Detection of Giardia and Cryptosporidium cysts/oocysts in watersheds and drinking water sources in Brazil urban areas

The protozoan parasites Giardia and Cryptosporidium have been described as important waterborne disease pathogens, and are associated with severe gastrointestinal illnesses. The objective of this paper was to investigate the presence of Giardia cysts and Cryptosporidium oocysts in samples from watershed catchments and treated water sources. A total of 25 water samples were collected and examined according to the US EPA—Method 1623, 2005, consisting of 12 from drinking water and 13 from raw water. Positive samples from raw water for Giardia cysts and Cryptosporidium oocysts were 46.1 and 7.6%, respectively. In finished water, positive samples were 41.7% for Giardia cysts and 25.0% for Cryptosporidium oocysts. Concentrations of Giardia cysts found in raw water samples ranged from “not detected” to 3.4 cysts/L, whereas concentrations of Cryptoporidium oocysts ranged from “not detected” to 0.1 oocysts/L. In finished water, Giardia concentrations ranged from “not detected” to 0.06 cysts/L, and Cryptosporidium, from “not detected” to 0.01 oocysts/L. Concentrations of Giardia cysts and Cryptosporidium oocysts were not high in the samples analyzed. Nevertheless, the results of this study highlight the need to monitor these organisms in both raw and drinking water.     

Screening of the municipal water system of La Plata, Argentina, for human intestinal parasites

The La Plata River, though severely contaminated by intestinal parasites through the discharge of tons of crude fecal material from a main sewage channel, nevertheless provides drinking water to two-thirds of La Plata, Argentina, after conventional purification at a processing plant. With intestinal parasitosis being endemic here, we investigated the importance of this water in transmitting such pathogens to the city's populace by means of standard methodology for sample acquisition and processing involving filter-concentration of waterborne particulates. Of 14 tap-water samples collected from the distribution network, 12 pertained to four zones (A-D) within the city center; while the remaining 2 were obtained near the processing plant, 15 kilometers outside the city. Although parasites were found within the samples derived from the four urban zones, none were detected in the specimens obtained near the plant. The four downtown areas differed from each other as to the quantity and nature of the parasites present in their water: whereas zones A and B registered similar lower levels of contaminants, C and D exhibited higher values significantly different from the former two and from each other. Given an average parasite count/l citywide of 0.38 and a probability of encountering a parasite within 11 of water of 0.32, the municipal network is seen to contribute to the transmission of intestinal parasites. A routine system of water-quality control is therefore needed throughout the city along with the establishment of infrastructures for locating and eliminating peripheral sources of contamination.     

Applicability of quantitative PCR for determination of removal efficacy of enteric viruses and Cryptosporidium by water treatment processes

This study aims to assess the applicability of quantitative PCR (qPCR) for removal studies of adenovirus, coxsackievirus, echovirus and Cryptosporidium by water treatment processes. Bench-scale coagulation jar tests were performed using the enteric viruses and Cryptosporidium. Standard methods (conventional cell-culture methods for the viruses and an immunofluorescence assay (IFA) for Cryptosporidium) were used to compare to qPCR. A significant correlation between microbial removals determined by qPCR and the standard detection methods and an approximate 1:1 correlation were observed for the challenge microorganisms. The results indicated that qPCR could be a satisfactory alternate for microbial removal studies using a relative quantification approach.     

Supply system factors associated with microbiological drinking water safety in regional New South Wales, Australia, 2001-2007

Aim: To determine factors associated with microbiological safety of public drinking water systems in regional New South Wales (NSW), Australia.     

Occurrence of Giardia and Cryptosporidium in water supplies of Russia and Bulgaria

The aim of the present study was to investigate water supplies in southern Russia and Bulgaria, in order to estimate the occurrence of Giardia and Cryptosporidium in drinking water resources from these countries. A total of 166 water samples of different origin (surface, tap, bottled, well, spring and waste water) were collected from Rostov (southern Russia), Sofia and Varna (Bulgaria) Greater Areas and screened for the detection of Giardia cysts and Cryptosporidium oocysts. The method incorporated concentration of water samples by filtration and flocculation, sucrose purification, (oo)cyst detection/identification by immunofluorescence test and differential interference contrast. Sixteen out of 166 samples (9.6%) were positive for Giardia and 30 (18.1%) positive for Cryptosporidium. Both Giardia and Cryptosporidium were detected in tap, river, well and waste waters. Giardia cysts were additionally detected in bottled water. Particularly some river, waste and well water samples were highly contaminated with (oo)cysts. This study has shown that drinking water supplies in Russia and Bulgaria are subject to contamination with Giardia and Cryptosporidium, with potential hazards for the public health.     

