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Acanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

We analyzed 2 batches of environmental samples after a microsporidial keratoconjunctivitis outbreak in Taiwan. Results indicated a transmission route from a parking lot to a foot washing pool to a swimming pool and suggested that accumulation of mud in the foot washing pool during the rainy season might be a risk factor.

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-induced gene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment., Author summary Comparative genomic analysis of pathogens is used to identify genes associated with disease manifestations. In the present study, this approach was used to identify genes, present in a strain of Entamoeba histolytica isolated from dysenteric patients, but absent in strains isolated from asymptomatic infected individuals. Strain KU27 is an “avirulent” strain, or less virulent as compared to the virulence of other strains. KU27 was isolated from an asymptomatic infected individual, and is incapable of producing abscesses in a hamster liver abscess model. A previous genotyping study, using tRNA-linked short tandem repeats as markers, also identified this Japanese strain as a genetically distinct low-virulence strain. We conducted a comparative genomic analysis of strain KU27 against virulent strain of E. histolytica. Our analysis identified an AIG1 gene as a potential determinant of virulence. Further, we demonstrated that the gene product is involved in the formation of protrusions on the plasma membrane and adherence to host cells. To the best of our knowledge, this is the first study to identify a virulence-associated gene by comparative genomics. Here, we demonstrate the causal connection of the identified gene with virulence by the analysis of cell morphology, adhesion competence, and distribution among clinical isolates.

Vittaforma corneae belongs to microsporidia, which include over 1500 species of opportunistic obligate intracellular fungi infecting almost all known animal taxa. Although outbreaks of ocular infections caused by waterborne V. corneae have been reported in recent years, little is known about the occurrence of this pathogen in aquatic environments. In this study, 50 water samples from rivers and reservoirs around Taiwan in two seasons were analyzed to explore the presence of this pathogen in natural aquatic environments. A high detection rate of Vittaforma-like amplicons (94%; 47/50) was observed in the water samples when examined by nested PCR with primer pairs specific to the small ribosomal subunit (SSU) rRNA gene. After electrophoresis, many lanes showed multiband patterns with expected molecular weights. After confirmation by DNA sequencing and by sequence alignment in the NCBI database, we identified a variety of Vittaforma-like microsporidia with weak sequence similarity, with approximately 85% identity to V. corneae, thus indicating high diversity of microsporidia in aquatic environments. Phylogenetic analysis showed clear-cut microsporidian clade classification and indicated that the most Vittaforma-like microsporidia in this study belong to clade IV and cluster into four major groups. The first group is similar to the microsporidia associated with ocular microsporidiosis. The second group is associated with the diarrheal pathogens, whereas the third and fourth groups are a novel group and a zoonotic group, respectively. This study provides abundant sequencing information, which will be useful for future molecular biological studies on microsporidia. Because microsporidia are important pathogens of animals and humans, it is urgently necessary to determine via a survey whether there are species with potential threats that have not yet been revealed.

Objectives We aimed to investigate the etiology of pulmonary complications of human immunodeficiency virus-(HIV)-1-infected patients in Taiwan in the era of combination antiretroviral therapy (cART). Methods From July 2009 to March 2012, a prospective observational study was conducted to identify the etiology of pulmonary complications in HIV-1-infected patients who sought HIV care at a university hospital in Taiwan. A stepwise diagnostic approach was adopted, which included radiography, serology, microbiology, bronchoscopy or video-assisted thoracoscopic surgery, and polymerase chain reaction assays for cytomegalovirus and Pneumocystis jirovecii . Results During the study period, a total of 203 episodes of pulmonary complications that occurred in 190 patients with a mean CD4 count of 123 × 10 6 cells/L were analyzed. Thirty-eight episodes (18.7%) occurred in patients with a CD4 count >200 × 10 6 cells/L, 71 (35.0%) between 50 and 200 × 10 6 cells/L, and 94 (46.3%) 6 cells/L. Pneumocystis pneumonia accounted for more than half of the complications in patients with a CD4 count 6 cells/L. In patients with a CD4 count >200 × 10 6 cells/L, the etiology of pulmonary complications was diverse, with bacterial infections (47.4%) being the most common, followed by tuberculosis (15.8%) and lung edema (13.2%). Pneumocystosis and cytomegalovirus pneumonitis were seen mostly or exclusively in patients with a CD4 count 6 cells/L and were the leading causes of interstitial pneumonitis. On the other hand, empyema, legionellosis, and lung edema were more commonly seen in patients with a CD4 count >200 × 10 6 cells/L. Conclusions The etiology of pulmonary complications in HIV-1-infected patients was diverse and varied with the categories of CD4 counts. Pneumocystosis remained the leading cause of pulmonary complications in patients with lower CD4 counts in Taiwan in the cART era.

