Genetic characterization of the Entamoeba moshkovskii population based on different potential genetic markers.

Entamoeba moshkovskii, according to recent studies, appears to exert a more significant impact on diarrhoeal infections than previously believed. The efficient identification and genetic characterization of E. moshkovskii isolates from endemic areas worldwide are crucial for understanding the impact of parasite genomes on amoebic infections. In this study, we employed a multilocus sequence typing system to characterize E. moshkovskii isolates, with the aim of assessing the role of genetic variation in the pathogenic potential of E. moshkovskii. We incorporated 3 potential genetic markers: KERP1, a protein rich in lysine and glutamic acid; amoebapore C (apc) and chitinase. Sequencing was attempted for all target loci in 68 positive E. moshkovskii samples, and successfully sequenced a total of 33 samples for all 3 loci. The analysis revealed 17 distinct genotypes, labelled M1­M17, across the tested samples when combining all loci. Notably, genotype M1 demonstrated a statistically significant association with diarrhoeal incidence within E. moshkovskii infection (P = 0.0394). This suggests that M1 may represent a pathogenic strain with the highest potential for causing diarrhoeal symptoms. Additionally, we have identified a few single-nucleotide polymorphisms in the studied loci that can be utilized as genetic markers for recognizing the most potentially pathogenic E. moshkovskii isolates. In our genetic diversity study, the apc locus demonstrated the highest Hd value and π value, indicating its pivotal role in reflecting the evolutionary history and adaptation of the E. moshkovskii population. Furthermore, analyses of linkage disequilibrium and recombination within the E. moshkovskii population suggested that the apc locus could play a crucial role in determining the virulence of E. moshkovskii.

Phylogenetic analyses of Chilomastix and Retortamonas species using in vitro excysted flagellates.

In vitro excystation of cysts of microscopically identified Chilomastix mesnili and Retortamonas sp. isolated from Japanese macaques and Retortamonas sp. isolated from small Indian mongooses could be induced using an established protocol for Giardia intestinalis and subsequently by culturing with H2S-rich Robinson's medium supplemented with Desulfovibrio desulfuricans. Excystation usually began 2 h after incubation in Robinson's medium. DNA was isolated from excysted flagellates after 4 h of incubation or from cultured excysted flagellates. Phylogenetic analysis based on their 18S rRNA genes revealed that two isolates of C. mesnili from Japanese macaques belonged to the same cluster as a C. mesnili isolate from humans, whereas a mammalian Retortamonas sp. isolate from a small Indian mongoose belonged to the same cluster as that of an amphibian Retortamonas spp. isolate from a 'poison arrow frog' [sequence identity to AF439347 (94.9%)]. These results suggest that the sequence homology of the 18S rRNA gene of the two C. mesnili isolates from Japanese macaques was similar to that of humans, in addition to the morphological similarity, and Retortamonas sp. infection of the amphibian type in the small Indian mongoose highlighted the possibility of the effect of host feeding habitats.

Investigating genetic polymorphism in E. histolytica isolates with distinct clinical phenotypes.

Amoebiasis is an infection caused by enteric protozoa, most commonly Entamoeba histolytica, and is globally considered a potentially severe and life-threatening condition. To understand the impact of the parasite genome on disease outcomes, it is important to study the genomes of infecting strains in areas with high disease prevalence. These studies aim to establish correlations between parasite genotypes and the clinical presentation of amoebiasis. We employ a strain typing approach that utilizes multiple loci, including SREHP and three polymorphic non-coding loci (tRNA-linked array N-K2 and loci 1-2 and 5-6), for high-resolution analysis. Distinct clinical phenotype isolates underwent amplification and sequencing of studied loci. The nucleotide sequences were analysed using Tandem Repeats Finder to detect short tandem repeats (STRs). These patterns were combined to assign a genotype, and the correlation between clinical phenotypes and repetitive patterns was statistically evaluated. This study found significant polymorphism in the size and number of PCR fragments at SREHP and 5-6 locus, while the 1-2 locus and NK2 locus showed variations in PCR product sizes. Out of 41 genotypes, two (I6 and I41) were significantly associated with their respective disease outcomes and were found in multiple isolates. We observed that I6 was linked with a symptomatic outcome, with a statistically significant p-value of 0.0183. Additionally, we found that I41 was associated with ALA disease outcome, with a p-value of 0.0089. Our study revealed new repeat units not previously reported, unveiling the genetic composition of E. histolytica strains in India, associated with distinct disease manifestations.

