T4 Genotype Research Articles

First report of free-living amoebae in sewage treatment plants in Porto Alegre, southern Brazil.

Free-living amoebae (FLA) are amphizoic protozoans with a cosmopolitan distribution. Some strains of species are associated with infections in humans. They feed on microorganisms by phagocytosis; however, some of these can become endocytobionts by resisting this process and taking shelter inside the amoeba. The whole world is experiencing increasing shortage of water, and sewage is being reused, so the study of this environment is important in public health context. The objective of this work was to identify FLA present in sewage treatment plants in Porto Alegre, Brazil. About 1 L samples were collected from eight stations (raw and treated sewage) in January, February, July, and August 2022. The samples were sown in monoxenic culture, and the isolated amoebae were subjected to morphological and molecular identification. Polymerase chain reaction results indicated the presence of the genus Acanthamoeba in 100% of the samples. Gene sequencing showed the presence of Acanthamoeba lenticulata and Acanthamoeba polyphaga - T5 and T4 genotypes - respectively, which are related to pathogenicity. The environment where the sewage is released can be used in recreational activities, exposing individuals to potential interactions with these amoebae and their potential endocytobionts, which may pose risks to public health.

Wheat Yield and Important Traits Influenced by Interaction of Potassium and Irrigation Levels Evaluated at Number of Locations in the Country by AMMI Analysis and Non-Parametric Measures

AMMI analysis of treatments consisted of levels of potassium with irrigations observed highly significant effects of locations, treatments, and T×L interactions for wheat yield. About 53.4% of the total variations in yield values was due to locations followed by 26.3% and 10.8% by treatments and interactions effects. Further analysis found 59.7% contributed by AMMI1 while 17.2% and 9.4% by AMMI2 and AMMI3 components for thousands grain weight as total of first two components cumulative to 76.9% of the total variation. The sums of squares for G×E signal and noise were 56.7% and 43.3% of interaction effects for grains per spike as the sum of squares of T×L signal was 2.58 times of treatments effects and IPC1 alone was 3.54 times the treatments effects. Measures ASV and ASV1 recommended T6, T5, T4 for wheat yield while measures utilized 81.6% of interaction sum of squares whereas MASV and MASV1 measures based on 98.4% identified T3, T5, T8, T4 treatments. Maximum average for thousands grains weight; GAI selected T8, T9, T6 whereas as per HM values treatments T5, T2, T8 would be more desirable. Grains per spike found the measures RPGV and HMRPGV settled for T8, T9, T5 treatments. Non parametric measures for yield observed Si1 selected T3, T2, T5 as opposed to T6, T4, T1 by Si2 values. T6, T4, T1 genotypes considered by Si3 Si4 measure considered T6, T4, T1 next Si5 for T6, T3, T4 and Si6 pointed towards T6, T4, T8 genotypes while Si7 favored T6, T1, T4 genotypes. Composite measures for thousands grains weight found NPi(1) for T3,T4,T7 while as per NPi(2) for T4,T3,T7, NPi(3) T4,T3,T2, NPi (4) found T4, T5, T7 as suitable treatment combinations. Multivariate hierarchical clustering as per Ward’s method for wheat yield observed first irrigation level with three potassium levels formed a cluster and other irrigation levels with potassium application remained in other one. At the first node of demarcation for thousands grains weight IPC5 exhibited MASV with MASV1, ASV1, IPC4, ASV, Si1 Si2 Si3 Si4 Si5 Si6 Si7 NPi(1), CV in one side and mean, GAI, PRVG, IPC1, HM, IPC1, NPi(2) NPi(3) NPi (4) on other side. The performance of treatments based on AMMI and non-parametric measures would be more meaningful for identification of suitable irrigation and potassium levels for wheat sustainable production.

