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# Introduction
Bats and toothed whales have independently evolved a sophisticated biosonar
system, allowing both clades to diversify and occupy many different niches.
Toothed whales constitute a morphologically and ecologically diverse group of
predators, inhabiting every ocean and several large, freshwater river systems.
Some species forage on deep-sea squid at mesopelagic depths (e.g. sperm whales),
others prey on large schools of fish sparsely distributed in oceanic habitats
(e.g. dusky dolphins) or on individual shrimp and fish encountered in shallow
river systems inhabited by several species of river dolphins, including
Irrawaddy and Ganges river dolphins. While the biosonar signals of many marine
toothed whales have been studied in detail, we know little about the
polyphyletic assembly of true river dolphins and how the biosonar of these old
lineages have evolved to their freshwater habitat.
Toothed whale biosonar signals have been studied in captivity over the last 60
years and increasingly also in the wild. Studies of captive animals have
contributed greatly towards our understanding of the biosonar performance
including dynamic biosonar control. Studies of free-ranging animals complement
laboratory studies by revealing how animals use echolocation in the wild, where
the natural habitat may have physical characteristics very different from
captive settings. Four different types of odontocete biosonar signals have been
identified: Sperm whales produce highly directional echolocation signals
characterized by low centroid frequency and very high peak-to-peak source level
(SL) exceeding 235 dB<sub>pp</sub> re 1 µPa @1 m, which enables them to
echolocate deep-sea squid or other prey at relatively long range. Whistling
delphinids use very short, broadband clicks with centroid frequencies above
60–80 kHz, and peak-to-peak SL of 210–228 dB. Beaked whales produce frequency-
modulated clicks centered around 45 kHz. Peak-to-peak source levels are slightly
lower than delphinid clicks, but due to their much longer duration, they contain
comparable amounts of energy. Lastly, a polyphyletic assemblage of porpoises,
six non-whistling delphinids of the Cephalorhynchus and Lagenorhynchus families,
pygmy sperm whales (*Kogia sp.*), and the Franciscana dolphin (*Pontoporia
franciscana*) all use Narrow Band High Frequency (NBHF) clicks where energy is
concentrated in a narrow frequency band around 130 kHz. These animals seem to
produce nearly as directional biosonar signals as delphinids, but at lower
source levels.
Despite the many studies quantifying sonar parameters for free-living, marine
toothed whales, much less variation in signal type or biosonar parameters has
been found compared to bats, especially among delphinids. However, most of the
delphinids studied to date forage in habitats that may differ less acoustically
than is the case for the different bat guilds. Instead it seems that an inverse
scaling of frequency with body mass to achieve a similar directionality may be a
major driving force across the toothed whale suborder. However, it is unclear
how these selective pressures for high amplitude, high source level biosonar
signals can be extrapolated to the acoustically complex, relatively shallow and
turbid environments inhabited by river dolphins.
To address this question, we studied two species of toothed whales that co-occur
in waterways of the Sundarbans mangrove forest of Bangladesh. Irrawaddy
dolphins (*Orcaella brevirostris*) are freshwater cetaceans living in shallow
coastal waters, generally associated with freshwater inputs, as well as far
upstream in three large, Indo-Pacific river systems. The extent of their inland
range in the Sundarbans varies with seasonal freshwater regimes and may be
influenced by the distribution of Ganges river dolphins. Ganges river dolphins
(*Platanista gangetica gangetica*) are obligate freshwater dolphins found in the
Ganges, Brahmaputra and Karnaphuli river systems where they exhibit a peculiar,
side-swimming form of locomotion. The extent of their downstream range in the
Sundarbans is also determined by seasonally dynamic freshwater flows, with the
Ganges river dolphin favouring low salinity, high turbidity and moderate depth.
Both Irrawaddy dolphins and Ganges river dolphins have relatively small bodies
comparable to small marine delphinids and porpoises. In the Sundarbans, they
inhabit geomorphically complex areas with extremely variable depth, salinity and
turbidity in contrast to the more stable characteristics of marine environments.
Given the complex acoustic environment and high amount of clutter and
reverberation, it may be hypothesized that Irrawaddy dolphins and Ganges river
dolphins employ echolocation signals characterized by low-amplitude, high
frequency sonar signals emitted at high repetition rates like small bat species
hunting in cluttered habitats.
In this study, we quantify the biosonar source parameters of Ganges river
dolphins and Irrawaddy dolphins to test this hypothesis. We show that these
animals use consistently lower source levels and higher repetition rates than
oceanic delphinids, possibly limited by high amounts of clutter and
reverberation. We demonstrate that Ganges river dolphins have a slightly broader
beamwidth than other toothed whales due to their very low centroid frequency but
that they achieve a higher directionality than expected from a direct scaling
with centroid frequency and size, possibly by using a novel set of bony plates
in the forehead. We conclude this study by discussing means to use acoustics to
help better understand the conservation needs of these highly endangered
freshwater toothed whales.
# Materials and Methods
## Study Area
Recordings were obtained in the waterways of the Bangladesh part of the
Sundarban mangrove forest where recording depths varied from 6.5 to 23 m, (mean
12.94 m). Recordings took place during daylight hours between the 4<sup>th</sup>
and16<sup>th</sup> of February 2010 from a 12 m long, wooden research boat. All
research was conducted under a research permit issued to the Bangladesh Cetacean
Diversity Project of the Wildlife Conservation Society by the Ministry of
Environment and Forest, Government of Bangladesh.
## Recording Equipment
A vertical array of four Reson TC4034 spherical hydrophones (Reson A/S,
Slangerup, Denmark) was formed by mounting hydrophones in a Perspex rod (4 cm
diameter, hollow) with 0.75 m spacing. The first hydrophone was positioned at 2
m depth while the last hydrophone was at 4.25 m depth. A buoy was attached to
the top of the array, and a 4 kg weight was fixed to the bottom to help maintain
the array vertical in the water. Signals were amplified 60 dB by a custom-made
amplifier and filter box (1 kHz 1-pole high-pass and 200 kHz 4-pole low-pass
filter), then digitized by two synchronized National Instruments USB-6251 A/D
converters (National Instruments, Texas, USA) at a sampling rate of 500 kHz per
channel and a resolution of 16 bits. The calibrated clip level of the recording
chain was 174 dB re µPa (peak), and the frequency response of the recording
chain was flat (±2 dB) from 2–180 kHz.
## Data Collection
Ganges river dolphins were recorded while foraging or resting at the
convergences of channels. Irrawaddy dolphins were recorded during different
behaviors (travelling, foraging, and socializing). The boat engine was turned
off and the array was lowered into the water once the animals were within about
100 m of the vessel. Data acquisition was initiated and terminated manually and
files were stored approximately every minute. Start and end time, position and
depth were recorded for every recording event, as well as group composition and
behavior.
## Click Analysis
Signal analysis was carried out with custom-written routines in Matlab 7.5 (The
Mathworks, Inc., Natwick, MA, USA). Each click series (also referred to in the
literature as a click train) was examined visually and discarded if more than
one animal was present to avoid underestimating interclick intervals.
Echolocation clicks were then located on the third hydrophone using an automated
click detector with a variable detection threshold chosen during visual
inspection of waveforms to exceed the background noise level and detect
individual click series. Each click was further analyzed only if detected on all
four channels.
## Acoustic Localization
Source location relative to the hydrophones was obtained through acoustic
localization techniques based on time-of-arrival differences of the same click
on the four receivers. To find the time of arrival differences, the signal
recorded on the top hydrophone was cross-correlated with the signals recorded on
the other hydrophones, excluding surface reflections. A sound speed of 1500 m/s
was measured in each recording habitat by emitting pulses with a portable
echosounder (Speedtech, Virginia, USA) at the position of the top hydrophone and
cross-correlating to find the time-of-arrival at the remaining hydrophones at
known distances. For each pair of hydrophones, the time-of-arrival difference
can be explained by the equation for a single hyperbola in the two-dimensional
plane of the array. Using four receivers, equations for three independent
hyperbolas can be generated, and the position of the sound source found by
solving the three equations with a least-squares method.
Acoustic localization with this array was calibrated in Aarhus Harbour, Denmark,
using artificial clicks (2 cycles at 70 kHz) generated by an omnidirectional
HS70 hydrophone (Sonar Products) connected to a waveform generator (model
33220A, Agilent Technologies, California, USA). Pulses were emitted from a depth
of 2 m and at distances from 5 m to 40 m from the array. Speed of sound during
this calibration was calculated using the Leroy equation from measured
temperature and salinity values.
## Source Parameter Estimation
The interclick interval (ICI) was defined as the time between each click and the
previous. Received levels were calculated as peak-peak (pp) and root-mean-square
(rms) sound pressure levels within a time window given by the −10 dB end points
relative to the peak of the amplitude envelope. The temporal duration of clicks
was defined as the length of the −10 dB time window. The energy flux density was
calculated for each click as the sum of squared sound pressure values within the
−10 dB analysis window. Subsequently, the click power spectrum was calculated as
the squared Fast Fourier Transform of a 32-point window centred on the peak
envelope of each signal. The power spectrum was then normalized and interpolated
with a factor of 100 using a low-pass interpolation. Peak frequency, centroid
frequency (defined as the frequency separating the power spectrum into two
halves of equal energy) and signal bandwidth (−3 dB power and −10 dB power) was
calculated from this power spectrum. Source levels (SL) were defined as the
back-calculated sound pressure level 1 m from the source on the acoustic axis,
and calculated from received levels by compensating for the transmission loss
(dB re. 1 m), estimated as the combination of spherical spreading and frequency-
dependent absorption (taken at the centroid frequency of the received click)
over the range from the source coordinates to the receiver.
## On-axis Criteria
Off-axis signals are subjected to distortion. This means that it is essential to
quantify the signal on or as close as possible to the acoustic axis when
investigating source parameters of highly directional biosonar signals. With a
linear array, the vertical angle of incidence can be estimated, but the
horizontal angle of incidence is unknown. To maximize the likelihood of
analyzing on-axis clicks, we selected only the highest-amplitude click in a
longer click sequences (scans) with clicks of increasing and decreasing
amplitude. These scans are most likely associated with the acoustic beam of the
animal passing across the axis of the array. Assuming the animal maintains the
same source level and directionality, the click with the highest amplitude has
the highest likelihood of being on-axis in the horizontal plane. The criteria
used to determine if the click was on axis is similar to that described in
previous studies with similar arrays, : (1) the click could be localized; (2)
the click had the highest received level in a scan (and thus assumed to be on-
axis in the horizontal plane); and (3) the highest received level was recorded
on one of the two central hydrophones, allowing for estimation of the angle of
incidence in the vertical plane.
## Implications for Passive Acoustic Monitoring
To evaluate the use of sound source parameters for passive acoustic monitoring
studies without the potential for identifying on-axis clicks, a set of click
series with only one clicking animal was identified. Each of these click series
was passed through an automatic click detector (described above) to find
accurate inter-click intervals for the two species. Subsequently, the power
spectrum of each click was analyzed to find the centroid frequency.
# Results
Irrawaddy dolphins were recorded on 16 different occasions during a total of 9
hours, 58 minutes of recordings. The median group size encountered during
recordings of Irrawaddy dolphins was 3 animals. During recordings, this species
was observed while foraging and travelling. Ganges river dolphins (median group
size 4 animals) were recorded in two different occasions and a total of 57
minutes of recordings were obtained from these encounters. In both recording
occasions, the Ganges river dolphins were located in channel convergences.
The hydrophone localization calibration indicated that clicks within 40 m were
localized with a resulting error in the transmission loss estimates of less than
3 dB, which was deemed acceptable in accordance with previous studies.
Consequently, only clicks recorded within a 40 m range of the hydrophone array
were used for the analysis of the source parameters.
A total of 15 Irrawaddy dolphin and 29 Ganges river dolphin clicks met the on-
axis criteria and were recorded within the localization range of 40 meters. Only
one click from each scan was used for analysis, and all recording areas were
well separated to prevent recording the same groups of animals repeatedly.
Clicks for both species were broadband transients similar to those of marine,
whistling delphinids. Mean click duration ± SD was 13.4±3.0 µs for Irrawaddy
dolphins and 21.7±2.2 µs for Ganges river dolphins, and Q ratios (defined as the
ratio of centroid frequency to RMS bandwidth) was 3.2±0.3 (mean±SD) for
Irrawaddy dolphins and 3.1±0.3 for Ganges river dolphins.
Ganges river dolphin click source levels were significantly lower than the
source levels of Irrawaddy dolphin clicks (Kruskal-Wallis: p\<0.0001). Peak-to-
peak source levels (mean±SD) were 194.5±3.6 dB re 1 µPa at 1 m for Irrawaddy
dolphins and 183.3±3.4 dB re 1 µPa at 1 m for Ganges river dolphins. For both
species, these source levels are significantly lower (Kruskal-Wallis: p\<0.0001)
than source levels produced by a marine delphinid, the Indopacific Bottlenose
dolphin (*Tursiops aduncus*) recorded in a 5–8 m shallow bay (mean peak-to-peak
source levels ± SD of 205±7 dB re 1 µPa at 1 m) and lower than published source
levels from most other free-ranging toothed whales with the exception of some
species producing narrow-band high-frequency clicks. Similarly, the mean source
energy flux density was 136.3 dB re 1 µPa<sup>2</sup>\*s at 1 m for Irrawaddy
dolphins and 126.6 dB re 1 µPa<sup>2</sup>\*s at 1 m for Ganges river dolphins.
There was no significant relationship between the recording range and the source
levels for either species (Kruskal-Wallis: p = 0.46 for Ganges river dolphins
and p = 0.45 for Irrawaddy dolphins). The centroid frequency (mean±SD) for
Irrawaddy dolphins was 94.6±9.7 kHz, with −3 dB bandwidth of 64.4±15.8 kHz.
Ganges river dolphins had a significantly lower centroid frequency (mean±SD) of
61.4±4.9 kHz (Kruskal-Wallis: p\<0.001) and correspondingly also a significantly
lower −3 dB bandwidth of 43.8±7.1 dB (Kruskal-Wallis: p\<0.001).
Interclick intervals were measured for both species for all on-axis clicks. The
ICI values for on-axis clicks were higher than ICI values measured across entire
click series. Interclick intervals (mean±SD) for Irrawaddy dolphin on-axis
clicks was 44.8±24.6 ms and for Ganges river dolphin on-axis clicks it was
35.0±18.4 ms. In addition, the ICI was measured for entire click series with
good signal-to-noise-ratio (SNR) and only one clicking animal at a time. A total
of 923 clicks across 41 click series were analyzed for the ICI values of
Irrawaddy dolphins and 614 clicks across 25 click series for Ganges river
dolphins. For the entire click series, ICI (mean±SD) for Irrawaddy dolphins was
33.5±13.5 ms, and for Ganges river dolphins it was 29.9±9.0 ms.
To test the potential for species discrimination in passive acoustic monitoring,
probability density functions for Ganges river dolphin and Irrawaddy dolphin
centroid frequencies were calculated using means and standard deviations from
this paper, and assuming a normal distribution. In addition, a normalized
probability density function for the Yangtze finless porpoise species
(*Neophocaena phocaenoides asiaeorientialis*) was calculated using peak
frequency (comparable to centroid frequency for narrowband high frequency
species) and standard deviations from Li et al.. An estimated best separation
criterion of 72.5 kHz provided a theoretical 98.7% correct classification of
Ganges river dolphin clicks and 98.9% correct classification of Irrawaddy
dolphin clicks, whereas an estimated best separation criterion of 112.35 kHz
provided 97.2% correct classification of Irrawaddy dolphins and 96.7% correct
classification of finless porpoises. For off-axis clicks, spectral distortion
increases low-frequency energy so centroid frequency estimates decrease. This
meant that the classification of Irrawaddy dolphins decreased to 72.7% (N = 971)
with the remainder being misclassified as Ganges river dolphins. Ganges river
dolphins, in contrast, were successfully classified 99.2% of the time (N = 641).
# Discussion
The study of toothed whale biosonar signals has developed rapidly during the
last decade. Most studies have focused on marine delphinids and have revealed
consistent high amplitude, highly directional echolocation signals from these
species. Here, we recorded two small toothed whale species inhabiting areas that
are more acoustically complex compared to the open ocean environments of many
delphinids to better understand the evolutionary factors shaping different
biosonar parameters of echolocating toothed whales.
Both species produce broadband echolocation clicks characterized by a short
duration and a low Q ratio of centroid frequency to RMS bandwidth of around 3. A
short, broadband echolocation click is characteristic of all whistling
delphinids, as well as sperm whales. The family platanistidae is an ancient
evolutionary lineage that diverged not long after physeteridae. Its use of
short, broadband clicks corroborates the hypothesis that the echolocation signal
evolved by the shared ancestor of toothed whales was a short, broadband click
that gradually evolved towards higher frequencies as greater high-frequency
hearing sensitivity co-evolved with the capacity for high-frequency sound
production.
Echolocating toothed whales normally wait until the echo from a potential target
has been received before producing a new click, meaning that the interclick
interval between clicks exceeds the two-way travel time plus a processing lag
time. When animals are searching, the interclick interval may also reflect the
limits of their environment, such as the back wall of a pool or for a deep-
diving animal, the altitude above the sea floor where the animal is operating.
The interclick interval is therefore often taken as a maximum estimate of the
acoustic search range of an echolocating animal. The two animals studied here
both had higher click repetition rates compared to Indo-Pacific bottlenose
dolphins (*Tursiops aduncus*) and even higher click repetition rates than
coastal harbor porpoises \[mean ICI: 80.5 ms, 47\] and riverine Yangtze finless
porpoises \[mean ICI: 60.4 ms, 47\]. This indicates that both Irrawaddy dolphins
and Ganges river dolphins were searching for prey within a shorter range than
most other studied odontocetes.
Concurrent with the higher repetition rates, the two species also produced
echolocation signals with much lower source level compared to similar sized
marine delphinids. Irrawaddy dolphins (mean source levels ± SD of 194.7±4 dB re
1 µPa pp at 1 m) and Ganges river dolphins (183.6±3.5 dB re 1 µPa pp at 1 m)
echolocate at more than 10 dB to 20 dB (respectively) lower source levels than
other small, oceanic delphinids such as free-ranging pygmy killer whales
(*Feresa attenuata*), bottlenose dolphins (*Tursiops sp.*), white-beaked
dolphins (*Lagenorhynchus albirostris*), spinner (*Stenella longirostris*) and
spotted dolphins (*Stenella attenuata*), and dusky dolphins (*Lagenorhynchus
obscurus*, max 210 dB pp). Common to these species is that they often forage in
an environment where background noise is the limiting factor that determines how
far away the faint echoes from prey organisms can be detected. In a noise-
limited echolocation scenario, the echo-to-noise ratio increases proportionally
with the source level so that a greater detection range can be achieved by
increasing the amplitude of the outgoing signals. For many of these exclusively
marine species, the detection range of sparse, patchily distributed prey is a
crucial parameter for survival. Selection for a long detection range would
therefore promote the evolution of high-amplitude echolocation signals within
the constraints provided by the size of the animal, principally the dimensions,
composition and biomechanics of the sound-generating nasal structures.