Prevalence and molecular characterization of Entamoeba moshkovskii in diarrheal patients from Eastern India.

BACKGROUND: Importance of the amphizoic amoeba Entamoeba moshkovskii is increasing in the study of amoebiasis as a common human pathogen in some settings. Limited studies are found on the genetic and phylogenetic characterization of E. moshkovskii from India; hence remain largely unknown. In this study, we determined the prevalence and characterized the E. moshkovskii isolates in eastern India. METHODS: A three-year systemic surveillance study among a total of 6051 diarrhoeal patients from ID Hospital and BC Roy Hospital, Kolkata was conducted for E. moshkovskii detection via a nested PCR system targeting 18S rRNA locus. The outer primer set detected the genus Entamoeba and the inner primer pair identified the E. moshkovskii species. The 18S rRNA locus of the positive samples was sequenced. Genetic and phylogenetic structures were determined using DnaSP.v5 and MEGA-X. GraphPad Prism (v.8.4.2), CA, USA was used to analyze the statistical data. RESULT: 4.84% (95%CI = 0.0433-0.0541) samples were positive for Entamoeba spp and 3.12% (95%CI = 0.027-0.036) were infected with E. moshkovskii. E. moshkovskii infection was significantly associated with age groups (X2 = 26.01, P<0.0001) but not with gender (Fisher's exact test = 0.2548, P<0.05). A unique seasonal pattern was found for E. moshkovskii infection. Additionally, 46.56% (95%CI = 0.396-0.537) were sole E. moshkovskii infections and significantly associated with diarrheal incidence (X2 = 335.5,df = 9; P<0.0001). Sequencing revealed that the local E. moshkovskii strains were 99.59%-100% identical to the prototype (GenBank: KP722605.1). The study found certain SNPs that showed a correlation with clinical features, but it is not necessarily indicative of direct control over pathogenicity. However, SNPs in the 18S rRNA gene could impact the biology of the amoeba and serve as a useful phylogenetic marker for identifying pathogenic E. moshkovskii isolates. Neutrality tests of different coinfected subgroups indicated deviations from neutrality and implied population expansion after a bottleneck event or a selective sweep and/or purifying selection in co-infected subgroups. The majority of FST values of different coinfected subgroups were <0.25, indicating low to moderate genetic differentiation within the subgroups of this geographical area. CONCLUSION: The findings reveal the epidemiological significance of E. moshkovskii infection in Eastern India as the first report in this geographical area and expose this species as a possible emerging enteric pathogen in India. Our findings provide useful knowledge for further research and the development of future control strategies against E. moshkovskii.

Prevalence and metronidazole resistance of Trichomonas vaginalis among Japanese women in 2021.

Objectives: Trichomonas vaginalis is the most prevalent sexually transmitted parasite worldwide. However, no surveillance system exists to monitor T. vaginalis cases and drug resistance in Japan. Methods: Cervical cytology vaginal swabs were collected from women with and without suspected symptoms of T. vaginalis infection; these swabs were used for the detection of T. vaginalis, human papillomavirus (HPV), and Candida albicans using specific polymerase chain reaction. Clinical isolates of T. vaginalis were subjected to metronidazole susceptibility tests using the previously reported minimal lethal concentration (MLC) and newly established half-maximal inhibitory concentration (IC50) values. Results: The prevalence of T. vaginalis in the study population was 4.2% (5/119; 95% confidence interval [Cl], 1.5-9.7). Additionally, asymptomatic infection constituted 60% (3/5) of all cases of T. vaginalis infection. All T. vaginalis-positive patients were coinfected with HPV but not C. albicans. Five clinical T. vaginalis isolates showed metronidazole susceptibility, which was evaluated using MLC values. The quantitative IC50 values revealed that two of these clinical isolates exhibited a decreased metronidazole susceptibility. Conclusion: This is the first study to demonstrate the prevalence of T. vaginalis in Japanese women. The IC50 values of metronidazole against T. vaginalis enabled the precise and quantitative evaluation of metronidazole-susceptible T. vaginalis.

Development of a simple PCR-RFLP technique for detection and differentiation of E. histolytica, E. dispar and E. moshkovskii.