Comparing cytotoxicity and efficacy of miltefosine and standard antimicrobial agents against Acanthamoeba trophozoites and cyst forms: An in vitro study

Acanthamoeba keratitis (AK) is an eye disease often occurring in contact lens wearers. AK treatment is prolonged and requires multiple drugs, which can lead to adverse effects. Our study aimed to compare the in vitro activities and safety of Miltefosine with that of conventional antimicrobial agents used to treat AK. Acanthamoeba castellanii genotype T4 was obtained from a patient with keratitis and subjected to in vitro susceptibility testing with various antimicrobial agents, including Chlorhexidine (CHX), Pentamidine isethionate (PI)Polyhexamethylene biguanide (PHMB), and Miltefosine to assess their efficacy against Acanthamoeba trophozoites and cyst. The cytotoxicity of the agents was evaluated in Vero cells, and their selectivity indexes (SI) were calculated. Chlorhexidine exhibited the highest amoebicidal activity with the highest selectivity index against the trophozoite and cyst, ranging from 1.17 to 8.35. The selectivity index of PHMB is slightly comparable to Chlorhexidine, exhibiting significant anti-Acanthamoeba activity.On the other hand, Pentamidine isethionate and Miltefosine displayed low SI among the compounds. Pentamidine isethionate was effective at high concentrations, which was toxic. Miltefosine exhibited the lowest cytotoxicity; nevertheless, due to the lowest anti-Acanthamoeba activity presented a low selectivity against the parasite. Further studies on more clinical samples and prolonged incubation time should be done to investigate the effectiveness and toxicity of drugs in both in vitro and in vivo conditions.

Molecular characterisation and potential pathogenicity analysis of Acanthamoeba isolated from recreational lakes in Peninsular Malaysia.

The present study aims to identify the Acanthamoeba genotypes and their pathogenic potential in three recreational lakes in Malaysia. Thirty water samples were collected by purposive sampling between June and July 2022. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. The samples were vacuum filtered through nitrate filter, cultured onto non-nutrient agar and observed microscopically for amoebic growth. DNAs from positive samples were extracted and made to react with polymerase chain reaction using specific primers. Physiological tolerance tests were performed for all Acanthamoeba-positive samples. The presence of Acanthamoeba was found in 26 of 30 water samples by PCR. The highest rate in lake waters contaminated with amoeba was in Biru Lake (100%), followed by Titiwangsa Lake (80%) and Shah Alam Lake (80%). ORP, water temperature, pH and DO were found to be significantly correlated with the presence of Acanthamoeba. The most common genotype was T4. Temperature- and osmo-tolerance tests showed that 8 (30.8%) of the genotypes T4, T9 and T11 were highly pathogenic. The presence of genotype T4 in habitats related to human activities supports the relevance of this amoeba as a potential public health concern.

Identification and quantification of Acanthamoeba spp. within seawater at four coastal lagoons on the east coast of Australia

Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic keratitis (AK), a painful and potentially blinding corneal infection. Major risk factors for AK have been linked to non-optimal contact lens hygiene practices and Acanthamoeba contamination of domestic and recreational water. This study investigated the incidence and seasonal variation of Acanthamoeba spp. within coastal lagoons located on the eastern coast of Australia and then examined the association between Acanthamoeba and water abiotic factors and bacterial species within the water.Water samples were collected from four intermittently closed and open lagoons (ICOLLs) (Wamberal, Terrigal, Avoca and Cockrone) every month between August 2019 to July 2020 except March and April. qPCR was used to target the Acanthamoeba 18S rRNA gene, validated by Sanger sequencing. Water abiotic factors were measured in situ using a multiprobe metre and 16S rRNA sequencing (V3-V4) was performed to characterise bacterial community composition. Network analysis was used to gauge putative associations between Acanthamoeba incidence and bacterial amplicon sequence variants (ASVs).Among 206 water samples analysed, 79 (38.3%) were Acanthamoeba positive and Acanthamoeba level was significantly higher in summer compared with winter, spring, or autumn (p = 0.008). More than 50% (23/45) water samples of Terrigal were positive for Acanthamoeba which is a highly urbanised area with extensive recreational activities while about 32% (16/49) samples were positive from Cockrone that is the least impacted lagoon by urban development. All sequenced strains belonged to the pathogenic genotype T4 clade except two which were of genotype clades T2 and T5. Water turbidity, temperature, intl1 gene concentration, and dissolved O2 were significantly associated with Acanthamoeba incidence (p < 0.05). The ASVs level of cyanobacteria, Pseudomonas spp., Candidatus spp., and marine bacteria of the Actinobacteria phylum and Acanthamoeba 18S rRNA genes were positively correlated (Pearson's r ≥ 0.14). The presence of Acanthamoeba spp. in all lagoons, except Wamberal, was associated with significant differences in the composition of bacterial communities (beta diversity).The results of this study suggest that coastal lagoons, particularly those in urbanised regions with extensive water recreational activities, may pose an elevated risk to human health due to the relatively high incidence of pathogenic Acanthamoeba in the summer. These findings underscore the importance of educating the public about the rare yet devastating impact of AK on vision and quality of life, highlighting the need for collaborative efforts between public health officials and educators to promote awareness and preventive measures, especially focusing lagoons residents and travellers.

Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30°C, 37°C, and 40°C) and osmotolerance (0.5M, 1M, and 1.5M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Identification and genotyping of Acanthamoeba spp. in the water resources of western Iran

BackgroundAcanthamoeba spp. is opportunistic amoeba that resides in water, soil, and air. Some pathogenic genotypes of the genus of Acanthamoeba can cause granulomatous amoebic encephalitis (GAE) in people with a defective immune system. The parasite can also cause Acanthamoeba keratitis (AK) among contact lens users. This study was conducted to isolate and identify the Acanthamoeba genotypes in water resources in Lorestan province, western Iran. MethodsCollected 72 water samples from surface and groundwater (springs and aqueducts) in Lorestan province. Samples were filtered and cultured in non-nutrient 1.5% agar medium covered with Escherichia coli (E. coli) at 25 °C. DNA extraction was done and the PCR reaction was performed to detect the Acanthamoeba spp. The positive PCR products were sequenced to determine the genotypes of Acanthamoeba. ResultsOut of 72 examined water samples, 23.61% were positive for Acanthamoeba sp. by PCR. From PCR-positive samples, 8 (47.05%) samples were T4 genotypes and others were other Acanthamoeba genotypes (T1-T23). Therefore, approximately half of the genotypes belong to the pathogenic T4 genotype. ConclusionsThe water examined samples in western provinces of Iran have the potential risk factor for public health. Therefore, the efforts of healthcare providers are needed to identify, train, and prevention from human infections.

Molecular identification of Acanthamoeba spp., Balamuthia mandrillaris and Naegleria fowleri in soil samples using quantitative real-time PCR assay in Turkey; Hidden danger in the soil!

Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri are pathogenic free-living amoeba (FLA) and are commonly found in the environment, particularly soil. This pathogenic FLA causes central nervous system-affecting granulomatous amebic encephalitis (GAE) or primary amebic meningoencephalitis (PAM) and can also cause keratitis and skin infections. In the present study, we aimed to determine the quantitative concentration of Acanthamoeba spp., B. mandrillaris, and N. fowleri in soil samples collected from places where human contact is high by using a qPCR assay in Izmir, Turkey. A total of 45.71% (n = 16) of Acanthamoeba spp., 20% (n = 7) of B. mandrillaris, and 17.4% (n = 6) of N. fowleri were detected in five different soil sources by the qPCR assay. The quantitative concentration of Acanthamoeba spp., B. mandrillaris, and N. fowleri in various soil sources was calculated at 10 × 105 - 6 × 102, 47 × 104 to 39 × 103, and 9 × 103 – 8 × 102 plasmid copies/gr, respectively. While the highest quantitative concentration of Acanthamoeba spp. and B. mandrillaris was determined in garden soil samples, N. fowleri was detected in potting soil samples. Three different genotypes T2 (18.75%), T4 (56.25%), and T5 (25%) were identified from Acanthamoeba-positive soil samples. Acanthamoeba T4 genotype was the most frequently detected genotype from soil samples and is also the most common genotype to cause infection in humans and animals. To the best of our knowledge, the present study is the first study to identify genotype T5 in soil samples from Turkey. In conclusion, people and especially children should be aware of the hidden danger in the garden and potting soil samples that come into contact most frequently. Public health awareness should be raised about human infections that may be encountered due to contact with the soil. Public health specialists should raise awareness about this hidden danger in soil.

