ABSTRACT
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.
INTRODUCTION
Typhoid fever is caused by ingestion of food or drink contaminated with Salmonella enterica serovar Typhi (S. Typhi). Symptoms include high fever, malaise, headache, abdominal pain, and constipation or diarrhea, and can lead to serious intestinal complications and death if untreated. Symptoms begin 1 to 6 weeks after exposure (average 2 to 4 weeks). Lack of improved sanitation and access to safe food and water among populations living in low- and middle-income countries is related to an elevated risk of typhoid fever infection (1); nearly 12 million cases and 130,000 deaths were estimated to have occurred in 2010, after adjusting for water-related risk factors (i.e., access to “improved water”) (2). High incidence of typhoid fever has been reported in 45 African countries (2) and there has been an increase in the number of reported outbreaks throughout the African continent over the last 10 years (3).
Kampala is the capital city and financial center of Uganda. The city is a dense urban environment with a population of more than 1.5 million people (4). In early 2015, a typhoid fever outbreak began in the downtown area and subsequently spread throughout the city and into adjacent districts. By June, more than 10,000 suspected cases had been reported throughout the five districts in Kampala. The attack rate was higher in males (11.0/1,000 residents) than in females (8.4/1,000 resident) and higher in adults 15 to 59 (12.0/1,000 residents) than in children under 15 (2.0/1,000 residents) (5). The World Health Organization (WHO) reported that contaminated drinking water and juices were the main sources of the outbreak and that most water sources tested were heavily contaminated with Escherichia coli, with groundwater being of the worst quality (see http://www.who.int/csr/don/17-march-2015-uganda/en/ ). This was supported by a small-scale study conducted in early April 2015 that detected evidence of fecal contamination in street-vended beverages and in unprotected spring water sources (5).
In response to a waterborne disease outbreak, public health officials often conduct an investigation to define the scope and potential causes of the outbreak and inform implementation of effective control measures to reduce the disease burden (6). One component of these investigations is monitoring free chlorine residual (FCR) in drinking water as an indicator of water treatment and proxy measure of the microbiological quality of water. During waterborne disease outbreaks, the WHO advises maintaining a free chlorine residual (FCR) of 0.5 mg/liter in drinking water for effective inactivation of most waterborne microbes (7). In low-resource settings, water testing for the pathogen responsible for an outbreak is generally not performed due to high costs, lack of prior training, and difficult field and laboratory methodologies. However, Escherichia coli is often used as a fecal indicator to assess microbiological quality of water. E. coli, which is relatively inexpensive and easy to detect, indicates the presence of fecal contamination and therefore the potential presence of enteric pathogens (8). WHO states that there should be no detectable E. coli in any 100 ml samples of water directly intended for drinking (9).
Despite the high incidence of typhoid fever globally, relatively few outbreak investigations incorporate drinking water testing (10–15) and routine water testing for S. Typhi is rarely done in regions where S. Typhi is endemic (16). While these studies have provided useful information, more comprehensive sampling and testing strategies are needed to identify drinking water sources that represent higher disease transmission risks and areas of focus for public health interventions. The objective of this study was to conduct an environmental survey of drinking water quality in affected areas in Kampala during the 2015 typhoid fever outbreak in order to identify drinking water sources that may have been contributing to the outbreak. The Centers for Disease Control and Prevention (CDC) partnered with Uganda's National Water and Sewerage Corporation (NWSC) and the Kampala Capital City Authority (KCCA) to identify potentially contaminated sources and to collect and test water samples. Grab samples and large-volume dead-end ultrafiltration (DEUF) samples were collected from multiple drinking water source types used in areas with the highest case numbers, and microbiological, chemical, and physical parameters were measured. Results from the survey were intended to identify both fecal- and S. Typhi-contaminated sources, and to provide measures of free chlorine residual (FCR) as an indicator of previous treatment and conductivity as an indicator of whether treated water sources were being improperly mixed with untreated groundwater.