Naegleria fowleri can cause primary amoebic meningoencephalitis, a rapidly developing and highly lethal infectious disease. The first confirmed case of primary amoebic meningoencephalitis in Taiwan was reported in November 2011, in which the patient visited a thermal spring recreational area 1 week prior to hospitalisation. Water sampling was performed to verify the presence of Naegleria at the facility. According to our results, 32% and 20% of recreational water samples were contaminated with Naegleria spp. and Acanthamoeba spp., respectively. The genotypes of Naegleria identified at the hot spring included N. fowleri, Naegleria australiensis and Naegleria lovaniensis. Using PCR, it was determined that the strain of N. fowleri in one sample possessed the same genotype 2 as the clinical isolate. Thus, the thermal spring was suggested to be the likely source of infection. This is the first known instance of simultaneously isolating N. fowleri from both a patient as well as from a hot spring in Taiwan. Following this initial study, the pools at the thermal spring recreational area were drained, scrubbed and disinfected, and a follow-up study was performed 1 month later. Naegleria fowleri was not detected in follow-up testing; however, other Naegleria spp. were identified. We postulate that the biofilm in the waterlines may have provided a reservoir for free-living amoebae. The presence/absence of Acanthamoeba and Naegleria spp. did not differ significantly with any measured parameters related to water quality; however, a high percentage of the thermal water pool samples were contaminated with Naegleria or Acanthamoeba. Thus, amoebic contamination may present a serious threat to the health of humans who engage in leisure activities at thermal springs.

The standard method for detecting Salmonella generally analyzes food or fecal samples. Salmonella often occur in relatively low concentrations in environmental waters. Therefore, some form of concentration and proliferation may be needed. This study compares three Salmonella analysis methods and develops a new Salmonella detection procedure for use in environmental water samples. The new procedure for Salmonella detection include water concentration, nutrient broth enrichment, selection of Salmonella containing broth by PCR, isolation of Salmonella strains by selective culture plates, detection of possible Salmonella isolate by PCR, and biochemical testing. Serological assay and pulsed-field gel electrophoresis (PFGE) can be used to identify Salmonella serotype and genotype, respectively. This study analyzed 116 raw water samples taken from 18 water plants and belonging to 5 watersheds. Of these 116, 10 water samples (8.6%) taken from 7 water plants and belonging to 4 watersheds were positive for a Salmonella-specific polymerase chain reaction targeting the invA gene. Guided by serological assay results, this study identified 7 cultured Salmonella isolates as Salmonella enterica serovar: Alnaby, Enteritidis, Houten, Montevideo, Newport, Paratyphi B var. Java, and Victoria. These seven Salmonella serovars were identified in clinical cases for the same geographical areas, but only one of them was 100% homologous with clinical cases in the PFGE pattern.

The protistan genus Acanthamoeba is a free-living amoeba existing in various environments. Within this protistan genus, there are some species recognized as potential human pathogens, which may cause granulomatous amoebic encephalitis, Acanthamoeba keratitis and chronic granulomatous lesions of the skin. In this study, 211 water samples were collected from two watersheds in southern Taiwan. We detected Acanthamoeba based on the PCR amplification with a genus-specific primer pair and investigation of Acanthamoeba in Puzih River and Kaoping River in southern Taiwan. Acanthamoeba species were detected in 34 (16.1%) samples. The presence or absence of Acanthamoeba within the water samples showed significant difference with the levels of water temperature and total coliforms. The most frequently identified Acanthamoeba genotype was T4 (n = 19), followed by T5 (n = 8), and then T15 (n = 3). Genotype T6, T7/T8, T11 and T12 were all detected once. Genotype T4, T5, T6, T11 and T15 of Acanthamoeba are responsible for Acanthamoeba keratitis and should be considered a potential health threat associated with human activities in environmental surface water watersheds.