Phenotypic performance of F3 progenies from crossbreeding of Sigupai and IRBB27

Sigupai is one of Aceh’s indigenous rice varieties originating from the Southwestern region of Aceh. This variety has advantages in the form of fluffier and good rice cooking performance and fragrant aroma, easy maintenance, and minimal fertilizer requirements. This variety has agronomic characteristics that need to be improved such as a harvesting age of up to six months with high plant stature that makes it easy to fall. A series of breeding processes and analyses have been conducted to improve the performance of this variety by crossing it with IRBB27. The phenotypic characters of the F3 progenies (genotypes T11, T22, and T25) were carried out. The purpose of this study was to identify the phenotypic character of the F3 progenies belonging to (genotypes T11, T22, and T25 ). These genotypes originated from the selected plant in F2 progenies by crossbreed between Sigupai and IRBB27. This research was carried out at the Experimental Farm and Genetics and Plant Breeding Laboratory, Faculty of Agriculture, Syiah Kuala University, Darussalam Banda Aceh, from December 2021 to June 2022. The planting medium used was a mixture of soil and manure with a ratio of 2:1. The seeds planted consisted of F3 progenies (genotype T11, T22, T25, as well as their parents Sigupai, and IRBB27. The results showed that there were 3 plants in the F3 Sg/IRBB27 prospective to be developed. These plants originated from genotype T11 (plant no. 6), genotype T22 (plant no. 6), and genotype T25 (plant no. 2). These prospective progenies have medium harvesting ages ranging from 122-132 DAS, and shorter plant heights compared to their mother parent ‘Sigupai’ (121-127 cm), with their products ranging from 5.13-7.40 tons ha−1.

Successive Acanthamoeba Corneal Isolates Identified in Poland Monitored in Terms of In Vitro Dynamics.

Amoebae of the genus Acanthamoeba cause a sight-threatening infection called Acanthamoeba keratitis. It is considered a rare disease in humans but poses an increasing threat to public health worldwide, including in Poland. We present successive isolates from serious keratitis preliminary examined in terms of the identification and monitoring of, among others, the in vitro dynamics of the detected strains. Clinical and combined laboratory methods were applied; causative agents of the keratitis were identified at the cellular and molecular levels; isolates were cultivated in an axenic liquid medium and regularly monitored. In a phase-contrast microscope, Acanthamoeba sp. cysts and live trophozoites from corneal samples and in vitro cultures were assessed on the cellular level. Some isolates that were tested at the molecular level were found to correspond to A. mauritanensis, A. culbertsoni, A. castellanii, genotype T4. There was variability in the amoebic strain dynamics; high viability was expressed as trofozoites' long duration ability to intense multiply. Some strains from keratitis under diagnosis verification and dynamics assessment showed enough adaptive capability to grow in an axenic medium, allowing them to exhibit significant thermal tolerance. In vitro monitoring that was suitable for verifying in vivo examinations, in particular, was useful to detect the strong viability and pathogenic potential of successive Acanthamoeba strains with a long duration of high dynamics.

Selected Gut Bacteria from Water Monitor Lizard Exhibit Effects against Pathogenic Acanthamoeba castellanii Belonging to the T4 Genotype.

Water monitor lizards (WMLs) reside in unhygienic and challenging ecological surroundings and are routinely exposed to various pathogenic microorganisms. It is possible that their gut microbiota produces substances to counter microbial infections. Here we determine whether selected gut bacteria of water monitor lizards (WMLs) possess anti-amoebic properties using Acanthamoeba castellanii of the T4 genotype. Conditioned media (CM) were prepared from bacteria isolated from WML. The CM were tested using amoebicidal, adhesion, encystation, excystation, cell cytotoxicity and amoeba-mediated host cell cytotoxicity assays in vitro. Amoebicidal assays revealed that CM exhibited anti-amoebic effects. CM inhibited both excystation and encystation in A. castellanii. CM inhibited amoebae binding to and cytotoxicity of host cells. In contrast, CM alone showed limited toxic effects against human cells in vitro. Mass spectrometry revealed several antimicrobials, anticancer, neurotransmitters, anti-depressant and other metabolites with biological functions. Overall, these findings imply that bacteria from unusual places, such as WML gut, produce molecules with anti-acanthamoebic capabilities.

