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Human cytomegalovirus (CMV) is associated with aspergillosis, but the simultaneous presence of CMV viral interleukin-10 (cmvIL-10) and aspergillosis has never been investigated. CmvIL-10 is produced by CMV-infected cells and acts as an immune modulator during CMV infection. The aim of this study was to evaluate cmvIL-10 levels in peripheral blood and its influence on the clinical outcomes of Aspergillus infection.Patients who visited or were admitted to the hospital with suspected Aspergillus infection, including invasive aspergillosis (IA) and chronic pulmonary aspergillosis (CPA), were prospectively enrolled. The cmvIL-10, human IL-10 (hIL-10), IL-1B, IL-6, IL-8, IFN-γ, and TNF-α levels in peripheral blood were measured.Patients with Aspergillus infection had a higher level of cmvIL-10 than the control group (158 ± 305 vs 27.9 ± 30.4 pg/ml, p .05). The level of cmvIL-10 was not correlated with CMV viremia or end-organ disease. The cmvIL-10 but not hIL-10 level was positively correlated with the IFN-γ level (p .05) and marginally negatively correlated with IL-1B and IL-8 levels (p .1). In patients with CPA, a high level of cmvIL-10 (≥100 pg/ml) was a poor prognostic factor for long-term survival (p .05). In contrast, CMV viremia or end-organ disease was associated with poor survival in patients with IA (p = .05).Aspergillus infection was associated with CMV coinfection with cmvIL-10 in blood. A cmvIL-10 concentration ≥100 pg/ml was a predictor for unfavourable outcome in CPA patients.

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.

Introduction Exact etiology and proper treatment of eczema are still unknown. The hygiene hypothesis and epidermal barrier dysfunction hypothesis attempted to give some plausible explanations for these issues but they still remain unclear. The identification of factors, including hygiene practices, related to eczema symptoms (ES) could shed some light on these matters. Therefore, this study aimed to determine risk factors related to ES and the ES prevalence in two disparate areas in terms of urbanization in Aceh, Indonesia. Methods A cross-sectional study with convenience sampling was conducted among schoolchildren living in urban and rural Aceh. Data on ES, socio-demographic characteristics, environmental factors, partial ablution and other hygiene related factors were collected by parental questionnaires. In addition, children's anthropometric measurements were also collected. Results The prevalence of current ES in the study population was 21%. When stratifying by residency, the prevalence of ES in urban and rural area was 20.93% versus 21.05%. Partial ablution was independently associated with a reduced risk of ES (OR = 0.36; 95% CI 0.13-0.96). Important risk factors for ES were paternal history of allergic disease (OR = 4.09%; 95% CI 1.51-11.11) and belonging to the older group of schoolchildren (10-13 years old) (OR = 2.57; 95% CI 1.03-6.40). Conclusions There were no significant differences in the prevalence of ES between urban and rural settings, and partial ablution had a protective effect on ES. These findings support the epidermal barrier dysfunction hypothesis as a possible pathway of eczema.

The aim of this study was to investigate the relationship of matriptase-2 expression with the clinicopathologic characteristics, the histologic grade, and patient survival in oral squamous-cell carcinoma (OSCC). Immunohistochemical analysis of matriptase-2 expression was performed in 102 surgical specimens from patients with OSCC. The immunohistochemical results were further verified by quantitative real-time reverse transcription-polymerase chain reaction. The immunostaining intensity was scored on a scale ranging from 0 (absence of staining) to 3 (intense staining). The distribution score was determined by the percentage of stained cells on a scale ranging from 0 (

National tuberculosis programmemanual. (DOCX 1617 kb)

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.