The overall body size of many oceanic delphinids is larger than the animals
studied here, and it is possible that this size difference could account for the
lower source levels of our animals. Indeed, large echolocating animals tend to
produce echolocation clicks at high source levels and scaling of source level
with body size might explain the low source levels produced by small species
such as dusky dolphins. However, Ganges river dolphins are about the same size
as dusky dolphins and spinner dolphins and produce similar biosonar clicks (as
characterized by short duration and low Q) but with a maximum measured source
level of 191 dB re 1 uPa (pp), about 20 dB lower than the maximum measured
source levels for the dusky dolphins. Irrawaddy dolphins are larger than both
dusky dolphins and Ganges river dolphins yet produce source levels on average
nearly 10 dB lower than dusky dolphins. Porpoises and other NBHF species have
also been thought particularly adapted to coastal environments, and these
species are mostly similar in size or smaller than the Ganges river dolphin. The
longer duration of NBHF signals compared to broadband delphinid signals means
that it is most appropriate to compare the click energy flux density between
species. Source levels of porpoises are comparable to the two species recorded
here, with source energy flux density (SL<sub>EFD</sub>)for harbor porpoises
(*Phocoena phocoena*) (mean SL<sub>EFD</sub>: 137 dB re 1 µPa<sup>2</sup>\*s)
similar to the source energy flux density of Irrawaddy dolphin clicks; Peale’s
dolphins (*Lagenorhynchus australis*) with somewhat intermediate source levels
(mean SL<sub>EFD</sub>: 133 dB re 1 µPa<sup>2</sup>\*s); and Commerson’s
dolphins (*Cephalorhynchus commersonii*) with source levels as low as Ganges
river dolphin (mean SL<sub>EFD</sub>: 125 dB re 1 µPa<sup>2</sup>\*s). However,
while porpoises and other NBHF species resemble the two study species here both
in size and source level, they echolocate at much higher peak and centroid
frequencies around 130 kHz. These species have seemingly undergone evolutionary
selection for a high-pass filtered biosonar signal, possibly to avoid predation
from other toothed whales such as killer whales (*Orcinus orca*). Ganges river
dolphins diverged out early in the evolution of *odontoceti*, and it is unlikely
that these animals ever risked predation by killer whales. However, the NBHF
signal type is a subsequently derived biosonar signal that comes at the cost of
a smaller bandwidth and thereby presumably less information about the acoustic
environment and it does not help explain why the two species in this study
produce source levels below those of oceanic delphinids.
One important challenge that these animals face is the task of locating and
catching food in an acoustic habitat with high reverberation and clutter levels.
Several studies have shown how close proximity to clutter or to the bottom may
interfere with the detection of targets. Reverberation from the bottom will
necessarily depend on signal frequency, grazing angle, bottom sediment type, and
especially depth. The two species here both forage for sparse prey through
relatively shallow environments (10–15 m in the Sundarbans). While it is
difficult to quantify both underwater clutter and reverberation, it is
reasonable to assume that a shallow, restricted river habitat provides more
challenging acoustic conditions than the open ocean. Unlike a noise-limited
situation, higher source levels do not help detect targets in either
reverberation or clutter limited conditions, as the backscattered echo from
clutter or bottom will be just as much greater as the echo from potential
targets. In addition, forward masking of the outgoing click may play an
increasingly important role for toothed whales echolocating at very close range.
Consequently, we argue that the acoustic properties of the shallow-water habitat
might have favored the use of clicks with relatively low source levels in
Irrawaddy and Ganges river dolphins.
If reverberation can play an important role in shaping the source levels of
echolocating toothed whales, this might also explain the lower source levels
found for the Indo-pacific bottlenose dolphins (*Tursiops aduncus*) in a shallow
coastal habitat, compared to deep-water common bottlenose dolphins (*Tursiops
truncatus*). While common bottlenose dolphins are capable of detecting a metal
target on a sandy bottom at up to 70 m range despite the clutter caused by the
environment, the typical prey of Irrawaddy dolphins and Ganges river dolphins
constitute small fish and shrimp. The low target strength and varied bottom
composition in shallow water may prove to be a more complex discrimination task
for the animals than detecting high target strength, metal objects. While
quantitative measurements of prey target strength and reverberation in different
river habitats are needed to support this, we hypothesize that both Irrawaddy
dolphins and Ganges river dolphins gain an advantage by using low source level
clicks for detecting and discriminating small prey items in shallow-water,
cluttered environments. This is not unknown among echolocating animals. Brinkløv
et al. demonstrated that the long-legged bat (*Macrophyllum macrophyllum*)
gradually decreased the source levels of its echolocation calls when operating
in three increasingly cluttered environments. Clutter-imposed constraints from
such habitats may have resulted in microchiropteran bats having specialized into
guilds inhabiting different foraging niches, with longer detection range
seemingly favored for open space foragers compared to bats hunting within dense
vegetation. This situation may be paralleled for source levels of toothed
whales: Oceanic delphinids use high source levels to find prey at long range in
open areas; Irrawaddy dolphins utilize coastal habitats and venture upriver
while using intermediate source levels for echolocation; and Ganges river
dolphins, which diverged early from the remaining toothed whales and evolved in
a spatially restricted freshwater habitat, received little advantage from long-
range echolocation and use the lowest measured source levels best suited for
echolocating prey at short range. It therefore seems that the selective
pressures that have favored the evolution of high frequency, high source level
biosonar signals in marine toothed whales cannot be extrapolated to the complex
acoustic habitats of freshwater cetaceans.
A central component in the high source levels of toothed whales is the
production of a narrow echolocation beam through partial collimation of the
acoustic energy. Evolution appears to have favored toothed whales with a high
directionality index that seems to be remarkably similar across species, with
horizontal −3 dB (half-power) beamwidths reported between 13.1 degrees for a
harbor porpoise to 6.5 degrees for a beluga (*Delphinapterus leucas*) and 6.2
degrees for a false killer whale (*Pseudorca crassidens*). Large odontocetes
(such as sperm whales or beaked whales) can achieve a certain directionality
with lower frequencies than smaller whales (such as porpoises or small
delphinids) , and this might explain the overall negative correlation between
biosonar frequency and body size in toothed whales. From this relationship
between body size and frequency, we would predict a relatively high centroid
frequency of around 80–100 kHz for the moderately sized Irrawaddy dolphins and a
higher centroid frequency of around 80–120 kHz for the small Ganges river
dolphins. While Irrawaddy dolphins produced clicks with a relatively high
centroid frequency (mean of 92 kHz), the Ganges river dolphins produced clicks
with a surprisingly low centroid frequency (a mean±SD of 61.4±4.9 kHz) compared
to their body size. Other toothed whales of similar size use biosonar centroid
frequencies of around 70–85 kHz (Pygmy killer whales), 80 kHz (Hawaiian spotted
dolphins and spinner dolphins), 90–100 kHz (Dusky dolphins) and around 130 kHz
for the many NBHF species. The measured centroid frequency and the small size of
the Ganges river dolphin would predict approximately half the directionality (6
dB smaller DI) and consequently a much broader beamwidth compared to delphinids
and porpoises. Using equations derived from Au et al. and Madsen and Wahlberg
(2007), the Ganges river dolphin should have a symmetric −3 dB beamwidth of some
20 degrees and a directionality index (DI) of some 19 dB. This prediction
conflicts with findings reported in the only paper investigating the
directionality of Ganges river dolphins: Bahl et al. reported that the −3 dB
beamwidths of the Ganges river dolphins were in the order of 10 degrees in the
horizontal plane and 14 degrees in the vertical plane. We find a similar, but
slightly higher value, when fitting the data from Bahl et al. (2007) with a
piston that best describes the variation in the data. The data indicate a
single-lobed sound beam like all other toothed whales studied so far rather than
the peculiar, double-lobed sound beam reported in the early literature. The
best-fitting piston model provides a composite beamwidth of 14.5 degrees in the
horizontal plane. Such a half power beamwidth corresponds to a DI of 22 dB which
is comparable to or slightly lower than the half power beamwidth of harbor
porpoises, but around 3 dB (50%) better directionality index than predicted from
the low frequency clicks and the small head size of the Ganges river dolphin.
Thus, somehow Ganges river dolphins seem to generate a beam directionality that,
albeit slightly lower than most toothed whales, is comparable to that of similar
sized toothed whales operating almost an octave higher in frequency. The reason
for this apparent discrepancy might well lie in the unusual head anatomy of this
species: Ganges river dolphins possess two unusual bony maxillary crests that
project anteriorly over the facial region and virtually encircle the melon. They
are asymmetrical and skewed to the left, and their ventral surfaces are
dominated by a thin network of air sacs that seem to have grown dorsally from
the pterygoid air sinus system. Purves and Pilleri and Pilleri and colleagues
proposed that the crests might function in directing the sound from the melon.
It is thus possible that these air-filled bony crests could help provide a
better directionality than expected from scaling, and hence explain why Ganges
river dolphins can produce clicks at centroid frequencies about an octave below
what should be predicted from their size and still achieve a sufficient
directionality. These findings support the notion that one of the evolutionary
drivers for the echolocation click frequency in toothed whales is indeed
directionality. The estimated beamwidth of Ganges river dolphins is still in the
broad end of measured toothed whale biosonar beams. While this might be
considered a more primitive condition, a slightly wider beam combined with the
greater short-range maneuverability of these animals (a consequence of having
completely free cervical vertebrae), may facilitate the capture of highly
maneuverable prey items at close range throughout a shallow, cluttered rivers
habitat.
The significant difference in frequency content for these two species might be
useful for acoustic species recognition such as seen in songbirds and other
animals, and arguably also for some sympatric delphinids. Passive acoustic
monitoring efforts may exploit such differences to locate critical species-
specific hotspots for these endangered species. The three toothed whale species
typically found in the coastal and river areas of the Sundarban National Forest
include *Platanista gangetica gangetica*, *Orcaella brevirostris* and
*Neophocaena phocaenoides*. The on-axis biosonar centroid frequencies of these
species are well separated, and spectral parameters may be a promising way of
both detecting and discriminating these animals acoustically. However, because
biosonar signals are somewhat distorted when recorded off the acoustic axis,
signals recorded away from the acoustic axis will have a lower frequency
emphasis ( B and C). Applying the centroid frequency criteria that best
separates on-axis clicks to a long series of clicks that would resemble what a
passive acoustic monitor could record, results in clicks from Ganges river
dolphins classified correctly nearly all the time (99.2% correct classification)
whereas clicks from Irrawaddy dolphins were classified less successfully (72.7%
correct classification). This results in some Irrawaddy dolphin clicks being
incorrectly classified as Ganges river dolphins. The same degree of spectral
distortion does not happen with NBHF clicks, whereby passive acoustic monitoring
would be able to detect the presence of both finless porpoises and Irrawaddy
dolphins reliably. Other criteria would be necessary to reliably classify Ganges
river dolphins and discriminate such detections from off-axis Irrawaddy
dolphins. One way of doing this would be to shift the separation criteria
slightly upwards, and to use only the maximum centroid frequency for a series of
clicks. For this dataset, reliable discrimination would be achieved based on the
maximum frequency of 11–15 clicks and evaluated using a separation criterion of
74 kHz. In addition to spectral species discrimination, source levels presented
here would be essential for estimating the detection function of an acoustic
monitoring system, providing the basis for quantifying abundance of these
threatened freshwater species.
Acoustic monitoring has proven to be a powerful method for determining range,
seasonality, and abundance of animals, and may prove essential for understanding
the population parameters of cryptic, aquatic animals such as beaked whales, or
finless porpoises. Freshwater dolphins all face significant extinction risks,
primarily due to habitat loss and fisheries interactions, which led to the
recent functional extinction of the Baiji (*Lipotes vexillifer*). Robust
acoustic discrimination mechanisms that allows for monitoring of Irrawaddy
dolphins and Ganges river dolphins could be especially helpful for managing
protected areas such as the three new wildlife sanctuaries that were established
by the Government of Bangladesh in the Sundarbans for the conservation of both
species and provide better information that can help prevent a continued decline
or extinction of these two threatened freshwater species.
## Conclusion
Irrawaddy dolphins and Ganges river dolphins within the river systems of the
Sundarban mangrove forest use high repetition rate, low source level
echolocation clicks compared to marine species of similar size. Whereas obligate
marine delphinids use high source level echolocation signals, Irrawaddy
dolphins, inhabiting coastal and upriver habitats, produce lower source levels,
with mean source levels of 194.7 dB (max 203 dB) re 1 µPa<sub>pp</sub> and
Ganges river dolphins, living exclusively in a shallow river habitat, produce
even lower source levels of 183.6 dB (max 191) re 1 µPa<sub>pp</sub>. The
ultimate cause of these low source levels may be a relaxed selection for long-
range echolocation inhabiting restricted, shallow, geomorphically complex river
systems, with limits on echolocation range imposed by reverberation and clutter.
Interestingly, the centroid frequency of the clicks used by Ganges river
dolphins is almost an octave lower than expected from their size. The unusual,
air-filled bony maxillary crests found in this species may compensate in part
for this lower frequency by providing a larger effective baffle and hence a more
directional sound beam than the biosonar frequency and head size would predict.
The beamwidth of Ganges river dolphins is still wider than most other toothed
whales, and it is possible that this may facilitate capture of highly
maneuverable prey items in shallow water. Acoustic discrimination between
freshwater odontocetes may facilitate acoustic monitoring efforts and may help
prevent a continued decline of these two threatened freshwater species.
This study was made possible through the logistical and field support of the
Bangladesh Cetacean Diversity Project of the Wildlife Conservation Society. The
study was conducted under a research permit issued by the Ministry of
Environment and Forest, Government of Bangladesh. E. Fordyce, A. Galatius, J.
Ososky, J. Mead, and C. Potter kindly provided pictures and helpful discussions
of internal skull morphology. J. S. Jensen and N. U. Kristiansen provided
technical support and K. Beedholm assisted with invaluable discussions and
technical help. Finally, three anonymous reviewers offered helpful comments and
constructive feedback to improve on the manuscript.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: FHJ AR PTM. Performed the
experiments: FHJ AR RMM BDS. Analyzed the data: FHJ AR. Contributed
reagents/materials/analysis tools: PTM VMJ. Wrote the paper: FHJ AR RMM BDS
VMJ PTM. |
# Introduction
*P. marneffei* is considered an indicator pathogen for AIDS. It mainly exits
endemically in area of South East Asia that causes fever, lymphadenopathy,
hepatosplenomegaly and cutaneous lesions. *P. marneffei* has the unique feature
among the species of *Penicillium* of being thermally dimorphic for diagnosis,
it grows as a mycelium at 25°C, and a soluble red pigment is produced, whereas,
at 37°C, it grows as a yeast form. The clinical features of *P. marneffei* in
AIDS patients have been well described, and like other opportunistic pathogens,
the infection of *P. marneffei* would exacerbate deterioration of the immune
response and accelerate AIDS disease progression, while the mechanism remains
elusive.
Dendritic cells (DCs) play pivotal roles in host defense by initiating innate
immunity and bridging adaptive immunity. DCs are widely distributed in the sub-
mucosa, yet have been believed to be involved in HIV-1 infection and
transmission. The migration property of DCs has been hijacked by HIV-1 for viral
dissemination to CD4<sup>+</sup> T cells by a process that is known as
*trans*-infection. The formation of DC-T-cell conjunction, or so-called
virological synapses, at which numerous intact viral particles and viral
receptors can be recruited, appears to be required for efficient viral transfer.
Upon activation by stimuli, such as the bacterial product LPS
(lipopolysaccharide), DCs could uptake much more viruses and recruit
significantly amounts of viral particles on the virological synapses for
enhancement of HIV-1 *trans*-infection. DCs express HIV-1 receptors and can
serve as targets for productive HIV-1 replication. Persistent infection of HIV-1
may generate the potential long-lived viral reservoirs in DCs. DCs appear to
take vital roles in HIV-1 infection and viral pathogenesis, and a better
understanding of the interactions between HIV-1 and DCs would facilitate the
elucidation of AIDS pathogenesis.
We hence isolated *P. marneffei* from the cutaneous lesions of AIDS patients and
analyzed its effects on HIV-1-dendritic cells interaction. We found that MDDCs
could be activated by both dimorphic forms of *P. marneffei* for significantly
promoting HIV-1 *trans*-infection of CD4<sup>+</sup> T cells, and the *Candida
albicans* (*C. albicans*), which has been proved to possess the similar
capacity, was used as control. Increased expression of intercellular adhesion
molecule 1 (ICAM-1) was observed on fungus-activated MDDCs, and the tighter DC-
T-cell conjunction recruited significant amounts of virus particles for viral
transfer. We also found that *P. marneffei*-activated MDDCs efficiently
activated resting CD4<sup>+</sup> T cells through cell-cell contact and thereby
could result in more susceptible targets for viral infection. Our findings
demonstrate that DC function and its interaction with HIV-1 have been modulated
by opportunistic pathogens such as *P. marneffei* for viral dissemination and
infection amplification, highlighting the importance of understanding DC-HIV-1
interaction for viral immunopathogenesis elucidation.
# Results
## P. marneffei stimulation promotes the activation of MDDCs
In current study, the *C. albican* which has been described previously for
induce DC activation was used as a control. *P. marneffe* and *C. albicans* were
isolated separately from the skin lesions or the tongues of AIDS patients and
cultured in Sabouraud agar plates. *P. marneffei* has the unique feature among
the species of *Penicillium* of being thermally dimorphic, it grows as a
mycelium at 25°C, similar to *Aspergillus* spp, and a soluble red pigment is
produced (PMm-i and-ii), whereas, at 37°C, it grows as a yeast form (PMy). *C.
albicans* was identified with sub-inoculation in CHROMagar Candida (, CA-ii), in
Corn Tween agar (CA-iii) and with the API 20C AUX yeast identification system.
These fungi were sub-cultured for amplification and harvested for heat
inactivation.
To investigate the potential activation of DCs by fungi, MDDCs were incubated
separately with heat-inactivated *C. albicans*, PMy and PMm at a ratio of 1∶10
of DCs to fungi. The phenotypes of MDDCs were examined by immunostaining of cell
surface markers; MDDCs showed high level of CD11c expression and were measured
for CD83, CD86 and HLA-DR expression levels. Fungal stimulation significantly
increased CD83, CD86 and HLA-DR expression compared with medium-treated cells,
indicating of fungus-induced MDDCs activation, whereas surface expression of a
C-type lectin, DC-SIGN (DC-specific intercellular adhesion molecule 3-grabbing
nonintegrin), was decreased in fungus-treated MDDCs. These data suggest that
stimulation of *P. marneffei* promotes DCs activation.