RESULTS
Median FCR was highest within the NWSC distribution system (Table 1). Two-thirds (n = 4) of NWSC reservoirs tested had an FCR ≥0.5 mg/liter (median 0.56 mg/liter) and the one NWSC booster station tested had an FCR of 0.85 mg/liter. The median FCR of all NWSC distribution system lines (premeter) was 0.37 mg/liter; 7 (70%) had FCR < 0.5 mg/liter. All non-NWSC reservoirs (n = 1) and non-NWSC taps (postmeter; n = 11) tested had an FCR of <0.5 mg/liter. All vended water tested had an FCR of <0.5 mg/liter. The median FCR for both manufacturer-packaged water bottles (n = 13) and refilled water bottles (n = 15) was 0.02 mg/liter and the median FCR for kaveras (n = 25) was 0.03 mg/liter. The median FCR for jerry cans was 0.03 mg/liter; one jerry can had an FCR of 0.54 mg/liter. FCR was not detected in protected springs (n = 13) nor in the protected well (n = 1).
Water quality results for drinking water samples collected during a typhoid fever outbreak in Kampala, Uganda, April 2015a
Electrical conductivity (EC) varied greatly across all samples (8 to 4,710 μS/cm) (Table 1). Median EC within the three components of the NWSC distribution system tested was 127 μS/cm (range, 105 to 141 μS/cm). Non-NWSC taps had a median EC of 131; the non-NWSC reservoir tested had an EC of 599 μS/cm. Within vended water source types tested, manufacturer-packaged water bottles had the lowest median EC (133 μS/cm), followed by refilled water bottles (218 μS/cm), and then kaveras (320 μS/cm). Jerry cans had a median EC of 371 μS/cm. Among samples with the highest electrical conductivity were two jerry can samples (694 and 799 μS/cm) and two kavera samples (805 and 4,710 μS/cm). Median EC was higher in protected springs (372 μS/cm) than in unprotected springs (237 μS/cm) and wells (293 μS/cm).
No total coliforms or E. coli were detected in the NWSC reservoirs, booster station, or water distribution system lines (pretap) (Table 2). The non-NWSC reservoir contained a high concentration of total coliforms (>2,419.6 most probable number [MPN]/100 ml), but no detectable E. coli. Approximately one-quarter of non-NWSC taps contained low mean concentrations of both total coliforms (5.2 MPN/100 ml) and E. coli (1.6 MPN/100 ml). Total coliforms were detected in two (15%) manufacturer-packaged water bottles (at 2 and 18.9 MPN/100 ml); the former contained 1 E. coli MPN/100 ml. Nearly all refilled water bottles (n = 14, 93%) and kaveras (n = 24, 96%) contained total coliforms. Twelve (80%) refilled water bottles contained E. coli, four of which (33%) contained >1,000 MPN/100 ml. Nearly two-thirds (n = 15, 60%) of kaveras contained E. coli with more than half (n = 13) at a concentration of 1 to 10 MPN/100 ml. All jerry cans contained total coliforms and 68% contained E. coli (mean, 24.2 MPN/100 ml); four of which contained >1,000 E. coli MPN/100 ml. All springs and wells contained total coliforms. The mean E. coli concentration in protected springs was 10.3 MPN/100 ml; 2 (15%) contained E. coli at >1,000 MPN/100 ml. All unprotected springs and wells contained E. coli, with approximately two-thirds of each containing >1,000 MPN/100 ml.
E. coli results by WHO risk category, for drinking water samples collected during a typhoid fever outbreak in Kampala, Uganda, April 2015
None of the DEUF concentrates submitted directly to PCR were positive for Salmonella spp. (data not shown). A total of six selenite cysteine (SC) enrichments (16%), representing four sources, were positive for Salmonella spp. by PCR (Table 3). These included one (8%) protected spring sample, one (25%) refilled water bottle sample, and samples from two (40%) unprotected springs. None of these six SC enrichments were positive for all three S. Typhi-specific gene targets. Two of the four presumptive-positive S. Typhi colony isolates sent to CDC displayed colonies suspected of Salmonella on Hektoen agar. Subsequent testing using the five-biochemical panel (see Materials and Methods) determined that only one was nontyphoidal Salmonella. This isolate, originating from an unprotected spring, was tested by molecular serotyping and determined to be in O-group C2, likely Salmonella enterica serovar Newport.
Salmonella and S. Typhi PCR results for drinking water samples collected during a typhoid fever outbreak in Kampala, Uganda, April 2015a
DISCUSSION
The results of this study identified high-risk drinking water sources that were being used by residents during the 2015 typhoid outbreak in Kampala, Uganda. Unregulated vended water sources, specifically single-use kaveras and refilled water bottles sold in markets and on the street, were of poor quality and may have contributed to disease transmission during the outbreak. A total of 15 (60%) kaveras contained detectable E. coli, with two (8%) containing E. coli at concentrations >100 MPN/100 ml. A case-control study conducted during this outbreak found that significantly more case patients than controls drank kavera water (5). Twelve (80%) refilled water bottles contained detectable E. coli, with more than half at concentrations >100 MPN/100 ml; 30% contained E. coli at concentrations >1,000 MPN/100 ml. FCR was not detected in any kaveras or refilled water bottles.