A matched case-control study was used to determine pathogens and risk factors associated with gastroenteritis in a Taipei Emergency Department. Viruses (40.0%) were the leading cause of gastroenteritis, with noroviruses the most prevalent (33.2%). Bacteria were found in 26.0% of all cases, mostly suspected diarrheagenic E. coli (22.2%), followed by Salmonella spp. (5.4%) and Vibrio parahaemolyticus (4.2%). Giardia lamblia was identified in 16.4% of all cases. Statistical significance was noted for seven risk factors: taking antacids before gastroenteritis (OR = 3.91; 95% CI, 2.13, 7.15), other household members with gastroenteritis (OR = 5.18; 95% CI, 2.09, 12.85), attending a banquet (OR = 1.93; 95% CI, 1.25, 2.98), eating out (OR = 2.35; 95% CI, 1.30, 4.23), drinking bottled water (OR = 1.72; 95% CI, 1.07, 2.75), eating honey peaches (OR = 3.26; 95% CI, 1.24, 8.58), and eating raw oysters (OR = 3.24; 95% CI, 1.02, 10.28). Eating out was identified as the highest risk behavior, as measured by population attributable risk fraction (PAR) (50.9%). Respective PAR values for drinking bottled water, attending a banquet and taking antacids before illness were 19.7%, 19.6% and 17.6%. Of these, additional research on bottled water appears to be the highest priority, because this is the first time it has been identified as a risk factor for gastroenteritis.

We characterized the complete nucleic and amino acid sequences of the Plasmodium inui circumsporozoite protein (Pincsp) gene and analyzed nucleotide diversity across the entire Pincsp gene by using 7 field isolates and strains Taiwan I and II obtained from Formosan macaques (Macaca cyclopis) in Taiwan. The length of the circumsporozoite protein ( CSP ) gene ranged from 1077 to 1125 bp. Size polymorphisms were due to variations in the number of tandem repeat units. The non-repetitive (NR) region exhibited high homology (99.1 ∼ 100 and 98.7 ∼ 100% at the nucleotide and amino acid levels, respectively) and was conserved among the variants (nucleotide diversities, π, of the 5'NR and 3'NR regions were 0.00364 and 0.00392, respectively). In the central repetitive (CR) region, we decomposed the sequences into 2 kinds of repeating amino acid motifs, i.e., a repeat unit R1, PA(P/A)(P/A)A(E)GG (n = 11-13), and a following repeat unit R2: P(A/G)(A/P/G)(P/Q)AQ(N/K) (n = 9-10). Analyzing these repeat sequences showed evidence of 3 genetic mechanisms for generating variations in the repeats of the Pincsp gene, i.e., point mutation, insertion, and recombination. These findings suggest that polymorphisms in the Pincsp gene are essentially limited to the CR region, which showed much greater variability in terms of length, number of repeats, and sequence.

Nelson medium and modified PYNFH medium were used for the axenic culture of the Naegleria fowleri clinical strain LDL to compare the effects of different temperatures on the organism's growth. In addition, Nelson medium supplemented with 1% peptone (N + pep) and modified PYNFH medium without peptone (PYNFH − pep), without yeast extract (PYNFH − yext), without folic acid (PYNFH − folac), and without yeast nucleic acid (PYNFH − yna) were used in order to compare the various effects of these medium components. In general, N. fowleri grew best at 37 C. The highest trophozoite densities per 10,000 μm2 were observed when N + pep and PYNFH − yext were used. At 25, 37, and 43 C, the growth density profile values were 50.5 ± 6.36 vs. 58 ± 1.41; 2,550 ± 494.97 vs. 2,100 ± 141.42; and 1,735 ± 21.21 vs. 1,800 ± 14.14, respectively. On the other hand, PYNFH − pep gave the lowest growth with its highest cell densities being 9 ± 1.41 at 25 C, 108 ± 7.07 at 37 C, and 169 ± 15.55 at 43 C. When the various medium ...

