Detection and quantification of enteric pathogens in aerosols near uncontained fecal waste streams in cities with poor sanitation

TL;DR: Vardef et al. as discussed by the authors detected and quantified enteric pathogen-associated gene targets in aerosol samples near open wastewater canals (OWCs) or impacted surface waters and control sites in La Paz, Bolivia; Kanpur, India; and Atlanta, USA via multiplex qPCR (37 targets) and ddPCR(13 targets).

Abstract: Urban sanitation infrastructure is inadequate in many low-income countries, leading to the presence of highly concentrated, uncontained fecal waste streams in densely populated areas. Combined with mechanisms of aerosolization, airborne transport of enteric microbes and their genetic material is possible in such settings but remains poorly characterized. We detected and quantified enteric pathogen-associated gene targets in aerosol samples near open wastewater canals (OWCs) or impacted surface waters and control sites in La Paz, Bolivia; Kanpur, India; and Atlanta, USA via multiplex qPCR (37 targets) and ddPCR (13 targets). We detected a wide range enteric pathogen-specific targets, some not previously reported in extramural urban aerosols, with more frequent detections of all enteric targets at higher densities in La Paz and Kanpur near OWCs. We report density estimates ranging from non-detects to 4.7 [x] 102 gc per m3air for targets including ST-ETEC, C. jejuni, EIEC/Shigella spp., Salmonella spp., adenovirus, and Cryptosporidium spp. An estimated 25%, 76%, and 0% of samples containing positive pathogen detects were accompanied by culturable E. coli in La Paz, Kanpur, and Atlanta, respectively, suggesting potential for viability of enteric microbes at the point of sampling. Airborne transmission of enteric pathogens merits further investigation in cities with poor sanitation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/21251650v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@13c817borg.highwire.dtl.DTLVardef@158e108org.highwire.dtl.DTLVardef@1e0626org.highwire.dtl.DTLVardef@125b214_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISWe detect and quantify molecular targets associated with important enteric pathogens in outdoor aerosols from cities with poor sanitation to assess the potential role of the aeromicrobiological pathway in enteric infection transmission in such settings.

Summary

INTRODUCTION

• With few exceptions, large cities in low- and middle-income countries have inadequate sanitation infrastructure1–3.
• In cities in LMICs, the transport of enteric pathogens in aerosols may be possible due to a confluence of inadequate sanitation infrastructure resulting in concentrated flows of fecal wastes, a high disease burden resulting in high-risk waste containing human enteric pathogens, high population density, and environmental conditions that may be conducive to the aerosolization of concentrated fecal wastes.
• The mechanisms behind aerosolization and transport of microorganisms from liquid surfaces and the microbial effects on droplet lifetime have been well-characterized under controlled conditions6,7.
• These phenomena and their implications are less well characterized for sanitation-related pathogens of public health importance.
• The majority of such studies are based on detection of fecal indicator bacteria including members of the coliform group54,58, partly because the presence of important enteric pathogens is unexpected outside high-burden settings.

METHODS

• In La Paz, the authors identified two control sites >1 km from known concentrated wastewaters or other contaminated sources: (1) Chacaltaya, a weather station and environmental observatory located at 5380 m in elevation and far from human habitation and (2) Pampalarama, an undisturbed site near the Choqueyapu headwaters.
• The authors treated the eluate with guanidine thiocyanate-based universal extraction (UNEX; Microbiologics, St. Cloud, MN, USA) lysis buffer in a 1:1 ratio, storing the mix in bead tubes for sample transport to the laboratory.
• Positive control sequences, primers, probes, and experimentally determined 95% LODs are detailed for each assay in Table S2.

RESULTS

• Of the 45 air samples the authors collected in Kanpur near OWCs (<1 km) and analyzed by culture, 61% had detectable E. coli with an average concentration and 95% CI of 1.5 ± 1.3 CFU/m3air across positive detections.
• The authors analyzed a subset of 40 high-volume samples from Kanpur, 23 high-volume samples from La Paz, and 13 high-volume samples from Atlanta for the presence and absence of 42 molecular targets including those specific to an a priori-defined list of globally important enteric viruses, bacteria, and protozoa.
• For all targets, at least one of the positive samples also had culturable E. coli.