As expected, untreated well and spring water sources were found to have the highest levels of fecal contamination, with many samples containing E. coli at concentrations above the WHO's high-risk threshold of 1,000 MPN/100 ml. Shortly after the start of the outbreak, KCCA banned the use of groundwater that was being stored in tanks and used to supplement NWSC-piped water. In addition, some shopping centers that used such water were forcibly closed. A number of groundwater wells were cemented over to prevent usage; however, some wells were accessible for sample collection and testing during this investigation.
EC is a measure of the ability of an aqueous solution to carry an electrical current. EC increases as the concentration of inorganic dissolved solids, such as those originating from the natural environment (e.g., soil, sediment, bedrock) or from discharges (i.e., runoff, wastewater intrusion), in the solution increases (see https://archive.epa.gov/water/archive/web/html/vms59.html ). In this study, median EC values of water collected directly from the NWSC distribution system ranged from 124 to 133 μS/cm. However, median EC values of kaveras (320 μS/cm) and refilled water bottles (218 μS/cm) were considerably higher and were similar to median EC values of springs and wells (range, 237 to 372 μS/cm). These elevated EC levels in unregulated vended water suggest that it originated from, or was mixed with, groundwater.
Nearly all water samples collected directly from distribution system components under the control of the municipal drinking water utility were found to have FCR levels recommended by the WHO for water system delivery points (0.2 mg/liter) and none contained indicators of fecal contamination (E. coli). However, 70% of distribution system lines (premeter) did not have an FCR of 0.5 mg/liter, as recommended by the WHO during waterborne outbreaks. Tap water was challenging to categorize in this setting. In some cases, water that originated from the distribution system was rerouted by private individuals to tanks and mixed with water from other sources, generally contaminated groundwater, before being piped to a tap. In addition, water that originated from illegal connections to the existing water distribution system infrastructure was piped into buildings; illegal tap-ins, especially when poorly constructed, can result in low pressure in the system, which can then draw in contaminants from the surrounding area. Waterborne outbreaks related to these types of contamination events are well-documented (18). Due to these circumstances, endpoint taps in Kampala are not the responsibility of the municipal water authority. E. coli was detected in more than one-quarter of tap water samples collected and only one sample had an adequate FCR concentration; in comparison with water quality of NWSC premeter water, this suggests intrusion of contaminants into premise plumbing, mixing of distribution system water with groundwater, or contamination of water during storage in tanks. Household-level treatment of water of unknown origin, as well as frequent storage tank cleaning, are recommended.
Water from jerry cans was obtained from residents on the street or from businesses (e.g., small restaurants). Whether originating from unsafe sources such as unprotected springs or wells or from the treated municipal water supply, water stored in jerry cans was generally of poor quality. More than 60% of jerry can water samples contained detectable E. coli, with many tests indicating the presence of E. coli at concentrations above 1,000 MPN/100 ml. Water storage in jerry cans has previously been shown to be a risk for consumers, whether due to their use for collecting unsafe water or their retention of contaminants from one batch of water to the next (19).
Salmonella spp. data from this study support the conclusion that unregulated water sources were likely a major contributor to the typhoid outbreak in Kampala. Residents were known and observed to obtain water for drinking from unprotected and protected springs. While S. Typhi was not detected in spring water samples collected for this investigation, nontyphoidal Salmonella was isolated from one spring water sample and DNA from Salmonella spp. was detected in multiple samples. The lack of S. Typhi detection in water samples collected for this study may have been due to the timing of the study (which was performed after the outbreak peaked), low concentration of S. Typhi in these water sources, or difficulty in culturing S. Typhi from water, as noted by other researchers (20). However, the Salmonella spp. data, in conjunction with E. coli data, are clear evidence that unprotected and protected springs were impacted by fecal contamination, likely of human origin in this dense urban setting, and therefore represented a high risk for typhoid transmission. In addition, the detection of Salmonella spp. in one refilled water bottle sample provided further evidence that this type of drinking water source represented a high risk for disease transmission to consumers in Kampala.