A single-tube multiprobe real-time PCR assay for simultaneous detection of Entamoeba histolytica and Entamoeba dispar was developed. One primer pair with 2 species-specific probes was designed based on new SSU RNA regions of the ribosomal DNA-containing episome. The sensitivity is 1 parasite per milliliter of feces and thus superior to the conventional nested PCR and comparable to other published real-time PCR protocols. The applicability for clinical diagnosis was validated with 218 stool specimens from patients. A total of 51 E. histolytica and 39 E. dispar positive samples was detected by the multiprobe real-time PCR compared to 39 and 22 by routine nested PCR diagnosis. The detection rate of Entamoeba species for the multiprobe real-time PCR assays was significantly higher than the nested PCR (40.8% vs. 28.0%, P0.01). The test did not show cross reactivity with DNA from Entamoeba moshkovskii, Giardia lamblia , Cryptosporidium sp., Escherichia coli , or other nonpathogenic enteric parasites. The multiprobe real-time PCR assay is simple and rapid and has high specificity and sensitivity. The assay could streamline the laboratory diagnosis procedure and facilitate epidemiological investigation.

Aims: Isolation and characterization of the clinically relevant amphizoic amoebas in vegetated farmlands, which may present a risk to farmers’ health. Methods and Results: Acanthamoeba species was isolated and characterized via morphological and molecular means in the rice field where the patient was exposed to rice paddy water which most probably was the point of infection. An Acanthamoeba sp. abundant in the rice field was identified. Genotyping showed the strain to be genotype T4, which was identical to the amoebic parasite found in patient’s cerebrospinal fluid. During the course of the study, three nonpathogenic free-living amoeba species were also isolated and characterized for the first time in Taiwan. Conclusions: This study successfully located a possible source of granulomatous amoebic encephalitis in a patient and provided the first evidence that Acanthamoeba genotype T4 may be a potential pathogen in Taiwan. Significance and Impact of the Study: The integration of field survey, clinical data and morphological and genetic examination represents a sound strategy for investigation of the possible role of free-living amoebae in causing human diseases. Future work should include investigating the potential contributory role of other nonpathogenic free-living protozoa in disease of livestock or even human.

Both Shigella spp. and enteroinvasive Escherichia coli (EIEC) are important human pathogens that are responsible for the majority of cases of endemic bacillary dysentery. However, they are difficult to identify and differentiate by biochemical tests or molecular methods alone. In this study, we developed a procedure to detect Shigella spp. and EIEC from environmental water samples using membrane filtration followed by nutrient broth enrichment, isolation using selective culture plates, and identification of the invasion plasmid antigen H (ipaH) gene by PCR amplification and DNA sequencing. Finally, we used a biochemical test and a serological assay to differentiate between Shigella and EIEC. Among the 93 water samples from nine reservoirs and one watershed, 76 (81.7%) water samples of culture plates had candidate colonies of Shigella and EIEC and 5 water samples were positive (5.4%) for a Shigella- and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the molecular method, the biochemical test, and the serological assay, 11 ipaH gene-positive isolates from 5 water samples were all identified as EIEC.

A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica , one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis.

Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal2, respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymor- phism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.

Background/Purpose Enterotoxigenic Bacteroides fragilis (ETBF) and toxin-encoding Clostridium difficile (TXCD) are associated with gastroenteritis. Routine anaerobic blood culture for recovery of these anaerobic pathogens is not used for the detection of their toxins, especially for toxin-variant TXCD. The aim of this study was to investigate the prevalence and risk factors of the genotypes of these anaerobes in patients with acute diarrheal illnesses. Methods The data and samples of 513 patients with gastroenteritis were collected in a Taipei emergency department from March 1, 2006 to December 31, 2009. Nonenterotoxigenic B. fragilis (NTBF) and ETBF and the toxin genotypes of TXCD were detected by molecular methods. Results The prevalence rates of NTBF, ETBF, and TXCD infections were 33.14%, 1.56%, and 2.34%, respectively. ETBF infections often occurred in the elderly (average age = 67.13 years) and during the cold, dry winters. TXCD infections were widely distributed in age and often occurred in the warm, wet springs and summers. The symptoms of ETBF-infected patients were significantly more severe than those of NTBF-infected patients. Conclusion This study identified and analyzed the prevalence, risk factors, and clinical presentations of these anaerobic infections. Future epidemiologic and clinical studies are needed to understand the role of ETBF and TXCD in human gastroenteritis.

