Author’s response to reviews
Title: Epidemic resurgence of dengue fever in Singapore in 2013-2014: A virological and
entomological perspective
Authors:
Hapuarachchige Chanditha Hapuarachchi
([email protected];[email protected])
Carmen Koo ([email protected])
Jayanthi Rajarethinam ([email protected])
Chee-Seng Chong ([email protected])
Cui Lin ([email protected])
Grace Yap ([email protected])
Lilac Liu ([email protected])
Yee-Ling Lai ([email protected])
Peng Lim Ooi ([email protected])
Jeffery Cutter ([email protected])
Lee Ching Ng ([email protected])
Version: 1 Date: 15 Feb 2016
Author’s response to reviews:
Reviewer reports:
We appreciate the efforts made by both reviewers in providing insightful guidance, comments
and concerns which have been helpful in improving the quality of the article. We have addressed
each of the concerns in the revised paper. An item by item response is given below.
Reviewer #1: Abstract
Absolute numbers or actual house index would be more informative than: "The proportion of Ae.
aegypti mosquito breeding among all Aedes immatures collected in residential premises was
59.5% and 54.6% in 2013 and 2014, respectively."
We originally chose to present the data as proportion of Aedes immature to show the increased
distribution of Aedes aegypti (the primary vector of DENV in Singapore), relative to Aedes
albopictus, which is ubiquitous. Absolute numbers may also not be appropriate as they are
highly dependent on inspection effort and availability of manpower.
However, we agree with the comment on using the house index and revised the sentence to “The
percentage of houses with Ae. aegypti breeding increased significantly (p<0.001) from 2012
(annual average of 0.07) to 2013 (annual average of 0.14), followed by a drop in 2014 (annual
average of 0.10)”. We report the Ae. aegypti house index instead of Aedes house index because
Ae. aegypti is the primary vector of DENV in Singapore.
I don't think the following conclusion can ever be made because there is no way of assessing
mitigation efficacy: "A multi-pronged approach backed by the epidemiological, virological and
entomological understanding paved way to mitigate dengue transmission through an integrated
vector management approach"
We agree with the comment on the use of term “mitigate” which may be inappropriate and overemphasizing. What we meant is that the vector control efforts put in place based on
epidemiological, virological and entomological data helped to control the case burden despite the
presence of a very high population density (7,697/km2) and a considerable proportion of
migrants who are potentially naive to DENV infection. According to the seroepidemiology data,
approximately 50% of the population in the country remains susceptible to DENV infection.
Therefore, we revised the sentence as follows
“A multi-pronged approach backed by the epidemiological, virological and entomological
understanding paved way to moderate the case burden through an integrated vector management
approach”
Introduction
"There is neither an effective antiviral agent nor a licensed vaccine against DENV". What about
the new Dengvaxia?
We revised the sentence as follows because the vaccine is yet to be approved in many endemic
countries.
“Except for the tetravalent vaccine (Dengvaxia®) approved recently in a few countries, there is
neither an effective antiviral agent nor a licensed vaccine against DENV in many endemic
settings where vector control remains as the sole strategy for epidemic control and prevention of
dengue.”
Results
"The relationship between the EHI diagnostic rate and total number of reported cases from 2012
to 2014 showed a positive correlation (r=0.65, P<0.05), with 7-11 weeks lag in the reported
cases." Why would this correlation be expected at such a long lag?
We are not able to explain the exact reason for the 7-11 weeks lag in the reported cases.
However, our correlation analyses have shown that an increase in diagnostic rate at EHI
diagnostics precedes that of the total number of reported cases. It is noteworthy that EHI
diagnostics reported only 10.2% and 13.9% of total case burden in 2013 and 2014 respectively.
The diagnostic services at EHI are solely targeted at dengue, chikungunya and malaria, and the
majority of samples received are highly suspected of having dengue. This bias may have
contributed to an early increase in the diagnostic rate among samples received at EHI before it is
reflected in the total number of cases reported island wide.
Based on the reviewer’s comments, we performed the same analysis from 2012 to 2014 on an
annual basis and observed that the lag period varies from year to year. Therefore, in the revised
version, we report the real time Spearman’s correlation instead of cross-correlation and thereby
remove the section on the lag period.
Figure 3b Y-axis should read 'cumulative' not 'accumulative'.
We revised the figure as per the suggestion.
"These findings suggested that favourable conditions for virus transmission prevailed in 2013
and sustained disease control measures facilitated the elimination of many DENV strains in
2014." I question the logic behind this interpretation. Why should control significantly reduce
heterogeneity but case numbers actually only decrease by 20% between 2013 and 2014? With
this statement, the authors are suggesting that control disproportionately operated on only a
subset of strains. Could another explanation be that the pool of susceptible individuals was
diminished following 2013, increasing strain selection pressure and reducing heterogeneity?
We thank reviewer for raising this point. We would like to clarify it further.
The statement did not mean that vector control was biased towards eliminating certain strains. As
shown in Table 1, a few strains were dominant and contributed to 70-80% of the cases. Those
dominant strains were widely distributed across the country, causing many disease clusters. On
the other hand, the “minor” strains were less well established and their transmission was
generally localized. Consequently, the effect of vector control efforts are presumably more
obvious on the “minor” strains than dominant strains, but the elimination of minor strains is
unlikely to have a profound effect on the case burden.