This study was subject to at least four limitations. First, this environmental investigation began in April, several months after the start of the outbreak and after the outbreak peaked. We may have had a better chance of detecting S. Typhi in water samples and therefore would have learned more about the sources of the outbreak, if we had been able to conduct the investigation earlier. In addition, regularly scheduled superchlorination of the municipal distribution system was performed before the investigation; sampling from the system before superchlorination would have provided characterization of water quality in the system before the additional treatment. Second, testing of water sources used by actual cases would have provided a stronger linkage between infection and water sources. Third, while this study has a larger sample size and more comprehensive set of water source types than previous environmental studies associated with outbreaks (10–15), data from additional samples, especially from unprotected springs, wells, and taps, would have allowed a more robust analysis of water quality. Fourth, S. Typhi is difficult to culture from environmental samples; improved culture methods are needed.
While consumption of unsafe drinking water is often implicated in waterborne disease outbreaks in low-income regions, well-timed drinking water quality testing is not always performed. Drinking water quality investigations may help identify sources with fecal contamination and possibly even those containing the etiologic agent implicated in the outbreak. This information can assist in prompt interventions (e.g., distribution of point-of-use water treatments) and policy changes (e.g., stricter regulations on the selling of water from unregulated sources). Comprehensive environmental evaluations such as this require strong partnerships with in-country collaborators, particularly with municipal water authorities and governmental organizations that oversee water supplies, as these groups provide valuable knowledge of water practices and location of (and access to) water sources.
MATERIALS AND METHODS
Sampling locations.A convenience water sampling strategy focused on areas surrounding four medical clinics where typhoid fever cases had been most numerous as of mid-April 2015, Kisenyi Clinic, Kawaala Clinic, Kisugu Clinic, and Kiswa Clinic (Fig. 1). More emphasis was placed on sampling in Kisenyi due to high case reports from that area. As the outbreak was still under investigation and information was limited, unofficial sources such as news reports were utilized to identify additional sites (e.g., New Taxi Park and Nakasero Market) for water sampling. Each sample was assigned a numeric sample identifier (ID) and the following information was recorded on a preprinted field data sheet: date, GPS coordinates at the site of collection or purchase, and site description. Additionally, a master log of all sample IDs and water source types was recorded. This investigation was part of a public health disease outbreak investigation and did not meet the definition of human subjects research under 45 CFR 46.102(d) and therefore did not require formal Institutional Review Board review.
Map of water sample locations and drinking water source types collected during a typhoid fever outbreak in relation to location of cases' residences and places of employment; Kampala, Uganda, April 2015. Map created using ArcGIS release 10.3.1.
Drinking water source types.Drinking water source types analyzed included NWSC distribution system reservoirs, boosters, and distribution lines (pre-water meter or official public taps); non-NWSC reservoirs; non-NWSC taps, which were located on distribution lines at some point after the utility-maintained water meter and therefore were not under the authority of NWSC; vended water, including manufacturer-packaged (plastic-sealed) water bottles, previously used water bottles that had been refilled and sold, and kaveras (drinking water sold in polyethylene bags designed for one-time use); water collected in PVC storage containers (i.e., jerry cans); and protected and unprotected springs; and protected and unprotected wells (Fig. 2). Source types and numbers of water samples collected each day were decided upon in collaboration with in-country partners based on logistical considerations.
Photographs of select drinking water source types collected during a typhoid fever outbreak in Kampala, Uganda, April 2015: (a) NWSC distribution line (pre-water meter), (b) kavera, (c) protected spring, (d) jerry can, and (e) unprotected spring.
Grab sample collection.Grab samples were aseptically collected into glass sample cells for free chlorine testing, sterile IDEXX bottles containing sodium thiosulfate for E. coli testing, and, for a subset of nonchlorinated sources, sterile WhirlPak bags for S. Typhi testing. For sources with taps, hosing (if present) was removed and the inside and outside areas of the tap were scrubbed with cleaning powder; water was allowed to flow for at least 30 s before sample collection. For reservoirs, a sampling device was lowered into the reservoir tank using a rope. The device was then thoroughly rinsed with water to be sampled, refilled, and poured into the collection container. Kaveras and bottled water were purchased and remained unopened in the field. Samples were stored in a cooler with ice packs during transport and were analyzed within 6 to 8 h of collection.