Molecular evidence for a new lineage within the Acanthamoeba T4 genotype.

Acanthamoeba is a widespread free-living amoeba capable of causing serious infections in humans and other animals, such as amoebic keratitis, disseminated infections, and fatal encephalitis. Strain identification is usually based on 18S rDNA sequencing, which allows the distinction of over twenty genotypes. Most sequences from environmental and clinical samples belong to the T4 genotype, which can be divided into seven subtypes, T4A to T4G, and by a nearly similar grouping of mitochondrial sequences into T4a to T4j subtypes. The co-clustering of nuclear and mitochondrial groups can be very useful for a better identification of lineages within the very rich T4 genotype. In this study, we provided molecular phylogenetic evidence for the delineation of a new nuclear subtype, hereafter labelled T4H, and its co-clustering with the mitochondrial T4j subtype. At least three cases of amoebic keratitis are due to strains belonging to this new group, present mainly in fresh water and detected in various countries (France, Iran, India and China).

Genotyping of Environmental Isolates of Acanthamoeba in Hamadan, West of Iran.

Different genotypes of Acanthamoeba have been abundantly isolated in environmental samples such as water, soil, and dust, as well as in different hospital departments and eyewash stations. This protozoan is a potential hazard for immunocompromised patients and contact lens wearers. The aim of the present study was isolation and genotyping of environmental and corneal isolates of Acanthamoeba in Hamadan, west of Iran. During 2018-2020, a total of 104 environmental samples including, water, soil, and dust and 16 corneal scraping samples were collected and investigated for the presence of Acanthamoeba using morphological and molecular identification tools. Genotypes were determined using sequence analysis of the diagnostic fragment 3 (DF3) from Acanthamoeba-specific amplimer S1 (ASA.S1) gene. Phylogenetic tree was constructed with the MEGA7 software using Neighbor-Joining method. The presence of Acanthamoeba spp. was determined in 87.5% of water, 53.1% of soil, and 25% of dust samples. From 30 dust samples collected from eight wards of three hospitals, 7 (23.3%) were contaminated with Acanthamoeba. Sequencing analysis of environmental samples revealed that the T4 genotype was the most prevalent (92.6%) one. Genotypes T2 (1.9%), T2/T6 (1.9%), and mixed T4 and T2/T6 (3.7%) were also identified in environmental samples. Acanthamoeba was seen in none of the examined corneal scraping samples from patients with suspected keratitis. The widespread occurrence of this potentially pathogenic amoeba in most hospital wards and environmental resources and areas of the region highlights a strong need to increase awareness regarding this ubiquitous amoeba among susceptible individuals, such as immunocompromised patients and contact lens wearers.

Assessing Drought Tolerance of Newly Developed Tissue-Cultured Canola Genotypes under Varying Irrigation Regimes

Drought is a major abiotic stress that greatly affects canola growth, production, and quality. Moreover, water scarcity is projected to be more severe and frequent as a result of climate change, in particular in arid environments. Thereupon, developing drought-tolerant and high-yielding canola genotypes has become more critical to sustaining its production and ensuring global food security with the continuing population growth. In the present study, ten canola genotypes comprising six developed tissue-cultured canola genotypes, two exotic genotypes, and two commercial cultivars were evaluated under four irrigation regimes. The applied irrigation regimes were well-watered (100% crop evapotranspiration, ETc), mild drought (80% ETc), moderate drought (60% ETc), and severe drought (40% ETc) conditions. Drought-stress treatments (80, 60, and 40% ETc) gradually reduced the chlorophyll content, relative water content, flowering time, days to maturity, plant height, number of pods, number of branches, seed yield, and oil percentage, and increased proline, phenolic, anthocyanin, and glycine betaine contents. The evaluated genotypes exhibited varied responses to drought-stress conditions. The developed tissue-cultured genotypes T2, T3, and T1, as well as exotic genotype Torpe, possessed the highest performance in all evaluated parameters and surpassed the other tested genotypes under water-deficit conditions. Overall, our findings elicited the superiority of certain newly developed tissue-cultured genotypes and exotic ones compared with commercial cultivars, which could be exploited in canola breeding under water-deficit conditions.