We report the first case of granulomatous amebic encephalitis caused by Acanthamoeba in a previously healthy farmer in Taiwan who fell into a ditch. The DNA amplicons of amebas identified by polymerase chain reaction in the cerebrospinal fluid specimen and the ditch water specimens were identical and shared strong homology with A. castellanii . He survived after treatment with amphotericin B, rifampin, and corticosteroids.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, andPseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.Key words: Gluconobacter orphans, recA gene, DNA repair, recA mutant, SOS box.

Among 345 persons who underwent indirect hemagglutination (IHA) serological assays and assays of specific amebic antigens in their stool samples, 24 of 36 (66.7%) who were seropositive for Entamoeba histolytica had intestinal amebiasis as determined by antigen assays compared with 2 of 309 (0.2%) who were seronegative (odds ratio, 307; 95% confidence interval, 64.9 to 1,451). The estimated cost to detect a case of intestinal amebiasis by serology followed by antigen assays ($52) could be reduced by 74.3% and 69.9%, respectively, compared with the costs of the concurrent use of both assays ($202) and the antigen assays alone ($173). Our finding suggests that IHA assays followed by specific-amebic-antigen assays can be cost-effective in the diagnosis of intestinal amebiasis among persons with or without human immunodeficiency virus infection who are at risk for E. histolytica infection.

After bathing at a hot spring resort, a 75-year-old man presented to the emergency department because of seizure-like attack with loss of conscious. This is the first case of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri in Taiwan. PAM was diagnosed based on detection of actively motile trophozoites in cerebrospinal fluid using a wet-mount smear and the Liu's stain. The amoebae were further confirmed by PCR and gene sequencing. In spite of administering amphotericin B treatment, the patient died 25 days later.

Toxoplasma gondii is an opportunistic, zoonotic pathogen with a worldwide distribution. There are large variations in the seroprevalence of T. gondii infection in different regions of the world. Although toxoplasmosis became a notifiable communicable disease in Taiwan in 2007, little is known about its epidemiology among the general population. This cross-sectional study aimed to survey the seroprevalence of T. gondii infection and its risk factors among healthy blood donors in Taiwan. Through collaborating with the Taiwan Blood Services Foundation, a total of 1,783 healthy blood donors from all six-branch blood service centers participated in this study. The blood samples were tested for the presence of T. gondii antibodies and DNA using enzyme immunoassays and real-time PCR, respectively. Structured questionnaires were used to gather information on risk factors for T. gondii infection. Of the 1,783 participants, 166 (9.3%) tested positive for anti-Toxoplasma IgG, while 5 (0.28%) tested positive for anti-Toxoplasma IgM. The five IgM positive donors had high avidity antibodies suggestive of past infection. No active parasitemia was detected by real-time PCR assays. Multivariate logistic regression showed that undercooked pork meat consumption (adjusted odds ratio [OR] = 2.9; 95% confidence interval [CI]: 1.3-6.5), raw mussels consumption (adjusted OR = 5.3; 95% CI: 1.5-19.1), having a cat in the household (adjusted OR = 2.0; 95% CI: 1.2-3.2), a lower education level (adjusted OR = 1.6; 95% CI: 1.1-2.3), and donation place in eastern Taiwan (adjusted OR = 2.5; 95% CI: 1.6-3.9) were independent risk factors for Toxoplasma seropositivity. These findings provide information on the seroprevalence and epidemiology of T. gondii infection among healthy blood donors in Taiwan.

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.

Some species of Legionella are recognized as opportunistic potential human pathogens, such as Legionella pneumophila, which causes legionnaires disease. Indeed, outbreaks of legionellosis are frequently reported in areas in which the organism has been spread via aerosols from contaminated institutional water systems. Contamination in hot tubs, spas and public baths are also possible. As a result, in this study, we investigated the distribution of Legionella at six hot spring recreation areas throughout Taiwan. Legionella were detected in all six hot spring recreation areas, as well as in 20 of the 72 samples that were collected (27.8%). Seven species of Legionella identified from samples by the direct DNA extraction method were unidentified Legionella spp., Legionella anisa, L. pneumophila, Legionella erythra, Legionella lytica, Legionella gresilensis and Legionella rubrilucen. Three species of Legionella identified in the samples using the culture method were L. pneumophila, unidentified Legionella spp. and L. erythra. Legionella species were found in water with temperatures ranging from 22.7 °C to 48.6 °C. The optimal pH appeared to range from 5.0 to 8.0. Taken together, the results of this survey confirmed the ubiquity of Legionella in Taiwan spring recreational areas. Therefore, a long-term investigation of the health of workers at hot spring recreational areas and the occurrence of Legionella in hot spring recreational areas throughout Taiwan are needed.

