key: cord-0285226-bidk3k4j
authors: Liu, P.; Ibaraki, M.; Kapoor, R.; Amin, N.; Das, A.; Miah, M. R.; Mukhopadhyay, A. K.; Rahman, M.; Dutta, S.; Moe, C.
title: Development of Moore Swab and Ultrafiltration Concentration and Detection Methods for Salmonella Typhi and Salmonella Paratyphi A in Wastewater and Application in Kolkata, India and Dhaka, Bangladesh
date: 2021-03-26
journal: nan
DOI: 10.1101/2021.03.20.21254025
sha: 766cda391c7709a3dc4c7d6bee917afb5e94dc91
doc_id: 285226
cord_uid: bidk3k4j

Enteric fever is a severe systemic infection caused by Salmonella enterica serovar Typhi (ST) and Salmonella enterica serovar Paratyphi A (SPA). Detection of ST and SPA in wastewater can be used as a surveillance strategy to determine burden of infection and identify priority areas for water, sanitation, and hygiene interventions and vaccination campaigns. However, sensitive and specific detection of ST and SPA in environmental samples has been challenging. In this study, we developed and validated two methods for concentrating and detecting ST/SPA from wastewater: the Moore swab trap method for qualitative results, and ultrafiltration (UF) for sensitive quantitative detection, coupled with qPCR. We then applied these methods for ST and SPA wastewater surveillance in Kolkata, India and Dhaka, Bangladesh, two enteric fever endemic areas. The qPCR assays had a limit of detection of 17 equivalent genome copies (EGC) for ST and 25 EGC for SPA with good reproducibility. In seeded trials, the Moore swab method had a limit of detection of approximately 0.05-0.005 cfu/mL for both ST and SPA. In 53 Moore swab samples collected from three Kolkata pumping stations between September 2019 to March 2020, ST was detected in 69.8% and SPA was detected in 20.8%. Analysis of sewage samples seeded with known amount of ST and SPA and concentrated via the UF method, followed by polyethylene glycol precipitation and qPCR detection demonstrated that UF can effectively recover approximately 8 log10 cfu, 5 log10 cfu, and 3 log10 cfu of seeded ST and SPA in 5 L, 10 L, and 20 L of wastewater. Using the UF method in Dhaka, ST was detected in 26.7% (8/30) of 20 L drain samples with a range of 0.11- 2.10 log10 EGC per 100 mL and 100% (4/4) of 20 L canal samples with a range of 1.02 - 2.02 log10 EGC per 100 mL. These results indicate that the Moore swab and UF methods provide sensitive presence/absence and quantitative detection of ST/SPA in wastewater samples, and these two methods can be used jointly or separately for Salmonella Typhi environmental surveillance.

Typhoid and Paratyphoid fevers are leading causes of severe febrile disease in low-income countries with poor access to safe water, food, and sanitation. 1 The etiologic agents, S. Typhi (ST) and S.

Paratyphi A (SPA), are human-specific pathogens transmitted through consumption of food and water contaminated by feces of an acutely or chronically infected person. 2 Previous studies have shown that food and water contaminated with human feces are associated with typhoid and paratyphoid outbreaks. 3, 4 In endemic urban settings, co-location of sewer pipes and poorly maintained water supply pipes facilitate cross-contamination and transmission of ST through the water system. Typhoid fever outbreaks 4, 5 have been associated with contaminated piped water and sewage-irrigated produce, emphasizing the importance of studying these pathways of disease transmission.

Detection of ST and SPA in drinking water, irrigation water, and environmental waters has usually been attempted in association with outbreak investigations 6, 7 and assessing risk of waterborne typhoid fever transmission in endemic settings. 8 Recently, we and other investigators have proposed detection of ST and SPA in wastewater from known catchment populations as a strategy to determine the burden of typhoid and paratyphoid fever in areas where clinic-based surveillance has limited sensitivity or is not feasible. 9, 10 However, environmental surveillance for typhoid and paratyphoid fever requires sensitive and specific methods to detect ST/SPA in wastewater, including samples where low pathogen concentrations are expected. It is usually necessary to concentrate ST/SPA in environmental samples in order to increase the sensitivity of detection. A wide range of methods have been used to concentrate bacterial pathogens from a variety of environmental waters. 10 Among these methods, Moore swabs and ultrafiltration (UF) are two important and distinct concentration methods that have been shown to be effective in recovering ST from water and wastewater. 11 The Moore swab was first introduced for Salmonella detection from sewage in 1948 in England during a paratyphoid epidemic. 6 Subsequently, this method has been successfully used to isolate Vibrio Cholerae 12 , poliovirus 13 and Burkholderia pseudomallei 14 from sewage. In contrast to the Moore swab method that only shows the presence or absence of a target pathogen, UF is a quantitative method that can simultaneously concentrate multiple pathogens from large volumes of water or wastewater, [15] [16] [17] and we recently reported the application of this method to detect S. Typhi in wastewater samples. 18 The Moore swab method offers several advantages over UF, specifically, Moore swabs are inexpensive, simple to use, and do not require collecting and transporting large volume samples of water or wastewater. However, UF can provide quantitative results with greater sensitivity which are valuable for microbial risk assessment or estimating infection prevalence. 18 The ability to detect and quantify ST/SPA in the environment is critical for monitoring and controlling transmission particularly in urban settings in low-and middle-income countries.