## HIV-1 infection of P. marneffei-activated MDDCs is blocked
To examine the effects of the stimulation of *P. marneffei* on HIV-1 infection
of MDDCs, fungus-treated MDDCs were inoculated with single-cycle, luciferase
reporter HIV-Luc/JRFL (CCR5-tropic), and HIV-1 infection was measured by
detecting the luciferase activity in cell lysates at 3-9 days post-infection.
HIV-1 infection was fully blocked in all fungus-treated MDDCs compared with
medium-treated controls, and the detected luciferase activities decreased by at
least 75% at 5, 7 and 9 days post-infection. The *C. albicans*, which has been
shown to inhibit HIV-1 replication in DCs, was used as a control. These data
suggest that HIV-1 infection in MDDCs is blocked after the stimulation of *P.
marneffei*.
## P. marneffei stimulation promotes DC-mediated HIV-1 transmission to CD4<sup>+</sup> T cells
To determine the capacity for DC-mediated HIV-1 transmission after *P.
marneffei* stimulation, MDDCs were treated with heat-inactivated fungi as above
and GFP-containing HIV-1 VLPs were used to measure viral transmission efficiency
using flow cytometry. HIV-1 VLP-loaded MDDCs were co-cultured with human
CD4<sup>+</sup> T-cell line Hut/CCR5 for 30 min, Hut/CCR5 cells with the
CD11c<sup>-</sup> staining were gated, and Gag-GFP-associated cells were
quantified. The level of GFP association on Hut/CCR5 cells increased from 11% in
medium-treated controls to 27–30% in fungus-activated MDDCs, the MFI values
showed an over 2-fold increase. Thus, the fungus, including *P. marneffei* and
the *C. albicans* control, can promote MDDC-mediated HIV-1 transfer to
CD4<sup>+</sup> T cells.
Viral *trans* infection was also quantified by using the DC-T-cell co-culture
system as described previously. Pseudotyped single-cycle, luciferase reporter
HIV-1 was used. Hut/CCR5 and activated autologous primary CD4 <sup>+</sup> T
cells were used as the target cells. Differently treated MDDCs loaded HIV-
luc/JRFL or HIV-luc/NL4-3 were co-cultured with target cells for 3 days, and
HIV-1 infection was monitored by measuring luciferase activity. Fungus-
stimulated MDDCs significantly enhanced the capacity to mediate HIV-luc/JRFL or
HIV-luc/NL4-3 *trans* infection of HutCCR5 cells, there was a 4.8- to 6.5-fold
increase in luciferase activity, when activated primary CD4<sup>+</sup> T cells
were used as target, fungus-stimulated MDDCs enhanced HIV-luc/NL4-3
*trans-*infection of primary CD4<sup>+</sup> T cells approximately 2-3- fold.
*C. albicans*, having been shown previously to promote DC-mediated HIV-1
transmission, was used as a positive control. Together, these results indicate
that MDDCs stimulated by PMm and PMy enhanced their capacity to mediate HIV-1
*trans* infection of CD4<sup>+</sup> T cells.
## Enhanced endocytosis and altered intracellular trafficking of HIV-1 in fungus-activated MDDCs
LPS-activated DCs potently enhance HIV-1 *trans* infection and the endocytosis
of large amounts of viruses, and the harboring of intact viruses in non-
classical multiple vesicular bodies might provide viruses with a means to escape
from intracellular proteolysis. To investigate whether fungal stimulation
similarly affect on viral endocytosis, fungus-stimulated MDDCs were pulsed with
HIV-1 VLPs for 2 h. Trypsin was used to strip cell-surface-bound virus
particles, and the internalized viral particles were quantified by detection of
Gag-GFP by flow cytometry. Numerous viruses were internalized by fungus-
stimulated MDDCs. Gag-GFP was demonstrated in 53.5–57.3% of MDDCs stimulated by
*C. albicans*, PMy or PMm, compared with 15.9% in medium-treated immature MDDCs,
and the calculated MFI of GFP also exhibited a two-fold increase relative to
that in medium-treated controls. The majority of MDDCs-associated viruses could
not be removed by trypsin digestion. Blocking with anti-DC-SIGN antibodies
before VLP pulsing did not inhibit viral uptake, suggesting a DC-SIGN-
independent endocytosis process. By contrast, as shown previously, immature
MDDCs bind HIV-1 mainly through surface-expressed DC-SIGN, which can be easily
removed by trypsin treatment.
To better understand intracellular trafficking in fungus-stimulated DCs, HIV-1
VLP-pulsed MDDCs were visualized by confocal microscopy with immunostaining.
Fewer viruses were evenly distributed on the surface or were internalized in
medium-treated immature MDDCs, whereas many viral particles were endocytosed and
concentrated into the CD81<sup>+</sup> DC-SIGN<sup>-</sup> compartments in
fungus-treated MDDCs. These results are consistent with previous observations
that LPS-stimulated MDDCs sequester intact HIV-1 in a specialized and
tetraspanin CD81<sup>+</sup> compartments. These data suggest that the
stimulation by PMy, PMm or *C. albicans* largely promotes HIV-1 endocytosis and
sequestration within the tetraspanin CD81<sup>+</sup> compartments of fungus-
activated DCs.
## Fungus-activated MDDCs increase ICAM-1 expression, and facilitate DC-T cell contact formation and viral concentration in virological synapses
The virological synapses have been demonstrated to provide the most efficient
route for HIV-1 transfers between contacting cells. The ICAM-1-LFA-1 interaction
has been proved to be involved in the formation of DC-T-cell conjunction and
contribute to efficient HIV-1 transfer. To investigate further the mechanism by
which fungal treatment enhances viral transfer, ICAM-1 expression on the cell
surface was measured. Stimulation with heat-killed PMy, PMm and *C. albicans*
enhanced ICAM-1 expression on MDDCs by 2.4- to 2.6-fold, which indicates the
potential for tighter cell conjunction formation. The formation of virological
synapses was visualized by confocal microscopy, and Hut/CCR5 or activated
primary CD4<sup>+</sup> T cells were used as target cells. Many viral particles
were efficiently concentrated at the fungus-stimulated DC-T cell contact sites
to form virological synapses. In more detailed analysis of the staining, the
tetraspanin molecule of CD81 was recruited to the contact sites, which suggests
a potential role of CD81 in HIV-1 trafficking. Taken together, these data
demonstrated that the enhanced ICAM-1 expression and virological synapses
account for increased HIV-1 *trans*-infection mediated by fungus-stimulated
MDDCs.
## Fungi facilitate DC-induced activation of resting CD4 <sup>+</sup> T cells and promote viral infection by providing more permissive cell targets
DCs can efficiently active naïve T cell, and the activated T cells can provide
more permissive targets for robust viral infection. To examine the potential
effects of fungi on DC-induced CD4<sup>+</sup> T cell activation and HIV-1
infection of T cells, MDDCs were pulsed with heat-killed fungi or control medium
for 2 h. After washing, MDDCs were co-cultured with allogeneic resting
CD4<sup>+</sup> T cells for an additional 48 h. T-cell activation was monitored
by detection of CD69 expression in gated CD3<sup>+</sup> cells. Overall, fungus-
pulsed MDDCs facilitated T-cell activation. In the presence of fungi-pulsed
MDDCs, CD69 was expressed on the surface of around 12% of T cells, compared with
4.8% of T cells cultured with MDDCs without fungi. In order to demonstrate the
DC-T cell direct contact is requirement for efficient T activation, the
transwell plates with an insert membrane size of 0.4 µm were used to separate
the MDDCs from T cells. As expected, much less or non- activation of resting T
cells was observed. Direct stimulation with heat-killed fungi alone induced very
little, transient expression of CD69 on resting T cells, or no expression at
all, which demonstrated the need for DCs for activation of resting T cells.
We investigated whether fungus-loaded MDDCs can facilitate T-cell susceptibility
to HIV-1 infection. T cells co-cultured with fungi-loaded MDDCs were purified
and plused with pseudotyped sing-cycle HIV-Luc/NL4-3 reporter virus, and viral
infection was detected 5 days later by measuring luciferase activity in cell
lysates. HIV-1 infection was significantly enhanced in primary CD4<sup>+</sup> T
cells activated by fungus-pulsed MDDCs compared with control MDDCs without
fungi. Moreover, direct cell-cell contact was required for initiating T-cell
susceptibility to viral infection. Direct treatment of heat-killed fungi did not
induce susceptibility of resting T cells to HIV-1 infection. PHA-activated
CD4<sup>+</sup> T cells were used as a positive control, which displayed around
30% cells expressed CD69 expression and supported efficient HIV-1 infection in
treated T cells. These data suggest that fungus-pulsed DCs facilitate the
activation of resting T cells and activate more permissive T cells targets for
robust HIV-1 replication.
# Discussion
Microbial translocation has been proposed as the cause of systemic immune
activation in chronic HIV-1 infection ; however, it has not been extensively
studied how these co-pathogens speed up deterioration of the immune response.
DCs appear to be the common targets for HIV-1 invasion and translocation of
other opportunistic pathogens at the mucosa. The functional compromise of DCs by
HIV-1 infection is associated with immunosuppression and lack of control of
microbial translocation. Given the pivotal roles of DCs in host immunity and
viral pathogenesis, the interactions of DCs with HIV-1 have been preferentially
targeted for exploiting the potential effects of opportunistic pathogens.
DCs treated with opportunistic pathogens, such as *Malaria hemozoin*,
*Mycobacterium tuberculosis*, and *C. albicans*, impairs degradative processing
and MHC-II presentation of HIV-1 antigens to CD4<sup>+</sup> T cells, and alters
cytokine secretion, the enhanced DC-mediated viral *trans* infection was also
observed during certain opportunistic infections. In those published studies,
the synthetic *hemozoin* products, the *M. tuberculosis* cell wall, or the heal-
killed *M. tuberculosis* or *C. albicans* laboratory strains was used.
Here, the effects of *P. marneffei* on HIV-1-DC interactions were investigated.
The difference is that the used *P. marneffei and the C. albicans* were directly
isolated from AIDS patients. Our results demonstrated that both thermally
dimorphic forms of *P. marneffei* activated DCs and promoted DC-mediated HIV-1
*trans*-infection of CD4<sup>+</sup> T cells. Moreover, *P. marneffei*
-stimulated DCs could further activate resting CD4<sup>+</sup> T cells to induce
more susceptible targets for HIV-1 infection. Our results have also shed light
on the detailed mechanisms for the enhancement of viral spread. We demonstrated
that heat-killed *P. marneffei*, along with *C. albicans*, promote viral uptake
in MDDCs, altered viral intracellular sequestration, and importantly,
facilitated MDDC-T cell contact by increasing ICAM-1 expression and efficiently
concentrating HIV-1 particles in virological synapses.
DC activation and altered viral intracellular trafficking are associated with
enhanced viral spread. Upregulation of HLA-DR, costimulatory molecules CD83 and
CD86, and intercellular molecules on fungus-activated DCs, in general, would
encourage DC-T cell conjugate formation. We and other groups have previously
reported that increased ICAM-1 expression on DCs correlates with promoted viral
transfer, due to stronger DC-T cell interactions through ICAM-1 binding to
T-cell-expressed LFA-1. Fungus-stimulated DCs accelerate viral uptake and
sequestrate intact viral particles in non-conventional, non-lysosomal
tetraspanin CD81<sup>+</sup> compartments. The harboring of intact virus into
the non-classical multiple vesicular bodies might provide virus a means to
escape from the cellular proteolysis. Upon encountering with CD4<sup>+</sup> T
cells, more viruses were recruited on the DC-T cell contacted sites. High levels
of endocytosis and altered intracellular trafficking of HIV-1 appear to account
for enhanced viral transmission mediated by fungus-activated DCs.
*P. marneffe*-stimulated DCs were less permissive for productive infection,
which is consistent with previous reports of LPS and *malaria hemozoin*
treatment. However, it remains to be clarified which fungal component(s) is
responsible for HIV-1 restriction and the underlying mechanisms. LPS-matured DCs
show dis-association of the susceptibility for HIV-1 infection with the capacity
for mediating HIV-1 *trans* infection. Post-entry restriction of HIV-1 infection
in LPS-induced mature DCs has been noted, and inhibition on the levels of
reverse transcription and post-integration have been further identified by using
real time PCR quantification of viral DNA and integration. Reduced gene
expression, such as for co-receptor CCR5, has been reported to be responsible
for impaired productive infection of HIV-1 in *malaria-hemozoin-*treated DCs.
Higher levels of APOBEC3G and APOBEC3F (for “apolipoprotein B mRNA-editing
enzyme, catalytic polypeptide-like 3G and 3F”) also have been shown to mediate
the post-entry block of HIV replication in DCs and LPS can upregulate the
expression of APOBEC3G/F,. The antiretroviral protein, namely SAMHD1 (SAM
domain HD domain-containing protein 1), has been recently identified to inhibit
the early step of HIV-1 replication in dendritic- and myeloid cells. It might be
possible that fungus-treated DCs increased the expression of these HIV-1
restriction factors and therefore become more resistant to HIV-1 infection.
DCs activate resting T cells and can provide more permissive targets for HIV-1
infection. We found that the stimulation of *P. marneffe* significantly
accelerated DC-induced activation of resting CD4<sup>+</sup> T cells, which
indicates the pivotal importance of DC-driven T-cell activation for the high
level of viremia and exacerbation of T-cell depletion in the late stage of HIV-1
infection. It would be interesting to confirm these *in vitro* observations in
HIV-1-infected individuals.
Our findings revealed that DC function and its interaction with HIV-1 have been
modulated by opportunistic pathogens for viral dissemination. Enhanced HIV-1
spread by DCs can target activated CD4<sup>+</sup> T cells, which could further
accelerate T-cell depletion and immunosuppression, leading to the lack of
control of both viral and fungal pathogens. Our results highlight the importance
of studying DC-HIV-1 interactions for understanding viral pathogenesis, and
might provide a new insight into the interventions against HIV-1 infection and
spread.
# Materials and Methods
## Ethics statement and fungi isolation and identification
This study was reviewed and approved by the Medical Ethics Review Committee of
Yunnan Province, Kunming, China. Written informed consent was provided by study
participants and/or their legal guardians. Fungi were isolated from skin lesions
or tongue of AIDS patients and cultured on Sabouraud agar plates. Fungus species
were identified, sub-cultured for amplification, then harvested and killed by
boiling for 1 hr. Fungal cell counts were determined under a light microscope
and diluted at 1×10<sup>8</sup>/ml in PBS. MDDCs were stimulated with fungi for
48 hrs at a 1∶10 ratio of cells.
## Cell culture
Human peripheral blood mononuclear cells (PBMCs) from healthy donors were
provided by the Blood Center of Shanghai, Shanghai, China. CD14<sup>+</sup>
monocytes and resting CD4<sup>+</sup> T cells were purified from PBMCs using
magnetic beads (BD Biosciences) as described before. CD14<sup>+</sup> monocytes
were cultured with granulocyte-macrophage colony-stimulating factor and
interleukin (IL)-4 for 6 days to generate the immature DCs. Resting
CD4<sup>+</sup> T cells were activated with 5 µg/ml of phytohemagglutinin-P
(PHA-P) (Sigma-Aldrich) for 48 h in the presence of 20 IU/ml of recombinant IL-2
(R&D). The human embryonic kidney cell lines HEK293T and the CD4<sup>+</sup>
T-cell line Hut/CCR5 (kind gifts from Dr. Vineet KewalRamani, National Cancer
Institute, USA) have been described previously.
## HIV-1 or virus-like particle stocks
Pseudotyped single-cycle HIV-1 stocks were generated by using calcium phosphate-
mediated co-transfection of HEK293T cells with the plasmid pLAI-Δ-env-Luc and
expression plasmids of either JRFL (R5-tropic) or NL4-3 (X4-tropic) envelope
glycoproteins as described previously. Virus like particles (VLPs), HIV-1-Gag-
GFP/JRFL, were generated by cotransfection of HEK293T cells with a plasmid
encoding HIV-Gag-GFP and with an expression plasmid of JRFL (kind gifts from Dr.
Vineet KewalRamani, National Cancer Institute, USA). Harvested supernatants of
transfected cells that contained HIV-1 particles were filtered and titrated with
p24<sup>gag</sup> capture ELISA.
## Flow cytometry
Cells were stained with specific monoclonal antibodies (McAbs) or isotype-
matched IgG controls. McAbs against the following human molecules were used for
staining (clone numbers and resources are given in parentheses), Phycoerythrin
(PE)-conjugated CD3 (UCHT1; eBioscience), PerCP-cy5.5-CD3 (OKT3, eBioscience),
PE-CD11c (3.9; eBioscience), APC-Alexa Fluor750-CD11c (B-ly6; BD Pharmingen),
PE-ICAM-1(CD54) (HA58;eBioscience), PE-CD69 (FN50; eBioscience), PE-CD83
(HB15e;eBioscience), PE-CD86 (IT2.2; eBioscience), PE-DC-SIGN (eB-h209;
eBioscience), and APC-cy7-HLA-DR (LN3; eBioscience). Stained cells were detected
with an LSRII flow cytometer (BD Pharmingen) and analyzed with FlowJo 7.6.1
software.
## HIV infection and transmission assays
The luciferase reporter system was adopted for assay of HIV-1 infection and
transmission as previously described. In brief, MDDCs were pulsed with 5 ng
p24<sup>gag</sup> amounts of pseudotyped HIV-luc/JRFL or HIV-luc/HXB2 for 2 h,
and cells were washed for culture or for co-culture with CD4<sup>+</sup> T
cells. Hut/CCR5 or PHA-P-activated primary CD4<sup>+</sup> T cells were used as
targets. Cells were harvested after 3 days post-infection, and the cell lysates
were measured for luciferase activity with a commercially available kit
(Promega).
DC-mediated HIV-1 transmission also was detected by flow cytometry. HIV-1 VLP
(HIV-Gag-GFP/JRFL) was used, and after 1-h co-culture of virus-loaded MDDCs with
Hut/CCR5 cells, the T cells were distinguished based on CD11c<sup>-</sup>
staining from the mixed cell culture, and the numbers of Gag-GFP associated with
T cells were measured.
