Detection and identification of Giardia species using real-time PCR and sequencing.

We report a specific region of Giardia spp. 18S ribosomal RNA (18S rDNA) that serves as an ideal target for quantitative PCR (qPCR) detection and sequencing to identify Giardia species, including the clinically-relevant G. duodenalis, in clinical and environmental samples. The presence of multiple copies of the 18S rDNA gene and variations in the selected 18S genomic region enabled the development of a rapid, sensitive qPCR screening method for the detection of Giardia spp. The analytical sensitivity of the Giardia qPCR assay was determined to be a cyst equivalent of 0.4 G. duodenalis cysts per PCR reaction. Amplicon sequencing of the PCR product confirmed Giardia spp. detection and among the 35 sequences obtained, 31, 3 and 1 isolates were classified as belonging to G. duodenalis, G. microti and G. muris, respectively. The TaqMan assay reported here may be useful for the detection of low levels of Giardia in clinical and environmental samples, and further enables the effective use of direct sequencing of the PCR product for Giardia confirmation and to identify major species of Giardia, including G. duodenalis.

Environmental Survey of Drinking Water Sources in Kampala, Uganda, during a Typhoid Fever Outbreak.

In 2015, a typhoid fever outbreak began in downtown Kampala, Uganda, and spread into adjacent districts. In response, an environmental survey of drinking water source types was conducted in areas of the city with high case numbers. A total of 122 samples was collected from 12 source types and tested for Escherichia coli, free chlorine, and conductivity. An additional 37 grab samples from seven source types and 16 paired large volume (20 liter) samples from wells and springs were also collected and tested for the presence of Salmonella enterica serovar Typhi. Escherichia coli was detected in 60% of kaveras (drinking water sold in plastic bags) and 80% of refilled water bottles; free chlorine was not detected in either source type. Most jerry cans (68%) contained E. coli and had free chlorine residuals below the WHO-recommended level of 0.5 mg/liter during outbreaks. Elevated conductivity readings for kaveras, refilled water bottles, and jerry cans (compared to treated surface water supplied by the water utility) suggested that they likely contained untreated groundwater. All unprotected springs and wells and more than 60% of protected springs contained E. coli Water samples collected from the water utility were found to have acceptable free chlorine levels and no detectable E. coli While S Typhi was not detected in water samples, Salmonella spp. were detected in samples from two unprotected springs, one protected spring, and one refilled water bottle. These data provided clear evidence that unregulated vended water and groundwater represented a risk for typhoid transmission.IMPORTANCE Despite the high incidence of typhoid fever globally, relatively few outbreak investigations incorporate drinking water testing. During waterborne disease outbreaks, measurement of physical-chemical parameters, such as free chlorine residual and electrical conductivity, and of microbiological parameters, such as the presence of E. coli or the implicated etiologic agent, in drinking water samples can identify contaminated sources. This investigation indicated that unregulated vended water and groundwater sources were contaminated and were therefore a risk to consumers during the 2015 typhoid fever outbreak in Kampala. Identification of contaminated drinking water sources and sources that do not contain adequate disinfectant levels can lead to rapid targeted interventions.

Relative insignificance of virus inactivation during aluminum electrocoagulation of saline waters.