Historically, culture-based isolation and identification methods are considered the gold standard because infectious bacteria can be detected. However, culture of S. Typhi from environmental samples is challenging. 19 Alternatively, PCR and qPCR technologies have been widely used to rapidly detect and quantify ST/SPA due to higher sensitivity and specificity and short turnaround time. Limitations of molecular detection methods include the inability to distinguish between All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The known EGCs for each standard were serially diluted, and a standard curve was incorporated in each real-time PCR assay. Ten-fold serial dilution of the standards was used to estimate the numbers of genome copies of the target pathogens in samples.

Large volume wastewater samples (5-20 L) were seeded with known amounts of ST and SPA (either 8 log 10 CFU, 5 log 10 CFU, or 3 log 10 CFU), and then each sample was concentrated by hollow fiber ultrafiltration ( Figure 1 ). The entire amount of the seeded wastewater sample was circulated through Polynephron TM Synthetic Hollow-Fiber Dialyzer (NIPRO All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Medical United States, Bridgewater, USA) using a peristaltic pump to achieve approximately 100 mL of retentate (concentrated sample) which was collected in a 500 mL bottle. Ultrafilter elution was then performed using 500 mL of PBS with 0.01% Tween 80, 0.01% sodium polyphosphate and 0.001% Antifoam Y-30 emulsion. The elution solution was recirculated for 5 mins, and the final eluate was merged into the concentrated sample. After elution, the ultrafilter was backwashed using 250 mL of PBS with 0.5% Tween 80, 0.01% sodium polyphosphate and 0.001% Antifoam Y-30 emulsion. The backwash fraction was added to the previous mixture of the concentrate and the eluate. Figure 2C ). Sewage samples were seeded with either 50 cfu/mL, 20 cfu/mL, 10 cfu/mL, 5 cfu/mL, 0.05 cfu/mL, or 0.005 cfu/mL of ST and SPA cells. The swab was submerged in the sewage to trap the ST and SPA cells while stirred continuously with an overhead spatula for 24 hours as shown in Figure 2C . The swabs were then transferred to 450 mL of universal pre-enrichment broth (EPA, All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. hrs with shaking. Then, a 20 mL volume of the pre-enrichment broth was filtered through a 0.45 µm filter and subjected to DNA extraction as described below after the addition of 1 mL of InhibitEX buffer ( Figure 2A ).

For each sample or blank control, 1 mL of the final sample suspension, mixed with the InhibitEX buffer, was transferred into a 2 mL microcentrifuge tube. For membranefiltered samples and controls, microcentrifuge tubes with the filter and 1 mL of InhibitEX buffer were used. After one minute of vortexing, the solution was centrifuged at maximum speed for 2-3 minutes, and the suspension was incubated at 95°C for 5 minutes, followed by full speed microcentrifugation for 15 seconds to pellet the sample particles. Subsequently, 600 µl of the supernatant was transferred into a 2 ml microcentrifuge tube containing 25 µL proteinase K, and then 600 µL of buffer AL was added. After vortexing for 15 seconds, the sample was incubated at 70°C for 10 minutes, and 600 µL of 100% ethanol was added to the lysate. A 600 µL volume of lysate was applied to a single QIAamp Mini column (Qiagen, Cat. # 51604, Hilden, Germany), and the column was centrifuged at full speed for 1 minute. This procedure was repeated two additional times with the same column in order to use up all of the lysate. Finally, TNA was eluted from the column with 150 µL of the supplied elution buffer. The extracted TNA samples were aliquoted and stored at -80°C until analyzed by qPCR. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. and SPA DNA standards, and the wastewater concentration factor. The quantification was expressed as log 10 EGC per 100 mL wastewater. Experiments evaluating the limit of detection of the Moore swab and UF concentration method were repeated three times for each volume and reported seeding level. Experiments for assessing ST and SPA recovery efficiencies were also repeated three times All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Where:

