Correction: Matthew et al. A Loop-Mediated Isothermal Amplification (LAMP) Assay Specific to Trichomonas tenax Is Suitable for Use at Point-of-Care. Microorganisms 2022, 10, 594.

In the original publication [...].

Roles of Cysteine Proteases in Biology and Pathogenesis of Parasites.

Cysteine proteases, also known as thiol proteases, are a class of nucleophilic proteolytic enzymes containing cysteine residues in the enzymatic domain. These proteases generally play a pivotal role in many biological reactions, such as catabolic functions and protein processing, in all living organisms. They specifically take part in many important biological processes, especially in the absorption of nutrients, invasion, virulence, and immune evasion of parasitic organisms from unicellular protozoa to multicellular helminths. They can also be used as parasite diagnostic antigens and targets for gene modification and chemotherapy, as well as vaccine candidates, due to their species and even life-cycle stage specificity. This article highlights current knowledge on parasitic cysteine protease types, biological functions, and their applications in immunodiagnosis and chemotherapy.

Hymenolepis diminuta, phylogenetic analyses of nuclear rRNA + ITS and mitochondrial cox1 and its infections in non-human primates.

Hymenolepis diminuta is a tapeworm commonly found worldwide in small rodents such as rats with occasional reports in other definitive hosts such as primates including chimpanzees and humans. It has not been reported in African green monkey (AGM, Chlorocebus sabaeus), and the parasite's molecular phenotype and phylogeny remain primitively sketchy. The aims of the current study were to determine if H. diminuta infected AGMs, to molecularly characterize H. diminuta and to review its infection in non-human primates. Feces of AGMs were examined visually for adult helminths and microscopically for eggs using centrifugation flotation. Total DNA extracted from eggs was amplified by PCR followed by DNA sequencing of targeted sequences of nuclear rRNA + internal transcribed spacers (ITS) and mitochondrial cox1. Phylogenetic analyses were performed. The DNA sequences of both nuclear rRNA + ITS and mitochondrial cox1 showed more than 98% and 99% identity to the known sequences respectively. Hymenolepis diminuta has been reported in various non-human primates with the highest prevalence of 38.5% in the white-headed capuchin monkey. The study presented here confirms that this tapeworm is capable of infecting various species of non-human primates with the first report of infections in AGM. Phylogenetic analyses of rRNA + ITS and mitochondrial cox1 demonstrated three separated clades I, II and III with the newly described AGM1 isolate belonging to the clade I. Whether these differences are at species level remains to be confirmed.

Trichomonas tenax: A Neglected Protozoan Infection in the Oral Cavities of Humans and Dogs-A Scoping Review.

Trichomonas tenax is a flagellated protozoan parasite found in the oral cavities of humans and animals and has been associated with periodontal disease, the most prevalent inflammatory disease affecting them all. Studies have shown that T. tenax can cause damage to mammalian cells and secretes virulent proteins, such as cysteine. It is presently considered zoonotic. Despite the few studies that have been done, the pathogenicity of this oral protozoan is still not fully understood. A database search was performed in July 2022 using PubMed and Google Scholar to retrieve data eligible for this study. PRISMA-ScR guidelines were followed to conduct this scoping review. A total of 321 articles were found with 87 included in this review after applying the exclusion criteria. Due to its increasing prevalence worldwide in both humans and dogs, detecting and elucidating the pathogenicity of this parasite is paramount for effective global control and prevention of periodontal disease. However, there is a paucity in the literature on this neglected zoonotic trichomonad, which is in large contrast to the closely related human pathogen T. vaginalis. Here, we comprehensively review the history, morphology and reproduction, host, prevalence, diagnosis, pathogenicity, control, and prevention of T. tenax. Hopefully, this article will call attention to both medical and veterinary professionals as well as epidemiologists on this most neglected and zoonotic protozoan. More epidemiological and clinical studies need to be conducted on T. tenax to gain a better understanding of its pathogenicity, to increase the chances of developing effective drugs to aid in the control of this oral parasite, and reduce the spread of periodontal disease worldwide.

A Loop-Mediated Isothermal Amplification (LAMP) Assay Specific to Trichomonas tenax Is Suitable for Use at Point-of-Care.

Trichomonas tenax is a flagellated protozoan that inhabits the human and canine oral cavity in patients with poor oral hygiene and periodontal disease. The loop-mediated isothermal amplification (LAMP) assay could provide clinicians with a quick, cheap and reliable diagnostic test used for the detection of T. tenax in various settings. In this study, we aimed to develop a LAMP assay that can detect T. tenax with high sensitivity and specificity. A set of LAMP primers were specifically designed to detect the ITS and 5.8S rRNA gene of T. tenax. The newly developed LAMP assay was 1000 times more sensitive than conventional PCR. The limit of detection of the LAMP assay was 10 fg of genomic DNA, or 0.2-1 cell. Moreover, the LAMP assay was specific, resulting in no cross-reaction even with a closely related protozoan T. vaginalis or other microorganisms (Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Candida albicans) used. The present LAMP assay can be performed directly without prior DNA extraction, making the assay an easy, fast, cheap, specific and sensitive diagnostic tool for the detection of T. tenax at the point-of-care of both medical and veterinary clinics in developed and developing countries.