Combined removal and inactivation of the MS2 bacteriophage from model saline (0-100 mM NaCl) waters by electrochemical treatment using a sacrificial aluminum anode was evaluated. Both chemical and electrodissolution contributed to coagulant dosing since measured aluminum concentrations were statistically higher than purely electrochemical predictions using Faraday's law. Electrocoagulation generated only small amounts of free chlorine in situ but effectively destabilized viruses and incorporated them into Al(OH)3(s) flocs during electrolysis. Low chlorine concentrations combined with virus shielding and aggregation within flocs resulted in very slow disinfection rates necessitating extended flocculation/contact times to achieve significant log-inactivation. Therefore, the dominant virus control mechanism during aluminum electrocoagulation of saline waters is "physical" removal by uptake onto flocs rather than "chemical" inactivation by chlorine. Attenuated total reflectance-Fourier transform infrared spectroscopy provided evidence for oxidative transformations of capsid proteins including formation of oxyacids, aldehydes, and ketones. Electrocoagulation significantly altered protein secondary structures decreasing peak areas associated with turns, bends, α-helices, ß-structures, and random coils for inactivated viruses compared with the MS2 stock. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements showed rapid initial RNA damage following a similar trend as plaque assay measurements of infectious viruses. However, ssRNA cleavage measured by qRT-PCR underestimated inactivation over longer durations. Although aluminum electrocoagulation of saline waters disorders virus capsids and damages RNA, inactivation occurs at a sufficiently low rate so as to only play a secondary role to floc-encapsulation during residence times typical of electrochemical treatment.

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp.

We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3'-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5'-end forms a hairpin structure. A fluorescent dye is attached to 5'-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3' dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000-0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource-limited environments.

Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).

RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.

Broadly reactive TaqMan assay for real-time RT-PCR detection of rotavirus in clinical and environmental samples. JIN2@cdc.gov.

Rotaviruses are enteric pathogens responsible for a significant burden of disease, especially in children, through person-to-person transmission and exposure to contaminated food and water. In the present study, a TaqMan probe-based real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and validated for sensitive and specific detection and quantification of rotavirus for the routine screening of clinical and environmental samples. The assay primers and probes were designed to target the non-structural protein region 3 (NSP3) of rotavirus. The rotavirus real-time RT-PCR assay was found to be specific to rotavirus, but broadly reactive to rotavirus genogroups 1-4, 9, 10 and 12. Specificity testing did not identify any cross-reactivity of the assay with a panel of 36 non-rotavirus enteric virus specimens. The sensitivity of the assay was determined using quantified rotavirus stocks and a plasmid DNA stock. Estimated detection limits in reagent-grade water were five genome equivalent copies (GEC) per reaction and two to four rotavirus particles per reaction. The sensitivity of the assay for detecting rotaviruses in environmental water samples was found to be six virus particles per reaction. The rotavirus real-time RT-PCR assay was effective in detecting rotavirus in all 79 stool specimens obtained from a hospital in India. The results of this study demonstrate that the real-time RT-PCR assay for rotavirus is broadly reactive, specific, and sensitive for detection of rotaviruses in clinical specimens and water samples.

Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays.

Rapid identification of the two major species of Cryptosporidium associated with human infections, Cryptosporidium hominis and Cryptosporidium parvum, is important for investigating outbreaks of cryptosporidiosis. This study reports the development and validation of a real-time PCR TaqMan procedure for detection of Cryptosporidium species and identification of C. hominis and C. parvum in stool specimens. This procedure comprised a generic TaqMan assay targeting the 18S rRNA for sensitive detection of Cryptosporidium species, as well as two other TaqMan assays for identification of C. hominis and C. parvum. The generic Cryptosporidium species assay can be duplexed with the C. parvum-specific assay. The generic Cryptosporidium species assay was able to detect ten Cryptosporidium species and did not cross-react with a panel of ten other protozoan parasites. The generic Cryptosporidium species assay could detect 1-10 oocysts in a 300 microl stool specimen, whilst each of the species-specific TaqMan assays had detection sensitivities that were approximately tenfold higher. The 18S rRNA assay was found to detect Cryptosporidium species in 49/55 DNA extracts from stool specimens containing either C. hominis or C. parvum. The C. hominis TaqMan assay correctly identified C. hominis in 24/31 validation panel specimens containing this species. The C. parvum-specific assay correctly identified C. parvum in 21/24 validation panel specimens containing this species. This real-time PCR procedure was used to detect and identify C. hominis and C. parvum in stool specimens from outbreak investigations in the USA and Botswana, resulting in identification of C. hominis and/or C. parvum in 66/67 stool specimens shown to be positive for these species using other techniques. From the outbreak specimens tested, the TaqMan procedure was found to have a specificity of 94%. This TaqMan PCR procedure should be a valuable tool for the laboratory diagnosis of cryptosporidiosis caused by C. hominis and C. parvum during outbreak investigations.

