Occurrence and distribution of Naegleria species from thermal spring environments in Taiwan.

UNLABELLED: Naegleria spp. is a free-living amoeba that can be found in the natural environment. A number of Naegleria spp. can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, water samples from various thermal springs were taken from four thermal spring areas. Naegleria spp. was detected via culture confirmation and molecular taxonomic identification. Among the 60 samples obtained, Naegleria spp. was identified in 26 (43·3%) samples. The identified species included Naegleria australiensis, Naegleria gruberi, Naegleria lovaniensis and Naegleria mexicana. The presence of living Naegleria spp. was significantly associated with elevated pH value in the water sample. SIGNIFICANCE AND IMPACT OF STUDY: In this study, we examined the presence of living Naegleria spp. in thermal spring waters in south-eastern Taiwan. Naegleria spp. was isolated and culture-confirmed from thermal spring water. Naegleria fowleri was not found in all water samples, and Naegleria australiensis was the most common Naegleria genotype.

Isolation and identification of Acanthamoeba species related to amoebic encephalitis and nonpathogenic free-living amoeba species from the rice field.

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.

Legionella prevalence in wastewater treatment plants of Taiwan.

Legionella is a bacterium that is ubiquitous in natural and artificial aquatic environments. Some species of Legionella are recognized as opportunistic potential human pathogens. We investigated the distribution of Legionella at seventeen WWTPs throughout Taiwan. Legionella were detected in 10 of the 17 WWTPs (58.8%) and 25 of 41 samples (61.0%). In the integrated, hospital, industrial and domestic wastewater systems were 13/18 (72.2%), 7/12 (58.3%), 2/7 (28.6%) and 3/4 (75.0%) of the samples were positive for Legionella, respectively. The most frequently encountered species were L. donaldsonii and uncultured L. spp., which were both found in 10 samples (24.4% of all samples), then followed by L. lytica (4.9%) and L. pneumophila (4.9%). L. anisa was detected in one sample (2.4%). The results of this survey confirm that Legionella are ubiquitous in WWTPs in Taiwan. Therefore, long-term investigations should be conducted to evaluate the overall occurrence of Legionella in WWTPs in Taiwan.

Molecular detection and prevalence of enterovirus within environmental water in Taiwan.

AIMS: To investigate the presence of enterovirus RNA in various samples of environmental water collected in Taiwan during 2004-2005, and to characterize the genotypes and distribution of the viruses identified in Taiwan. METHODS AND RESULTS: Total 131 environmental samples were screened using the reverse transcription polymerase chain reaction (RT-PCR) for the highly conserved 5'-nontranslated regions (5'-NTR). Among these enterovirus RNA-positive samples, 32, 15 and 6 of the samples were recovered from surface water, ground water and sewage water respectively. However, the total positive detection rate increased to 40.5% with the application of seminested PCR. Sequencing revealed that the majority of isolates belonged to the following genotypes: coxsackie A2 (35.8%), coxsackie A6 (13.2%) and enterovirus (EV)71 (11.3%); echovirus 11, porcine EV9 and coxsackie A16 isolates were also observed. CONCLUSIONS: This study confirms that the major epidemic genotypes of enterovirus are prevalent in the surface and ground water of Taiwan. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is helpful in understanding the significance and epidemiology of the virus within and beyond the study area. Moreover, it was possible to predict the enterovirus genotype and evaluate possible correlations between water contamination and viral sequences found among clinical samples.

Performances of the immunomagnetic separation method for Cryptosporidium in water under various operation conditions.

Immunomagnetic separation (IMS) has been specified as a standard method for the measurement of Cryptosporidium in some countries. In this study, the IMS method was evaluated on the basis of the recovery efficiencies of Cryptosporidium oocysts at various IMS operation conditions. The average recovery for different Cryptosporidium concentrations in deionized water was 82.6 +/- 18.2% (n = 52). No significant change in recovery was observed by altering the debris ratio of the water samples. The efficiency was increased by prolonging the reaction time, and by increasing the amount of immunomagnetic beads. The recoveries of oocysts seeded in an Eppendorf with a small reaction volume were similar to those seeded in glass tubes with 10 times the reaction volume. The recovery efficiency of oocysts was reduced significantly when the reaction buffer was replaced by PBS. In conclusion, this method has good reproducibility and high recovery.