Since the 1970s, no information on simian malaria has been documented in Taiwan, an area that is free from human malaria. To update the prevalence of simian malaria, a molecular-based survey was performed. Blood samples from 286 Formosan macaques ( Macaca cyclopis ) were tested for Plasmodium species by microscopy and nested polymerase chain reaction. Furthermore, the field isolates were characterized by sequencing the 42-kDa fragment of the merozoite surface protein 1 (MSP-1(42)). Of the 286 blood samples analyzed, 7 (2.4%) were positive by microscopy and nested PCR. All malaria-infected Formosan macaques were those collected from southern Taiwan, whereas no evidence of malarial parasites was observed among monkeys from eastern and northern Taiwan. Molecular and phylogenetic analyses based on the asexual stage small subunit ribosomal RNA (SSU rRNA) gene clearly identified these samples as a single infection with Plasmodium inui . Furthermore, phylogenetic analysis of the MSP-1(42) gene showed that the 7 field isolates were closely related to P. inui strains Taiwan I and II, which were obtained from Formosan macaques in 1963. These findings indicate that P. inui is the only cause of simian malaria in Taiwan, has been circulating in Formosan macaques at least for 46 yr, and has a geographic preference for southern Taiwan.

Aims This study aimed to assess the prevalence of amoebiasis among patrons visiting gay saunas in Taiwan. Methods A cross-sectional survey was conducted using questionnaire interview and indirect hemagglutination assays and specific Entamoeba histolytica antigen assays of blood and rectal swab specimens, respectively, among patrons visiting 10 gay saunas between September 2006 and December 2006. Results During the three-month study period, 208 blood and 120 rectal swab specimens were tested for E. histolytica infection. Amoebiasis was detected among 3.8% and 3.3% of the patrons by serologies and antigen assays, respectively. During the latest sexual encounter, more than 70% of the patrons had oral-anogenital sex, and only 20% used condoms during oral-anogenital contact. Conclusion Our findings suggest that there is a potential risk of E. histolytica transmission among the patrons visiting gay saunas who do not practise safe sex consistently in Taiwan.

BackgroundBecause the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor β1 (TGF-β1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain (NF-L), tissue transglutaminases (tTGs), β-amyloid precursor proteins (AβPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae ofToxocara caniscan invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT.MethodsBIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250T. canisembryonated eggs was investigated from 3 days (dpi) to 8 weeks post-infection (wpi) by Western blotting and RT-PCR.ResultsResults revealed that at 4 and 8 wpi,T. canislarvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-β1, S100B, NF-L, tTG, AβPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain.ConclusionFurther studies are needed to determine whether there is an increased risk of CT progression into neurodegenerative disease because neurodegeneration-associated AβPP and phosphorylated tau emerged in the brain.