## HIV-1 binding and internalization assay
HIV-1 binding and internalization were quantified by flow cytometry using the
VLPs (HIV-Gag-GFP/JRFL). MDDCs were incubated with 40 ng p24<sup>gag</sup>
amounts of viruses for 2 h at 37°C and washed. The numbers of Gag-GFP associated
with MDDCs were quantified by flow cytometry, and mean fluorescence intensity
(MFI) was calculated. When indicated, anti- human DC-SIGN specific McAbs (10
µg/ml) (120507; Abcam) were used for pre-incubation with MDDCs before viral
pulsing, or 5 min treatment with 0.25% trypsin (without EDTA) was used after
viral-loading to remove surface-bound HIV-1.
## T-cells activation assay and viral infection
Various fungus-stimulated MDDCs or heat-killed fungi species were used to
coculture with or treat allergenic resting CD4<sup>+</sup> T cells for 48 h at
the same ratio of cells. The T cells were gated based on CD3-positive
populations, and the activation was monitored by detecting the transient
expression of CD69 by flow cytometry. For viral infection, the activated T cells
from DC-T cell co-cultures were purified by magnetic beads and then challenged
with 5 ng p24<sup>gag</sup> amounts of HIV-1/NL-43 for 2 h. After washing, the
cells were further cultured for 5 days, and HIV-1 infection was detected with
luciferase activity assay as mentioned above. Transwell culture plates with a
membrane size of 0.4 µm were used to separate MDDCs and T cells.
## Confocal microscopy and image analysis
HIV-1 intracellular trafficking and the formation of virological synapses were
observed by confocal microscopy. MDDCs were pulsed with HIV-1 VLPs, or virus-
loaded MDDCs were co-cultured with CD4<sup>+</sup> T cells for 30 min. Cells
were seeded on the poly-L-lysine coated microscope slides and fixed with 4%
paraformaldehyde (Sigma-Aldrich) for 1 h at 4°C. Cells were immunostained with
anti-CD81 McAbs (M38; Abcam), anti-DC-SIGN McAbs (120507; R&D system), or anti-
βactin (AC-15, Sigma), followed by staining with Alexa-Fluor 555-labeled goat
anti-mouse IgG (Invitrogen). Nuclei were stained indicated with DAPI. Slides
were mounted with Fluorescent Mounting Medium (Dako) and observed using a laser
scanning confocal microscope (Leica SP5).
## Statistical analysis
Statistical analysis was performed using paired *t* test with the SigmaStat
program.
We thank Dr. Vineet KewalRamani for generous reagents.
[^1]: Conceived and designed the experiments: YQ KL JW. Performed the
experiments: YQ YL WL RT QG. Analyzed the data: YQ YZ KL JW. Contributed
reagents/materials/analysis tools: YL SL HL DZ LW. Wrote the paper: LW JW.
[^2]: The authors have declared that no competing interests exist. |
# Introduction
## Background
The SARS-CoV-2 (COVID-19) pandemic is posing major challenges for health care
systems across the world. Throughout the pandemic, the primary goal has been to
protect the population from infection and provide medical care for infected
persons. In the first peak of infections in spring 2020, the German federal
government, in consultation with the federal states, enacted containment
measures for the general public, including social distancing, the isolation of
positive or suspected cases, a ban on admissions to nursing homes and a ban on
visitation in hospitals, nursing homes and hospices. Although these were helpful
strategies to reduce infection and mortality, by March 2021, more than 70,000
people had died from or with COVID-19 in Germany.
Throughout the pandemic, people have continued to require end-of-life care for
cancer and other advanced chronic diseases. However, in Germany as all over the
world, adequate palliative care for the severely ill and dying (with and without
COVID-19), and their loved ones, has not been available at all times and in all
settings during the pandemic. This has caused psychological, social and
spiritual distress for patients, thereby compromising their quality of life.
Palliative care aims at maintaining patients’ quality of life. It can be
provided on at least two levels: general and specialist. When administering
palliative care, GPs maintain close contact with patients and their relatives.
Frequently, they form significant and long-term relationships with patients,
initiating and coordinating care and further treatment with other health care
providers. Thus, in Germany, GPs represent a key provider of general palliative
care, and the COVID-19 pandemic has served to underline their significance in
this respect.
## Study aim
This paper aims at describing GPs’ experiences, challenges and perspectives
relating to general palliative care during the COVID-19 pandemic in Germany.
# Materials and methods
The present study, based on an online survey with GPs in Germany, is part of the
German collaborative project "National Strategy for Palliative Care of Severely
Ill and Dying People and their Relatives in Pandemics (PallPan) in Germany," led
by the National Research Network of University Medicine on COVID-19. PallPan
aims at developing and achieving consensus on a national strategy for the care
of seriously ill, dying and deceased adults (with and without COVID-19) and
their relatives during a pandemic.
## Pre-study
Recent subjective field experiences were explored through two informal
conversations with resident GPs in July and August 2020. The topics that emerged
in these conversations were further discussed and explored in September 2020
within an online focus group involving three GPs, as well as telephone
interviews with two GPs. The focus group and interviews were audio-recorded and
transcribed verbatim. Main reported experiences and challenges were for instance
limited home visits, restricted physical closeness to patients and less visits
from relatives, less body-related therapies, and an increased isolation of
patients.
## Survey development and pre-test
Between September and November 2020, a standardized questionnaire was developed
using the synthesized findings from our pre-study. The questionnaire was pre-
tested by six GPs using the online survey tool Unipark, with special attention
paid to the questionnaire’s structure and coherence, comprehensibility,
technical aspects and duration.
Information was collected on the following sections:
1. sociodemographic data on the study population;
2. patient contact;
3. telephone contact;
4. video consultation;
5. cooperation with other health care providers;
6. psychosocial aspects; and
7. needs and suggestions for managing end-of-life care in the context of a
pandemic.
The questionnaire used 4- and 5-point verbal rating scales (i.e. *totally
agree*, *rather agree*, *rather disagree*, *fully disagree*) to determine the
extent of (dis)agreement with the presented statements, which reflected
subjective experiences, challenges and perspectives pertaining to end-of-life
care during COVID-19. Free-text options were also provided to allow for
respondents’ comments on their individual provided statements.
## Recruitment of the study population
In November and December 2020, the information and invitation letter, including
a direct, non-personalized link to the GP survey in Unipark, was sent to:
1. nine university institutes for general practice in seven federal states
(Mecklenburg-Western Pomerania, Berlin, Lower Saxony, Hessen, North Rhine-
Westphalia, Baden-Wuerttemberg, Bavaria), for distribution to their teaching
and research networks;
2. three GP Associations in Lower Saxony and Bremen;
3. the German College of General Practitioners and Family Physicians;
4. the German Association for Palliative Medicine; and
5. the Competence and GP Training Center of Lower Saxony, for distribution
to their members.
The research team had no direct access to the distribution lists of the
abovementioned parties and cannot quantify the number of GPs who were contacted.
However, we estimate that the survey was distributed to at least 3,000 GPs. In
the invitation letter, participants were asked to forward the letter to other
interested parties, thereby triggering a snowball effect to maximize the study
population. Each participant was asked to complete the questionnaire only once.
Participation was completely anonymous. The survey was open from November 23 to
December 18, 2020.
## Data analysis
The SPSS 26 statistical software package was used to calculate descriptive
statistics (mean value, standard deviation, minimum, maximum) and the absolute
and percentage frequencies of the questionnaire data. Outliers were treated with
the full dataset. Missing data are reported explicitly.
The qualitative analysis of the pre-study data and free-text comments was based
on content analysis (according to Kuckartz), using MAXQDA version 18. The main
categories of the qualitative interview guide were used as the basis for the
questionnaire content domains.
## Ethical requirements
A written positive ethics vote (No. 9232_BO_K\_2020 of 24.07.2020) for the
project was issued by the Ethics Committee of the Hannover Medical School.
Prior to a subject’s participation in the online questionnaire, the participants
had to confirm a check box that they have read and understood the written
informed consent form concerning ethics and data protection and accept the
regulations. Without this confirmation the participation was not possible.
# Results
## Sociodemographic data on the study population
The survey was completed by 410 GPs, comprising an approximately equal number of
women and men. Their average age was 54 years (range 31–73 years), and they
represented all 16 federal states in Germany. Approximately half of the GPs
(51.5%) had completed additional training in palliative care. On average, they
required 23 minutes to complete the questionnaire. Almost all of the GPs were
experienced palliative care providers and reported that they had seen patients
with COVID-19 in their practice.
## Experiences and challenges during the pandemic
The following results for patient contact, telephone contact, video
consultation, cooperation with other health care providers, psychosocial
aspects, and needs and suggestions for end-of-life care in the context of a
pandemic refer exclusively to GPs’ experiences caring for severely ill and dying
patients during the first peak of the pandemic, in spring 2020.
### Patient contact
The majority of respondents assessed the quality of their patients’ end-of-life
care as consistent (61.5%), while 36.8% reported a decrease in quality relative
to the pre-pandemic period. Of the GPs who made private home visits to severely
ill and dying patients, 61.4% reported a consistent number of visits, 28.5%
fewer home visits, and 9.1% more home visits (1.1% missing data) compared to the
pre-pandemic situation. Similar results were found with respect to nursing home
visits. The most frequently cited reason for reduced visits was restricted
access.
### Telephone contact
In total, 62.7% of the GPs reported increased telephone contact to replace
personal contact with severely ill and dying patients. Of these, 36.2% indicated
that the quality of end-of-life care worsened due to the lack of personal
contact, because there were no physical examinations, communication was
challenged and they were less able to provide emotional support. Similar
problems were reported for telephone contact with relatives.
### Video consultation
In total, 36.1% of the GPs offered video consultation in lieu of face-to-face
contact with severely ill and dying patients and their relatives, which was
generally only realized in individual cases by primary care physicians at
private homes and nursing homes. Many GPs stated that video consultation was not
used or requested by severely ill and dying patients. Their cited reasons for
this included technical difficulties, lack of user competence on the part of the
patient and poor quality of care. Video consultation was, however, used to
support relatives of severely ill and dying patients.
### Cooperation with other health care providers
The respondents rated their cooperation with other GPs, community nursing
services and nursing homes as good. In contrast, they evaluated their
cooperation with physio, occupational and other therapeutic professions, medical
specialists, hospitals and health authorities as satisfactory, and thus slightly
worse. Local health authorities, described as overburdened, were criticized
primarily for their lack of accessibility.
The GPs described nursing homes’ hygiene concepts as inconsistent. In the free-
text fields, some GPs (n = 15) reported that they were challenged in their
attempts to access nursing homes. The main reason for this was that nursing home
stakeholders were uncertain of how to interpret and apply hygiene-related
contact restrictions. In addition, the GPs also reported problems admitting
severely ill patients to nursing homes.
According to the GPs evaluation, many relatives could have been restricted
(48.5%) or prohibited from visiting (33.4%) patients in nursing homes. The GPs
perceived deterioration in the physical and mental health of patients in private
homes and nursing homes as a consequence of this restricted contact.
The GPs also perceived that relatives saying goodbye to their loved ones was
only possible to a very limited extent (91.7%) or not at all (56.1%).
### Psychosocial aspects of severely ill and dying patients and their relatives
The GPs observed an increased fear of loneliness among severely ill and dying
patients in nursing homes (91.9%), private homes (87.3%) and hospitals (86.1%).
With regard to the psychosocial burden on relatives, the majority of the GPs
reported increased distress due to relatives’ reduced receipt of information
about the patient (85.9%) and inability to support them with their physical
presence (99.3%).
### Needs and suggestions for end-of-life care in the context of a pandemic
The GPs identified social contact with relatives and face-to-face contact with
physicians as the most important aspects of patient care.
In total, 92.4% of the GPs (fully/rather) agreed that GPs should be involved in
local crisis teams, and 79.5% (fully/rather) agreed that palliative care
physicians should also be involved. These local crisis teams were imagined to
improve the exchange between outpatient and inpatient care providers and
facilitate efficient, decentralized coordination and decision making at the
local level.
# Discussion
The present study administered a nationwide online survey to collect GPs’
experiences, challenges and perspectives with respect to caring for severely ill
and dying patients and their relatives during the COVID-19 pandemic. Almost all
of the participating GPs had treated patients with COVID-19 in their practice.
Throughout the pandemic, despite many efforts to adapt their individual practice
management, the GPs felt challenged in their ability to administer high quality
palliative care.
Of note, the GPs reported deterioration in patients’ physical and mental health
in both private and nursing homes, due to contact restriction. This concerning
trend has also been observed by the German Association for Palliative Medicine
and other professional organizations.
In the present study, the GPs reported an increased fear of loneliness in their
patients, as well as greater psychological distress in patients’ relatives, due
to an inability to support their loved ones in person or to say goodbye. Girum
et al., in a systematic review of 22 studies, demonstrated that quarantine and
isolation measures have been effective in controlling the spread of COVID-19.
Thus, protective measures (e.g. social distancing) are recommended, especially
for those at greater risk of infection. However, while these measures may be
important for managing the wider spread of the pandemic, the present study and
other research has highlighted their serious physical and psychological
consequences for severely ill and dying patients. To prevent these negative
consequences, the GPs in our study recommended that social contact be maintained
for patients receiving palliative care. The long-term effects of contact
restriction and isolation on vulnerable groups must be investigated in future
studies.
Germany’s Federal Government Commissioner for Long-Term Care and the Federal
Minister of Health addressed this challenge in December 2020, proposing
regulations for visitation to care facilities. They emphasized the central role
of social relationships for residents and listed basic measures to enable
relatives to safely visit during the pandemic, with as few restrictions as
possible.
In contrast, the German College of General Practitioners and Family Physicians
advised, in its “Action Recommendation on the New Coronavirus,” reduced face-to-
face contact between GPs and nursing home residents, where possible. A reduction
in home visits across both private and nursing homes was confirmed by
approximately one-third of our GP respondents.
In the event of reduced home visits, the German College of General Practitioners
and Family Physicians recommended that GPs should reduce personal patient
contacts, but engage in telephone and video consultation. They also recommended
that these methods be widely applied in GP practices, to ease pressure. Our
survey showed that this occurred in almost two-thirds of our respondents’
practices, with the GPs increasing the frequency of their telephone
consultations with patients relative to the pre-pandemic period. Saint-Lary et
al. showed a similar trend for increased use of telephone communication with
patients in their observational survey with French GPs.
In the present study, video consultation was offered by slightly more than one-
third of the GP practices, for individual treatment. In contrast, another study
found that 81% of GPs in Norway offered video consultation and found it suitable
for maintaining patients’ continuity of care. In our study, the GPs connected
their minimal use of video consultation to their reservations about technical
implementation, user competence and reduced quality of care (due to a lack of
physical examination). Similar attitudes were found by Randhawa et al., in their
qualitative study of 12 GPs in London. Hawley et al. and Lieneck et al. reported
further barriers to the use of this technology, including uncertainty among
patients, concerns over data protection and lack of access to mobile devices
among older patients.
While reservations towards video consultation should not exclude the expansion
of digital communication in health care, they reveal the need for training,
broader implementation in nursing homes and clarity around data protection. In
its draft “Digital Care and Nursing Modernization Act” of January 20, 2021, the
Federal Cabinet addressed some of the abovementioned technical and
infrastructure issues. As further guidelines on digital communication are
developed, they must consider the views and experiences of health care
providers, patients and their relatives.
Based on their research in Atlanta, Kuntz et al. see great potential for video
communication with relatives of palliative care patients. Drawing on data from
their online survey with 67 caregivers and 10 semi-structured telephone
interviews, the authors evaluate digitally-mediated family meetings as feasible
and efficient. In addition, they conclude that video meetings might allow
relatives to understand both the health and the care of the patient and to
express their thoughts and feelings.
## Limitations
As we did not have access to the distribution lists used for our survey, we
cannot comment on the response rate or non-responder characteristics, and we
cannot fully exclude the possibility that some individual GPs participated in
the survey more than once. Furthermore, due to the cross-sectional study design,
we can only provide data related to changes over time during the pandemic. Since
this survey was based on the GPs’ recall of their experiences, there is the
potential for recall and confirmation bias. Finally, it can be assumed that,
among the study participants, GPs with a particular interest in palliative care
were disproportionately represented. Therefore these findings may not be fully
representative of primary palliative care and end of life care provision by GPs
across Germany.
# Conclusion
The present work provides insights into the nationwide pandemic management of a
representative group of GPs in Germany. The findings may support the development
of a national strategy for palliative care during a pandemic. We conclude that,
during a pandemic, the preservation of face-to-face visits by relatives and the
development of feasible and safe video communication should be prioritized.
Finally, to address end-of-life care issues appropriately, GPs and palliative
care specialists should be involved in COVID-19 task forces on the micro, meso,
and macro levels of health care.
We thank all of the GPs who participated in the survey. We also acknowledge
Valerie Appleby’s excellent editorial scrutiny of the present research article.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Diabetes mellitus is one of the most common chronic diseases worldwide, with an
increasing incidence in most countries. There were more than 382 million
people with diabetes mellitus in 2013 and this is forecasted to reach 592
million people by 2035.\[–\] Most of the deaths among patients with diabetes are
not due to diabetes itself but due to the complications associated with it. This
includes cardiovascular disease which can cause about 50% of the deaths among
patients with diabetes. In Saudi Arabia, diabetes mellitus has become an
overwhelming health problem with an overall prevalence among adults of
approximately 23.7%. The complications that are usually associated with
diabetes mellitus are due to macrovascular or microvascular disease.\[–\] The
prevalence of these complications in Saudi Arabia is quite high. In one study
done in that country, the prevalence of complications were: myocardial
infarction (14.3%), retinopathy (16.7%), acute coronary syndrome (23.1%) and
nephropathy (32.1%).
Erectile dysfunction (ED) is a common association of diabetes and is caused by a
neuropathy or vasculopathy.\[–\] ED is defined as “the persistent inability to
attain and maintain an erection that is sufficient to permit satisfactory sexual
performance”. There is some evidence that suggests that low testosterone
might be involved in both the development of type 2 diabetes and the subsequent
complication of ED. Several epidemiological studies have shown that both type 1
and type 2 diabetes are associated with higher risks of ED. Also, it has
been recognized that ED can be found even in preclinical or newly diagnosed
diabetes. The prevalence of ED among men with diabetes ranges from 35% to 90%
depending on the method used to identify it. In Saudi Arabia, studies have
shown that the overall prevalence of ED in men with diabetes range from 63.5% to
83%.\[–\] There is a threefold increased risk of ED in men with diabetes
compared to men without diabetes. Furthermore, even after adjusting the risk
of ED for age in men with diabetes, the risk is still double compared to those
without the disease.
Moreover, ED in men with diabetes occurs 10–15 years earlier than in men without
diabetes. It has been shown that quality of life is reduced in men with diabetes
who are suffering from ED. In addition, ED is considered a predictor for
cardiovascular events and can be associated with silent myocardial ischemia
among men with type 2 diabetes mellitus.\[–\] In addition to that, ED in people
with diabetes can be the first sign of future cardiovascular events. The
existence of ED in men with diabetes is a reason to screen for other diabetic
complications caused by microangiopathy in target organs.