Evaluation of immunomagnetic separation method for detection of Giardia for different reaction times and reaction volumes.

Immunomagnetic separation (IMS) has been specified as a standard method for the measurement of Giardia. In this study, Dynal IMS was evaluated on the basis of recovery efficiencies of Giardia cysts for various IMS operational conditions. The average recoveries for Giardia in deionized, treated and raw water samples were 82.6 +/- 12.2% (n = 6), 75.6 +/- 15.2% (n = 3), and 70.6 +/- 18.2% (n = 3), respectively. Significant changes in recovery were observed by altering the debris ratio and the debris components of water samples. Changing the reaction volume within the same vessel had no significant effect on cyst recovery efficiencies. However, prolonging the reaction time did increase recovery efficiencies.

Evaluation of two concentration methods for detecting Giardia and Cryptosporidium in water.

The cartridge filtration method and membrane filtration method based on the fluorescent antibody procedure were evaluated for their recovery efficiencies and detection limits of Giardia and Cryptosporidium. We assessed the performances of the two concentration methods for water samples collected from Taiwan water treatment plants. The membrane filtration method was characterized by higher recovery rate and detection limit comparing with the cartridge filtration method. The occurrences of both parasites, and the relationships of parasite concentrations with indicator microorganisms show inconsistency between the two methods. It was discovered that water turbidity reduced the recovery efficiencies, and raised the detection limits for both parasites regardless of the method used.

Analysis for Giardia cysts and Cryptosporidium oocysts in water samples from small water systems in Taiwan.

Giardia and Cryptosporidium have emerged as waterborne pathogens of concern. Twenty-six water samples were collected from small water systems in Taiwan and checked for the occurrence of both parasites. Water quality parameters and characteristics of the sampling sites were also recorded. The frequencies of occurrence for Giardia and Cryptosporidium were 46.2% for each; and their mean concentrations were 79.5 cysts/100 l and 22.1 oocysts/100 l, respectively. The concentrations of oocysts and heterotrophic bacteria exhibited the highest correlation and followed the concentrations of the two protozoa. The water samples from sites with filtration devices had a lower oocyst concentration than those from sites without filtration devices, while no significant difference was found for cysts. The level of each parasite had no direct relationship with altitude. The cyst concentrations increased proportionally with the consumer population using the water systems. Risk assessment of the parasitic infections suggests that setting up disinfection devices in the small water systems would be needed.

Filtration behaviors of Giardia and Cryptosporidium--ionic strength and pH effects.

The laboratory-scale filtration tests of Giardia cysts and Cryptosporidium oocysts in both 2 mm-phi glass beads and 2 mm-phi polystyrene beads filters were conducted to investigate their filtration behaviors. The protozoan parasites were used as target particles, while the chemical system altered by changing the electrolyte concentration and pH. The results significantly indicate that ionic strength have a positive effect on the removal efficiencies for Giardia cysts and Cryptosporidium oocysts. The removal efficiency of two filters for Giardia cysts slightly decreased from pH 2.4 to 8.7 and decreased significantly in pH as pH up to 8.7, while that for Cryptosporidium slightly rippled beyond pH 8.7, and with the decrease in pH up to pH 8.7. The experimental collision efficiencies from the interactions between colloids and the filter media were calculated with a semi-empirical approach of the single sphere model and clean-bed filtration theory. The results also indicated that experimental collision efficiencies for (oo)cysts corresponded to the (oo)cysts removal efficiencies in all trials, and oocysts exhibits higher collision efficiencies than cysts.

The prevalence of Giardia and Cryptosporidium in Taiwan water supplies.

Giardia and Cryptosporidium have emerged as waterborne pathogens of concern. Thirty-one water samples were collected from nine potable water treatment plants in Taiwan and investigated for the presence of Giardia cysts and Cryptosporidium oocysts. The immunofluorescence assay was used for the simultaneous detection of cysts and oocysts. The frequency of occurrence of cysts was 77.8% for Giardia and 72.2% for Cryptosporidium in 18 raw water samples. Ten out of 13 samples collected from treated water samples showed the presence of cysts, while in 5 out of 13 treated water samples oocysts were detected. The risk assessment for adverse human effects arising from the presence of cysts and oocysts indicates the possibility of waterborne transmission of Giardia and Cryptosporidium infection in Taiwan if water is not adequately treated.