Background The causative pathogen is rarely identified in the emergency department (ED), since the results of cultures are usually unavailable. As a result, antimicrobial treatment may be overused. The aim of our study was to investigate the pathogens, risk factors of acute gastroenteritis, and predictors of acute bacterial gastroenteritis in the ED. Methods We conducted a matched case-control study of 627 stool samples and 612 matched pairs. Results Viruses (41.3%) were the leading cause of gastroenteritis, with noroviruses (32.2%) being the most prevalent, followed by bacteria (26.8%) and Giardia lamblia (12.4%). Taking antacids (adjusted odds ratio [aOR] 4.10; 95% confidence interval [CI], 2.57-6.53), household members/classmates with gastroenteritis (aOR 4.69; 95% CI, 2.76-7.96), attending a banquet (aOR 2.29; 95% CI, 1.64-3.20), dining out (aOR 1.70; 95% CI, 1.13-2.54), and eating raw oysters (aOR 3.10; 95% CI, 1.61-5.94) were highly associated with gastroenteritis. Elders (aOR 1.04; 05% CI, 1.02-1.05), those with CRP >10 mg/L (aOR 2.04; 95% CI, 1.15-3.62), or those who were positive for fecal leukocytes (aOR 2.04; 95% CI, 1.15-3.62) or fecal occult blood (aOR 1.97; 95% CI, 1.03-3.77) were more likely to be hospitalized in ED. In addition, presence of fecal leukocytes (time ratio [TR] 1.22; 95% CI, 1.06-1.41), abdominal pain (TR 1.20; 95% CI, 1.07-1.41), and frequency of vomiting (TR 0.79; 95% CI, 0.64-0.98) were significantly associated with the duration of acute gastroenteritis. Presence of fecal leukocytes (aOR 2.08; 95% CI, 1.42-3.05), winter season (aOR 0.45; 95% CI, 0.28-0.74), frequency of diarrhea (aOR 1.69; 95% CI, 1.01-2.83), and eating shrimp or crab (aOR 1.53; 95% CI, 1.05-2.23) were highly associated with bacterial gastroenteritis. The area under the receiver operating characteristic curve of the final model was 0.68 (95% CI, 0.55-0.63). Conclusions Acute bacterial gastroenteritis was highly associated with season, frequency of diarrhea, frequency of vomiting, and eating shrimp or crab.

We report on a novel application of real-time reverse transcription-loop-mediated isothermal amplification (real-time RT-LAMP) to identify the presence of a specific body fluid using blood as a proof-of-concept model. By comparison with recently developed methods of body fluid identification, the RT-LAMP assay is rapid and requires only one simple heating-block maintained at a single temperature, circumventing the need for dedicated equipment. RNA was extracted from different body fluids (blood, semen, saliva, menstrual blood, sweat, and urine) for use in real-time RT-LAMP reaction. The 18S rRNA locus was used as the internal control and hemoglobin beta (HBB) as the blood-specific marker. Reverse transcription and LAMP reaction were performed in the same tube using a turbidimeter for real-time monitoring the reaction products within a threshold of 60 min. HBB LAMP products were only detected in blood and not in any of the other body fluid, but products from the 18S rRNA gene were detected in all the tested body fluids as expected. The limit of detection was a minimum of 10(-5) ng total RNA for detection of both 18S rRNA and HBB. Augmenting the detection of RT-LAMP products was performed by separation of the products using gel electrophoresis and collecting the fluorescence of calcein. The data collected indicated complete concordance with the body fluid tested regardless of the method of detection used. This is the first application of real-time RT-LAMP to detect body fluid specific RNA and indicates the use of this method in forensic biology.

In this study, antibiotic resistance and major phenol and genotypes of non-typhoid Salmonella spp. from riversheds in Taiwan were examined. In 236 water samples tested, 54 (22.9%) contained Salmonella spp. Fifteen Salmonella serovars were identified from the Salmonella isolates, and some common serovars are associated with infections of human and livestock, including Albany (27.8%), Newport (14.8%), Bareilly (13.0%), Derby (11.1%), and Typhimurium (7.4%). Various environmental factors may also affect the presence and proportion of different serovars in the receiving waters. In contrast, serovars with narrower range of hosts, e.g., Dublin, were rarely detected. The Salmonella isolates were subjected to eight antibiotics for drug resistance, and 51.9% of the samples were resistant to at least one tested antibiotics. Tetracycline and sulfadiazine were the two most ineffective antibiotics against the Salmonella isolates, and the results were indicative of long-term antibiotics abuse as fodder supplements in animal husbandry. The more commonly detected serovars such as Albany, Derby, and Typhimurium were also more likely to be resistant to multiple antibiotics. Finally, a significant correlation was observed between resistance to chloramphenicol and the resistance gene cmlA, suggesting that the resistance genotypes could persist in the environment even long after prohibition of the drug use. The high prevalence of antibiotic-resistant Salmonella spp. infers elevated infection risks that must be further examined.

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.