Doctors can diagnose ED by several methods. A detailed medical history and
physical examination can give a good idea about its causes or degree of
severity. Obviously, the most important step to start with is to simply ask
men with diabetes about this problem during a routine clinical review. The UK
NICE guidelines for diabetes mellitus recommend that “Review the issue of
erectile dysfunction with men annually”.
Surprisingly, few doctors ask men with diabetes about ED and this problem is
frequently overlooked. For example, in one study done in England only 9% of men
with diabetes were asked by their physicians about ED. In another study done
in the United states, physicians initiated the discussion about ED with only 18%
of their patients with diabetes. On the other hand, very few studies have
addressed the issue of the discussion of ED from the perspective of the patient
with diabetes, for example, whether they are willing to be asked about ED by
their physicians. For example, a study done in Taiwan showed that 56.6% of
patients with diabetes wished to discuss ED with their physicians.
The literature indicates that barriers that might prevent health professionals
from asking about sexual problems such as ED include lack of time or knowledge,
lack of training among physicians, false beliefs about sexuality, thinking that
this is a job for another physician, patients not being ready to discuss these
issues, believing it is not an important subject, fear of increasing patient
anxiety and patients being too ill or too old to be asked.
In the Middle East, the literature is lacking in studies on the proportion of
men with diabetes who have been asked about ED or their willingness to discuss
ED with their physicians. Also, in searching the literature, no studies were
found that described the barriers faced by patients with type 2 diabetes to
discuss ED with their physicians.
This study was aimed primarily to find out the proportion of Saudi men with type
2 diabetes who have been asked about ED in the last year by their physicians in
hospital-based primary care clinics in Riyadh. We also aimed to determine the
willingness of Saudi men with type 2 diabetes to discuss ED with their
physicians and the factors that either increase or reduce their willingness to
discuss this issue.
# Methods and materials
## Study design
This study employed a cross-sectional survey using a quantitative self-
administered questionnaire investigating the proportion of Saudi men with type 2
diabetes who have been asked by their physicians about ED in the last year,
their willingness to discuss ED with their physicians, and the factors that may
be related to their willingness to discuss ED with their physicians.
## Study site
The study was conducted in hospital-based primary care clinics at King Khalid
University Hospital, Riyadh, Saudi Arabia. These primary care clinics consist of
8–10 clinic sessions each day, led by approximately 40 staff specialized in
family medicine. Each clinic has about 30 patients daily with the majority of
the patients having diabetes.
## Eligibility criteria
The participants were included in this project if they were married, adult (i.e.
\> 18 years), diagnosed with type 2 diabetes mellitus, having at least one year
of follow-up in the clinics, and could read and write Arabic. We excluded any
participant with anatomical penile deformities, past history of spinal cord
injury or past history of prostate diseases or prostate surgery.
## Patient enrolment
Patients were approached at the reception desk after they completed their review
with their physician and asked about the inclusion and exclusion criteria. Those
who met our eligibility criteria were included in the study. The participants
were informed about the study’s objectives. They were asked to enter the study
and for those who accepted this request, written consent was obtained.
Confidentiality of their information was assured. The data were collected by a
family physician (the principle investigator) from July to September 2015
## Instrument development
The questionnaire consisted of 26 items divided into 4 sections. The first
section of the questionnaire collected information about the socio-demographic
background of the patients. The second section contained two questions, the
first one regarding the proportion of patients with type 2 diabetes who have
been asked about ED. The responses were either yes, no, or I can’t remember. The
second section measured the degree of willingness to discuss ED by the patients
with their physician (i.e. unwilling, slightly willing, moderately willing, and
very willing).
The third section consisted of self-reported statements about 11 barriers
preventing patients from discussing ED with their physicians. The responses to
these statements were recorded on a five point Likert scale ranging from
strongly agree to strongly disagree. These statements were developed after
reviewing the literature. The last section of the questionnaire comprised a
brief survey to assess the presence of ED in respondents by using a validated
Arabic translation of the Index of Erectile Function (IIEF-5) questionnaire.
IIEF-5 is a well-known tool to screen for ED and it has been used extensively in
previous studies. It comprises only five questions. Also, it categorizes ED
according to its severity as follow: severe ED (1–7), moderate ED (8–11), mild
to moderate ED (12–16), mild ED (17–21), and no ED (22–25).
Apart from the last section of the questionnaire which was adopted from the
previously validated Arabic version, the majority of the questionnaire was
developed in English, translated by an accredited translator in Arabic, and then
back-translated by another accredited translator into English. The mismatches
between the two English versions, the original and back-translated versions,
were discussed and resolved by the primary author and the translators.
The questionnaire was pretested and piloted on 30 monolingual patients with type
2 diabetes to ensure the comprehensibility and readability of the final Arabic
version. The participants in the pilot study were recruited from medical out-
patient clinics to prevent contamination with the main sample for the current
study.
## Sample size calculation
A sample size calculation was based on a pilot study with 30 participants which
showed that 15% of patients with type 2 diabetes had been asked about ED in the
last year. So, a sample of 306 patients with type 2 diabetes was required to
obtain a 95% confidence interval of +/- 4% around the prevalence estimate of
15%. Assuming 10% of questionnaires in the pilot study were incomplete or not
returned, a total of 336 questionnaires was required.
## Statistical analysis
Descriptive statistics were used to describe the study sample characteristics
and the participants’ identified barriers to discussing ED with their
physicians. To test the association between patients who were willing to discuss
(i.e. very willing, moderately willing, and slightly willing) and unwilling to
discuss ED with the physicians, and the participants’ socio-demographic and
clinical characteristics, chi-square tests were used for categorical variables.
In one variable (current occupation), we collapsed some groups together to meet
the conditions of the Chi-square test. For continuous variables, the normality
of the data was checked by the Kolmogorov-Smirnov test. For normally distributed
data, an independent sample t test was used to compare means. For skewed data,
the Mann-Whitney U test was used to compare medians.
Chi-square tests were used to compare the association between the willingness to
discuss ED with the physician and the different participants’ barriers, after
removing the neutral response variable. The Fisher exact test was used to test
the association between participants having ED and their willingness to discuss
but were not yet asked by their physicians in the last year, as the conditions
of the Chi-square test were not met.
Multivariable logistic regression analyses were performed to predict the
willingness to discuss ED with the physicians by using the participants’
barriers, socio-demographic and clinical characteristics as covariates. For the
logistic regression analysis, participants’ willingness to discuss ED was
categorized as a binary variable, comparing those who reported any degree of
willingness (very willing, moderately willing, and slightly willing) with those
who were unwilling.
The data were analysed using the statistical software package IBM SPSS
Statistics for Windows, Version 22.0 (IBM Crop., Armonk, NY, USA). A p-value
of less than 0.05 was considered to be statistically significant for all
analyses.
## Ethics
The study protocol was reviewed and approved by the Monash University Human
Research Ethics Committee and the Institutional Review Board of King Khalid
University Hospital, Riyadh, Saudi Arabia, where the data were collected. The
participants were informed about the study’s objectives and their permission to
enter the study was requested. Written consent was obtained from the
participants. Confidentiality of their information was assured.
# Results
## Participants’ socio-demographic and clinical characteristics
Out of the 336 distributed questionnaires, 309 were completed and returned. The
response rate was therefore 92%. The median age of the respondents was 60 years
and the median duration of diabetes among the respondents was 10 years, with
over half (59.2%) on tablets alone as treatment for this condition. Few (9.7%)
had been asked by their physicians about ED in the last year although most
(84.8%) were willing to discuss this problem with them. The presence of ED among
the respondents was 89% with one third of them (28.2%) suffering from severe ED.
The remaining socio-demographic and clinical characteristics are shown in.
## Prevalence of identified participants’ barriers to discussing ED with their doctors
shows the distribution of participants’ barriers to discussing ED with their
physicians. The most prevalent barriers among these respondents were having sex
is not important to me (49.5%) and the treatment is too expensive (24.6%).
## Willingness to discuss erectile dysfunction (ED) and participants’ socio-demographic and clinical characteristics
shows the association between the willingness of respondents to discuss ED with
their physicians, and the respondents” socio-demographic and clinical
characteristics. The participants who were willing to discuss ED with their
physicians were younger with the mean age being 59.3 compared to the mean age of
65 in unwilling participants (P\< 0.001). Participants with low monthly incomes
(i.e. \<5000 SR) (53.2%) were unwilling to discuss ED with their physicians (P =
0.03). Also, among participants who have ED, those who were complaining of
severe ED (63.1) were unwilling to discuss it with their physicians. There were
no significant associations between a willingness to discuss ED with the
physicians and the highest education level, location of residency, current
occupation, smoking status, duration of diabetes, type of diabetes treatment,
and presence of ED.
Multivariable logistic regression analysis was used to predict the participants’
willingness to discuss ED by their socio-demographic and clinical
characteristics. After adjusting for the educational level, location of
residency, monthly income, current occupation, smoking status, duration of
diabetes, type of diabetes treatment, presence of ED, two participants’
characteristics were associated with willingness to discuss ED with the
physicians. These characteristics were age above 60 (OR = 0.25, 95% CI:
0.11–0.55), and having severe ED (OR = 0.26, 95% CI: 0.08–0.85). No significant
association has been found between the participants’ willingness to discuss ED
and the other socio-demographic and clinical characteristics.
## Participants’ willingness to discuss ED and identified barriers
shows the comparison between participants’ willingness to discuss ED with their
physicians and the identified barriers. Comparing the ‘unwilling’ participants
to the ‘willing’ ones revealed that the barriers which provide the main
obstacles to discussing ED with the doctors are: embarrassing my doctor (63.9%,
P \< 0.001), ED is a personal issue (60.6%, P \< 0.001), too old now (59.4%, P
\< 0.001), feeling embarrassed to talk about it (57.1%, P \< 0.001), too sick
now to address ED issues (55.9%, P \< 0.001), no effective treatment is
available (54.8%, P \< 0.001), and my doctor is too young to discuss my ED with
him (54.8%, P \< 0.001).
## Predicting participants’ barriers to their willingness to discuss ED
shows the multivariable logistic regression analysis which was used to predict
the participants’ willingness to discuss ED by their identified barriers. After
adjusting for the age and severity of ED as possible confounders, two
participants’ barriers were associated with willingness to discuss ED with the
physicians. These barriers were “it may embarrass my doctor” (OR = 0.04, 95% CI:
0.01–0.2), and “It is a personal issue” (OR = 0.05, 95% CI: 0.01–0.28).
## Participants who have not been asked about ED and their willingness to discuss it
shows that among the respondents who have not yet been asked about ED in the
last year by their physicians, 91% of them have ED and would be willing to
discuss it with their physicians (P = 0.02). Even if they do not have ED, twice
as many are willing to discuss this matter as unwilling.
# Discussion
This survey has shown that few (9.7%) patients with type 2 diabetes mellitus
have been asked about ED in the past year by their physicians, in spite of the
majority (84.8%) being willing to discuss it. Further, the presence of ED was
high (89%) among these patients, with one third of them (28.2%) suffering from
severe ED.
In spite of guidelines recommending physicians to enquire about ED in patients
with diabetes, this does not take place in most cases. Grant et al found
that only 9% of the patients with diabetes have been asked about ED in their
last diabetes review consultation. In addition, Perttula found that
physicians discussed ED with just 18% of their patients who have diabetes.
This is in spite of the prevalence of ED being high in these patients.
Also, the low rate of asking about ED in patients who have diabetes by
physicians who work in primary care settings is similar to what happens in
specialty practices where patients are at high risk for ED, such as those seen
by cardiologists. Nicolai et al found that only 16% of cardiologists admitted to
discussing sexual function regularly with their patients. So, there is a
wide gap between recommendations and what takes place in practice. A study done
in Bulgaria has shown that this gap reflects, in part, physicians’ beliefs that
patients with ED rarely share this problem with their physicians,
Overall, a large majority of patients (84.8%) were willing to discuss this
topic. Unfortunately, to date few studies have examined ED-related issues from
the patient perspective. Jiann et al showed that 56.6% of patients with type 2
diabetes wished to discuss ED with their physicians while Lo et al found that
76.1% of patients with type 2 diabetes would want to receive treatment for ED
from their physicians. However, most patients think that the discussion
should be initiated by the physicians. At the same time, most physicians
seem to assume that patients do not like to be asked about sexual problems.
The study’s findings suggest that two main factors were associated with a
willingness to discuss ED: age and severity of ED. The patients above 60 years
were 70% less willing to discuss ED with their physicians compared to the
patients less than 60 years old. In addition, the patients who do have severe ED
were 75% less willing to discuss ED with their physicians compared to the
patients who have mild ED.
As mentioned above, we found that elderly people were less willing to discuss ED
with their physicians compared to younger patients in spite of the majority of
the elderly population remaining interested in sexual activity. This group needs
to be given more attention by their physicians as they have a very high
prevalence of ED. In addition to that, ED is often underreported and
underdiagnosed in the older male population. It has been shown that physicians
are not proactive in discussing and managing the sexual health of elderly
people. In a study done by Harding and Manry in the United States among health
care providers, it was found that only 28% of the surveyed health care providers
would usually asses the sexual health of elderly patients. Also, a negative
attitude has been found in a study done to examine American psychologists’
willingness to assess the sexual health of older adults.
In addition to the age of patients with diabetes as a predictor for willingness
to discuss ED, the level of ED severity plays a major role, with this study
showing that patients who have diabetes with severe ED are less willing to
discuss this with their physicians compared to those with mild ED. This is
particularly important as the literature suggests that diabetes is associated
with more severe forms of ED.
Men with diabetes also require more aggressive therapy to treat ED. In a study
done by Walsh et al. it was found that men with diabetes were likely to need
more aggressive therapy, and most went on to second line therapy (i.e. penile
prosthesis surgery) for ED as these patients were less responsive to first line
therapy (oral agents). It is important to identify the group who have diabetes
and suffer from severe ED to optimise diabetes control and treat the ED as best
one can. This should lead to improvement in both their sexual function and
depressive symptoms, as shown by the SUBITO-DE study, an Italian multicentre
study.
The main barriers contributing to an unwillingness to discuss ED were:
embarrassing the doctor, ED is a personal issue, too old, too sick to address ED
issues now, no effective treatment available, and the doctor is too young to
discuss ED with. Jiann BP et al found that patients’ embarrassment and false
beliefs about ED treatment being either ineffective or harmful accounted for
three quarters of the reasons why patients with diabetes will not discuss ED
with their physicians. Embarrassment was the key factor preventing this
discussion according to Rutte et al. as also shown by Gott M and Hichliff S.
Other studies have revealed the importance of other barriers including
differences in patient characteristics, i.e. their age, lack of knowledge, and
difference in their culture. These differences in patients’ barriers found by
various studies can be explained by difference in customs, traditions, culture,
and health systems.
The study findings also suggest that most (91%) of the patients who have not yet
been asked about ED in the last year actually have ED and are willing to discuss
it. This is contrary to what has been reported by Smith et al. who found that
sexually active men are more likely to discuss sex with their physicians. These
discrepancies in findings might be related to differences in sociocultural
factors including social norms and attitudes.
The current study has several implications for clinical practice. Firstly, ED is
a major problem among patients with type 2 diabetes and this is frequently
ignored by physicians even though a majority of these patients are willing to
discuss this problem. Physicians who are involved in treating these patients
should initiate the discussion. Secondly, patients with diabetes who are
older and suffer from severe ED are less likely to discuss ED with their
physicians. Targeting this sub-group of patients through education and the
building of better relationships between physicians and their patients should
help. Thirdly, there are multiple barriers that prevent patients with type 2
diabetes discussing ED with their physicians which could be reduced by better
patient education and the addressing of psychological factors.
There are several limitations to this study. The sample was taken from one
hospital and may therefore not be generalizable. Also, the patients were taken
from primary care clinics affiliated to a teaching hospital so that they might
have more severe diabetes, and be older than patients in other primary care
clinics. In addition to that, and due to the nature of the study design, the
results revealed associations and not necessarily causal relationships between a
range of factors and willingness to discuss ED. However, we believe that our
findings shed an important light on this very sensitive issue among patients
with type 2 diabetes. Also, no comparable work has been done in this country,
and so it is of importance within this health care system.
# Conclusions
ED is a highly prevalent condition among patients who have type 2 diabetes. Most
of these patients are not asked about ED within the last year of attending a
clinic, even though most are willing to discuss it with their physicians. Many
patients’ barriers to discussing ED have been identified, including being older
and suffering from more severe ED, with these patients being less willing to
discuss this with their physicians. Further research is needed to explore the
barriers which prevent physicians from discussing ED with their patients who
have diabetes.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Tuberculosis (TB) remains a global threat with an estimated 1.8 million deaths
in 2015; approximately 14 percent attributed to multi-drug resistant (MDR-TB)
and rifampicin resistant (RR-TB) tuberculosis. Advances in case detection and
treatment regimens have dramatically reduced incidence and mortality; yet
lengthy and complex treatment regimens continue to take a toll on treatment
completion and success rates. Patients who default on TB treatment continue to
contribute to the infectious disease burden of the community, increase their
risk of developing MDR-TB, and are at an increased risk for TB-related
mortality.
According to the WHO 2016 report, Ukraine is among the top 20 highest drug-
resistant TB burden countries in the world. National survey data estimate 25% of
new incident cases and 58% of previously treated cases are MDR/RR-TB cases,
resulting in approximately 22,000 cases per year. Driving these increasing
MDR/RR-TB rates is an unchecked treatment default rate. Among the 2014 national
TB cohort, treatment success was only 72% compared to 83% globally.
Patient predictors for treatment default vary across Europe and Central Asia. In
Spain, Cayla and colleagues found that immigrants, patients living alone,
patients previously treated, and injection drug users (IDU) were at higher risk
for default; whereas in Russia, the homeless, unemployed and alcoholics were at
highest risk; in Moldova patients who were homeless, living alone, less
educated, or living for extended periods outside the country were at risk; and
in Estonia alcohol abuse, unemployment, MDR-TB, urban residence and previous
incarceration increased risk. Timing of default was found to be heterogeneous
across a large meta-analysis by Kruk and colleagues. In Uzbekistan, the first
two-month intensive phase led to high rates of default, in Moldova the risky
period was between intensive and continuation treatment, while data overall
suggest that lengthy treatment during the continuation phase is the most likely
period for defaulting.