Free-living amoebae, Legionella and Mycobacterium in tap water supplied by a municipal drinking water utility in the USA

Legionella and Mycobacterium can proliferate within free-living amoebae (FLA) where they are protected from disinfectants at concentrations that can kill bacteria but not protozoa. Despite effective treatment of drinking water, microbes can enter water utility distribution systems (DS) and hence the plumbing within building premises. Additionally, biofilm formation may account for the persistence of microbes in the DS. In the present study a domestic water tap in north-central United States (USA) was sampled in March and September 2007 and analysed for FLA, Legionella and Mycobacterium. Identification of organisms was determined by growth on specific culture media, light and electron microscopy, and amplification of DNA probes specific for each organism. In both the spring and fall samples, amoebae, Legionella and Mycobacterium were detected. However, Acanthamoeba was prominent in the spring sample whereas Vahlkampfia and Naegleria were the amoebae detected in the autumn. Bacterial proliferation in laboratory cultures was noticeably enhanced in the presence of amoebae and biofilms rapidly formed in mixed amoebae and bacteria cultures. It is hypothesized that temperature affected the dynamics of FLA species population structure within the DS and that pathogenic bacteria that proliferate within FLA, which are themselves opportunistic pathogens, pose dual public health risks.     

Cryptosporidium oocysts in water for human consumption

A study was carried out on the presence of oocysts ofCryptosporidium spp. in water for human consumption in the Province of Salamanca, Spain, using four different staining techniques: Ziehl-Neelsen, auramine, indirect immunofluorescence with monoclonal antibodies (Meridian, USA) and direct immunofluorescence with monoclonal antibodies (Pasteur, France). The presence of oocysts was detected in the water used for the washing of sand filters in the four water purifying plants and in the drinking water of two of the six geographical areas studied. The Ziehl-Neelsen and auramine staining techniques were found to have excellent diagnostic correlation. Immunofluorescence stainings with monoclonal antibodies proved to be less sensitive.     

Residual microbiological risk after simple chlorine treatment of drinking ground water in small community systems

In order to assess the residual health risk after mere disinfectionof drinking ground water, an epidemiological study was conductedin 24 French villages, among 2,033 schoolchildren aged 7–11years. Thirteen villages which delivered drinking water thatdid not need to be treated because the water naturally met microbiologicalstandards were used as a reference. Eleven other villages wereselected, which were required to disinfect the spring waterbefore distribution because of microbiological contamination.However, after a well-conducted chlorine-only treatment, tapwater also complied with microbiological standards. Acute gastrointestinal(Gl) conditions (diarrhoea and/or vomiting) were recorded dailyduring 8 months using standard questionnaires. The bacteriologicalquality of the water was assessed bi-monthly. The crude incidencerate of diarrhoea was 1.4 times more frequent among childrendrinking treated water than among controls (95% confidence interval:1.30–1.50). This rate ratio was not significantly influencedby the chlorine concentration after treatment. The excess riskwas associated with the occurrence of small epidemics. However,when discarding the epidemic events, the background endemicmorbidity also differed between the 2 groups. This study showsthat after a disinfection-only treatment that produces finishedwater meeting the current bacteriology quality and treatmentefficiency indicators, a low but significant risk of acute Glinfection persists, probably associated with the survival ofsome chlorine-resistant micro-organisms. Effective drinkingwater management should primarily aim at protecting the qualityof natural water. If this does not suffice, this study suggeststhat the treatment of contaminated water should encompass multiplebarriers, not merely disinfection.     