Two singleplex TaqMan real-time qPCR assays were evaluated for the detection of ST and SPA species using primers/probe previously described by Kaykey et al. 21 To evaluate the analytical sensitivity of the two qPCR assays, DNA from ST strain (ATCC (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 20 L wastewater samples with a mean total of 3.49 log 10 cfu ST resulted in the recovery of an average 2.81 log 10 EGC ST ( Figure 4A) . Similarly, seeding an average total of 7.94 log 10 cfu SPA led to a mean total recovery of 5.80 log 10 EGC SPA. When an average total of 5.0 log 10 cfu SPA was seeded, an average total of 3.62 log 10 EGC SPA was recovered. Seeding with a mean total of 4.07 log 10 cfu SPA resulted a total of 3.32 log 10 EGC SPA recovery ( Figure 4B ). These results indicate that UF followed by PEG concentration can effectively recover different concentrations of ST and SPA seeded in large volumes of sewage, but there is about a 2 log 10 loss of the target bacteria during the concentration, TNA extraction, and qPCR process.

To examine if sample volume affects ST and SPA recovery using UF, an average total of 9.23 log 10 , 8.54 log 10 , and 9.13 log 10 cfu ST were seeded into 5 L, 10 L, and 20 L sewage samples, respectively. Mean totals of 7.57 log 10 , 6.13 log 10 , and 6.84 log 10 EGC ST were recovered ( Figure 5A ), respectively. Similarly, when an average total of 8.73 log 10 , 8.74 log 10 , and 7.94 log 10 cfu SPA was seeded into 5 L, 10 L, and 20 L of sewage water, respectively, a mean total of 6.82 log 10 , 5.97 log 10 , and 5.70 log 10 EGC SPA was recovered, respectively ( Figure 5B ). These results indicate that sample volumes in the range of 5-20 liters did not affect the efficiency of ST and SPA recovery by UF.

In the study neighborhood in Dhaka, wastewater from toilets shared by multiple households (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254025 doi: medRxiv preprint EGC/100 mL with a mean of 0.82 log 10 EGC/100 mL. In the canal samples, the estimated ST concentrations ranged from 0.71 to 2.14 log 10 EGC/100 mL (mean 1.43 log 10 EGC/100 mL) (Table 3 ). These results indicate that ultrafiltration is an effective quantitative method for ST detection in wastewater samples. (Table 4 ). These results suggest that ST and SPA infections are endemic in the city wards in Kolkata that are served by these pumping stations, and that the Moore swab method is an effective low-cost method for ST and SPA sewage surveillance.

Environmental surveillance offers a low-cost, non-invasive, and sensitive strategy to characterize the burden of infection in specific populations for pathogens that may be challenging to diagnose through collection and analyses of clinical specimens -such as poliomyelitis, typhoid fever, and recently, COVID-19. [22] [23] [24] [25] The goal of this study was to develop and validate sensitive and specific methods for concentration and PCR detection of ST and SPA in wastewater for typhoid environmental surveillance and also in other environmental samples that could serve as vehicles of ST and SPA transmission. We report here that UF followed by PEG precipitation allowed detection of 1000 cells of ST and SPA seeded in 20 L (0.05 cells per ml) of sewage. Application All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In typhoid-endemic areas of Nepal and Bangladesh, previous investigators have reported the detection of ST in grab samples of drinking water that were concentrated by membrane filtration, followed by DNA extraction from the filter and PCR analyses. 21, 26 Moore swabs were instrumental in the detection of ST in sewage canals near Santiago, Chile that were used to irrigate vegetable crops typically eaten without cooking. 6 These findings indicate risk of ST transmission via drinking water and raw produce, but these samples and methods do not provide quantitative information that could be used to estimate the burden of typhoid infection in the population. Quantitative detection of ST and SPA in wastewater samples collected from sites where the population catchment can be estimated allows modeling approaches to estimate prevalence of typhoid and paratyphoid infection in the catchment population. 9 This goal requires careful selection of sample collection sites and reliable methods to detect ST and SPA in samples that represent excreta from a defined population -typically wastewater samples. Ideally, methods to detect ST and SPA in wastewater for environmental surveillance are quantitative, sensitive, specific, low-cost, and feasible for laboratories in low-resource settings.