Strategies to improve treatment adherence and success center on directly
observed therapy short-course (DOTS) with adaptations to different clinical
service and social environments. DOTS has been implemented in clinical settings,
through home visits by medical personnel, use of community volunteers, and DOTS
by family members. Patient incentives are sometimes included to improve
adherence, most commonly periodic food packages, transportation vouchers, and
cash payments. However, evaluation of the impact of these strategies on
treatment success remains inconclusive.
The standard TB treatment for smear-positive patients in Ukraine covers 2–4
months of intensive inpatient therapy at the central TB dispensary. Once the
patient tests smear-negative, (s)he is referred to a TB cabinet or polyclinic
closest to their home for 2–5 months of outpatient continuation therapy.
Directly observed therapy is the standard of care, requiring direct contact
between patients and providers to administer the TB medication. According to the
National TB Program (NTP), adherence to therapy is insufficiently controlled,
with a 7.6% national default rate in 2010.
In 2010, the Ukraine Red Cross Society (URCS), funded by the United States
Agency for International Development (USAID), piloted a community-based social
support program designed to improve TB treatment adherence during outpatient
continuation therapy. URCS provided DOTS to a limited number of patients in
their homes. Additionally, incentive food packages, psychological and career
counseling, and/or vouchers for transportation or other necessities were
provided periodically based on client needs. TB physicians managing patients’
continuation treatment at the outpatient facility made case-by-case referrals to
URCS based on identification of the patient as high risk for defaulting on
treatment according to established criteria for program inclusion. In 2011, the
URCS program was suspended due to insufficient funds. By 2012, the program was
again active and expanded to cover 10 oblasts in eastern and southern Ukraine
with reported default rates ranging from 6.1–12.7%.
This study measures the effect of the URCS social support program on the rate
of treatment default among those at risk for defaulting during continuation
therapy. Given the high rate of treatment default and the growing problem of
treatment-resistant TB strains, identifying effective strategies for treatment
adherence is critical.
# Methods
## Study settings and sample population
Three oblasts in Ukraine were purposively chosen for this study due to their
high TB caseloads and high treatment default rates. In 2010, Dnipropetrovsk
reported 1,077 TB cases and 12.4% default rate; Kharkiv reported 738 cases with
11.1% default; and Odessa reported 789 cases and 9.4% default rate. The study
population was composed of patients receiving TB continuation treatment in these
three oblasts in 2011 and 2012. Patients were classified as high-risk for TB
treatment default per program criteria covering eleven self-reported risk
factors: HIV positive, alcoholism, injection drug use (IDU), a contact to a TB
case, a co-morbidity, homeless, unemployed, a health care worker, a migrant, a
refugee or immigrant, an ex-prisoner, and room to record other risk factors that
a provider might deem noteworthy. Low-risk patients did not report these risk
factors with the exception of unemployment. Facility staff revealed that TB
patients routinely report being unemployed. This is to avoid stigma in the
workplace, as workplace TB screening is routinely undertaken after a case is
diagnosed. Consequently, we considered a patient as low risk during sample
selection if the only reported risk factor was unemployment. During data
cleaning, minimal corrections were made for misclassification of risk status at
time of data entry. All study patients completed TB intensive treatment,
initiated TB continuation treatment, and had a TB treatment outcome recorded in
their medical record.
Five patient cohorts were sampled: high-risk (HR) patients enrolled in the URCS
social support program January 1 –May 31, 2012 (henceforth 2012 HR-
Intervention); high-risk patients not enrolled in the social support program in
January 1 –May 31, 2012 (henceforth 2012 HR-Comparison); low-risk (LR) patients
not enrolled in the social support program in January 1 –May 31, 2012
(henceforth 2012 LR-Comparison); high-risk patients not enrolled in the social
support program in January 1 –May 31, 2011 (henceforth 2011 HR-Comparison); and
low-risk patients not enrolled in the social support program in January 1 –May
31, 2011 (henceforth 2011 LR-Comparison). A cohort of 2012 HR-Intervention
patients (n = 409) was randomly sampled from a complete listing of URCS
enrollees, stratified and proportionate in size to the TB patient population by
oblast. These patients served as the index cases. Each TB facility where an
index case was receiving continuation therapy served as the facility match point
to ensure controls experienced similar service environments to the randomly
selected cases. In order to obtain a group of controls not exposed to the
program, four patients from these facilities’ TB registries were matched to the
index patient: one 2012 HR-Comparison patient; one 2012 LR-Comparison patient;
one 2011 HR-Comparison patient; and one 2011 LR-Comparison patient. The primary
comparison group for this analysis was the 2012 HR-Comparison cohort; however,
additional cohorts were sampled to explore selection. The second matching
variable was the start date for TB continuation treatment for the index case in
order to control for seasonality of TB and services. Additional matching on sex
and age was done if more than one match was eligible. Data from 1630 TB patients
across the five cohorts were collected. Sampling weights were generated to
account for sampling proportionate to the varying size of TB caseload per oblast
and non-response. During data cleaning, minimal corrections were made for
misclassification of risk status at time of data entry. Data entry
misclassifications included: dropping cases who received the intervention in
2012 but had no reported risk factors (n = 12); reclassifying those whose only
risk factor was unemployment from high risk to low risk (n = 16); and
reclassifying another 33 cases as high risk based on a risk factor reported.
A survey of 50 TB polyclinics and cabinets that provided continuation therapy to
the study population was also completed to provide details on the referral and
treatment practices at these facilities (see report for details).
## Data collection and definitions
For each study patient, retrospective data were abstracted from TB medical
records (national form TB01). The data abstracted included basic demographics,
sex, age, employment status, urban or rural residence; and TB diagnosis,
treatment, interruptions to intensive treatment and treatment outcomes. Standard
WHO definitions were used for TB classification (e.g., first diagnosis, re-
initiated treatment, treatment failure, relapse, and referral); for clinical TB
(e.g., pulmonary or extra-pulmonary); for WHO diagnostic categories to indicate
treatment regimens; and for treatment outcomes (e.g., success, death, treatment
failure, treatment interrupted, and transferred). A patient’s outcome was
successful if the full course of prescribed treatment was completed or follow-up
testing indicated patient was cured. Treatment default included anyone who
missed treatment for more than 60 consecutive days per WHO standards. Additional
data from the TB records were abstracted from form TB01-01, the risk screening
form used by providers to identify a patient’s risk for defaulting on treatment.
For the 2012 HR-Intervention group, data on social support services received
such as home visits, food packages, clothing, transport vouchers, monetary
incentives, and counseling were abstracted from the URCS records and merged with
the patient TB record.
## Data analysis
Descriptive statistics were generated to compare demographics, TB disease
characteristics, and reported risk factors for treatment default across the five
cohorts. Logistic regression models with average marginal effects (AME) were
estimated to test the study questions. All analyses used data weighted for
sample selection; reported standard errors are clustered at the facility level.
To validate risk factors predictive of treatment default, the social support
program criteria risk factors were regressed on treatment default among patients
receiving continuation therapy. Dichotomous variables for seven individual risk
factors (HIV positive, alcoholic, IDU, contact to a case, co-morbidity,
homeless, and unemployed) were included. Very few patients reported being a
health care worker, migrant, refugee, or ex-prisoner; hence, these were combined
with the unspecified risk factor as “other”. A dichotomous variable indicating
the presence of more than one risk factor was also added. All risk factors were
run simultaneously first (Model A). Next, we controlled for basic demographics
and four dichotomous disease and treatment characteristics due to their
hypothesized role in TB treatment adherence and outcome: first time TB
diagnosis, pulmonary TB, WHO Category I and more than 2 interruptions in care
during intensive therapy (Model B). To identify the salient risk factors for
default in the absence of an intervention, this analysis was restricted to data
from 2011 when the URCS program was not operating.
To evaluate the impact of the social support program on treatment default, the
second regression analysis was limited to the 2012 HR-Intervention and the 2012
HR-Comparison cohorts. Prior to estimating impact, balance between the
intervention and comparison groups was examined. The final model estimated the
impact of the social support program on treatment default, controlling for risk,
disease status, and demographics.
Analyses were produced using Stata SE version 13 (College Station, TX). This
study was approved by the Office of Human Research Ethics at the University of
North Carolina at Chapel Hill and the ethical review board of the F.H. Yanovskyi
Institute of Phthisiology and Pulmonology, Academy of Medical Sciences of
Ukraine. Both review committees waived the requirement for informed patient
consent. Data collection was performed by the IFAK Institut in Ukraine. Data
collectors had access to patient names in order to track patients from registry
entries to patient records; however, names were not recorded on data collection
tools nor reported to the researchers.
# Results
## Study population
The final dataset included 1,618 records from TB patients across the five
cohorts: 2011 LR-Comparison (n = 308), 2011 HR-Comparison (n = 340), 2012 LR-
Comparison (n = 262), 2012 HR-Comparison (n = 311), and 2012 HR-Intervention (n
= 397). The study populations shared similar demographic profiles across risk
cohorts and years. Approximately two-thirds of the patients were male in every
risk group, just over three-quarters were under fifty years of age, and a large
majority lived in urban areas. Over half of all patient cohorts were unemployed,
ranging from 55–72%. Half of the study population received TB continuation
treatment in Dnipropetrovsk (50.0%), with the remainder evenly divided between
Kharkiv (25.4%) and Odessa (24.6%).
Among the three HR cohorts, unemployment was the most common reported risk
factor (48–62 percent), followed by alcoholism (34–44 percent), co-morbidities
(33–36 percent) and being HIV-positive (21–47 percent). A majority (63–72
percent) reported between 2 and 3 factors putting them at risk for treatment
default, while 3–7 percent reported four or more. Notably, the proportion of HR
patients who reported injection drug use in their medical records was small,
ranging from 5–12 percent. In discussions with facility staff, it was noted that
information on IDU status and treatment is not routinely recorded in the TB
charts nor shared across cabinets due to concerns of confidentiality. As
expected over half of the LR patients reported no risk factors for treatment
default, while the remaining reported unemployment.
Overall, 81.1 percent of the TB patients were undergoing treatment for a first
diagnosis, although among the HR cohorts, a higher percentage re-initiated
treatment after earlier failure or relapse compared to the LR cohorts (7–12
percent versus 3–5 percent). Ninety-three percent of all cases were pulmonary
TB, a majority was classified as WHO Category I (63.8 percent), and 81.3 percent
reported only one or fewer interruptions in intensive treatment.
TB treatment outcomes in 2011 were significantly different between the LR and HR
cohorts on treatment adherence. Treatment default among the 2011 LR-Comparison
cohort was 4.2 percent compared to 13.3 percent in the 2011 HR-Comparison cohort
(p\<0.000); while 90.6 percent of the LR-Comparison cohort reported treatment
success compared to only 74.3 percent of the HR cohort (p\<0.000). Similar
differences were measured in 2012 when comparing the LR-Comparison and HR-
Comparison on default, 4.6 percent and 10.6 percent (p\<0.006), and success,
87.0 percent and 69.8 percent (p\<0.000) respectively. The 2012 HR-Intervention
cohort fared better than the 2012 HR-Comparison on default (1.3 and 10.6
respectively, p\<0.000) and on success (88.4 and 69.8 respectively, p\<0.000).
However, comparisons between the 2012 LR-Comparison and the 2012 HR-Intervention
on default (4.6 and 1.3 percent respectively) and success (87.0 and 88.4 percent
respectively) found no statistical differences. Lastly, statistical differences
were found between the 2011 HR-Comparison and the 2012 HR-Intervention cohorts
for both treatment default and treatment success (p\<0.000); while no
statistical differences were found between the 2011 HR-Comparison and 2012 HR-
Comparison groups. These comparisons across cohorts highlight that the
difference in outcomes between the 2012 HR and LR comparison cohorts is similar
to the differences between the 2011 HR and LR cohorts. This supports the impact
identification strategy employed in our evaluation of the program.
## Predicting treatment default
The URCS social support program was designed to target those at highest risk of
treatment default and provide support to improve treatment adherence. The
official eligibility criteria for program support cover eleven risk factors.
Among patients from 2011, only those who reported being an alcoholic (p = 0.002)
or an IDU (p = 0.043) were more likely to default on TB continuation treatment,
while a patient reporting a co-morbidity was less likely to default (p = 0.028).
Additionally, those patients enrolled in continuation care who had two or more
interruptions recorded during intensive treatment were more likely to default
during outpatient treatment (p = 0.028). Estimated marginal effects predict that
an individual’s probability of default increased by 0.08 (p = 0.017) if an
alcohol abuser and by 0.05 (p = 0.043) if prior treatment interruptions were
noted; yet one’s probability of default decreased by 0.06 (p = 0.043) if
reporting a co-morbidity.
## Evaluating program impact
Primary impact analyses were limited to the 2012 HR-Intervention and the 2012
HR-Comparison cohorts. Mean differences between the two groups were tested for
18 variables; seven (38 percent) were unbalanced at standard statistical levels
(p\<0.05). Looking at the intervention group, a higher proportion of patients
were alcoholics or unemployed, and were 18–29 years of age, while the comparison
group had a higher proportion of persons with HIV, homeless, undergoing WHO
treatment category 1, and who were male. All risk factors, disease
characteristics, patient demographics and treatment oblast were controlled for
in the final impact model. Measuring impact, results indicate that the HR
patients receiving the social support program decreased their probability of
treatment default by 0.101 (p\<0.000) compared to the comparison cohort.
A second analysis, comparing outcomes for the 2012 HR-Intervention cohort to the
2011 HR-Comparison cohort, produced similar results. Five of 18 variables (28
percent) were not balanced between the cohorts. Controlling for all variables,
the 2012 HR patients receiving the social support intervention were
significantly less likely to default (p\<0.000) compared to the 2011 HR-
Comparison cohort, and the probability of default decreased by 0.120 (p\<0.000)
(data not shown). However, alcoholism remained a significant risk factor with
the probability of default 0.069 (p\<0.014) higher among alcoholics compared to
non-alcoholics. Additionally, the probability of default among those with more
than two treatment interruptions during intensive care was higher at 0.047 (p =
0.017).
# Discussion
In 2012, TB patients receiving social support provided by URCS in Ukraine
reduced their probability of defaulting on continuation treatment by 10
percentage points compared to high-risk patients who did not receive social
support in 2012 or 2011. Treatment success rates for the high-risk patients
receiving social support were comparable to the low-risk cohorts and
significantly improved over the high-risk comparison cohorts. This result was
found despite the heterogeneity of the patient population and the services
provided.
Although treatment oblast was not predictive of success or default in 2012,
routine implementation of DOTS and social support varied by study oblast.
According to reported practices in 2014, 89 percent of surveyed facilities in
Dnipropetrovsk provided facility-based DOTS and a majority of these facilities
required daily DOTS visits (83 percent). Among the sites offering home-based
DOTS, half provided weekly or bi-weekly visits. In contrast, 88 percent of the
facilities in Odessa provided home-based DOTS and the majority of home visits
were daily. This increase in home-based services may reflect a growing
recognition in Odessa that facility DOTS is insufficient to assure compliance.
Variation across oblasts may reflect patient population needs or facility
capacity. Further investigation into best practices for DOTS and social support
in Ukraine is warranted.
URCS was the only provider of social support in Kharkiv and Odessa in 2012 and
the primary provider in Dnipropetrovsk. In 2011, only 23 percent of the
facilities referred patients for social support, increasing to 94 percent by
2012. In all oblasts the primary point of referral was the city or raion TB
physician. This is in keeping with URCS’ policy to only provide social support
to smear-negative patients who successfully completed intensive TB treatment and
initiated continuation treatment. This focus on continuation patients ignored
patients who defaulted during intensive treatment, which could be substantial.
In Russia, Jakubowiak et al. found that 44 percent of TB treatment defaulters
exited treatment during the intensive regimen. In Moldova, the highest default
rates were recorded during the first month of inpatient intensive treatment. Our
data did not include patients who defaulted during inpatient treatment, however
in 2011, patients with more than two treatment interruptions during inpatient
care increased their probability of defaulting during outpatient care by 5
percentage points. This is similar to findings by Jakubowiak in Russia and
Santha in India, where gaps in intensive treatment were associated with future
treatment default. Prioritization for support services may benefit those who had
difficulty during the intensive phase.
Alcoholism was the one risk criteria predictive of defaulting among the 2011
cohorts, increasing the probability of default by 5 percentage points. Neither
positive HIV status nor reported injection drug use were statistically
associated with higher default rates. Under-reporting of these two risk factors
may be one explanation for their lack of significance. In Ukraine, sharing of
confidential patient information between service delivery clinics is limited.
The risk factor information documented on a patient’s TB form is all self-
reported and possibly under-reported due to fear of stigmatization. For example,
a patient seeking HIV-related services may not report their status to the TB
physician. Unless an infectious disease specialist is overseeing services for
both TB and HIV patients, this case of co-infection may go undetected by the
individual clinics, despite best practices of routine HIV screening among TB
patients. According to the facility survey, only 32% of facilities providing
DOTS also provided ART for persons living with HIV. For IDUs the challenge may
be two-fold. First, the availability of drug-substitution therapy for IDUs in
Ukraine is scarce; only 16% of the outpatient TB facilities reported offering
this service. Without adequate substitution therapy, many IDUs may drop out of
service during the intensive, inpatient TB treatment phase, excluding them from
our sample. Second, the stigma for drug addiction may discourage IDUs from
revealing this risk to their TB physician. In either scenario, the risk of
default among IDUs may not be adequately reflected in our data.
Patients with reported co-morbidities reduced their probability of defaulting by
almost 6 percentage points in 2011, possibly due to additional support received
from providers caring for the co-morbidities. For all other risk factors, no
statistical associations were found with default. Whether this is due to the
small numbers of patients with these other risks or because these factors do not
increase one’s risk of default is undetermined in this study. Interestingly,
almost 20% of the high-risk cohort receiving the intervention had an
undetermined or “other” risk factor recorded. Provider interviews suggested that
compliant patients in our study sites may have been referred to URCS as a reward
for their adherence. If widespread, this preferential referral of adherent
patients could create selection bias, affecting results. This is one of the
limitations of retrospective data analysis, it is difficult to measure the
fidelity of program implementation retrospectively. However, if there was
widespread selective referral one would expect the 2012 HR-Comparison group to
have reported a higher default rate than the 2011 HR-Comparison group. The
comparability of the default rates among these two cohorts suggests that very
little selection bias exists.
In an era of declining health resources and increasing drug-resistant TB,
refining and standardizing the referral criteria for additional social support
may reduce the national default rate, but not without a cost. This study shows
that social support is effective in reducing default rates but whether or not
that means it should or can be scaled-up depends on the cost of wider
implementation and the cost relative to other potential interventions that might
also reduce default rates.