Epidemiological studies on amoebic infections are complicated by morphological overlap between the pathogenic E. histolytica, the commensal E. dispar and the amphizoic E. moshkovskii, necessitating molecular identification. The present study developed a simple and economical 18S PCR-RFLP method for the simultaneous detection and differentiation of the three species. PCR products were differentiated by Tat1 restriction digestion generating three different RFLP patterns. Validation was conducted by screening 382 faecal samples from human patients from Kolkata, India, hospitalized for diarrhoea. Analysis indicated that the PCR-RFLP could successfully differentiate between the three species and was confirmed by sequence analysis. This method could prove useful for clinical and epidemiological studies of amoebiasis.

Gene expression of axenically-isolated clinical Entamoeba histolytica strains and its impact on disease severity of amebiasis.

The severity of Entamoeba histolytica infection is determined by host immunology, pathogen virulence, and the intestinal environment. Conventional research for assessing pathogen virulence has been mainly performed using laboratory strains, such as a virulent HM-1: IMSS (HM-1) and an avirulent Rahman, under various artificial environmental conditions because of the difficulties of axenic isolation of the clinical strains. However, it is still unclear whether scientific knowledge based on laboratory strains are universally applicable to the true pathogenesis. Hereby, we performed transcriptomic analysis of clinical strains from patients with different degrees of disease severity, as well as HM-1 under different conditions. Even after several months of axenization, Clinical strains show the distinct profile in gene expression during in vitro passage, moreover, difference between any 2 of these strains was much greater than the changes on the liver challenge. Interestingly, 26 DEGs, which were closely related to the biological functions, were oppositely up- or down regulated between virulent Ax 19 (liver abscess) and avirulent Ax 11 (asymptomatic carrier). Additionally, RNAseq using laboratory strain (HM1) showed more than half of genes were differently expressed between continuously in vitro passaged HM1 (in vitro HM1) and periodically liver passaged HM1 (virulent HM1), which was much greater than the changes on the liver passage of virulent HM1. Also, transcriptomic analysis of a laboratory strain revealed that continuous environmental stress enhances its virulence via a shift in its gene expression profile. Changes in gene expression patterns on liver abscess formation were not consistent between clinical and laboratory strains.

Molecular evidence suggests the occurrence of Entamoeba moshkovskii in pigs with zoonotic potential from eastern India.

Entamoeba moshkovskii Tshalaia, 1941 is prevalent in developing countries and it is considered to be primarily a free-living amoeba, which is morphologically indistinguishable, but biochemically and genetically different from the human infecting, pathogenic Entamoeba histolytica Schaudinn, 1903. The pathogenic potential of this organism is still under discussion. Entamoeba moshkovskii in human stool samples has been reported in different countries such as the United States, Italy, Australia, Iran, Turkey, Bangladesh, India (Pondicherry), Indonesia, Colombia, Malaysia, Tunisia, Tanzania and Brazil, but no data are available about the occurrence of E. moshkovskii in farm animals. This study provides data on the occurrence of E. moshkovskii in pigs in a total of 294 fresh faecal samples collected from five different regions in Kolkata, West Bengal, India. Stool samples were tested by nested PCR using primers targeting SSU rDNA of E. moshkovskii. The amplified PCR products were further confirmed by RFLP technique. Purified nested PCR products were also sequenced and identified via BLAST program run on the NCBI website to confirm species along with their genetic characteristics of the E. moshkovskii isolates. Overall 5.4 % samples were identified as E. moshkovskii positive. Results of this study demonstrate that swine can host E. moshkovskii and should be considered as a potential natural reservoir for E. moshkovskii. However, the occurrence of E. moshkovskii infection in pigs was not statistically associated with their faecal consistency, sex and developmental stage.

Genotyping of Entamoeba nuttalli strains from the wild rhesus macaques of Myanmar and comparison with those from the wild rhesus macaques of Nepal and China.