DISCUSSION

• Open sewers conveying domestic, institutional, commercial, and industrial effluent are common in cities in LMICs.
• They may pose risks, however, both to downstream communities and also people in close proximity to open urban wastewater flows.
• The health risk implications of the presence of aerosolized enteric microbes in these settings are unknown but merit further study.
• The authors observed comparable prevalence of Aeromonas spp. associated nucleic acids in Atlanta, La Paz, and Kanpur with 8, 9, and 7 positive detections per 1000 m3air at each site respectively.
• There is some epidemiological evidence that proximity to concentrated fecal waste streams in urban areas can be related to enteric infection risk.

Figures

Figure 3. Positive detects via TAC per 2000 m3 air and the fraction of samples positive for culturable E. coli.

Figure 2. Size distribution of culturable E. coli and total coliform in La Paz and Atlanta. The mean standard error bars show 95% confidence intervals for densities in each size range. We did not collect ACI samples in Kanpur, so size-resolved E. coli detections by culture are not available for that site.

Figure 4. Mean densities of gene targets associated with enteric microbes with mean standard error bars as observed among the distribution of positive detects in gene copies per cubic meter of air. Densities were censored according to the assay-specific 95% LOD.

Figure 5. Percentage of positive detects (via TAC or ddPCR) in sample sets for each site versus straightline distance from impacted water flows, where densities were censored according to the assay-specific

Figure 1. Aerosol sampling sites in La Paz, Bolivia; Kanpur, India; and Atlanta, USA. Sites located <1 km from OWCs are represented by triangles and sites located >1 km from OWCs are represented by circles. Control sites outside the city of La Paz not shown.

Full text

1
Detection and quantification of enteric pathogens in aerosols near open wastewater canals in cities
with poor sanitation
Olivia Ginn
1
, Lucas Rocha-Melogno
2
, Aaron Bivins
3
, Sarah Lowry
1
, Maria Cardelino
1
, Dennis Nichols
4
,
Sachi Tripathi
5
, Freddy Soria
6
, Marcos Andrade
7,8
, Mike Bergin
2
, Marc A. Deshusses
2
, Joe Brown
9*
1
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA
2
Department of Civil and Environmental Engineering, and Duke Global Health Institute, Duke University, Durham,
NC, 27708, USA
3
Department of Civil and Environmental Engineering and Earth Science, University of Notre Dame, Notre Dame,
Indiana, 46656, USA
4
Rollins School of Public Health, Emory University, Atlanta, GA, 30322, USA
5
Department of Civil Engineering & Centre for Environmental Science and Engineering, Indian Institute of
Technology − Kanpur, India
6
Centro de Investigación en Agua, Energía y Sostenibilidad, Universidad Católica Boliviana “San Pablo”, La Paz,
Bolivia
7
Laboratory for Atmospheric Physics, Institute for Physics Research, Universidad Mayor de San Andres, La Paz,
Bolivia
8
Department of Atmospheric and Oceanic Sciences, University of Maryland, College Park, MD, USA
9
Deparment of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North
Carolina, Chapel Hill, North Carolina, 27599-7431, USA
*Corresponding author: Department of Environmental Sciences and Engineering, University of North Carolina, 135
Dauer Drive, Chapel Hill, NC, 27599-7431, USA. Tel: 404 385 4579. Email: joebrown@unc.edu
SYNOPSIS
We detect and quantify molecular targets associated with important enteric pathogens in outdoor aerosols
from cities with poor sanitation to assess the potential role of the aeromicrobiological pathway in enteric
infection transmission in such settings.
ABSTRACT
Urban sanitation infrastructure is inadequate in many low-income countries, leading to the presence of
highly concentrated, uncontained fecal waste streams in densely populated areas. Combined with
mechanisms of aerosolization, airborne transport of enteric microbes and their genetic material is possible
in such settings but remains poorly characterized. We detected and quantified enteric pathogen-associated
gene targets in aerosol samples near open wastewater canals (OWCs) or impacted surface waters and
control sites in La Paz, Bolivia; Kanpur, India; and Atlanta, USA via multiplex qPCR (37 targets) and
ddPCR (13 targets). We detected a wide range enteric pathogen-specific targets, some not previously
reported in extramural urban aerosols, with more frequent detections of all enteric targets at higher densities
in La Paz and Kanpur near OWCs. We report density estimates ranging from non-detects to 4.7 x 10
2
gc
per m
3
air
for targets including ST-ETEC, C. jejuni, EIEC/Shigella spp., Salmonella spp., norovirus,
and Cryptosporidium spp. An estimated 25%, 76%, and 0% of samples containing positive pathogen
detects were accompanied by culturable E. coli in La Paz, Kanpur, and Atlanta, respectively, suggesting
potential for viability of enteric microbes at the point of sampling. Airborne transmission of enteric
pathogens merits further investigation in cities with poor sanitation.
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.14.21251650doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