# Conclusions
This study demonstrates the positive impact of providing social support to those
at-risk for treatment default. Targeting services to those who will most benefit
is critical to reduce continuing TB transmission. Further research is
recommended to differentiate the costs and benefits from home-based DOTS versus
additional services offered through social support programs. Prospective cohort
studies could refine targeting of programming, evaluate social support program
fidelity, identify which populations respond best to select services, and what
barriers might still exist to achieving better adherence. With that information,
tailoring programs to most effectively reach and serve clients in a patient-
centered approach may reap substantial rewards for Ukraine. Prioritizing support
services for clients who struggle with alcohol or drug addictions or struggle
with adherence to intensive inpatient treatment regimens, may improve treatment
success. Identifying approaches to assure intensive treatment completion and
flagging those upon completion for additional follow-up during continuation
treatment, has the potential to further reduce program defaults and improve
outcomes.
MEASURE Evaluation is funded by the U.S. Agency for International Development
(USAID) under Cooperative Agreement GHA-A-00-08-00003-00 and is implemented by
the Carolina Population Center at the University of North Carolina at Chapel
Hill, in association with The Palladium Group, ICF International; John Snow,
Inc.; Management Sciences for Health, and Tulane University.
We are grateful for the Carolina Population Center (R24 HD050924) for general
support. We also thank the F.H. Yanovskyi Institute of Phthisiology and
Pulmonology, Academy of Medical Sciences of Ukraine for their support in
conducting this study in Ukraine.
[^1]: The authors have read the journal's policy and have the following
conflicts: Stephanie Mullen is employed by John Snow Inc. This affiliation
does not alter our adherence to PLOS ONE policies on sharing data and
materials.
[^2]: Current address: Office of Family and Community Health Improvement,
Washington Department of Health, Olympia, Washington, United States of
America |
# Introduction
Ferulic acid (FA) is a phenolic substance and an important active ingredient
that is common in various plants. It occurs at high concentrations in food
ingredients such as coffee, grain hulls, vanilla beans, wheat bran, and rice
bran. The FA molecule is a 4-hydroxy-3-methoxy cinnamic acid (C<sub>10</sub>
H<sub>10</sub> O<sub>4</sub>), and FA exhibits various biological activities and
physiological functions and has low toxicity. Importantly, FA has been shown to
exhibit antioxidant and anti-inflammatory properties and is an effective
modulator of multidrug resistance in cancer. FA can prevent acute liver injury
due to sepsis by attenuating the inflammatory response, and it improves
corticosterone-induced liver damage. Moreover, FA also improves corticosterone-
induced depressive behaviour and oxidative stress in mice, and enhances the
antibacterial activity of quinolone antibiotics. Geese is a nutritious and
healthy food resource. the short reproductive periods, low hatchability, and
high embryo mortality of geese. it is reported that the content of protein and
trace elements in the meat of goose is higher than in other poultry products.
So, geese breeding industry has broad prospects. Intestine plays a key
supporting role in the growth of animals. and early growth and development of
the gastrointestinal tract are critical to optimizing the growth of poultry. In
the early stages of goose growth and development, a large amount of nutritional
support is required. After the intestinal structure and function are damaged, it
will affect the individual’s early nutritional absorption, causing irreversible
damage to the body’s growth and development in the early stages.
Lipopolysaccharide (LPS) is a pathogenic compound that occurs in the outer
membrane of the cell wall of all gram-negative bacteria, and it can elicit
multiple signaling events in the cell. LPS can act as a strong inflammatory
mediator and is widely used in animal studies to simulate bacterial infection.
Peng et al. studied inflammation in bovine endometrial epithelial cells
caused by LPS and found that FA exerted an anti-inflammatory effect by
inhibiting the release of cytokines. Chen et al. reported that 30 mg/kg FA
increased the antioxidant capacity of the liver and repaired liver damage,
reducing hepatocyte death due to LPS in blunt-nosed sea bream. Further, FA plays
a positive role in reducing renal injury in HFD/STZ-induced DN mice by enhancing
autophagy and inhibiting inflammation.
However, to date, the effects of FA treatment in geese after triggering
oxidative stress have not been studied, nor has the oxidative stress damage
encountered during the development of the geese industry been addressed, which
would lead to mortality in geese, and could adversely affect the development of
the geese industry. In the present study, FA was added to the diets of Jilin
white geese at different concentrations, and LPS was injected intraperitoneally
at 14 and 21 days of age to investigate the effects of FA on the growth
performance and intestinal antioxidant capacity of LPS-stressed Jilin white
geese. Our findings show that FA can protect animals under oxidative stress
conditions and provide a scientific basis for the implementation of FA as an
antioxidant agent.
# Materials and methods
## Experimental design
This study was carried out in strict accordance with the recommendations of the
Guide to Nursing and Use of Experimental Animals of Jilin Agricultural
University. The study was verbal approval by the Experimental Ethics Committee
of Jilin Agricultural University. All operations were performed under
pentobarbital sodium anesthesia, and every effort was made to reduce pain.120
male Jilin white geese of similar weight at 7 d of age were used. There was a
pre-feeding period of 7 d, and the trial period was 21 d. After the pre-feeding
period, the 120 Jilin white geese were randomly divided into six groups with
five replicates (four birds in each group). Groups F1 (60 mg/kg feed FA), F2
(120 mg/kg feed FA), F3 (180 mg/kg feed FA), F4 (240 mg/kg feed FA), and L were
given intraperitoneal injections of LPS (500 μg/kg BW) on days 14, 17, and 20.
The doses and routes of LPS administration referred to the previous studies.
Group C was given intraperitoneal injections of normal saline (0.5 mg/kg BW).
The test was carried out by establishing an oxidative stress model. shows the
composition of rations. During the test period, all geese had access to feed and
water ad libitum throughout the trial. Water was provided in a half-open plastic
cylindrical water tank, and the feed was provided in feeders on one side of each
pen. The geese were reared indoors conditions (temperature: 26.0°C ± 3.0°C;
relative humidity (RH): 60.5 ± 5.0%; lighting period: 16 h; space allocation:
0.49 m2/gander), and the feed intake and body weight were recorded daily. On day
21, 10 animals in each group were randomly selected for slaughter, and tissue
samples of the heart, liver, spleen, kidney, bursa of fabricius, and thymus
organs, duodenum, jejunum, and ileum were collected.
1\) The premix provided the following per kg of diets: VA 2500 IU,
VD<sub>3</sub> 1000 IU, VE 3100 mg, VK<sub>3</sub> 200 mg, VB<sub>1</sub> 100
mg, VB<sub>2</sub> 1 200 mg, VB<sub>6</sub> 200 mg, VB<sub>12</sub> 2 mg,
Nicotinic acid 600 mg. Pantothenic acid 1 700 mg, Folic acid 200 mg, Biotin 20
mg, Fe (as ferrous sulfate) 6 000 mg, Cu (as copper sulfate) 300 mg, Mn (as
manganese sulfate) 15 000 mg. Zn (as zinc sulfate) 8 500 mg, I (as potassium
iodide) 10 mg, Se (as sodium selenite) 30 mg.
## Test materials
### Test animals and reagents
The test animals were purchased from Jilin Yuhong Ecological Agriculture
Technology Co. We also used *E*. *coli* lipopolysaccharide (Sigma Chemical Co.,
St. Louis, MO, USA), FA (97%) (Shanghai Maclean Biochemical Technology Co.,
Ltd), and Malondialdehyde assay (MDA)kit, Total Antioxidant Dismutase Assay
(SOD) Kit, Glutathione peroxidase assay (GSH-PX) kit, Hydrogen peroxidase assay
(CAT) kit, Total Antioxidant Capacity Assay(T-AOC) Kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China).
## Sample collection and indicator determination
Growth performance. The initial weight, day 14 weight, and final weight of the
geese were recorded. The average daily gain (ADG), average daily feed intake
(ADFI), and feed conversion ratio (F/G) in the stages of days 1–14, 15–21, and
1–21 in the trial were calculated.
### Visceral index
All surgeries are performed under pentobarbital sodium anesthesia, and every
effort was made to reduce pain. Slaughter was carried out on day 21 of the
trial. The organs of the geese (heart, liver, spleen, bursa, and thymus) were
removed after slaughter and were weighed after removing excess fat.
Measurement of intestinal oxidative stress indicators. Determination of MDA,
SOD, GSH-PX, CAT and T-AOC in the intestinal tract of geese.
## Data analysis
The raw data were processed using Microsoft Excel. A one-way ANOVA and Duncan’s
multiple comparison test were performed using SPSS 26.0 software. Results are
presented as mean ± standard error (SE), with *P \<* 0.05 indicating a
significant difference.
# Results
## Growth performance
The LPS damage model was developed to observe the change in growth performance
of each group before and after oxidative stress. Between days 1 and 14, final
body weight (FB) and ADG were significantly higher in group F3 than in group L
(*P \<* 0.05), and ADFI was significantly higher in groups F2 and F3 than in
groups C and L (*P* \< 0.05). The FCR was significantly lower in group F3 than
in group L (*P* \< 0.05). On days 15–21, FB was significantly higher in the
groups with FA added than in group L (*P* \< 0.05). ADG was significantly higher
in the groups with FA added than in group L and significantly lower than in
group C (*P* \< 0.05). ADFI was significantly higher in the groups with F1 and
F4 than in group L and significantly lower in the groups with FA added and L
than in group C (*P* \< 0.05). FCR was significantly lower in the groups with FA
added than in group L and significantly higher in groups F1, F2, F3, and L than
in group C (*P \<* 0.05). From days 1 to 21, ADG was significantly higher in the
groups with FA added than in group L and significantly lower in groups F1 and L
than in group C (*P \<* 0.05). ADFI was significantly higher in groups C, F2,
and F3 than in group L (*P \<* 0.05). FCR was significantly higher in group L
than in the other groups (*P \<* 0.05).
## Organs indices
To reflect the changes in organ weight in geese after oxidative stress, samples
were taken at 21 days and the organs indices was measured. The thymic indices
was significantly higher in group F4 than in groups C (*P \<* 0.05).
## Antioxidant activity in the duodenum
We measured the changes in antioxidant activity in the duodenum of geese after
oxidative stress to reflect the antioxidant activity of the duodenum. Groups F1,
F3, F4, and C showed a significant decrease in MDA compared to group L (*P \<*
0.05). Group F1 showed a significant decrease in SOD compared to group C and
Group F4 showed a significant reduce in GSH-Px compared to group C (*P \<*
0.05). Group C showed a significant increase in SOD and GSH-Px compared to group
L (*P \<* 0.05). Groups L, F1, F2, and F4 showed a significant decrease in CAT
compared to group C (*P \<* 0.05).
## Antioxidant activity in the jejunum
We measured the changes in antioxidant activity in the jejunum of geese after
oxidative stress to reflect the antioxidant activity in the duodenum. MDA was
significantly lower in groups F3 and F4 than in group L and in group F4 than in
group F1 (*P \<* 0.05). SOD was significantly higher in group C than in groups L
and F1 (*P \<* 0.05). GSH-Px was significantly higher in group F3 than in groups
L, C, and F1 (*P \<* 0.05). The CAT content of group L was significantly lower
than that of all other groups (*P \<* 0.05).
## Antioxidant activity in the ileum
We measured the changes in antioxidant activity in the jejunum of geese after
oxidative stress to reflect the antioxidant activity in the duodenum. MDA was
significantly lower in groups F2, and C than in group L, (*P \<* 0.05). GSH-Px
was significantly higher in group C than in groups L and F1, F2, F4 and
significantly higher in group F2, F3, F4 than in group L and significantly
higher in group F3 than in group F1 (*P* \< 0.05).
# Discussion
During the process of goose breeding, oxidative stress can lead to a decrease in
gse growth performance and a loss of economic benefits. LPS, a major component
of the cell wall of gram-nega-tive bacteria, is a pathogenic compound. In this
study, we obtained equivalent experimental results: LPS injection at 0.5 mg/kg
body weight significantly reduced both the body weight gain of geese at d 15 to
21 and the feed intake during, and, but increased the FCR of geese on d 15 to
21. These initial results indicated that the oxidative stress model was
established successfully and was suitable for subsequent investigation of the
effects of FA on geese health. The addition of 6 mg/kg of FA to the feed
increased beef the tenderness, juiciness, flavor intensity, and amount of some
fatty acids. Supplementation with 180 mg/kg of FA in the present study enhanced
growth performance and reversed the decline in growth performance brought about
by oxidative stress, indicating that ferulic acid can alleviate the damage
caused by oxidative stress.
The liver, as the main metabolic and detoxification organ in the body, is
closely related to various physiological functions and circulation, and the
effects of immune organs such as the liver on oxidative stress occur through the
recruitment of immune cells. In the present study, the addition of 240 mg/kg FA
increased the thymic index, probably because FA mitigates the effects of
oxidative stress from LPS by affecting the weight of the thymus and increasing
body immunity.
Reported that FA has cytoprotective capacity and may promote gastrointestinal
health and microbial protein synthesis. In mice with high oxidative stress
levels, insulin synthesis was improved in pancreatic β-cells. In the present
study, the addition of 180 mg/kg of FA significantly alleviated the intestinal
damage caused by oxidative stress, may be related to the enhancement of
intestinal epithelial cell activity by ferulic acid, enhances cell activity to
resist LPS induced damage.
FA also inhibited the activation of the ROCK/NF-κB signaling pathway, thereby
improving the dysregulation of oxidative stress and inflammation and exerting an
effective hepatoprotective effect. The above growth performance and intestinal
oxidative indexes were due to excessive reactive oxygen species production from
LPS-induced oxidative stress, which indirectly affected the changes in
physiological indexes and enzyme activities. This may be related to antioxidant
signaling pathways, such as Nrf2/HO-1 and ROCK/NF-κB. Administering FA after
LPS-induced oxidative stress can alleviate the effects of oxidative stress on
growth performance and reduce intestinal antioxidant activity.
# Conclusion
1\. Adding 180 mg/kg of FA can increase the body weight of geese and promote
their growth. Adding 60 mg/kg FA can improve the thymus index, alleviate the
damage to immune function caused by stress, and reduce the negative effects of
stress.
2\. Adding 180 mg/kg FA can alleviate oxidative stress damage in the duodenum,
ileum, and jejunum, reduce cell membrane damage, maintain the homeostasis of the
membrane lipid bilayer, and protect cells from oxygen ions.
In conclusion, adding 180 mg/kg of FA promoted the growth of geese and
alleviated the effects of oxidative stress and the damage caused by oxidative
stress in the duodenum, jejunum, and ileum.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Computational fluid dynamic (CFD) simulations of biological valves have steadily
improved over the years; however, procedures accounting for the formation of
actual solid aggregates, such as calcifications or clots, have not been
implemented yet. At the same time, researchers have also devised mathematical
models for clot formation and growth; however, these models have been developed
independently and are not usually associated to the dynamics of the valve. We
propose a particle-based method that, by taking advantage of its mesh-free
nature, can compute the fluid dynamics, together with valve deformation and
formation of solid aggregates.
In general, the simulation of biological valves, where a solid structure
interacts with the surrounding flow, constitutes a fluid-structure interaction
(FSI) problem. The algorithms to solve FSI problems may be broadly classified
into two categories: conforming mesh methods and non-conforming mesh methods.
Conforming mesh methods divide the computational domain in two parts: (i) a part
occupied by the liquid where the Navier-Stokes equations are solved, and (ii) a
part occupied by the structure where the stress-deformation equations are
solved. Since the structure moves and/or deforms with time, re-meshing is needed
as the solution advances. Non-conforming mesh methods, most notably the Immersed
boundary methods (IBM), treat the interface between the fluid and the structure
as a constraint and the force exerted by the structure to the fluid becomes a
source term in the momentum equation. As a result, the fluid and solid equations
are solved independently and re-meshing is not necessary. Both methods, however,
have difficulties handling phenomena such as calcification and clotting that
involve some sort of transition where part of the liquid transforms into a
solid. In general, attempts to account for the formation of solid aggregates in
CFD/FSI studies are based on ‘numerical artifices’ such as fluids with higher
viscosities to mimic clotting, or membranes with higher stiffness to mimic
calcification.
For a different approach see.
On the other hand, modelling of clot formation and growth had followed an
independent path that, in some cases, has brought to particle-based techniques
such as Lattice-Boltzmann or Coarse grained Molecular Dynamics. In general,
however, these models assume simple hydrodynamic conditions and/or refer to
straight blood vessels with not moving/deforming parts. Additionally, coupling
fluid dynamics and solid deformation can be implemented with the Smoothed
Particle Hydrodynamics method. But, it can’t easily account for other phenomena
such as contact mechanics and agglomeration.
In order to account for the fluid dynamics, the valve deformation, and the
formation of solid aggregates at the same time, we propose *discrete multi-
physics*: a mesh-free approach, where computational particles are employed for
both the flow and the structure. With this method, the distinction between
liquid and solid depends exclusively on the types of forces that act on each
particle: pressure and viscous forces characterize liquid particles, while
elastic forces characterize solid particles. By changing the type of forces on
specific groups of particles, we can change their status form liquid to solid
and vice versa. In, we used this idea to model melting and solidifying flows; in
this paper, we extend it to the formation of agglomerates in biological valves.
This article is organized as follows. Initially, we discuss the basic ideas
behind our discrete multi-physics technique and describe the geometry used in
the simulations. Next, we validate the model against both traditional modelling
techniques and experimental data. Finally, we introduce the formation of solid
aggregates at the membrane surface and in the flow.
The objective of this paper is to apply discrete multi-physics to biological
valves in general. For this reason, we do not focus on a specific type of valve
at this stage. However, in order to test our model in the most challenging
scenario, we chose dimensions and velocities similar to those occurring in
aortic valves. We consider these conditions to be the most challenging scenario
because (i) they involve higher velocities, which generate complex recirculation
patterns, and (ii) they involve higher stresses, which generate
larger membrane deformations. From this point of view, the fact
that we simulate bicuspid valves, while the aortic valve has three leaflets, is
not a limitation. Given the same mechanical stress, in fact, deformations are
higher in bicuspid valves than in tricuspid valves. Therefore, by forcing
velocities that are typical of aortic valves in bicuspid valves, we test our
model under conditions that are even more critical (i.e. produce higher
deformations) than those occurring in aortic valves.
# Modelling
## Discrete multiphysics
Our discrete multi-physics approach is based on the so-called discrete multi-
hybrid system (DMHS). This technique combines various mathematical models to
achieve a representation of fluid-structure interactions and solid-liquid
systems that is not attainable with each model separately. Elsewhere, we showed
that the linkage of different models is mathematically complex and
computationally time consuming. In order to facilitate this, the DMHS combines
models that share a common discrete (particle-based) paradigm, such as SPH
(Smoothed Particle Hydrodynamics), CGMD (Coarse-grained Molecular Dynamics), DEM
(Discrete Element Method) or BD (Brownian Dynamics).
