaijan Province, northwest of Iran between July 2015 and September 2016. Five hundred and sixty oropharyngeal swab smears were examined, 297 (53%) of which were identified to be positive for T. gallinae. Also, the results obtained from blood smears examination indicated that 115 (20.5%) cases were found to be infected with H. columbae. The rates of T. gallinae and H. columbae infection infemale pigeons (58.3% and 21.3%, respectively) were higher compared to those in males. There were 76 (26.1%) and 210 (72.4%) cases of H. columbae and T. gallinae infection in adolescent birds, respectively, which were higher than those in other age groups. We found high prevalence rates of H. columbae and T. gallinae infections in domestic pigeons. Therefore, more attention should be focused on controlling the spread of T. gallinae and H. columbae infection in domestic pigeons.

Prevalence and molecular characterization of Trichomonas gallinae from domestic pigeons in Beijing, China.

Trichomonas gallinae is a globally distributed protozoan parasite, mainly affect the upper avian digestive tract and can bring huge economic losses to pigeon industry. The objective of the present study was to determine the prevalence and genotypes of T. gallinae in Beijing, China. A total of 569 samples of throat swabs of pigeon were collected from pigeon farms in Shunyi District, Fangshan District, Daxing District and Miyun District of Beijing. The overall prevalence was 28.30%. The significant difference in infection rates was not observed between regions, but was found between age groups. The highest prevalence was nestling pigeons (33.16%), followed by adolescent pigeons (30.05%) and breeding pigeons (20.59%). Moreover, genotype A and B of T. gallinae were identified by sequencing the ITS1/5.8S/ITS2 regions and phylogenetic analysis. To our knowledge, this is the first report to display the prevalence and genotype of T. gallinae from Beijing, China.

Three Pentatrichomonas hominis Cases Presenting with Gastrointestinal Symptoms.

We aimed to demonstrate that Pentatrichomonas hominis may also be an agent, although rare, in diarrheal episodes. Stool samples were first examined macroscopically and microscopically during routine parasitological examinations. Samples were then evaluated by Native-Lugol and formol-ethyl acetate centrifugation method. To exclude other pathogenic bacterial agents, a bacteriological culture method was applied. Samples were evaluated using a qualitative immunochromatographic test kit for rotavirus and adenovirus. We presented three cases of 77-year-old and 10-year-old male and 9-year-old female patients. Cases 1 and 2 were admitted to the hospital with complaints of diarrhea, abdominal pain, and weakness in July 2013. Leukocytes and active P. hominis trophozoites were detected. No bacterial and other parasitic and viral agents were found in their stool specimens. Oral metronidazole treatments were administered to the patients. In Case 3, P. hominis trophozoites were detected in the cellophane band in the plastic locked bag which could survive for 48 hduring a field survey in May 2012. Case 3 was contacted and advised to visit a pediatrician. P. hominis is a rare parasitic zoonosis, and we believe that it should not be ignored among diarrheal agents.

Prevalence of two Entamoeba gingivalis ST1 and ST2-kamaktli subtypes in the human oral cavity under various conditions.

Advances in molecular biology have facilitated analyses of the oral microbiome; however, the parasites role is poorly understood. Periodontal disease is a multifactorial process involving complex interactions among microorganisms, the host, and environmental factors. At present, the precise composition of the mouth parasites microbiota is unclear. Two protozoan species have been detected in the oral microbiota: Trichomonas tenax and Entamoeba gingivalis, and a new variant, E. gingivalis-ST2-kamaktli, was recently identified by us. In this study, both E. gingivalis and the new E. gingivalis-ST2-kamaktli variant were detected in the oral cavities of people with healthy periodontium, individuals undergoing orthodontic treatment, and patients with periodontal disease. In the group with healthy periodontium, the prevalence of E. gingivalis-ST1 was 48.6% and that of E. gingivalis-ST2-kamaktli 29.5%, with a combined prevalence of 54.3%. In patients undergoing orthodontics treatment, 81.2% carried both amoebas, with 47.5% having E. gingivalis-ST1 and 73.8% E. gingivalis-ST2-kamaktli. In people with periodontal disease, the prevalence of E. gingivalis-ST1 was 57.8%, and that of E. gingivalis-ST2-kamaktli 50.0%, with a combined prevalence of 73.5%; hence, E. gingivalis-ST1 and E gingivalis-ST2-kamaktli were detected in all three groups. The question arises, what are E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli doing in the oral cavity? Although, the answer remains unclear, our results suggest that each amoeba subtype is genetically distinct, and they exhibit different patterns of infectious behavior. We hypothesize that E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli may represent separate species. Our data contribute to better understanding of the roles of E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli in the oral microbiota.