Entamoeba nuttalli found in macaques is phylogenetically the closest species to Entamoeba histolytica and is potentially pathogenic. In this study, the prevalence of Entamoeba infections was examined in wild rhesus macaques by examining 73 and 90 fecal samples collected from two sites, Popa Taung Kalat (PTK) and Pho Win Taung (PWT), in Myanmar. The positive rates of E. nuttalli detected using PCR were 49% and 31% in PTK and PWT, respectively, but no infections of E. histolytica and E. moshkovskii were found. Entamoeba dispar was detected in 6% of samples only from PWT. Positive rates of E. chattoni and E. coli were both 70% in PWT and 67% and 79% in PTK, respectively. Six E. nuttalli strains from PTK and eight from PWT were obtained in the culture with xenic medium and then, one and two strains, respectively, were axenized and finally cloned. The genotypic analysis of serine-rich protein genes revealed two genotypes each in both sites. The genotypes found in five of six strains from PTK were similar to those from the strains found in Nepal, whereas the remaining one from PTK and two from PWT were similar to those obtained from macaques in China. The sequence of the 18S rDNA of strains with these four genotypes was identical to that of the strains from China. Six loci of tRNA-linked short tandem repeats were analyzed for further genotyping of the strains. Although there were two types in locus A-L in PTK isolates, one of each type for PTK and PWT was found in the other loci, including locus A-L in PWT strains. These results demonstrated that the E. nuttalli strains from Myanmar are closer to the strains from macaques in China rather than those from macaques in Nepal.

Analysis of D-A locus of tRNA-linked short tandem repeats reveals transmission of Entamoeba histolytica and E. dispar among students in the Thai-Myanmar border region of northwest Thailand.

Intestinal parasitic infections, including those caused by Entamoeba species, are a persistent problem in rural areas of Thailand. The aims of this study were to identify pathogenic Entamoeba species and to analyze their genotypic diversity. Stool samples were collected from 1,233 students of three schools located in the Thai-Myanmar border region of Tak Province, Thailand. The prevalence of Entamoeba infection was measured by polymerase chain reaction (PCR) using species-specific primers. Thirty-one (2.5%) positive cases were detected for E. histolytica, 55 (4.5%) for E. dispar, and 271 (22.0%) for E. coli. Positive samples for E. histolytica and E. dispar were exclusively obtained from a few school classes, whereas E. coli was detected in all grades. No infections caused by E. moshkovskii, E. nuttalli, E. chattoni, and E. polecki were detected in the students studied. The D-A locus of tRNA-linked short tandem repeats was analyzed in samples of E. histolytica (n = 13) and E. dispar (n = 47) to investigate their diversity and potential modes of transmission. Five genotypes of E. histolytica and 13 genotypes of E. dispar were identified. Sequences of the D-A were divergent, but several unique genotypes were significantly prevalent in limited classes, indicating that intra-classroom transmission has occurred. As it was unlikely that infection would have been limited within school classes if the mode of transmission of E. histolytica and E. dispar had been through the intake of contaminated drinking water or food, these results suggest a direct or indirect person-to-person transmission mode within school classes. Positive rates for three Entamoeba species were 2-fold higher in students who had siblings in the schools than in those without siblings, suggesting that transmission occurred even at home due to heavy contacts among siblings.

Near-chromosome level genome assembly reveals ploidy diversity and plasticity in the intestinal protozoan parasite Entamoeba histolytica.

BACKGROUND: Amoebozoa is a eukaryotic supergroup composed of unicellular and multicellular amoebic protozoa (e.g. Acanthamoeba, Dictyostelium, and Entamoeba). They are model organisms for studies in cellular and evolutionary biology and are of medical and veterinary importance. Despite their importance, Amoebozoan genome organization and genetic diversity remain poorly studied due to a lack of high-quality reference genomes. The slime mold Dictyostelium discoideum is the only Amoebozoan species whose genome is available at the chromosome-level. RESULTS: Here, we provide a near-chromosome-level assembly of the Entamoeba histolytica genome, the second semi-completed Amoebozoan genome. The availability of this improved genome allowed us to discover inter-strain heterogeneity in ploidy at the near-chromosome or sub-chromosome level among 11 clinical isolates and the reference strain. Furthermore, we observed ploidy-independent regulation of gene expression, contrary to what is observed in other organisms, where RNA levels are affected by ploidy. CONCLUSIONS: Our findings offer new insights into Entamoeba chromosome organization, ploidy, transcriptional regulation, and inter-strain variation, which will help to further decipher observed spectrums of virulence, disease symptoms, and drug sensitivity of E. histolytica isolates.

Phylogenetic position of Nyctotherus teleacus isolated from a tortoise (Astrochelys radiata) and its electron microscopic features.

A commensal ciliate was isolated from the stool of a tortoise (Astrochelys radiata). The ciliate was classified as Nyctotherus teleacus, according to its basic morphological features. Electron microscopic observations using cultured N. teleacus (NictoT1 strain) revealed many spherical hydrogenosomes and methanogen-suspected bacteria, together with a characteristic triangular macronucleus containing many spherical chromosomes in the cytoplasm of NictoT1. The results of phylogenetic analysis showed that NictoT1 was included in the cluster of Nyctotheroides spp. (family Nyctotheridae). Nyctotheroides spp. commonly infest amphibians, which are taxonomically closely related to reptiles, including the tortoises evaluated in the present study.