2
INTRODUCTION
With few exceptions, large cities in low- and middle-income countries (LMICs) have inadequate sanitation
infrastructure
1–3
. Unsafe water and sanitation enable the transmission of enteric pathogens from infected
individuals to susceptible hosts via direct contact or through the environment in multiple interconnected
pathways
4,5
. While a rich and rapidly growing body of literature describes microbial risks associated with
direct or indirect exposure to fecal contamination in a wide variety of settings, relatively few studies have
examined the potential for transmission of enteric pathogens via the aeromicrobiological pathway in high-
risk settings. In cities in LMICs, the transport of enteric pathogens in aerosols may be possible due to a
confluence of inadequate sanitation infrastructure resulting in concentrated flows of fecal wastes, a high
disease burden resulting in high-risk waste containing human enteric pathogens, high population density,
and environmental conditions that may be conducive to the aerosolization of concentrated fecal wastes. The
aerosolization, transport, and deposition of microbial pathogens in cities lacking good sanitation could lead
to exposure either through inhalation or through ingestion via other pathways (e.g., food, water, direct
contact).
Aerosolization of biological material is known to be possible via several mechanisms including bubble
bursting
6–8
, evaporation, raindrop impaction
9,10
, and others
11–14
. The creation and persistence of bioaerosols
can be associated with a range of variables related to environmental conditions and the built environment
including rain events
15–18
, meteorological conditions
19–21
, urban surface waters and water features
22–24
,
wastewater treatment unit processes that include mechanical mechanisms
25,26
, and other infrastructure. The
mechanisms behind aerosolization and transport of microorganisms from liquid surfaces and the microbial
effects on droplet lifetime have been well-characterized under controlled conditions
6,7
. Laboratory studies
have revealed that bubbles in contaminated water surfaces may experience conditions manipulated by
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.14.21251650doi: medRxiv preprint