Clinical and environmental distribution of Legionella pneumophila in a university hospital in Italy: efficacy of ultraviolet disinfection

The molecular epidemiology of Legionella pneumophila in the 'V. Monaldi' University Hospital was studied. Seven cases of nosocomial Legionnaires' disease were diagnosed between 1999 and 2003. Two clinical legionella strains obtained from two patients in the adult cardiac surgery unit (CSU) and 30 environmental legionella strains from the paediatric and adult CSUs, neonatal intensive care unit (NICU) and the cardiorespiratory intensive care unit (CR-ICU) were serotyped and genotyped. L. pneumophila serogroup 1/Philadelphia with an identical pulsed-field gel electrophoresis (PFGE) profile A was isolated from two patients in the adult CSU, and from three and one water samples taken in the adult CSU and the paediatric CSU, respectively, from 2001 to 2002. Furthermore, L. pneumophila serogroup 3 with an identical PFGE profile B was identified in 20 environmental strains from all wards, L. pneumophila serogroup 3 with PFGE profile C was identified in a single environmental strain from the CR-ICU, and non-pneumophila Legionella with identical PFGE profile D was identified in five environmental strains from the adult CSU, paediatric CSU and NICU. Ultraviolet irradiation was effective in disinfection of the hospital water supplies in the adult and paediatric CSUs contaminated by L. pneumophila clone associated with nosocomial Legionnaires' disease. In conclusion, these data demonstrate that two cases of nosocomial legionellosis were caused by the persistence of a single clone of L. pneumophila serogroup 1/Philadelphia in the hospital environment, and that disinfection by ultraviolet irradiation may represent an effective measure to prevent nosocomial Legionnaires' disease.     

Sporicidal activity of sodium dichloroisocyanurate, peroxygen and glutaraldehyde disinfectants against Bacillus subtilis

The activity of sodium dichloroisocyanurate (NaDCC), peroxygen and glutaraldehyde disinfectants against spores of Bacillus subtilis NCTC 10073 was evaluated using suspension tests. The activity of aqueous solutions of NaDCC disinfectants increased with the level of available chlorine (av.Cl) but was considerably reduced by low levels of blood. Five percent Titan Sanitizer (1200 ppm av.Cl) achieved a > 10⁵-fold reduction in spore count (kill) in 3 h in the absence of blood but no kill in 3 h with 2% blood present. One percent Presept (3180 ppm av.Cl) achieved a > 10⁵-fold kill in 1 h in the absence of blood and in 2 h with 2% blood present. One percent Haz-Tab (5750 ppm av.Cl) achieved a >10⁵-fold kill in 5 min in the absence of blood and in 30 min with 2% blood present. One percent Virkon (peroxygen) achieved a 10⁵-fold kill in 2–3 h in the absence of blood but little kill in 3 h with 2% blood present. Nu-Cidex (peracetic acid) was rapid in action and tolerant of organic matter. A 24 h old solution achieved a > 10⁵-fold kill in 5 min with 10% serum present. Cidex Long-Life (glutaraldehyde) worked much slower; a 28-day-old solution achieved a >10⁵-fold kill in 2 h with 4% blood present. Neat sporicidin (glutaraldehyde-phenate) was slightly superior to Cidex Long-Life but in a 1 in 8 dilution exhibited markedly reduced activity; a 30-day-old solution achieved a 10⁴-fold kill in 10 h in the absence of blood but only a 10²-fold kill in 10 h with 2% blood present.     

An outbreak of Escherichia coli 0157 and campylobacteriosisassociated with contamination of a drinking water supply

Stream water into which treated sewage discharged contaminated the public water supply of a Fife village with a population of about 1100. Of 765 residents and workers who reported illness 711 had gastrointestinal symptoms and 633 were defined as cases. Mains water analysis revealed high faecal coliform counts. An outbreak of Campylobacter and E coli 0157 infection was bacteriologically confirmed in eight and six people respectively. Two of the latter developed haemolytic uraemic syndrome. All affected individuals made full recoveries. Human normal immunoglobulin was offered to those aged between six months and 40 years of age and 618 individuals were immunised. Salivary tests four months after the incident confirmed that no Hepatitis A outbreak had occurred. In a follow-up questionnaire residents generally expressed happiness with the medical response to the incident although many reported that they still had worries. The two major medical issues which emerged from this outbreak were the importance of effective communications and the appropriateness of offering post-exposure human normal immunoglobulin.     