We agree that other factors suggested by the reviewer may also affect the virus heterogeneity.
However, as explained in the manuscript, the seroepidemiology data indicates the presence of a
large pool of susceptible individuals despite Singapore being hyperendemic to dengue. Only
about 50% of individuals between 18 and 79 yrs have previously been infected with DENV,
leaving a susceptible pool of approximately 2.5 million individuals. The exposure level among
the young is the lowest. Therefore, an epidemic of the scale described in 2013 may still leave a
substantial number of susceptible human hosts.
Unlike in human-to-human transmitted viruses, the sustainability of vector-borne virus
transmission is also affected by the vector density. When the mosquito population size is small,
the dominant strains are likely to saturate the infectious mosquito pool relatively fast, leaving
little opportunity for minor strains to settle down. On the other hand, when the mosquito
population expands, especially during peak periods of transmission, the chances for more virus
strains to establish transmission also increase. We have observed that virus diversity correlates
positively with case numbers during 2013-2014, implying that transmission intensity plays a role
in maintaining the viral diversity (manuscript under review).
As the control measures are targeted to suppress the Aedes population, their numbers fluctuate
and virus populations are expected to go through bottlenecks during periods of low Aedes
density. Virus strains better adapted to local Aedes populations are likely to survive during such
events and the selection pressure may even generate variants with high fitness. Importantly, the
vector density fluctuations are not universal across the country as areas with higher number of
disease clusters, especially major clusters, require more effort and time to bring down the
mosquito density than in areas with localized transmission. In fact, the dominant strains that
contribute to 70-80% of the cases (Table 1) are widely distributed across the country, causing
many disease clusters. On the other hand, the “minor” strains are less well established and their
transmission is generally localized. Consequently, it is likely that the effect of vector control
efforts are more profound on the “minor” strains than dominant strains.
The entomological data indicated doubling of the Aedes house index in 2013 as compared to
2012 and a gradual reduction in 2014 from the peak reached in 2013. Therefore, it was evident
that vector control measures were effective (at least partially) in bringing down the Aedes
population. The fluctuation in case numbers (Revised Figure 6) followed that of Aedes
population and the drop in total cases in 2014 was associated with the disappearance of many
“minor” strains (Table 1). Therefore, we believe that sustained vector control measures
facilitated the elimination of many DENV strains, especially those with a minor contribution to
case burden as the epidemic continued.
We have included an extended explanation in the revised manuscript.
Discussion
"The lower prevalence of antibodies reactive to DENV-2 than that of DENV-1 across multiple
age groups in a cross-sectional study among healthy individuals aged 16-60 years during 200910 also testified that a higher proportion of individuals, especially those within the age group of
16-30 years, are susceptible to DENV-1 than DENV-2 [19] ." I have struggled with this
sentence. Lower prevalence of antibodies reactive to DENV-2 implies less exposure to DENV-2
implies greater susceptibility to DENV-2…please clarify.
We apologise for the mistake. The sentence was corrected as follows.
“The lower prevalence of antibodies reactive to DENV-1 than that of DENV-2 across multiple
age groups in a cross-sectional study among healthy individuals aged 16-60 years....”
The section entitled "Epidemic Response" reads as methodological more than discussion;
consider repositioning this section.
In this section, we wanted to introduce the approach and strategies adapted to manage the
epidemic crisis. The section does not entail a detailed description on each strategy/approach as
expected in a methodology section, instead briefly describes the activities. We believe that
information presented in this section is useful to readers as an example of integrated measures to
control dengue epidemics, especially in urban settings.
Reviewer #2: Overall comments
The authors present a comprehensive descriptive report of the Dengue epidemic from 2013 to
2014 in Singapore. The data presented includes human cases with the different serotypes,
phylogenetic analyses of the viral lineages, and entomological data. This is a valuable
manuscript that shows many of the operational insights to how Dengue epidemics are monitored
and controlled in Singapore by the National Environment Agency.
Overall one conclusion that I find confusing is the statements, in multiple locations, that the
management actions mitigated dengue transmission. One topic relevant to this is that it is
difficult to actually see what management activities were actually done. I see the program where
the public was warned by different color-coded banners that represent different dengue risk, but
was there specific efforts at source reduction, larvicides, adulticides, etc.? Also, I don't really see
specific data or analyses that would help judge these control actions and how they affected
dengue transmission in terms of the number or incidence of dengue cases or an entomological
risk index over time. Did DENV-1 re-emerge, displace other serotypes, and then simply wane
through natural herd immunity processes or natural seasonal patterns in mosquito abundance or
did the NEA control program actually have a benefit? This needs to be addressed since the final
sentence of the abstract and the entire manuscript say that the multi-faceted management strategy
increased understanding and facilitated the reduction in dengue transmission through integrated
vector management.