3
microorganisms, allowing for smaller, more numerous, and higher velocity droplets to transition from water
to air
8
. Similarly, it has been shown that plant pathogens may be released from plant surfaces through
raindrop impaction and such releases escape the laminar boundary layer of leaves, allowing for long-
distance, airborne transport of some pathogens
9,10
. Although the aerosolization and transport of plant
pathogens and respiratory viruses (in indoor environments) have been studied, these phenomena and their
implications are less well characterized for sanitation-related pathogens of public health importance.
Studies in high-risk, extramural (outdoor) settings in the USA and in other high-income countries have
revealed that bioaerosols containing enteric microbes are common where concentrated fecal waste and one
or more mechanisms for aerosolization exist. Enteric microbes in aerosols have been best characterized in
ambient air surrounding wastewater treatment plants
27–37
and in the context of land application of
biosolids
38–48
; several studies have examined bioaerosols surrounding composting facilities
49,50
, meat
markets
51
, urban areas
52–54
, and concentrated animal feeding operations
55–57
. The majority of such studies
are based on detection of fecal indicator bacteria including members of the coliform group
54,58
, partly
because the presence of important enteric pathogens is unexpected outside high-burden settings. A small
number of studies have reported relative abundance of potentially pathogenic genera in 16S sequencing
studies
53,59
. No previously reported studies have captured a broad range of enteric pathogens in bioaerosols
from high-risk outdoor settings and no studies have conducted absolute quantification of enteric pathogens
in bioaerosols in cities of LMICs, a necessary step in further assessment of the potential public health
relevance of this poorly understood pathway of transmission. Based on previous literature on the presence
of enteric microbes in aerosols from well-studied settings in wealthy countries, we hypothesized that
aerosolized enteric pathogens could be present and quantifiable where urban sanitation is lacking. We
assessed this hypothesis in two cities with poor sanitation and in one city with established and maintained
wastewater infrastructure as a reference site.
METHODS
Sampling locations. We conducted sampling in Kanpur, India (May – July 2017); La Paz, Bolivia (March
2018, June 2018, March 2019, June and July 2019); and Atlanta, Georgia, USA (March 2018-January
2019). Kanpur has distinct dry (October to June) and rainy (July to September) seasons; we sampled from
May to August to capture both periods. Similarly, we intentionally sampled in La Paz during both rainy
(December to March) and dry (May to August) seasons.
Kanpur is densely populated (Nagar district: 4.6 million people, population density of 1500 persons/km
2
)
60
with a majority of untreated industrial, agricultural, and sewage waste conveyed via a system of uncovered
canals (open wastewater canals, OWCs) discharging to the Ganges River
61,62
. In La Paz, a network of rivers
receive untreated sewage discharge, industry effluent, and stormwater runoff; most of the waterway flows
in a series of engineered channels
63,64
, also characterized as OWCs. The largest of these is the highly
impacted Choqueyapu River, flowing through central La Paz (population: 900,000, 900 persons/km
2
)
65,66
where it is joined by tangential tributaries including the Orkojahuira, Irpavi, and Achumani rivers. In past
studies, this river system and its basin – eventually flowing into the Amazon – has been shown to contain
a diverse and rich array of enteric microbes indicating high levels of fecal contamination
63,66,67
. As a
reference site, Atlanta is characterized by having an established and maintained subsurface wastewater
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4
infrastructure, although urban surface waters in Atlanta’s watershed experience elevated levels of fecal-
associated pathogens
68–70
due to nonpoint source pollution and combined sewer overflows
71,72
. The city of
Atlanta’s population density is an estimated 1500 persons/km
2 73
, though sampling locations near impacted
streams were in suburban locations at lower than mean population density.
We identified 18 sites in Kanpur, 37 sites in La Paz, and 8 sites in Atlanta meeting the following criteria:
(1) proximity to sources of bioaerosols (<1 km) containing enteric microbes, OWCs in the cases of India
and Bolivia and impacted surface waters in Atlanta; (2) public and ground level accessibility; and (3)
unintrusive to members of the community during multi-hour sampling events. In Kanpur, we selected a
control site greater than 1 km away from known OWCs and located on the Indian Institute of Technology
(IIT)-Kanpur’s campus. The campus is a controlled private area with limited access to non-students and
non-faculty, is less densely populated, has underground piped sewerage, and has a much lower animal
presence. In La Paz, we identified two control sites >1 km from known concentrated wastewaters or other
contaminated sources: (1) Chacaltaya, a weather station and environmental observatory located at 5380 m
in elevation and far from human habitation and (2) Pampalarama, an undisturbed site near the Choqueyapu
headwaters. In Atlanta, we sampled at eight sites adjacent to impacted streams and rivers in Atlanta’s
watershed: the Chattahoochee River, Proctor Creek, Foe Killer Creek, and South Fork Peachtree Creek.
Additionally, we sampled on the roof of our laboratory and at ground level on Georgia Tech’s campus
(located in Midtown Atlanta), >1 km from surface waters.
Figure 1. Aerosol sampling sites in La Paz, Bolivia; Kanpur, India; and Atlanta, USA. Sites located <1
km from OWCs are represented by triangles and sites located >1 km from OWCs are represented by
circles. Control sites outside the city of La Paz not shown.
Bioaerosol sampling, extraction and analysis. We used a combination of high-volume filtration and
aerosol impaction in sampling across sites. We used the ACD-200 BobCat Dry Filter Continuous Air
Sampler (InnovaPrep, Drexel, MO, USA) with 52 mm electret filters and a flow rate of 200 L/min for
downstream molecular analysis post extraction. We applied a single-use wet foam carbon compressed
elution kit (InnovaPrep, Drexel, MO, USA) to flush the filter following the manufacturer’s instructions,
yielding approximately 6 mL of liquid eluate
74
. We treated the eluate with guanidine thiocyanate-based
universal extraction (UNEX; Microbiologics, St. Cloud, MN, USA) lysis buffer in a 1:1 ratio, storing the
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.14.21251650doi: medRxiv preprint