Growth-promoting effects of the hydrogen-sulfide compounds produced by Desulfovibrio desulfuricans subsp. desulfuricans co-cultured with Escherichia coli (DH5α) on the growth of Entamoeba and Endolimax species isolates from swine.

Certain Desulfovibrio sp. (anaerobic sulfate-reducing bacteria) are indigenous to swine cecum and colon, which are also common habitats for parasitic amoebae such as Entamoeba polecki and Entamoeba suis. In this study, we evaluated the growth-promoting effects of D. desulfuricans co-cultured with Escherichia coli (DH5α) and its products [e.g., hydrogen sulfide (H2S) and certain iron-sulfide (FeS) compounds] using Robinson's medium, on the 4 amoeba isolates from swine-Entamoeba polecki subtype (ST)-1, E. polecki ST-3, Entamoeba suis, and Endolimax sp., and, consequently, a continuous culture system for these amoebae was established. However, this novel culture system was required to regulate the excess H2S dissolved in the medium by increasing air space as amoeba isolates thrive only in large air spaces (30-40%). The effects of air space and H2S and FeS compounds on the growth of E. polecki ST-1 (TDP-5) were determined. E. polecki ST-1 (TDP-5) thrived well in culture bottles with an air space of 30-40% (aerobic) (H2S: ~250-400 µmoles/L), but did not grow at all in an air space < 5% (microaerobic) ( H2S:~800 µmoles/L) and in anaerobic vessels (H2S: 20-30 µmoles/L). In both H2S-depleted and FeS compound-depleted conditions, the amoebae sp. could not thrive either. It was hypothesized that an appropriate concentration of H2S and FeS compounds might function as important physiologically active components of electron carriers such as FeS and ferredoxin.

Correction: Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches.

[This corrects the article DOI: 10.1371/journal.pone.0185233.].

Deep Venous Thrombosis in a Patient with Duplication of the Common Iliac Vein.

A congenital abnormality of the inferior vena cava is said to be an anatomical risk factor for venous thromboembolism. In this report, we present a case of a patient with a left duplicated common iliac vein who developed a venous thromboembolism following total abdominal hysterectomy and bilateral salpingo-oophorectomy. Only 2 items were risk factors for thromboembolism: age of ≥40 years and open surgery duration of ≥30 min; no congenital abnormalities of the inferior vena cava or thrombotic factors were observed. Thus, it was suspected that the duplicated common iliac vein could have caused the venous thromboembolism.

Efficacy of innovative polyglycolic acid sheet device delivery station system: a randomized prospective study.

BACKGROUND: Although there have been several reports of treating large post-endoscopic submucosal dissection (ESD) ulcers by covering them with a polyglycolic acid sheet (PGAs), this approach presents problems regarding PGAs delivery. This study assessed the usefulness of a device delivery station system (DDSS) to evaluate the appropriate and rapid PGAs coating method with DDSS. METHODS: Thirty-nine of 41 patients who were diagnosed with early gastric cancer over 20 mm in diameter and pathologically diagnosed with well-differentiated adenocarcinoma were randomly allocated to the following two groups according to delivery method: the conventional PGAs delivery group (C group) (n = 19) and the new DDSS group (DDSS group) (n = 20). The primary outcome was the coating area per minute in the C group and DDSS group (cm2/min). RESULTS: There were significant differences in the coating time (min), with values of 34.1 (15.0-60.7) vs. 16.85 (11.5-27.2) min for the C group and DDSS group, respectively (p = 0.001). There was also a significant difference in coating area per minute, with values of 0.261 (0.02-1.00) and 0.96 (0.173-2.06) cm2/min for the C group and DDSS group, respectively (p = 0.001). There were four cases of post-ESD bleeding (1-7 days after ESD) in the C group compared with 0 in the DDSS group, which represented a significant difference (p = 0.030). CONCLUSIONS: The DDSS was very useful for rapidly delivering and tightly attaching a PGAs to control post-ESD bleeding. TRIAL REGISTRATION: University Hospital Medical Network (UMIN) 000026377.

Intestinal Inflammation-Mediated Clearance of Amebic Parasites Is Dependent on IFN-γ.