The aim of this study was to investigate the impact of hMLH1 polymorphisms on treatment outcomes in patients with oral squamous cell carcinoma (OSCC). Genotypings were performed by direct DNA sequencing in peripheral blood leukocytes from 185 male OSCC patients. Patients received primary surgery with or without adjuvant radiotherapy. Two hMLH1 tag single nucleotide polymorphisms (SNPs)—rs1800734 (−93G>A in the promoter) and rs1540354 (in the third intron)—were chosen from the HapMap project. Overall survival (OS) and disease-free survival (DFS) were compared between different genotypes. The hMLH1 rs1800734 and rs1540354 polymorphisms were in weak linkage disequilibrium (r 2 = 0.456). OSCC patients with the rs1800734 AA genotype had a significantly poor prognosis in both OS and DFS. This SNP can also predict the outcomes of OSCC patients with postoperative adjuvant radiotherapy, especially in advanced stage; however, no significant differences in patient outcomes were found for the hMLH1 rs1540354 genotypes. Our results demonstrate that the hMLH1 −93G>A SNP is found to be associated with patient outcomes in OSCC. This SNP can also predict their treatment outcome of radiotherapy.

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.

Vittaforma corneae is an obligate intracellular fungus and can cause human ocular microsporidiosis. Although accumulating reports of V. corneae causing keratoconjunctivitis in both healthy and immunocompromised persons have been published, little is known about the organism's occurrence in aquatic environments. Limitations in detection sensitivity have meant a large sampling volume is required to detect the pathogen up to now, which is problematic. A recent study in Taiwan has shown that some individuals suffering from microsporidial keratitis (MK) were infected after exposure to the pathogen at a hot spring. As a consequence of this, a survey and analysis of environmental V. corneae present in hot springs became an urgent need. In this study, sixty water samples from six hot spring recreation areas around Taiwan were analyzed. One liter of water from each sample site was filtered to harvest the fungi. The positive samples were detected using a modified nested PCR approach followed by sequencing using specific SSU rRNA gene primer pairs for V. corneae. In total fifteen V. corneae-like isolates were identified (25.0% of sites). Among them, six isolates, which were collected from recreational areas B, C and D, were highly similar to known V. corneae keratitis strains from Taiwan and other countries. Furthermore, five isolates, which were collected from recreation areas A, C, E and F, were very similar to Vittaforma-like diarrhea strains isolated in Portugal. Cold spring water tubs and public foot bath pools had the highest detection rate (50%), suggesting that hot springs might be contaminated via untreated water sources. Comparing the detection rate across different regions of Taiwan, Taitung, which is in the east of the island, gave the highest positive rate (37.5%). Statistical analysis showed that outdoor/soil exposure and a high heterotrophic plate count (HPC) were risk factors for the occurrence of V. corneae. Our findings provide empirical evidence supporting the need for proper control and regulations at hot spring recreational waters in order to avoid health risks from this pathogen. Finally, we have developed a small volume procedure for detecting V. corneae in water samples and this has proved to be very useful.

Summary Entamoeba histolytica infection (amoebiasis) is the second leading cause of death from parasitic diseases. Epidemiological studies from developed countries have reported an increasing prevalence of amoebiasis and of invasive infections, such as amoebic colitis, among men who have sex with men (MSM) who engage in oral–anal sex. Although most infections with E histolytica are asymptomatic, clinical manifestations of invasive amoebiasis mainly include amoebic colitis and amoebic liver abscess, which are associated with substantial morbidity and medical cost. Laboratory diagnosis of amoebiasis should be based on detection of E histolytica by use of tests with high sensitivity and specificity, such as specific amoebic-antigen or PCR-based assays. Microscopy used in routine clinical laboratories is not sensitive or specific enough for detection of E histolytica . Metronidazole or tinidazole remains the mainstay of treatment for invasive amoebiasis, followed by treatment with luminal agents to prevent relapse and transmission of E histolytica to sexual partners or close contacts.

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.

Japanese encephalitis virus (JEV) infection in mosquitoes was monitored at Guandu Nature Park in Taipei City from September 2002 to December 2004. In total, 30,386 female mosquitoes consisting of six genera and 14 species were processed for virus in 1,229 pools by using Flavivirus NS5 gene sequences detected by reverse transcription-polymerase chain reaction (PCR) and nested PCR assay. Overall, 101 pools were positive, including 95, 1, 4, and 1 for Culex tritaeniorhynchus Giles, Culex sitiens Wiedemann, Culex rubithoracis (Leicester), and Aedes vexans noctunmus (Theobald), respectively.