Background Incidence of Entamoeba histolytica infection and clinical manifestations and treatment response of invasive amebiasis (IA) in HIV-infected patients have rarely been investigated before. Methodology/Principal Findings At the National Taiwan University Hospital, medical records of HIV-infected patients who received a diagnosis of IA between 1994 and 2005 were reviewed. The incidence of amebiasis was investigated in serial blood and stool samples from 670 and 264 HIV-infected patients, respectively, using serological and specific amebic antigen assays. DNA extracted from stool samples containing E. histolytica were analyzed by PCR, sequenced, and compared. Sixty-four (5.8%) of 1,109 HIV-infected patients had 67 episodes of IA, and 89.1% of them were men having sex with men (MSM). The CD4 count at diagnosis of IA was significantly higher than that of the whole cohort (215 cells/µL vs. 96 cells/µL). Forty episodes (59.7%) were liver abscesses, 52 (77.6%) colitis, and 25 (37.3%) both liver abscesses and colitis. Fever resolved after 3.5 days of metronidazole therapy (range, 1–11 days). None of the patients died. The incidence of E. histolytica infection in MSM was higher than that in other risk groups assessed by serological assays (1.99 per 100 person-years [PY] vs. 0 per 100 PY; p, Author Summary Entamoeba histolytica, morphologically identical to but genetically different from E. dispar and E. moshkovskii, is the causative agent of amebiasis. Recently there have been reports of increased risk for amebiasis among men who have sex with men (MSM) due to oral-anal sexual contact in several developed countries. In this longitudinal follow-up study, the incidence of amebiasis was determined among HIV-infected patients using serological and specific amebic antigen assays. DNA extracted from stool samples containing E. histolytica were analyzed by PCR, sequenced, and compared. Clinical manifestations and treatment response of invasive amebiasis in HIV-infected patients were reviewed. The results demonstrated that HIV-infected MSM were at significantly higher risk of amebiasis than patients from other risk groups. Clustering of E. histolytica isolates by sequencing analyses from geographically unrelated patients suggested person-to-person transmission. Despite immunosuppression, amebic liver abscesses and colitis responded favorably to metronidazole therapy. It is important to investigate in areas of high incidence of both amebiasis and HIV (sub-Saharan Africa) how generalizable these findings are.

A total of 141 independent strains of Pseudomonas aeruginosa with different heterogeneities in the exo gene ( exoS , exoT , exoU , and exoY ) background were examined for their pathogenic roles. Results indicated that the exoU gene is the major contributor to cytotoxicity in Madin-Darby canine kidney cells but is not related to bacterial colonization in mice.

Here we show that blood-stage Plasmodium falciparum organisms accumulate a high mass of triacylglycerol and diacylglycerol. However, we failed to detect cholesterol esters, a second neutral lipid species reported to be important for a related apicomplexan, Toxoplasma gondii . Evidence for P. falciparum and T. gondii homologues of acyl coenzyme A:diacylglycerol acyltransferase suggests that acylglycerols may be the conserved neutral lipids in apicomplexans.

Although human toxoplasmosis is a notifiable disease in Taiwan since 2007, little is known about its risk factors. This study aimed to investigate the risk factors for acute Toxoplasma gondii diseases in Taiwan. We conducted a nationwide population-based case-control study. Cases of acute human toxoplasmosis notified to the Taiwan Centers for Diseases Control (Taipei, Taiwan) during 2008-2013 were compared with controls that were randomly selected from healthy T. gondii-seronegative blood donors who participated in a nationwide T. gondii seroepidemiologic study during 2009-2010. Cases and controls were matched according to age, gender and residency at an 1:8 ratio. Structured questionnaires were used to gather information regarding risk factors. A total of 30 laboratory-confirmed acute T. gondii disease cases and 224 controls were enrolled. The most common clinical manifestation of the cases was flu-like symptoms (n = 20), followed by central nervous system disease (n = 4), ocular diseases (n = 3), abortion (n = 2), and congenital infection (n = 1). Multivariate conditional logistic regression showed that raw clam consumption (adjusted odds ratio [OR] = 3.7; 95% confidence interval [CI] = 1.4-9.9) and having a cat in the household (adjusted OR = 2.9; 95% CI = 1.1-7.9) were two independent risk factors for acute T. gondii disease. We conclude that raw shellfish consumption and domestic cat exposure were risk factors for acquiring acute T. gondii diseases in Taiwan. This finding may guide future research and control policies.

Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum , the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein ( gfp ) reporter and the other expressing a drug resistance marker ( Tgdhfr-ts M23), allowed selection of a population in which about ∼30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7–14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5′hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum .

Keywords: Cell Cycle Proteins ; Guanine Nucleotide Exchange Factors Reference EPFL-ARTICLE-148125doi:10.1016/S0166-6851(98)00107-8 Record created on 2010-04-07, modified on 2016-08-08

A process for production, isolation, and purification of melanin produced by the fermentation of Streptomyces lividans 66 harboring a recombinant plasmid pIJ702-bearing tyrosinase gene has been developed. The efficacy of melanin in the protection of mosquito larvacidal activity of Bacillus thuringiensis var. israelensis against uv light has been studied. Results obtained by the live cell counts and the bioassay of residual mosquitocidal activity of B. thuringiensis var. israelensis after exposure to uv radiation showed that melanin is an excellent photoprotective agent.