Development and evaluation of a broadly reactive TaqMan assay for rapid detection of hepatitis A virus.

Primers and a TaqMan probe for the 5'-untranslated region (UTR) of the hepatitis A virus (HAV) genome were designed and evaluated. The assay detected 0.5 infectious units of HAV and 40 copies of a synthetic transcript and provides an important screening tool for rapid quantitative HAV detection in clinical or environmental samples.

Air pollution in stone crushing industry, and associated health effects.

Stone crushers are small scale industries in the unorganised sector. They provide basic material for road and building construction. They are highly labour intensive. The various unit operations involved in stone crushing viz., size reduction, size classification and transfer operations have the potential to emit process and fugitive dust. A detailed air pollution survey was conducted at Pammal, 26 km to the southwest of Chennai. High volume and respirable particulate samplers were deployed at seventeen locations to monitor SPM and PM10 levels in ambient air. The particle size analysis indicates high percentage of finer particles and silica content posing serious health problems to the people exposed for longer duration. Personal samplers were employed to quantify the total dust and respirable particulate fraction in the work environment, which was found significantly high, when compared to the occupational safety and health standards. Fine inhalable particulate matter (PM2.5) which has more associated human health problems was found high in the work place of stone crushers. Health survey viz., Pulmonary function test, blood sample test, general clinical evaluation was conducted to assess the extent of the damage caused to the workers. This study indicates that most of the people are having respiratory problems. The measurements show that good house keeping practice is essential for effective control of dust, in addition to National Productive Council's (NPC) measures.

Isolation of coliphages specific to enterotoxigenic E. coli (ETEC).

Bacteriophages specific to Enterotoxigenic E. coli (ETEC) are reported for the first time. Out of 15 isolated phages only 10 were specific to strains of ETEC. All ten phages of dsDNA could be grouped into three different genotypes based on their RAPD patterns observed and it is likellly that they belong to only 3 different strains. The three phages yielded clear plaques on 10 strains of ETEC within 4-6 h at 37 degrees C.

A field test for the assessment of faecal contamination of potable water.

A simple, sensitive, rapid, inexpensive paper strip impregnated with Salmonellal E. coli medium (SEM) was formulated, and placed in a test tube. When potable water of 10 ml was added to the test tube it detected the faecal contamination of water samples within 16-48 h when incubated at room temperature from 20 to 35 degrees C. The positive results were indicated when the medium turned black (hydrogen sulfide production) for the presence of Salmonella sp. and/or the formation of a red ring (free indole from tryptophan) when a few drops of Kovac's reagent was added for the presence of coliform bacteria (E. coli). More than 600 water samples were tested with the new test (SEM) and results showed 99% agreement with that of the standard most probable number (MPN) coliform test and also proved highly successful in the field when it was employed to detect both Salmonella and E. coli. Different water testing laboratories involved in a water quality monitoring programme and governmental agencies evaluated the test media and reported that the test was user friendly, reliable and simple to perform in the field and will be especially useful for screening of both urban and rural water supplies for routine monitoring of bacteriological contamination.

Duplex RT-PCR for simultaneous detection of hepatitis A and hepatitis E virus isolated from drinking water samples.