Acanthamoeba castellanii Genotype T4: Inhibition of Proteases Activity and Cytopathic Effect by Bovine Apo-Lactoferrin.

Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity.

Molecular detection and genotype analysis of Kudoa septempunctata from food poisoning outbreaks in Korea.

Concerns about foodborne illnesses caused by Kudoa septempunctata are steadily growing, but reports of K. septempunctata in clinical and food specimens related to food poisoning in Korea are limited. This study aimed to genetically identify K. septempunctata in patients with acute diarrhea and in clinical and food samples related to food poisoning caused by sashimi consumption. Both real-time and nested polymerase chain reaction assays were performed to detect K. septempunctata 18S and 28S rDNA genes in the stools of 348 patients with acute diarrhea, 11 samples (6 stool and 5 rectal swab samples) from patients with food poisoning, and 2 raw Paralichthys olivaceus samples collected from a restaurant where a food poisoning incident occurred. K. septempunctata was identified in 5 clinical specimens (4 stools and 1 rectal swab) and 1 P. olivaceus sashimi sample. All detected K. septempunctata were of genotype ST3. This is the first study to identify K. septempunctata in both patients and food samples with epidemiological relevance in Korea, providing evidence that it is a pathogen that causes food poisoning. Also, this is the first study to confirm the presence of K. septempunctata genes in rectal swabs. Despite continuing suspected occurrences of Kudoa foodborne outbreaks, the rate of identification of K. septempunctata is very low. One reason for this is the limitation in obtaining stool and vomit samples for the diagnosis of Kudoa infection. We strongly suggest the inclusion of rectal swabs among the diagnostic specimens for Kudoa food poisoning.

Sub-Genotyping of Acanthamoeba T4 Complex: Experience from North India

The Acanthamoeba genus comprises the free-living amoebae that are ubiquitously present as opportunistic pathogens. They cause serious human diseases—for instance, Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), cutaneous acanthamoebiasis and disseminated infections. The traditional method for classifying Acanthamoeba was based on the morphological examination of cysts. However, this method was less consistent as the morphology of cysts changes with the culture conditions. After the advent of molecular techniques, genotyping is considered an essential tool in accurately identifying Acanthamoeba at the species level and is further helpful in classification up to the sub-genotype level. The most recommended and currently used methods for Acanthamoeba genotyping are 18S and 16S rDNA gene sequencing. Based on these two genes, Acanthamoeba is classified into 23 genotypes. Out of these, it is the T4 genotype that is most commonly associated with clinical disease and isolation from environmental samples. The T4 genotype contains more than ten species within it. Differences in geographical distribution, virulence, pathogenesis and drug susceptibility profile have been observed among different genotypes. However, whether such differences exist within sub-genotypes/species under T4 is yet unknown. In the present study, 11 Acanthamoeba isolates, which were already characterized as the T4 genotype by the hypervariable region of diagnostic fragment 3 (DF3) of the 18S rDNA, were sub-genotyped using the 16S rDNA mitochondrial sequence. Nine of these were isolated from patients with AK and two from water samples. Phylogenetic analysis of these isolates attributed them to four sub-genotypes (T4a (n = 6), T4b (n = 1), T4Neff (n = 2) and T4d (n = 2)). The study highlights the potential use of 16S in the sub-genotyping of Acanthamoeba T4. The 16S rDNA sequences of two isolates, one from an Acanthamoebic keratitis (AK) patient and one environmental, were found to group with A. mauritaniensis (T4d). This group was believed to be a non-pathogenic environmental Acanthamoeba and the identification of the AK isolate may be confirmed by whole-genome sequencing.