Expression of pertussis toxin subunit S4 as an intracytoplasmic protein in Bacillus subtilis

The expression and secretion of pertussis toxin subunits S1 to S5 in Bacillus subtilis by the aid of a bacillar signal sequence has been reported. While secretion of subunit S1 was high, that of others was low. Ways have now been explored to improve the yield, using S4 as an example. The addition of a protease inhibitor was found to increase the amount of S4 in the culture supernatant, but the final amount was still much below that of S1. However, intracellular expression of S4 gave a high yield (500 mg I⁻¹) and the aggregated protein could easily be isolated in a few simple steps.     

Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine

Heat killed spores of the Gram-positive bacterium Bacillus subtilis have been evaluated as a vaccine delivery system with mucosal adjuvant properties for influenza. Killed spores were able to bind H5N1 virions (NIBRG-14; clade 1) and, when intra-nasally administered to mice, resulting immune responses, both humoral and cell mediated, were enhanced compared to immunization with the virion alone. Levels of both systemic IgG and mucosal sIgA specific to the virion were elevated. Levels of IgG2a (a Th(1) antibody type) were strongly enhanced when the virion was co-administered with killed spores. Cytokine induction in stimulated splenocytes was also apparent indicating balanced T(h)1 and T(h)2 responses. Evidence of cross-neutralization of clade 2.2 viruses was shown. In a challenge experiment mice dosed two times with spores adsorbed with just 20 ng HA (hemagglutinin) of inactivated NIBRG-14 were fully protected against challenge with 20 LD(50) of H5N2 virus. Interestingly, partial protection (60%) was observed in animals dosed only with killed spores. Mice dosed only with killed spores were shown to be fully protected against H5N2 (5 LD(50)) infection indicating that innate immunity and its stimulation by spores may play an important role in protection. Supporting this killed spores were (i) shown to stimulate TLR-mediated expression of NF-κB, and (ii) able to recruit NK cells into lungs and induce maturation of DCs. This work demonstrates the potential and underlying mechanism for the use of bacterial spores as an adjuvant for H5N1 vaccination.     

Evaluation of the efficacy of ultraviolet irradiation for disinfection of hospital water contaminated by Legionella

We evaluated the efficacy of the ultraviolet irradiation on hospital water colonized by Legionella pneumophila serogroup 3, by inserting a lamp system on a hot water pipe supplying a small area. Cultures were performed for four months from 5 L samples of water, collected before and after the ultraviolet treatment at the lamp unit and from two distal points. Irradiation was effective immediately after disinfection (<10 cfu/L), even when the incoming water was highly contaminated. One distal point showed little or no contamination (<10–20 cfu/L), while the other showed little to moderate contamination (<10³ cfu/L). We conclude that ultraviolet irradiation is useful to protect the water system in small area; however, because of the lack of residual activity, it should be combined with other methods of disinfection. Maintenance of the water system is also necessary in order to reduce biofilm formation and Legionella recolonization.     

饮水复合材料除砷滤芯的研制与效果评价

目的　研制饮水复合材料除砷滤芯并在高砷饮水地区进行模拟现场效果评价。方法　以载铁活性炭粉与超细聚乙烯树脂为主要成分制备复合材料除砷滤芯 ,现场使用加标高砷饮水为原水 ,测定了滤芯的安全性、除砷效果以及其它净水作用。结果　以等量载铁活性炭粉和超细聚乙烯树脂制备的复合材料除砷滤芯可安全用于饮用水处理对砷含量为 0 4mg L的实验原水 ,除砷容量可达 2 5 6mg g ,同时对水质浊度和耗氧量指标有明显改善。结论　以载铁活性炭粉与超细聚乙烯树脂为主要成分制备复合材料除砷滤芯适合于农村高砷饮水的除砷     

The effect of anti-spore antibody responses on the use of spores for vaccine delivery

Among the different types of bacteria being exploited as live vaccines Bacillus endospores have a number of distinct advantages most importantly being their heat stability. Typically antigens are displayed on the spore surface or expressed in the germinating spore, i.e., the vegetative cell. How antigens are delivered by spores can significantly affect the nature of the resulting immune response. When antigens are expressed in the germinating spore, then, after the first dose of recombinant spores, ensuing humoral responses are abruptly arrested. We have investigated this phenomenon by first demonstrating that while immune responses against the encoded antigen are impaired this is not the case for anti-spore responses that progressively increase until hyperimmunity is reached. We reasoned that anti-spore IgG or sIgA could be responsible for inhibiting spore germination, thus preventing expression of the vegetatively expressed antigen. In this work we have demonstrated that antisera from both immunised and naïve animals can bind to spores non-specifically yet only IgG or sIgA from immunised animals can bind specifically. Both immune and naïve antibodies were found to inhibit germination in vitro and most probably non-specific binding could account for this. On the other hand only immune antibodies were capable of opsonising spores and enhancing their uptake by macrophages and we reason that specific antibody-spore binding is required. Opsonophagocytosis could be an important tool to reduce spore germination and subsequent outgrowth. With regard to use for heterologous antigen delivery though, this severely curtails the use of a repetitive dosing regime.     