Human squamous carcinoma cells express complex defects in the control of proliferation and differentiation.

Four human squamous carcinoma cell (SCC) lines (SCC-9, SCC-13, SCC-15, and SCC-25) were studied to characterize their relative ability to control proliferation and differentiation. These experiments were based on previous data that established that in normal human keratinocytes three distinct and sequential steps are involved in the integrated control of proliferation and differentiation: 1) reversible growth-arrest at a predifferentiation state, 2) irreversible loss of proliferative potential, and 3) terminal differentiation. The current results show that SCC can show changes in the culture conditions required to undergo reversible growth-arrest and SCC can express partial or complete defects in their ability to irreversibly growth-arrest or terminally differentiate. For example, SCC-9 and SCC-25 cannot irreversibly growth-arrest or terminally differentiate, SCC-13 can irreversibly growth-arrest but cannot terminally differentiate, and SCC-15 can irreversibly growth-arrest and terminally differentiate to a moderate extent. These results therefore extend previous data by establishing that the malignant transformation of human epithelial cells does not simply result from defects in the control of terminal differentiation but rather from a combination of complex defects in the regulation of proliferation and differentiation.

Two subtypes of reversible cell cycle restriction points exist in cultured normal human keratinocyte progenitor cells.

Three methods can induce reversible arrest of the growth of cultured human keratinocytes in the G1 phase of the cell cycle. These include the incubation of cells in medium containing transforming growth factor (TGF)-beta or ethionine, or in isoleucine-deficient medium. The current studies were performed to determine if the growth arrest induced by these methods occurs at a common or at a distinct G1 state(s). We first evaluated the relative time interval required for arrested cells to initiate DNA synthesis after growth restimulation with mitogenic medium. The results show that ethionine arrested cells require 22 to 28 hours to initiate DNA synthesis and that a maximum rate of DNA synthesis occurs at 46 hours. Cells arrested by isoleucine deficiency required 10 to 12 hours to initiate DNA synthesis with peak DNA synthesis occurring at 24 hours. Finally, TGF-beta arrested cells require only 6 to 8 hours to initiate DNA synthesis and show a maximum rate of DNA synthesis at 18 hours. We next evaluated if cells that were growth arrested at these states were differentially capable of initiating DNA synthesis in different types of potentially mitogenic medium. The results show that if TGF-beta arrested cells were refed TGB-beta free serum containing medium with ethionine or similar medium with isoleucine deficiency, no DNA synthesis occurred. In contrast, if cells whose growth was arrested in ethionine-containing medium or in isoleucine-deficient medium were refed mitogenic medium containing TGF-beta, significant DNA synthesis was detected. These results suggest that least two different types of reversible growth arrest states exist in cultured human keratinocytes. One appears to be mediated by receptor-dependent processes, such as that induced by TGF-beta, and the other appears to be mediated by other types of metabolic events, such as those induced by ethionine treatment or by isoleucine deficiency.

Biologic mechanisms for the regulation of normal human keratinocyte proliferation and differentiation.

Normal human keratinocytes can be grown in serum-free medium, and the integrated control of their proliferation and differentiation can be modulated experimentally. The growth of cultured human keratinocytes can also be specifically arrested at either reversible or irreversible growth arrest states. Reversible growth arrest is induced by culture in medium containing TGF-beta or ethionine or in medium deficient of isoleucine. Irreversible growth arrest is induced by culture in razoxane-containing medium or by routine passage of keratinocytes until senescence results. The current studies were performed to determine from which growth arrest states keratinocyte differentiation occurs. Cells were therefore growth-arrested at each state, and they were then incubated in several different differentiation-promoting culture conditions. The results show that differentiation, as determined by morphologic, cytochemical, and immunofluorescent assays, can be induced from multiple reversible and irreversible growth arrest states by a series of complex biologic mechanisms. More specifically, at least three distinct stages appear to be involved in the process of keratinocyte differentiation. First, cells arrest their growth at a reversible predifferentiation state. Second, cells irreversibly lose their proliferative potential. Finally, cells express the terminally differentiated keratinocyte phenotype.