A duplex reverse transcription polymerase chain reaction (RT-PCR) protocol for simultaneous detection of hepatitis A virus (HAV) and hepatitis E virus (HEV) in water samples has been developed and demonstrated. Both HAV and HEV were concentrated from drinking water samples through a one-step concentration protocol. Different cDNA could be produced in the RT step carried out with a random primer in a single reaction tube. Two different sets of primers specific for HAV-cDNA and HEV-cDNA were used for PCR amplification. Amplified DNA products representing HAV and HEV were identified by gel electrophoresis at 247 and 327 bp (base pair) sequences, respectively. Specific sets of primers amplified a single type of virus and no cross-reactivity of the primers was noticed in duplex RT-PCR. The protocol was used for direct isolation and detection of HAV and HEV from 23 water samples in urban areas of Chennai city. Out of these, nine water samples were positive for HAV, and three for HEV. All three samples positive for HEV were also positive for HAV. The test provides a rapid and economical means of water quality surveillance to specifically detect HAV and HEV.

Removal of Escherichia coli O157:H7 from surface tissues of beef carcasses inoculated with wet and dry manure.

Beef tissues were contaminated with wet and dry manure. The manure was previously inoculated with Escherichia coli O157:H7 GFP, genetically modified with a plasmid encoding a protein that fluoresces green when exposed to long-wave ultraviolet light. After incubation at 37 degrees C for 5 days, the wet manure was spread on the surface of beef tissues at an average E. coli O157:H7 GFP level of 6.62 log CFU/cm2. Dry manure was obtained by subjecting wet manure to natural drying (simulating dry manure adhering to the hides of cattle) and was also applied to the surfaces of beef tissues. The degree of removal of E. coli O157:H7 GFP by washing was compared to the removal of cells of the same strain that had been inoculated as a suspension. The E. coli O157:H7 mixed into feces of cattle adhered more strongly to meat surfaces than that applied as a suspension, complicating the removal by conventional washing procedures. The fate of the bacterium mixed into wet or dry manure was evaluated. An initial decrease of the inoculated population was observed; this was probably an effect of the changed environment represented by the manure. After adaptation, the inoculated bacteria grew in the wet manure; a maximum population was reached in 5 days at 37 degrees C; levels declined with drying. The use of the GFP marker was of great value, since it allowed enumeration of E. coli O157:H7 in the presence of the natural flora of manure.

Immunomagnetic capture PCR for rapid concentration and detection of hepatitis A virus from environmental samples.

We studied the concentration of hepatitis A virus (HAV) from environmental samples by membrane filter-based urea-arginine phosphate buffer and its detection by using immunomagnetic capture (IC) reverse transcription (RT)-PCR (IC PCR). Magnetic beads coated with anti-HAV rabbit antibodies were used for enrichment and concentration of HAV from environmental samples. IC PCR is sensitive enough to detect as few as 0.04 PFU of cell culture-adapted HAV in inoculated water and sewage samples. IC PCR is specific and does not yield positive reactions with poliovirus 1, HAV RNA, or selected bacteriophages. IC concentrates viruses suspended in small volumes to microliter volumes that can be used directly in RT-PCR. IC concentration of viruses from sewage samples without concentration of inhibitory substances is important for successful RT-PCR detection. In a field trial, 2 of 18 raw sewage samples tested by IC PCR were positive for HAV.

Elution and reconcentration of coliphages in water from positively charged membrane filters with urea-arginine phosphate buffer.

Coliphages in drinking water and waste water samples have been adsorbed onto positively charged membrane filters, eluted with urea-arginine phosphate buffer (UAPB), reconcentrated, and detected with Escherichia coli C (ATCC 13706). The proposed membrane filter-based UAPB method for concentration and detection of coliphages compares favorably with the beef extract elution and reconcentration procedure and also with the proposed coliphage detection procedure described in Standard Methods for the Examination of Water and Wastewater. The higher recovery of coliphages with UAPB elution from positively charged membrane filters is attributed to testing the whole volume of concentrated sample, rather than partial analysis of the sample as in the procedure described in Standard Methods for the Examination of Water and Wastewater, especially when the titre is very low.

A simple device for the concentration and detection of enterovirus, hepatitis E virus and rotavirus from water samples by reverse transcription-polymerase chain reaction.