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Currently, two types of samples have been used for ST and SPA wastewater surveillance: grab samples and trap samples using Moore swabs. 10 Grab samples are typically concentrated through some type of filtration or centrifugation, sometimes allowed to incubate in an enrichment broth, and then analyzed by culture or PCR. Grab samples can be collected either in small volume or large volume, but microbial concentration from large volume samples can improve the sensitivity of detection. However, the volume of a grab sample that can be feasibly processed may be limited by the turbidity of the wastewater or environmental water that can clog filters UF is an effective technique for concentrating multiple microbes simultaneously. Laboratory studies that seeded selected viruses, bacteria, and parasites into large-volume samples of tap All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254025 doi: medRxiv preprint water and reclaimed wastewater (100 L) reported mean recovery rates of five bacteria, protozoa, and viruses between 38 to 130%. 17, 27 Two UF techniques, hollow-fiber ultrafiltration (TFUF) using tangential-flow 28 and dead-end ultrafiltration (DEUF), 29 provide effective recovery of diverse microbes from different types of water and wastewater, and both methods have showed similar recovery efficiencies in previous studies. 28, 29 The main difference between the DEUF technique and TFUF configuration is that one of the ultrafilter ports is closed so that the water/wastewater sample must pass through the membrane. In contrast, the TFUF technique allows particles and other constituents larger than the membrane pore to be circulated and concentrated into a smaller volume. The DEUF technique is easier to perform than the TFUF because the water passes through the ultrafilter only once. However, the DEUF approach is more likely to clog the ultrafilter than the TFUF configuration and therefore is more appropriate for samples with low turbidity (e.g., tap water or reclaimed water). In this study, we used the hollow-fiber TFUF technique to concentrate turbid samples (e.g., from open drains and canals).

With this method, particles and microbes are maintained in the ultrafilter cartridge under pressure, and there is a low risk of the ultrafilter clogging. 28 Although the UF method has been described previously, 17, 27 our recent deployment of this technique to process field samples in Dhaka, Bangladesh demonstrated the feasibility and value of this method to detect a range of pathogens, including S. Typhi, in high proportions of drain, canal, and flood water samples in a low-resource setting. 18 "Moore swabs" have been used for decades by public health professionals around the world to detect and isolate enteric pathogens from wastewater and environmental waters, and their use to recover typhoidal Salmonella bacteria has recently been reviewed by Sikorski and Levine. 6 Consisting of a strip of cotton gauze tied with string and suspended in flowing water, this All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254025 doi: medRxiv preprint sampling method acts as a trap that allows collection of pathogens over an extended period of time, especially when pathogen shedding is intermittent and in sites with smaller catchment populations. The Moore swab was first deployed by Brendan Moore in 1948 to trace Salmonella Paratyphi B from sewage in North Devon, England to determine the sources of contamination responsible for sporadic outbreaks of paratyphoid fever. Since the first application to detect typhoidal Salmonella in sewage, the Moore swab method has been utilized in several studies throughout the world to detect Salmonella Typhi in irrigation water, surface water, municipal sewers, and storm drains. 11, [30] [31] [32] Given its effectiveness, simplicity, and affordability, we adapted this method for use in typhoid environmental surveillance of wastewater in Kolkata, India. We found that the Moore swab method has several advantages over UF in terms of greater sensitivity, simplicity, shorter processing time, less labor, and lower cost. However, the Moore swab method also has several limitations. First, this method only provides results on the presence or absence of ST and SPA in the sample and does not allow a quantitative assessment of ST or SPA concentration in the sample. Second, Moore swab sample collection requires two trips to place and later retrieve the swab, whereas grab samples only require a single collection trip.

Third, deployment of Moore swabs for environmental surveillance has not been standardized in terms of sampling frequency, duration of immersion, and swab processing. This study addresses some of these information gaps by providing a standardized processing method for ST and SPA detection in wastewater with some benchmarks for limit of detection in wastewater matrices. We recognize that wastewater is a complex and highly variable matrix and that the limit of detection for this method will vary by setting.

The Moore swab method described in this study provides a low-cost, simple, and sensitive approach for presence/absence detection of ST and SPA in wastewater samples and is feasible to All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 21 5A. Black bars represent the mean input ST seeded into 5 L, 10 L and 20 L samples in three replicate experiments, and gray bars represent the mean recovered detected by qPCR. 5B. Black bars represent the mean SPA seeded, and gray bars represent the mean recovered SPA detected by qPCR.

2 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

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