In this study, models for solid contact/collision (i.e. DEM), or for fluctuating
hydrodynamics (i.e. BD) are not necessary; consequently, the coupling is limited
to SPH (liquid phase) and CGMD (solid phase). For the numerical solution, the
model was implemented in LAMMPS. A mathematical introduction to SPH and CGMD and
the approach used to couple the models is given in. Specific details of the DMHS
and other mesh-free hybrid techniques can be found in. In previous DMHS
publications, there is an interchangeable use of the terms CGMD and Mass-Spring
Model (MSM). This depends on the fact that these articles cover different
scales. Articles dealing with microscopic scales use CGMD, whereas articles
dealing with macroscopic scales use MSM. Mathematically, however, the two
techniques are equivalent. In the main text, we prefer MSM, which is more
consistent with the scale under investigation. In, we use CGMD, which is more
consistent with the original DMHS formulation.
## Geometry
We use a 2D simplified geometry for modelling a generic bicuspid valve as
illustrated in. The channel half-thickness is *Z* = 0.0125 m, the length of the
membrane is *L* = 0.016m and the radius of the circular area is *R* = 0.0215 m.
As mentioned above, the DMHS combines various particle-based modelling
techniques. In this study, the simulations are based on two models and three
types of particles: SPH particles for the fluid, fixed SPH particles for the
walls and MSM particles for the flexible leaflets (membrane). Periodic boundary
conditions are used at the inlet/outlet. To model the Young modulus *E* and the
flexural rigidity *F* of the membrane, the MSM particles are joined together by
numerical ‘springs’ and ‘hinges’, as described in. The relation between the
spring (*k*<sub>*b*</sub>) and hinge (*k*<sub>*a*</sub>) constants and the
actual Young modulus and the flexural rigidity is given in.
## Pulsatile flow
In order to test the model in the most critical conditions, we target high
velocities typical of cardiac valves such as the aortic valve. The flow is
pulsatile and corresponds to a normal cardiac output of 5.5 L min<sup>-1</sup>,
a beat rate of 72 bpm and an aortic pressure of 100 mmHg. This frequency gives a
peak velocity of around 0.9 m s<sup>-1</sup>. In the simulation, we force the
flow by means of a sinusoidal pressure *P* gradient $$\frac{dP}{dx} =
A\text{sin}(\frac{2\pi}{T}t),$$ where *A* is the force amplitude and *T* the
period. To obtain this pressure gradient in the simulations, we impose to each
liquid particle the acceleration $$g = g_{0}\text{sin}(2\pi ft),$$ with
*g*<sub>*0*</sub> = 500 m s<sup>-2</sup> and oscillation frequency *f* = 1/*T*
(*T* = 1 s). Under these conditions, we reach high velocities but the flow
remains laminar. We focus on the laminar regime for two reasons: (i) blood flow
under normal conditions is laminar, and (ii) we want to test, at this stage, the
accuracy of our model without dealing with the additional complexity of
turbulence.
## Dimensionless analysis
In Section *Membrane deformation*, we compare our simulations with experimental
data. The comparison is based on specific dimensional groups that are defined in
this section.
Dimensional analysis bring to three fundamental groups Re (Reynolds Number),
N<sub>f</sub> (dimensionless frequency) and Λ (geometric ratio), defined as
$$\text{Re} = \frac{\rho UZ}{\mu},$$ $$N_{\text{f}} = \frac{\rho
f^{2}d^{5}}{F/L},$$ and $$\Lambda = \frac{Z}{L},$$ where *ρ* is the density of
the fluid, *U* is a reference velocity (here we use the max velocity in the
channel), *Z* the half-thickness of the channel, *μ* the fluid viscosity, *f*
the oscillation frequency, *d* the membrane thickness, *F* the flexural
rigidity, and *L* the length of the membrane. The computational particles used
in our simulations are point particles; strictly speaking, they do not have an
actual thickness. Their thickness is the result of the repulsive forces acting
on the particles to impose no-penetration boundary conditions. The value of *d*
in, therefore, is calculated from *k*<sub>*a*</sub> and *k*<sub>*b*</sub> as
discussed in. The value of the Young modulus *E* does not compare explicitly in
any of the dimensionless numbers above; this is due to the fact that *F* and *E*
are interchangeable as discussed in. In theory, we should also account for
another dimensionless group based on *R* (radius of the convex area). In
practice, however, this group is not necessary as discussed in Section *Membrane
deformation*.
Each dimensionless number provides specific information about the geometric
constants and the physical forces acting in the system. Re indicates the extent
of the inertial forces with respect to the viscous forces. N<sub>f</sub>
indicates the membrane resistance with respect to the stress generated by the
oscillating flow. Λ indicates the geometric ratio between the channel thickness
and the membrane length. In comparing the simulations with the experimental data
(see Section *Membrane deformation*), we found that the group
$$\text{N}_{\text{R}} = \text{Re}.\text{N}_{\text{f}} =
\frac{\rho^{2}f^{2}d^{5}UZ}{\mu F/L}$$ is particular relevant. This
dimensionless number compares the effect of the forces that tend to deform the
membrane (numerator) with those that tend to oppose the deformation
(denominator). In Section *Membrane deformation*, we show that different
geometries and flow conditions generate the same type of membrane deformation if
N<sub>R</sub> is the same.
# Results
There are two types of parameters required for the simulations: model parameters
and simulation parameters. The first group consists of internal parameters used
by the SPH and MSM solvers; the second refers to the operative conditions. This
Section focuses on the second group (i.e. *Z*, *L*, *R*, μ, *ρ* and *F*); the
internal parameters (e.g. *k*<sub>*a*</sub>, *k*<sub>*b*</sub>, number of
particles, time step, smoothing length, etc.) can be found in.
The geometric parameters *L*, *Z*, *R* are given in Section *Geometry* (see also
). All the simulations assume blood as liquid medium. Blood is a viscoelastic
fluid, but in flow simulations is often considered Newtonian. In our
calculations, we also use the Newtonian approximation with *ρ* = 1056 kg
m<sup>-3</sup> and *μ* = 0.0035 Pa s. We consider membranes with different
flexural rigidities, the specific value of *F*, for each case, is given in
Section *Membrane deformation*.
This section is divided in three parts. The first part is dedicated to the flow,
and we validate our results against traditional CFD simulations. The second part
is dedicated to the membrane, and we validate our results against experimental
data. The third part focus on the formation of solid aggregates, and highlights
the main advantages of the DMHS in modelling biological valves.
## Hydrodynamics
We compare results obtained with our model with traditional CFD simulations
performed with Abaqus 6.14<sup>®</sup> with the same geometry and under similar
flow conditions. In these simulations, the membrane is fixed in order to focus
solely on the hydrodynamics. This is done on purpose: if at this stage we had
considered both the fluid and the membrane together, we could not have
distinguished whether potential errors originated from the fluid dynamics or the
membrane mechanics.
Calculations are run at two constant inlet velocities, 0.2 m s<sup>-1</sup> and
0.9 m s<sup>-1</sup>. Because of the different nature of the two modelling
techniques, the inlet/outlet boundary conditions (b.c.) are not the same. The
DMHS uses periodic inlet/outlet b.c., while in the CFD simulation the inlet has
constant velocity and the outlet fixed pressure. shows the CFD results; shows
the DMHS results.
Comparison between Figs and shows a good agreement between the CFD and the DMHS
calculations. Both models, in particular, capture the recirculation zones in the
circular chamber in the centre. For the velocity, minor differences (2–5%) can
be found at the tip of the valve. These differences depend on the different
inlet conditions between the DMHS and the CFD model and the nature of the
discretization method (particles vs. mesh).
Another important variable often reported in literature is shear stress (Figs).
But also in this case CFD and DMHS results are similar. Both models, in
particular, identify a region of high stress near, but not exactly at, the end
of the leaflets.
## Membrane deformation
In this section, we account for the flexibility of the leaflets and calculate
both the flow and the membrane dynamics. For validation purposes, we compare the
membrane deformation observed during the simulations with those obtained
experimentally by. In both simulations and experiments the valve has two
leaflets and the flow is pulsatile. The geometric conditions, however, are not
exactly the same: in, in fact, *L* = 0.0263 m, *Z* = 0.015m, and the channel is
straight without the circular chamber in the centre (this is why, in Section
*Dimensionless analysis*, we did not introduce a forth dimensionless number).
Additionally, our simulations are 2D and based on blood, while employ water in a
rectangular channel with depth *w* = 0.05 m. gathers all the parameters used in
the simulations and in the experiments. The values of *k*<sub>*a*</sub> and
*k*<sub>*b*</sub> corresponding to a specific *F* in the simulations are given
in. The values of *d* for the simulations are calculated according to the
procedure described in.
After running a large number of simulations and observing how the membrane
deforms under various flow conditions, we realized that the fundamental group
that affects the membrane dynamics is N<sub>R</sub>. Therefore, we chose
specific values of *f*, *g*<sub>0</sub> (which gives *U*), *k*<sub>*a*</sub> and
*k*<sub>*b*</sub> (which give *F* and *d*) to obtain in our simulations the same
N<sub>R</sub> of the experiments.
We consider three cases that we call, soft membrane, intermediate membrane, and
hard membrane. We start with the case of the intermediate membrane: the ‘normal’
case, which subsequently is compared to the soft and the hard membrane.
shows the comparison between simulations and experiments in the case of the
intermediate membrane. The overall dynamics is very similar, but there are same
noticeable differences. In the simulation, the maximum opening of the membrane
is wider. This is due to the absence of the central chamber in the experimental
set-up. Another minor difference occurs at the end of the cycle when the
backpressure closes the valve completely. When closed, the experimental valve
has a more elongated shape since the leaflets are longer. This is a consequence
of the fact that, besides the main group N<sub>R</sub>, also Λ has a (minor)
effect on the membrane. Experiments and simulation have the same N<sub>R</sub>,
but not exactly the same Λ; some (minor) differences, therefore, are expected.
shows the comparison between simulations and experiments in the case of the soft
membrane. The soft membrane can be considered defective since its position is
completely reversed by the backflow. As in the previous case, there are some
minor differences, but overall the membrane behaviour is well captured by the
model. Similar deformation profiles have also been by other studies.
shows the comparison between simulations and experiments in the case of the hard
membrane. Also the hard membrane can be considered defective since it does not
completely opens. In this case, the comparison focuses on the membrane’s tip. At
this location, the two leaflets slide one over the other and symmetry is lost.
This phenomenon is captured in both the simulations and the experiments.
The loss of symmetry suggests that the simulations should account for the whole
geometry and not only half of it (considering only one leaflet).
Besides validating our model, this section also highlights the importance of
N<sub>R</sub>. The values of Re, Λ and N<sub>f</sub> between the simulations and
in the experiments are different, but, since N<sub>R</sub> is the same, the
membrane behaviour in both cases is similar (with the little caveat about Λ as
discussed above).
## Formation of solid aggregates
This section introduces solid aggregation in the DMHS. We consider two cases:
solid deposits at the membrane surface, and formation of aggregates in the main
flow. We generally indicate the first case as ‘calcification’ and the second as
‘clotting’. Our focus, however, is not to the formation and the evolution of
actual calcifications and clots. These are very complicated biochemical
phenomena and their full dynamics is beyond the scope of this article. The goal
is here to illustrate how, given a criterion for aggregation, this can be
implemented in our model. Once this has been achieved, more complicated
agglomeration models can be implemented.
Both calcification and clotting imply the formation of solid aggregates
developing from the liquid. In the DMHS framework, this can be achieved by
changing the forces acting on certain particles from SPH to MSM. describes the
algorithm used in the simulations. The procedure starts from an agglomeration
seed. In our simulations, the seed is chosen arbitrarily, but it can depend on a
specific criterion; for example, when local shear stress exceeds a threshold
value, the particle at that location becomes a seed. Once the position of the
seed is known, the algorithm propagates the agglomerate. Every *N* time-steps,
it identifies all the particles within a distance *R*<sub>MAX</sub> from the
seed, and, with a certain probability, transforms some of the liquid-particles
in solid agglomerate-particles by (i) changing the forces acting on the
particles from SPH to MSM, (ii) and creating a bond between the seed and the
newly created agglomerate-particle. The strength of the new bond determines the
material properties of the agglomerate. In our simulations, the probability of
transforming a liquid-particle in an agglomerate-particle has been related to a
fixed value, but, as mention before, it can be associated to a specific
criterion (e.g. shear stress threshold).
The algorithm repeats the above procedure iteratively to propagate the
agglomerate further and new agglomerate particles create bonds only to other
fluid particles and not to existing agglomerate particles. At the next time
step, the previously generated agglomerate-particles become seeds; these seeds
create new agglomerate-particles and so on as illustrated in.
We can affect the final shape of the agglomerate by changing, as time
progresses, the behaviour of the seeds. If the seeds are active all the time,
they continue to create new agglomerate-particles around them until they are
fully surrounded by the agglomerate. In this case, the overall shape of the
agglomerate tends to be circular. If the seeds remain active only for one time
step, the agglomerate propagates in one preferential direction and tends to
assume a filiform (thread-like) shape.
shows three types of simulations where the algorithm is applied to three
different configurations. The simulation parameters (*N*, *R*<sub>MAX</sub>,
agglomeration probability, etc.) for all three cases are gathered in. With the
goal of obtaining a sizable aggregate in a few cycles, we accelerated the
agglomerate formation by using higher aggregation probabilities. This is a
typical technique used to study phenomena with very different timescales as
those occurring in pipelines erosion.
In the first case (called ‘calcification’), the initial seed is located in the
region between the membrane and the wall and the deposit propagates following
the circular agglomeration algorithm illustrated in. An interesting feature of
the simulation is that, as time progresses, the agglomerate makes increasingly
difficult the movement of the lower leaflet until it stops almost completely.
In the second case (called ‘free clot’), the initial seed is located in the flow
and the agglomerate propagates following the circular algorithm. The presence of
a solid aggregate alters the hydrodynamics as indicated in. Once a liquid
particle transform into a solid particle, the fluid streamlines must change
direction to account for the new solid-liquid boundaries. This feature would not
be possible with mesh-based algorithms and highlights one of the advantages of
discrete multi-physics.
The third case (called ‘filiform clot’) is similar to the previous case. This
time, however, the initial seed is located at the tip of the leaflet and the
agglomerate propagates according to the filiform algorithm. As the filiform
aggregate grows, it moves alternately on the right and on the left of the
membrane due to the oscillating flow.
We can emphasize another advantage of using a particle-based technique by
introducing fragmentation. The drag between the fluid and the agglomerate
creates internal stresses in the solid. These stresses tend to pull apart the
agglomerate-particles that respond with a stronger binding force (according to
equation J). At this point, we can slightly modify the algorithm and introduce a
criterion for break-up: if the force between two agglomerate-particles exceeds a
certain value, the bond breaks. In, we used a threshold force of
1.3·10<sup>−7</sup> N. At a certain point of the simulation, the threshold force
is exceeded and the agglomerate breaks in two parts. One part remains attached
to the leaflet; the other becomes free and moves unrestricted with the flow.
# Conclusions
Mesh-free methods are usually considered viable alternatives to traditional
modelling, but have never enjoyed the same popularity of mesh-based techniques.
Many mesh-free methods have been developed only in relatively recent years and
offer, to the potential user, less available information, experience and
software. On the other hand, a specific sub-set of mesh-free algorithm (e.g.
SPH, DEM, CGMD, BD etc.) share a common particle-based framework that makes
particularly easy their linkage in multi-physics problems. We call this approach
*discrete multi-physics* and, in this paper, we show that, in certain
circumstances, it is more than a mere alternative to traditional modelling.
Discrete multi-physics can tackle, with relatively little effort, problems that
are considered very challenging with mesh-based multi-physics. Elsewhere, we
focused on solid-liquid flows where the dispersed phase is made of deformable,
breakable, dissolving, melting or solidifying particles. Here, we apply the same
approach to biological valves including the formation of solid aggregates in the
flow and at the membrane surface. To the best of our knowledge, this is the
first study to directly account for the hydrodynamics, the membrane deformation
and the formation of solid aggregates at the same time and, as such, it has the
potential to open a new prospective to the modelling of biological valves.
# Supporting information
[^1]: The authors from LivaNova didn't provide any financial support to
these works. Commercial affiliation of authors from LivaNova does not alter
our adherence to PLOS ONE policies on sharing data and materials.
[^2]: **Conceptualization:** MA M. Bussone FG AA. **Data curation:** MA AA.
**Formal analysis:** MA MHA M. Bussone FG FB M. Barigou AA. **Funding
acquisition:** M. Barigou AA. **Investigation:** MA MHA AA. **Methodology:**
MA AA. **Project administration:** M. Barigou AA. **Resources:** M. Bussone
FG FB M. Barigou AA. **Software:** MA MHA. **Supervision:** AA.
**Validation:** AA. **Visualization:** MA AA. **Writing – original draft:**
MA AA. **Writing – review & editing:** MA AA.
[^3]: ‡ These authors also contributed equally to this work. |
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Dataset Card for Only Clean Data (OCD)
If you are training base language models and want the cleanest sources available, OCD was built just for you.
Dataset Details
Dataset Description
It is without question that the quality of a language model rests on the quality of its training data. OCD is a meticulously curated and cleaned corpus of text documents, ensuring the highest quality text from a variety of sources. Part of this process includes manually inspecting (and sometimes manually fixing) thousands of documents. Whenever problem documents are found (from e.g. conversion errors, or spam that got through), they are fixed for the next release.
Dataset Sources
OCD currently consists of 3 subsets:
- Web data originating from C4. This subset was heavily filtered to remove a lot of spam, templates, and other low quality data. It consists of approximately 18M documents, which is roughly 5% of the original 365M documents.
- Peer reviewed research originating from PLOS. Documents were normalized to markdown from the original JATS XML, and were processed to remove captions and references to missing figures. A large number of these documents were manually inspected to remove irrelevant files (e.g. journal announcements, letters to the editor, and short comments).
- Non-fiction books from CCOpenBooks. This will be expanded soon, as the original collection was quite small. However, it contains only high quality text books, all of which have non-restrictive cc-by compatible licenses.
This dataset is actively in development, and will continue to be extended to include books, research, and other documents from various domains, code, documentation, and more.
License
OCD is released under the cc-by-4.0 license. Note that this is directly compatible with PLOS and CCOpenBooks (cc-by-4.0).
Documents originating from C4 were released under the ODC-BY license as noted here. As this subset was derived from an enormous Common Crawl corpus, there is a possibility that it contains documents not compatible with this license. However, the heavy filtering applied as part of the OCD project greatly reduces this possibility. Additionally, any opt-out requests from content authors will be respected.
Uses
The primary intended use of this dataset is for training base language models.
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