A simultaneous concentration of enteroviruses, hepatitis E virus, and rotavirus from drinking water samples through a filtration column filled with granular activated carbon (GAC) was achieved. Urea-arginine phosphate buffer (UAPB) as an eluent at pH 9.0 was used for effective desorption and elution of viruses from GAC. Further concentration of viruses with magnesium chloride enabled nucleic acid extraction, cDNA synthesis, amplification with a specific set of primers for enterovirus, hepatitis E virus and rotavirus. Polymerase chain reaction (PCR) products were then confirmed by Southern transfer and hybridization with the relevant probes. The efficacy of the protocol was established with 100 1 of water samples seeded with poliovirus-1, providing 74% recovery in granular activated carbon based UAPB-RT-PCR. The GAC-based method for concentration of viruses from water samples was preferred, despite its somewhat lower efficacy compared to 80% in membrane filter based UAPB-RT-PCR protocol, due to the specific requirements of short-time and savings in cost of analyses. The protocol was used for the detection of waterborne viruses from 24 drinking water sources in urban areas of New Delhi. Direct isolation of viruses from water samples revealed that the 4 samples were positive for enteroviruses, two for hepatitis E virus, and 10 samples for rotavirus. One sample was positive for both hepatitis E virus and rotavirus, and another for all the 3 types of viruses.

Detection of hepatitis E virus in raw and treated wastewater with the polymerase chain reaction.

The main objective of this study was to determine the applicability of the polymerase chain reaction (PCR) to detection of hepatitis E virus (HEV) in sewage treatment plants and establishment of the prevalence of hepatitis viral diseases in a population. Epidemics of HEV infection because of inadequate public sanitation have been reported in several developing countries. A procedure for concentration of HEV in sewage samples through adsorption to membrane filters, elution with urea-arginine phosphate buffer, and subsequent reconcentration with magnesium chloride enabled us to concentrate HEV to volumes in the microliter range. HEV-specific cDNA was prepared by reverse transcription of the total RNA extracted from samples. Specific DNA amplification by PCR in combination with slot blot hybridization was used to demonstrate the presence of HEV in sewage samples from the inlets and outlets of three sewage treatment plants. The assay was specific for HEV, and a 240-bp amplified product was visualized by ethidium bromide fluorescence. Sewage samples adjusted to pH 5.0 for adsorption of viruses to membrane filters were PCR positive, while samples adjusted to pH 3.5 were PCR negative.

Anoxygenic degradation of aromatic substances by Rhodopseudomonas palustris.

Three strains of the phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris were isolated from different environments and were evaluated for their aromatic degradative potential under phototrophic conditions. All three strains (PFR1, PNR4, and MRL1) utilized benzoate, 4-hydroxybenzoate, 4-aminobenzoate, 4-aminophenol, cinnamate, ferulate, phloroglucinol, and 4-dimethylaminobenzaldehyde in the absence of exogenous CO2. 4-Aminobenzoate and 4-aminophenol served as a carbon and nitrogen source for all the three strains. Utilization of 4-aminophenol was enhanced in the presence of 4-hydroxybenzoate. Salicylate was utilized by PFR1 and MRL1 strains, and phenol was utilized by the MRL1 strain only in the presence of exogenous CO2.

Rapid detection of waterborne viruses using the polymerase chain reaction and a gene probe.

We describe a membrane-filter-based urea-arginine phosphate buffer method for concentrating waterborne viruses from large volumes of water to microlitre volumes, and their subsequent detection by the polymerase chain reaction (PCR). The detection step involves the extraction of RNA, synthesis of complementary DNA, amplification by PCR of target DNA with specific primers, and confirmation through nucleic acid hybridization with a radiolabelled oligonucleotide probe. The PCR technique detected the presence of enteroviruses in spiked as well as in contaminated water samples. The technique is sensitive and detects as few as 120 waterborne viral particles. PCR is simple, rapid, sensitive, specific and adaptable for water quality surveillance in less developed countries.