Large-volume sample collection and processing.At a subset of nonchlorinated sources, 20 liters of water was concentrated by DEUF using REXEED 25S ultrafilters (Asahi Kasei Medical Co, Ltd., Tokyo, Japan). Filters were stored in a cooler with ice packs during transport and were backflushed within 6 to 8 h of collection. Each ultrafilter was backflushed with 500 ml of solution containing 0.5% Tween 80, 0.01% sodium polyphosphate (Sigma-Aldrich, St. Louis, MO, USA), and 0.001% Antifoam Y-30 emulsion (21). A 200-ml volume of backflush was submitted to S. Typhi culture, as described below. The remaining backflush volume was stored at 4°C and shipped to CDC, where samples were centrifuged at 4,000 × g. The pelleted material was then added to an equal volume of lysis buffer and tested directly for Salmonella spp. by PCR, as described below.
Free chlorine residual testing.Free chlorine residual (FCR) was measured onsite for reservoirs, booster stations, taps, jerry cans, protected springs, and protected wells and in the laboratory for manufacturer-packed and refilled bottled water and kaveras, using a digital colorimeter and N,N-diethyl-p-phenylenediamine (DPD) methodology (Hach, Loveland, CA, USA), according to the manufacturer's instructions. FCR was not measured in water from unprotected springs or unprotected wells.
Electrical conductivity testing.Electrical conductivity (EC) was measured at the NWSC Central Laboratory, Kampala within 6 h of collection according to the electrolytic method, using a SensION+ benchtop meter (Hach, Loveland, CA, USA).
Quantification of total coliforms and E. coli.A total of 122 grab samples (100 ml) were collected from 12 source types, including NWSC reservoirs (n = 6), an NWSC booster (n = 1), NWSC distribution lines, premeter (n = 10), a non-NWSC reservoir (n = 1), non-NWSC taps (n = 11), manufacturer-packaged water bottles (n = 13), refilled water bottles (n = 15), kaveras (n = 25), jerry cans (n = 19), protected springs (n = 13), unprotected springs (n = 5), a protected well (n = 1), and unprotected wells (n = 2). Total coliforms and E. coli were quantified using IDEXX QuantiTray/2000 (Westbrook, ME, USA) most probable number (MPN) methodology and Colilert-18 media, according to the manufacturer's instructions. In order to calculate geometric means, a value of 0.5 MPN/100 ml was assigned to samples that fell below the QuantiTray/2000 lower detection limit of <1 MPN/100 ml and a value of 4,839.2 MPN/100 ml was assigned to samples that were above the detection limit of 2,419.6 MPN/100 ml.
S. Typhi culture.A total of 37 grab samples (100 ml) were collected from seven source types, including non-NWSC taps (n = 2), refilled water bottles (n = 4), kaveras (n = 6), jerry cans (n = 4), unprotected springs (n = 5), protected springs (n = 13), an unprotected well (n = 1), and a protected well (n = 1). Paired DEUF samples were collected from 16 of these sites, including from unprotected springs (n = 4), protected springs (n = 10), an unprotected well (n = 1), and a protected well (n = 1).
Grab samples and DEUF concentrates were analyzed for S. Typhi following the United States Environmental Protection Agency (USEPA) Standard Analytical Protocol for Salmonella Typhi in Drinking Water (22), with slight modification due to logistical constraints (e.g., unavailability of premade selective media in-country). A 100-ml volume of each grab sample was added to 100 ml of 2× Universal Pre-enrichment (UP) broth (BD, Franklin Lakes, NJ, USA) and a 200-ml volume of DEUF backflush was added to 200 ml of 2× UP broth. Inoculated broths were incubated at 35.0 ± 0.5°C for 24 ± 2 h. Preenrichments were subcultured into selenite cysteine (SC) broth (BD, Franklin Lakes, NJ, USA) and were incubated at 35.0 ± 0.5°C for 18 ± 2 h. All SC enrichments were streaked for isolation on both bismuth sulfite (BS) and xylose lysine deoxycholate (XLD) agars (BD, Franklin Lakes, NJ, USA). Additionally, 700 μl of each SC enrichment was added to 700 μl of lysis buffer, stored at ambient temperature, and shipped to CDC for further testing. Following incubation, plates were then assessed for typical S. Typhi colonies (green-black colonies with metallic sheen on BS agar; clear colonies with pinpoint black center on XLD agar); presumptive positive colonies were then subcultured on nonselective Mueller-Hinton (MH) agar (BD, Franklin Lakes, NJ, USA) to increase cell density. Pure cultures were stored in agar dram vials and shipped to CDC for subsequent testing.