Acanthamoeba keratitis: Molecular typing of Acanthamoeba species directly from ocular tissue

This report explores the molecular profiling of Acanthamoeba spp. from individuals in the UK suffering from a debilitating, sight-threatening disease of the cornea known as Acanthamoeba keratitis (AK). Seventy ocular samples from individuals undergoing investigations for AK were sent to the Scottish Microbiology Reference Laboratories (SMiRL), Glasgow during 2017–2019, and subjected to DNA extraction followed by in-depth molecular typing using a nested PCR/bi-directional sequencing approach. Of the 70 samples tested, 40 were PCR-positive. Of these, 32 were successfully sequenced and assigned to two of 23 existing genotypes termed T1 to T23. Molecular profiling of the 32 samples highlighted two genotypes, namely T3 (n = 3) and T4 (n = 29). For those 29 samples identified as the T4 genotype, a sub-genotype (T4A-T4H) was recorded: T4A (n = 18); T4B (n = 5); T4C (n = 1); T4E (n = 4); and T4F (n = 1). This study highlights that the T4 genotype and T4A subtype are the predominant molecular variants to cause ocular disease in the UK. Gaining in-depth information on the molecular profiling of Acanthamoeba spp. is essential to increase our understanding of the source(s) of infection, transmission pathways, and potential associations with clinical outcomes for this rare, yet potentially debilitating ocular disease.

Detection of Acanthamoeba spp. in groundwater sources in a rural area in the Philippines.

Research on free-living amoebae (FLA) and its public health implication as an etiologic agent of parasitic infection in humans has recently gained traction in the Philippines. This study aimed to identify potential FLAs in collected groundwater samples from Masinloc, Zambales, Philippines. Fifty-four (54) water samples were collected in 250-mL sterile polyethylene containers by purposive sampling from selected groundwater sources in six (6) barangays of Masinloc. The samples were vacuum filtered through a 1.2-μm pore glass microfiber filter, cultured onto non-nutrient agar (NNA) lawned with Escherichia coli, and observed microscopically for amoebic growth for 14 days using light microscopy. Amoebic growth was observed in 11.1% (6/54) of water samples. DNAs from positive samples were extracted and were made to react with polymerase chain reaction using Acanthamoeba-specific JDP1 (5'-GGCCCAGATCGTTTACCGTGAA-3') and JDP2 (5'-TCTCACAAGCTGCTAGGGAGTCA-3') primers, and universal primer Euk A (5'-AACCTGGTTGATCCTGCCAGT-3') and Euk B (5'-TGATCCTTCTGCAGGTTCACCTAC-3'). The presence of Acanthamoeba genotypes T4, T7, and T11 was confirmed using molecular and phylogenetic analysis. Our results confirmed that groundwater sources from two of six sampling sites (33.3%) in Masinloc, Zambales, were contaminated with potentially pathogenic FLAs. Proper identification of risk factors that may cause contamination consequently leads to the implementation of programs that will prevent future infections.

Nucleotide Sequence Assessment of Four ORFs of Citrus Tristeza Virus: Evidence of Recombination

Citrus Tristeza Virus (CTV), usually occurs in nature as a mixture of genotypes. Six naturally infected citrus (Citrus sinensis) trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt (Sharqia, Qalyubia and Garbia). In this study, RT-PCR, Single-Strand Conformation Polymorphism (SSCP) and nucleotide sequence analysis were used for four independent CTV genomic regions (p65, p18, p20, and p23) to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates. RTPCR products (650 bp) for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing. SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns. Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7% with T36 isolate from USA, Florida. Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate (T36 isolate group), suggesting that they may have originated from closely related ancestors. Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang, p18, p20 and p65, amplified from isolate A3, Sharqia governorate, revealed that the p18, p65, and p20 genes were related to the T3-KB isolate from South Africa with 99%–100% sequence homology. Phylogenetic relationship analysis for p65, p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group. The recombination analysis identified three of six isolates from Sharqia, and Garbia as potential recombinant for p23 gene. The isolates T36 and T3 were identified as major donors for recombination events in isolate A3. Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event. The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.