5
mix in bead tubes for sample transport to the laboratory. As a process control prior to extraction, we spiked
the mix with 5uL of Inforce 3 Bovine Vaccine (Zoetis, Parsippany, NJ) containing bovine respiratory
syncytial virus (BRSV) and bovine herpes virus (BoHV). After DNA and RNA lysis, purification, and
elution of nucleic acids following the manufacturer’s protocol
75
, we stored extracted nucleic acids in 50-75
µL of 10 mM Tris-1 mM EDTA (pH 8) at -80°C until further analysis. In total, we collected 75 high-volume
air samples from La Paz (71 collected near OWCs and 4 collected from reference sites >1 km from OWCs),
53 high-volume air samples from Kanpur (45 collected near OWCs and 8 collected from one reference site
>1 km from OWCs), and 15 high-volume air samples in Atlanta.
For all high-volume air samples in Kanpur (n=53), we applied 1 mL of BobCat eluate to Compact Dry-EC
(CD-EC) plates (Hardy Diagnostics, Santa Maria, CA, USA)
76
for culture of total coliform and E. coli.
Concurrent with high-volume air sampling in La Paz (n=31) and in Atlanta (n=15), we simultaneously used
the Six-Stage Viable Andersen Cascade Impactor (ACI) with plates in six partitioned chambers at a flow
rate of approximately 28.5 L/min for 1 hour to collect size-resolved bioaerosols in the size range of 0.65 to
>7 µm (ACI, Thermo Scientific
TM
, USA)
77
. We used AquaTest medium (Sisco Research Laboratories PVT.
LTD., India) in the ACI to detect E. coli
78–80
. All culture samples were incubated at 37 °C and counted per
the manufacturer’s instructions after 18-24 hours for colony forming units (CFUs).
Enteric pathogen screening: multiplex qPCR. As a first step in screening enteric targets, we analyzed
high-volume aerosol samples using a custom multiplex qPCR-based TaqMan Array Card (TAC) for the
presence or absence of selected targets including enteric viruses (pan-adenovirus, pan-astrovirus, pan-
enterovirus, norovirus GI/II, rotavirus A-C, and sapovirus I/II/IV/V), bacteria (Aeromonas spp.,
Campylobacter coli, Clostridium difficile, numerous genes of Escherichia coli (SI Table 1), Enterococcus
faecalis, Enterococcus faecium, Mycobacterium tuberculosis, Salmonella spp., Shiga toxins, Vibrio
cholerae, Yersinia spp.), protozoa (Cryptosporidium parvum, Entamoeba histolytica, Giardia duodenalis),
and helminths (Trichuris trichiura, Ascaris lumbricoides) as well as multiple internal controls
81
. We
summarize methods previously described below
82
and further detail them in Supporting Information (SI).
Each TAC included eight ports: a no template control (NTC) in the first port, six samples in ports two
through 7 and a positive control (PC) in port eight. We used a total reaction mixture of 100 µL distributed
across each row that included 50 uL of template DNA and 50 uL of qScript XLT 1-step RT-qPCR
ToughMix that includes (Quantabio, MA, USA). For the NTC we used molecular water extracted using
the same protocol as the samples. For the PC template, we used a single-use aliquot mixture of nucleic acid
for each target (gene targets inserted into plasmids) (IDT, Coralville, IA) which were developed using
methods previously described
83
. Amplification under Ct=40 was counted as a positive detection given the
following criteria were met: (1) there was no amplification in the NTC row except for the internal positive
control, (2) the internal positive control column amplified for each row, and (3) there is amplification in the
PC row for all targets in all wells. The threshold of amplification was set for each individual assay at the
point of inflection and we interpreted samples as positive if there was a clear distinction between the
positive and negative amplification curves. A complete description of methods, descriptive statistics,
targets, specific classifications of strains and types included in these assays, and their pathogen relevance
are detailed in SI Text and SI Table 1.
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Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Detection and quantification of enteric pathogens in aerosols near open wastewater canals in cities with poor sanitation" ?

The authors detected a wide range enteric pathogen-specific targets, some not previously reported in extramural urban aerosols, with more frequent detections of all enteric targets at higher densities in La Paz and Kanpur near OWCs. The authors report density estimates ranging from non-detects to 4. It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. An estimated 25 %, 76 %, and 0 % of samples containing positive pathogen detects were accompanied by culturable E. coli in La Paz, Kanpur, and Atlanta, respectively, suggesting potential for viability of enteric microbes at the point of sampling. Airborne transmission of enteric pathogens merits further investigation in cities with poor sanitation. While a rich and rapidly growing body of literature describes microbial risks associated with direct or indirect exposure to fecal contamination in a wide variety of settings, relatively few studies have examined the potential for transmission of enteric pathogens via the aeromicrobiological pathway in highrisk settings. 