At CDC, culture material from dram vials was streaked onto Hektoen agar for isolation. Suspect colonies (i.e., clear or green colonies with dark centers) were subjected to an abbreviated panel of tests (23). This panel is appropriate for detecting Salmonella or Shigella spp. and for biochemically differentiating Salmonella serovars Typhi and Paratyphi A from other serovars of Salmonella. The biochemical tests included triple sugar iron agar, lysine iron agar, urea agar (BD, Franklin Lakes, NJ, USA), motility-indole-ornithine agar, and Simmons citrate agar (CDC, Atlanta, GA). Suspect colonies were then tested by the xMAP Salmonella serotyping assay (Luminex Corporation, Austin, TX).
Molecular analyses.Nucleic acids in SC enrichments and backflush secondary concentrates were extracted (24) at CDC. First, a broadly reactive real-time PCR was performed for detection of Salmonella spp. (fimA gene) (25). Next, S. Typhi-specific real-time PCR was performed, targeting the fliC-d (phase-1 flagellin gene for d antigen H:d of Salmonella serovar Typhi), tyv (tyvelose epimerase), and viaB (Vi antigen) genes (Table 4). To improve the PCR amplification efficiency and thus increase the sensitivity of the assay, primers were designed to amplify short template regions for fliC-d, tyv, and viaB genes while retaining the TaqMan probes designed by Hyytia-Trees (26). Standard curves were generated using an S. Typhi genomic DNA standard to evaluate the efficiency of each assay. The real-time PCR assay amplification efficiencies were calculated from the slope of the standard curve and had an efficiency of 85.6% for fimA, 86.4% for fliC-d, 81.4% for tyv, and 93.5% for viaB.
Salmonella spp. and S. Typhi target genes and primer and probe sequences used to test drinking water samples collected during a typhoid fever outbreak in Kampala, Uganda, April 2015
A 2-μl volume of nucleic acid extract was analyzed in duplicate in a 20-μl reaction volume, using Applied Biosystems TaqMan Environmental master mix 2.0 (Life Technologies, Carlsbad, CA). The following amplification protocol was used for real-time PCR testing using an Applied Biosystems 7500 thermocycler: denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s and annealing, extension, and fluorescence acquisition at 60°C for 30 s. CT values of <41 were considered positive for the fimA gene target. Due to cross-reactions with nontarget Salmonella species, CT values of <38 were considered positive for each S. Typhi-specific gene target. All gene targets were required to be positive for a S. Typhi classification.
ACKNOWLEDGMENTS
We appreciate NWSC for the local logistical support and specifically wish to acknowledge Rose Kaggwa and Alex Gisagara for coordinating the joint monitoring activities, as well as the staff from the Water Quality Management Department at Central and Gaba Laboratories in Kampala, who helped with the field work and laboratory analytical tests.
We acknowledge the CDC Global Disease Detection Operations Center Outbreak Response Contingency Fund for financial support.
We are grateful for contributions from Ray R. Arthur, Sudhir Bunga, Catherine C. Chow, Kira A. Christian, Serena Fuller, C. Jason (CJ) McKnight, Rossanne M. Philen, and Myron Schultz from the Global Disease Detection Operations Center, Division of Global Health Protection, Center for Global Health, Centers for Disease Control and Prevention.
We acknowledge Matthew Mikoleit, Center for Global Health, and Blake Dinsmore, Enteric Diseases Laboratory Branch, for their contributions to this work, including molecular serotyping. We thank Ellaine Hallisey of the Geospatial Research, Analysis and Services Program, a division of the Agency for Toxic Substances and Disease Registry at CDC, for assistance with map creation. We also acknowledge the Robert Downing Uganda Virus Research Institute and personnel from the Uganda Central Public Health Laboratories for their assistance in the investigation.
The use of trade names and commercial sources is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention (CDC) or the U.S. Department of Health and Human Services. The findings and conclusions in this presentation are those of the authors and do not necessarily represent those of the CDC.
FOOTNOTES
- Received 4 August 2017.
- Accepted 30 August 2017.
- Accepted manuscript posted online 29 September 2017.
- Copyright © 2017 American Society for Microbiology.