Intestinal amebiasis is a major cause of diarrhea. However, research on host-amebae interactions has been hampered owing to a lack of appropriate animal models. Recently, a mouse model of intestinal amebiasis was established, and using it, we reported that Entamoeba moshkovskii colonized the intestine in a manner similar to that of the pathogenic Entamoeba histolytica In this study, we evaluated the protective mechanisms present against amebae using this model. CBA/J mice infected with E. histolytica had a persistent infection without apparent symptoms. In contrast, E. moshkovskii-infected mice rapidly expelled the ameba, which was associated with weight loss, diarrhea, and intestinal damage characterized by apoptosis of intestinal epithelial cells (IECs). Expression of NKG2D on intestinal intraepithelial lymphocytes (IELs) and IFN-γ-producing cells in Peyer's patches were significantly induced after infection with E. moshkovskii but not with E. histolytica IFN-γ-deficient mice infected with E. moshkovskii showed no obvious symptoms. Notably, none of these mice expelled E. moshkovskii, indicating that IFN-γ is responsible not only for intestinal symptoms but also for the expulsion of amebae. Furthermore, apoptosis of IECs and expression of NKG2D on IELs observed in E. moshkovskii-infected mice did not occur in the absence of IFN-γ. In vivo blocking of NKG2D in mice infected with E. moshkovskii enabled ameba to survive longer and remarkably reduced apoptotic IECs. Our results clearly demonstrate a novel protective mechanism exerted by IFN-γ against intestinal amebae, including induction of cytotoxicity of IELs toward IECs.

One-to-One Correspondence Locations of Resected Polyps after Endoscopic Resection Using Catcher Tagged Method: A Randomized Prospective Study.

BACKGROUND/AIMS: Multiple colorectal polyps with a diameter in the range of 10-19 mm are unable to be retrieved through a 3-mm endoscopic channel by the aspiration method. This study aims to assess the usefulness of Catcher Tag retrieval, which not only allows the accurate identification of the resected location but also enables the easiest retrieval in a short time without any special device. METHODS: One hundred thirty five patients (483 polyps) were diagnosed with colorectal neoplasm, and 64 patients (225 polyps) were enrolled and randomly allocated into the Net forceps group (NET) and the Catcher Tagged group (TAG). In TAG, 3 types of colored ring-threads were used to retrieve resected polyps. After local injection of natural saline, ring-threads were placed close to polyps. The primary outcome was the number of one-to-one correspondence locations (UMIN000020826). RESULTS: There was significant difference in one-to-one correspondence (p = 0.004). The average retrieval procedure time was 13.56 ± 3.47 (min) in NET and 3.55 ± 1.68 in TAG (p = 0.006). In NET, 1 polyp in each of 4 cases was lost during endoscopic mucosal resection and 2 polyps were lost in 1 case. In TAG, no polyp was lost (p = 0.016). CONCLUSION: The Catcher Tagged method is very useful for accurate one-to-one correspondence locations and pathological evaluation, and easy-to-retrieve multiple resected specimens.

Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches.

Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA) from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba.

Allovahlkampfia spelaea Causing Keratitis in Humans.

BACKGROUND: Free-living amoebae are present worldwide. They can survive in different environment causing human diseases in some instances. Acanthamoeba sp. is known for causing sight-threatening keratitis in humans. Free-living amoeba keratitis is more common in developing countries. Amoebae of family Vahlkampfiidae are rarely reported to cause such affections. A new genus, Allovahlkampfia spelaea was recently identified from caves with no data about pathogenicity in humans. We tried to identify the causative free-living amoeba in a case of keratitis in an Egyptian patient using morphological and molecular techniques. METHODS: Pathogenic amoebae were culture using monoxenic culture system. Identification through morphological features and 18S ribosomal RNA subunit DNA amplification and sequencing was done. Pathogenicity to laboratory rabbits and ability to produce keratitis were assessed experimentally. RESULTS: Allovahlkampfia spelaea was identified as a cause of human keratitis. Whole sequence of 18S ribosomal subunit DNA was sequenced and assembled. The Egyptian strain was closely related to SK1 strain isolated in Slovenia. The ability to induce keratitis was confirmed using animal model. CONCLUSIONS: This the first time to report Allovahlkampfia spelaea as a human pathogen. Combining both molecular and morphological identification is critical to correctly diagnose amoebae causing keratitis in humans. Use of different pairs of primers and sequencing amplified DNA is needed to prevent misdiagnosis.