Q2. What have the authors stated for future works in "Detection and quantification of enteric pathogens in aerosols near open wastewater canals in cities with poor sanitation" ?

Their quantitative estimates of specific pathogens are an initial step toward further work in understanding the implications of the presence of these microbes in air, including fate and transport modeling and risk assessment. Further work is needed on methods for source-tracking of bioaerosols, including via sequencing approaches. Further studies of specific pathogen transport under specific controlled conditions are needed to fully describe mechanisms of aerosolization, transport, deposition, viability and persistence in aerosols, and risk of exposure to humans. The concurrent detection of culturable E. coli in many samples from La Paz and Kanpur suggests that some of these important pathogenic bacteria ( including pathogenic E. coli ), viruses, and protozoa the authors detected may have been viable at the point of sampling. 

Q3. What are the main factors that affect the persistence of bioaerosols?

The creation and persistence of bioaerosols can be associated with a range of variables related to environmental conditions and the built environment including rain events15–18, meteorological conditions19–21, urban surface waters and water features22–24, wastewater treatment unit processes that include mechanical mechanisms25,26, and other infrastructure. 

Q4. What is the role of aerosols in the spread of enteric pathogens?

Aerosols may allow for transport of enteric pathogens between and among media, contributing to the spread of fecal contamination and associated microbes, resulting in potential for greater exposure via contact, inhalation, or ingestion either directly or indirectly following deposition on a surface, food, water or other subsequent exposure pathway24,122. 

Q5. How is airborne transport of enteric microbes possible?

Combined with mechanisms of aerosolization, airborne transport of enteric microbes and their genetic material is possible in such settings but remains poorly characterized. 

Q6. How many aerosol samples were found in the city of La Paz?

In La Paz, the authors quantified heat-stable enterotoxigenic E. coli (ST-ETEC) in two aerosol samples at densities of 28 gc/m3air and 150 gc/m3air. 

Q7. How many high-volume air samples were collected in Kanpur?

In total, the authors collected 75 high-volume air samples from La Paz (71 collected near OWCs and 4 collected from reference sites >1 km from OWCs), 53 high-volume air samples from Kanpur (45 collected near OWCs and 8 collected from one reference site >1 km from OWCs), and 15 high-volume air samples in Atlanta. 

Q8. What is the epidemiological evidence that proximity to concentrated fecal waste streams in urban areas?

There is some epidemiological evidence that proximity to concentrated fecal waste streams in urban areas can be related to enteric infection risk. 

Q9. What was the threshold of amplification for each individual assay?

The threshold of amplification was set for each individual assay at the point of inflection and the authors interpreted samples as positive if there was a clear distinction between the positive and negative amplification curves. 

Q10. What were the five viral targets detected in the air samples?

Among these positive detections were five viral targets (adenovirus 40/41, panadenovirus, pan-astrovirus, pan-enterovirus, and norovirus GII), and nine bacterial targets (Aeromonas spp., EAEC, ST-ETEC, LT-ETEC, EIEC/Shigella spp., Enterococcus faecium, Salmonella spp., and Yersinia spp.). 

Q11. What is the role of enteric microbes in bioaerosols?

Studies in high-risk, extramural (outdoor) settings in the USA and in other high-income countries have revealed that bioaerosols containing enteric microbes are common where concentrated fecal waste and one or more mechanisms for aerosolization exist. 

Q12. How many previous studies have reported detection of enteric protozoans in air samples?

The authors are aware of only one previous study reporting detection of enteric protozoan parasites in air samples, from rural Mexico, by microscopy; the study reported 8 of 12 samples positive for Cryptosporidium and 10 of 12 samples positive for Giardia, possibly via aerosolization of soil112. 

Q13. Why are studies based on detection of enteric bacteria?

The majority of such studies are based on detection of fecal indicator bacteria including members of the coliform group54,58, partly because the presence of important enteric pathogens is unexpected outside high-burden settings. 

Q14. How many uL of template DNA was used for the qScript XLT?

The authors used a total reaction mixture of 100 µL distributed across each row that included 50 uL of template DNA and 50 uL of qScript XLT 1-step RT-qPCR ToughMix that includes (Quantabio, MA, USA).