Cephalexin synthesizing enzyme (CSE) of Gluconobacter oxydans ATCC 9324 was purified up to about 940-fold at a yield of 12%. CSE biosynthesis in G. oxydans was found inducible in the presence of D-phenylglycine but not its substrate phenylglycine methyl ester. The purified enzyme was shown homogeneous on SDS-PAGE and exhibited a specific activity of 440 U per mg protein. The apparent molecular mass of the native enzyme was estimated to be 70 kDa over a Superdex 200 gel filtration column and 68 kDa on SDS-PAGE, indicating that the native enzyme is a monomer. Its isoelectric focusing point is 7.1, indicating a neutral character. The enzyme had maximal activity around pH 6.0 to 6.5, and this activity was thermally stable up to 40 degrees C. Synthesis of cephalexin from D-phenylglycine methyl ester and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) by the purified CSE was demonstrated. Its L-enantiomer was not accepted by CSE. Apart from cephalexin, ampicillin was also synthesized by the purified CSE from its acyl precursors and 6-aminopenicillanic acid (6-APA). Substrate specificity studies indicated that the enzyme required a free alpha amino group and an activated carboxyl group as a methyl ester of D-form phenylglycine. Interestingly, the purified enzyme did not catalyze hydrolysis of its products, e.g., cephalexin, cephradine, and ampicillin, in contrast to enzymes from other strains of Pseudomonadaceae.

Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the alpha-amino-group of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity. Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both. The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa. Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule. The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration. Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA. However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity.

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.

Background Sexually transmitted Entamoeba histolytica infection (EHI) has been increasingly recognized among men who have sex with men (MSM). We used the National Disease Surveillance Systems (NDSS) to identify prevalent and incident HIV diagnoses among adults with EHI and to determine the associated factors. Methodology The NDSS collect demographic, clinical, and behavioral characteristics of case patients through physician reports and public health interviews. EHI was confirmed by polymerase-chain-reaction assays, histopathology, or serology with documented liver abscess. We linked NDSS databases to identify prevalent and incident HIV diagnoses among noninstitutionalized Taiwanese adults with confirmed EHI during 2006–2013. Cox proportional-hazards analysis was used to determine associated factors. Principal findings Of noninstitutionalized adults with EHI, we identified prevalent HIV diagnosis in 210 (40%) of 524 males and one (1.7%) of 59 females, and incident HIV diagnosis in 71 (23%) of 314 males. MSM accounted for 183 (87%) and 64 (90%) of prevalent and incident HIV diagnoses in males, respectively. From 2006–2009 to 2010–2013, the prevalence of HIV diagnosis increased from 32% to 45% (P = 0.001) while the incidence of HIV diagnosis increased from 5.4 to 11.3 per 100 person-years (P = 0.001) among males with EHI. Incident HIV diagnosis was independently associated with a younger age, residing in metropolitan areas, hospitalization, previous syphilis, and engagement in oral, anal, or oral–anal sex before illness onset. Conclusions/significance Prevalent and incident HIV diagnoses were increasingly identified among adult males in Taiwan, preferentially affecting younger urban MSM. Surveillance and risk-reduction interventions are recommended against the interplay of HIV epidemic and sexually transmitted EHI., Author Summary Although sexually transmitted Entamoeba histolytica infection has been increasingly recognized among men who have sex with men (MSM) in Australia, Japan, Korea, and Taiwan, particularly those with HIV infection, no studies have examined E. histolytica infection in relation to HIV and attribution of sexual transmission at a national level. We analyzed the national surveillance data in Taiwan and demonstrated that the prevalent and incident HIV diagnoses increased among E. histolytica-infected adult males from 2006–2009 to 2010–2013, which were associated with previous syphilis and engagement of oral, anal, oral–anal sex. The findings indicate sexual transmission might have replaced traditional risk factors as the major route of E. histolytica transmission among adult males in Taiwan. E. histolytica infection associated with prevalent and incident HIV diagnoses preferentially affects younger urban MSM, suggesting new targets of surveillance and public health interventions for this sexually transmissible protozoan infection.

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.

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...

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-typerecA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.Key words: Gluconobacter oxydans, recA gene, recombination, SOS function, interference.