Dissemination of Bacillus cereus in a paediatric intensive care unit traced to insufficient disinfection of reusable ventilator air-flow sensors

Over a two-week period in November 2006, vancomycin-resistant Bacillus cereus was isolated from respiratory samples from six ventilated paediatric intensive care unit (PICU) patients. To investigate the possibility of a common source and extent of the dissemination, all procedures related to mechanical ventilation were monitored and surveillance cultures performed. B. cereus was isolated from reusable air-flow sensors, before and after on-site disinfection with 70% alcohol. The organism was also isolated from respiratory samples from three other ventilated patients and from two ventilation grids in the ceiling of PICU, as well as from the alcohol solution itself. Using amplified fragment length polymorphism (AFLP) typing, B. cereus strains from the six PICU patients together with isolates recovered from the air-flow sensors and the alcohol solution were shown to be closely related. Isolates from the ventilation grids demonstrated different AFLP patterns to the outbreak strain. Intervening measures, including disinfection by autoclaving all reusable air-flow-guiding parts and the use of disposable non-autoclavable parts, resulted in rapid termination of the outbreak. B. cereus infections can cause significant morbidity, particularly in intensive care patients. Disinfection of all air-flow-guiding reusable parts for mechanical ventilation should be addressed with great care and should include effective autoclaving in order to eradicate spores.     

Dynamics of Aeromonas species isolated from wastewater treatment system

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of São Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism's dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.     

Disconnecting central hot water and using electric showers to avoid colonization of the water system by Legionella pneumophila: an 11-year study

Legionella spp. can be difficult to control in hospitals. The objective of this study was to describe an 11-year experience with the use of electric showers in the control of Legionella pneumophila. From June 1989 to March 1990 there was an outbreak of pneumonia caused by L. pneumophila in a 20-bed renal transplant unit in a university-associated tertiary-care hospital. Control measures included hyperchlorination, heating and flushing of the water system with limited results. In November 1993 the central hot water was disconnected and water for bathing was heated using electric showers. From January 1992 to June 1995 water was collected from showers and water faucets and cultured for L. pneumophila every two weeks. Surveillance cultures were then collected every month until May 1999. During this seven-year surveillance period, 1115 samples of water were cultured. Water cultures were positive on 24 of 429 occasions (without cases of legionellosis) during the pre-shower period (22 months). In the post-shower period (67 months) only one of 686 cultures was positive. Subsequently there have been no new cases of nosocomial pneumonia by L. pneumophila although surveillance continues. In conclusion, disconnecting the central hot water was effective in avoiding colonization of the water system by L. pneumophila. Heating was possible by using electric showers, which are effective, easy to maintain and cheap.     

Seasonal prevalences of Cryptosporidium and Giardia infections in children attending day care centres in Salamanca (Spain) studied for a period of 15 months

Five studies were carried out in children younger than 4 years old attending regularly day care centres in order to determine the intestinal parasitization by Cryptosporidium and Giardia. The percent of children parasitized by Cryptosporidium was 10% (17 children out of 170 studied). Cryptosporidiosis was more frequent in winter than in other seasons (difference statistically significant). Giardia intestinulis which was the most frequent parasite, was identified in 25.3% (43 children out of 170 studied). Giardiasis was more frequent in autumn and in the low SEL (difference statistically significant in both cases). The frequency of giardiasis increased from 8% in children studied during 1–2 seasons, to 34% in children studied for 3–5 seasons (difference statistically significant), whereas the frequency of cryptosporidiosis did not show variation with increased number of specimens studied per child. The 35% of children parasitized by Cryptosporidium sp. showed gastrointestinal symptoms but only 14% of children with Giardia intestinalis.