Prevalence of Feline Immunodeficiency Virus and Toxoplasma gondii in Feral Cats on St. Kitts, West Indies.

Toxoplasma gondii (T. gondii) is a cosmopolitan protozoan parasite that infects all warm-blooded species including humans. The definitive hosts of T. gondii are felid vertebrates including the domestic cat. Domestic cats shed oocysts for approximately two weeks in their feces after the primary infection. It has been shown that feline immunodeficiency virus (FIV) positive cats have a higher prevalence of and a higher titer of antibodies to T. gondii than those of FIV-negative cats. The main purposes of this study were to determine FIV prevalence and to investigate the oocysts shedding in FIV-positive and FIV-negative feral cats on St. Kitts. Fecal samples were collected from feral cats while their FIV statues were determined using a commercial SNAP kit. Total fecal DNA of each cat was tested for the presence of T. gondii DNA using a polymerase chain reaction (PCR) consistently detecting one genome equivalent. A FIV-positive status was detected in 18 of 105 (17.1%, 95% confidence interval (CI): 9.9%-24.3%) feral cats sampled. Furthermore, males were three times more likely to be FIV positive than females (p = 0.017) with an odds ratio of 3.93 (95% CI: 1.20-12.89). Adults were found to have at least twice the prevalence of FIV compared to cats younger than one year of age (p = 0.056) with an odds ratio of 3.07 (95% CI: 0.94-10.00). Toxoplasma gondii DNA was not detected in the feces of any of the 18 FIV-positive (95% CI: 0%-0.18%) and 87 FIV-negative cats (95% CI: 0%-0.04%). A follow-up study with a much bigger sample size is needed to prove or disprove the hypothesis that FIV-positive cats have a higher prevalence of shedding T. gondii oocysts than FIV-negative cats.

Failure to detect Tritrichomonas foetus in a cross-sectional survey in the populations of feral cats and owned outpatient cats on St Kitts, West Indies.

OBJECTIVES: For over two decades, feline trichomonosis caused by Tritrichomonas foetus has been recognized as a large-bowel protozoan disease of the domestic cat. It has a wide distribution, but no reports exist in the Caribbean. The objectives of this study were to detect the presence of T foetus and its prevalence in the domestic cat on St Kitts, West Indies. METHODS: A cross-sectional study was performed between September 2014 and December 2015. This study recruited 115 feral cats from a trap-neuter-return program and 37 owned cats treated as outpatients at the university veterinary clinic. Fresh feces were inoculated in InPouch culture medium, as per the manufacturer's instructions. In addition, PCR was performed using primers for T foetus. DNA extraction with amplification using primers of a Feliscatus NADH dehydrogenase subunit 6 was used as a housekeeping gene for quality control. RESULTS: Only two owned cats had reported diarrhea in the preceding 6 months. None of the 152 samples were positive on InPouch culture microscopic examination. Only 35/69 feral cat fecal DNA samples were positive for the housekeeping gene, of which none tested PCR positive for T foetus. CONCLUSIONS AND RELEVANCE: T foetus was not detected by culture and PCR in feral cats and owned cats on St Kitts. A high proportion of PCR inhibitors in the DNA samples using a commercial fecal DNA kit can lead to underestimating the prevalence, which should be taken into consideration when a survey on gastrointestinal pathogens depends exclusively on molecular detection.

Parasites of small Indian mongoose, Herpestes auropunctatus, on St. Kitts, West Indies.

Herpestes auropunctatus, the small Indian mongoose, is an invasive omnivore introduced to the Caribbean, including the island of St. Kitts over 150 years ago. It has played a role in changing native fauna and can carry zoonotic pathogens of public health importance. The aim of the current study was to estimate the prevalence of parasites harbored by mongooses. In total, 87 mongooses trapped from April to July 2015 were examined for parasites using (1) hair plucks (N = 79), ear swabs (N = 79), and general coat and skin examination (N = 87) for mites, ticks, lice, and fleas; (2) dissection of the trachea, bronchi, and lungs for lungworms and flukes (N = 76); (3) a double centrifugation fecal flotation method for parasites of the gastrointestinal tract (N = 75); and (4) PCR of heart homogenates for Toxoplasma gondii (N = 60). The only ectoparasite seen was Ctenocephalides felis (79.3%; 69/87), with most mongooses having > 10 fleas (based on a subjective assessment) but insufficient numbers to result in signs of pruritus or anemia. On fecal flotation, coccidial oocysts were found with a prevalence of 69.3% (52/75). Neither T. gondii, lungworm, nor fluke infections were detected with the methods used. The high number of C. felis-infested mongooses and the infestation level of the individual mongooses suggest that they could serve as a reservoir for these potential vectors of pathogens. No evidence was found to support that mongooses are a component of T. gondii cycles on St. Kitts, although this finding needs to be confirmed with a larger sample size from other geographic locations.

Freedom from Tritrichomonas foetus infection in cattle in St. Kitts.

Trichomonosis is an endemic disease in cattle that are reared under extensive conditions and bred by natural mating. It causes profound economic losses to the producers by increasing calving interval, increasing embryo losses, and decreasing pregnancy rates. The aim of this study was to determine whether Tritrichomonas foetus infections were absent from cattle in St. Kitts. Using the modified hypergeometric method, preputial samples from bulls (n = 78) were tested using the InPouch™ culture for presence of T. foetus. Results highlighted an absence of trichomoniasis in bulls on St. Kitts with a 95% confidence.

Molecular species identification of Trichuris trichiura in African green monkey on St. Kitts, West Indies.

The population of African green monkeys (AGM, Chlorocebus aethiops sabaeus) on St. Kitts, West Indies is believed to be as large as or greater than the human population. Interactions with humans are frequent and the pathogens carried by AGM, such as Trichuris spp., may pose a risk to humans. The objectives of this study were to assess the use of molecular methods for diagnosing Trichuris spp. in AGM and compare its DNA sequences to those of Trichuris spp. found in other non-human primates and humans. Fecal samples were collected from trapped and individually housed AGM between January and December 2015 and analysed using fecal flotation with Sheather's sugar flotation solution and PCR amplification and DNA sequencing of 18S rRNA and ITS2 fragments. Phylogenetic analysis was performed. 91% (81/89) and 55.4% (31/56) were Trichuris spp. positive by fecal flotation and PCR, respectively. Both AGM-NADH1 gene and T. trichiura-18S rRNA gene showed no variations in sequence and were 100% identical to corresponding sequences deposited in GenBank. Nevertheless Trichuris ITS2 showed some diversities among 12 sequences, which was <5%. Phylogenetic analysis of ITS2 put Trichuris spp. in Kittitian AGM into the same clades of T. trichiura found in human and other non-human primates in many other geographical regions. These data confirm that AGM are reservoirs for T. trichiura in humans. We suggest a one health approach to curtail enteric parasitic infections in human populations in the insular country.

First case of canine leishmaniasis on the island of St. Kitts, West Indies.

Leishmaniasis caused by Leishmania infantum is an emerging/re-emerging zoonosis with dogs as the main reservoir. The first autochthonous case of canine leishmaniasis in St. Kitts was diagnosed, and had cutaneous involvement. This discovery expands the distribution of leishmaniasis to include this insular country in the West Indies and dictates further characterization of the disease in this region.

Multiple products of the Leishmania chagasi major surface protease (MSP or GP63) gene family.

The major surface protease (MSP or GP63) of the Leishmania spp. protozoa facilitates parasite evasion of complement-mediated killing, phagocytosis by macrophages, and intracellular survival in macrophage phagolysosomes. Immunoblots of several Leishmania species have shown there are distinct MSP isoforms, but the biochemical bases for these differences are unknown. Northern blots show that transcripts of the three tandem gene classes encoding Leishmania chagasi MSP (MSPS, MSPL, MSPC) are differentially expressed during parasite growth in vitro. Cell-associated MSPs increase in abundance during growth, correlating directly with parasite virulence. We examined whether distinct products of these >18 MSP genes are either differentially expressed or differentially processed during parasite growth. Two-dimensional gel electrophoresis and immunoblots delineated more than 10 MSP isoforms in stationary phase L. chagasi, distributed between pIs of 5.2-6.1 and masses of 58-63 kDa. Post-translational modifications including N-glycosylation, GPI anchor addition and phosphorylation did not account for all differences among the isoforms. MALDI-TOF mass spectrometry demonstrated that at least some L. chagasi MSPs were the products of different MSP genes. One isoform was not available for surface biotinylation, suggesting it could be located internally. Parasites in logarithmic growth expressed only four MSP isoforms, and an attenuated strain of L. chagasi (L5) did not express one of the MSP classes (MSPS). These data demonstrate that the products of individual MSP genes are differentially expressed during Leishmania development. We hypothesize they may play different roles during parasite migration through its two hosts.