We agree with the comment on the use of term “mitigate” which may be inappropriate and overemphasizing. What we meant is that the vector control efforts put in place based on
epidemiological, virological and entomological data helped to moderate the case burden despite
the presence of a very high population density (7,697/km2) and a considerable proportion of
migrants who are potentially naive to DENV infection. According to the seroepidemiology data,
approximately 50% of the population in the country remains susceptible to DENV infection.
We have briefly described the following activities implemented to manage the epidemic (in the
section on “epidemic response”).
1. Enhanced case surveillance measures to increase the diagnostic coverage and to enable
prompt vector control response
2. Statistical model-based projection of case numbers to facilitate stockpiling of diagnostic
reagents and to accommodate increased demand on the healthcare system
3. Expansion of virus surveillance activities to facilitate resource allocation for targeted
vector control
4. Early launch of the dengue campaign to promote community awareness of dengue
situation and prevention of transmission
5. Enhanced source reduction for mosquito breeding through an accelerated premise
inspection programme
6. Launch of gravitrap surveillance to monitor the fluctuations of adult Aedes population
7. Integrated vector management activities aligned with the whole-of-government’s effort
8. Guiding resource allocation for targeted vector control based on a spatial risk map
It is difficult to directly measure the impact of control measures on the overall disease
transmission. However, as vector control remains the mainstay of dengue control, we can
indirectly measure the success of the control programme by comparing vector population and
case trend patterns. In the revised version, we modified Figure 6 to compare Aedes
population/breeding data and case data over time to demonstrate that the mosquito population
trend preceded that of case incidence on a weekly basis from 2012 to 2014.
We also modified the text to highlight these points and replaced the term “mitigate” with
“moderate” to avoid the over-emphasis on the success of the control activities.
Minor comments
Page 4 Ln 8: This first sentence could use a citation.
A reference was inserted.
Page 5 Ln 50 to 58: These 4 main pillars are not very clear so perhaps numbers (1), xxxxxx, 2),
xxxxx, etc.) could help.
The section was revised as per the suggestion.
Page 10 Ln 39 to 47: While observing more strain diversity in 2013 than in 2014, are you really
able suggest that control measures reduced many DENV strains? Couldn't this loss of diversity
simply been the population sweeps commonly observed among the various DENV strains and
independent of control measures?
We agree that other factors such as population sweeps affect the diversity of virus populations.
The sweep occurred at the beginning of the epidemic when DENV-1 genotype III replaced the
existing dominant strain (DENV-2 cosmopolitan clade III). Besides, we detected many other
virus strains of all four serotypes emerging during the epidemic. As shown in Table 1, many of
them had a minor contribution to the overall case burden. All these strains emerged independent
of the sweeping event that occurred in early 2013. As shown in Table 1, several of those “minor”
strains could not continue the transmission throughout the study period.
Unlike in human-to-human transmitted viruses, the sustainability of vector-borne virus
transmission is affected by the availability of a susceptible human pool and an adequate vector
density. As described in the manuscript, the seroepidemiology data indicates that approximately
50% of individuals between 18 and 79 yrs remain susceptible to infection. In a population of
more than 2.5 million susceptible individuals, an epidemic of the scale described here may not be
enough to limit the availability of susceptible human hosts. This human factor is uncontrollable
in the absence of a licenced vaccine for dengue in Singapore.
In contrast, the survival of various virus strains, especially “minor” strains, is likely to be
dependent on the vector density. When the mosquito population size is small, the dominant
strains are likely to saturate the infectious mosquito pool relatively fast, leaving little opportunity
for minor strains to settle down. On the other hand, when the mosquito population expands,
especially during peak periods of transmission, the chances for more virus strains to establish
transmission also increase. We have observed that virus diversity correlates positively with case
numbers during 2013-2014, implying that transmission intensity plays a role in maintaining the
viral diversity (manuscript under review).
As the control measures are targeted to suppress the Aedes population, their numbers fluctuate
and virus populations are expected to go through bottlenecks during periods of low Aedes
density. Virus strains better adapted to local Aedes populations are likely to survive during such
events and the selection pressure may even generate variants with high fitness. Importantly, the
vector density fluctuations are not universal across the country as areas with higher number of
disease clusters, especially major clusters, require more effort and time to bring down the
mosquito density than in areas with localized transmission. In fact, the dominant strains that
contribute to 70-80% of the cases (Table 1) are widely distributed across the country, causing
many disease clusters. On the other hand, the “minor” strains are less well established and their
transmission is generally localized. Consequently, the effects of vector control efforts are more
likely to be profound on the “minor” strains than dominant strains.
The entomological data indicated doubling of the Aedes house index in 2013 as compared to
2012 and a gradual reduction in 2014 from the peak reached in 2013. Therefore, it was evident
that vector control measures were effective (at least partially) in bringing down the Aedes
population. The fluctuation in case numbers (Revised Figure 6) followed that of Aedes
population and the drop in total cases in 2014 was associated with the disappearance of many
“minor” strains (Table 1). Therefore, we believe that sustained vector control measures
facilitated the elimination of many DENV strains, especially those with a minor contribution to
case burden as the epidemic continued.
We have included an extended explanation in the revised manuscript.
Page 17 Ln 57; Page 18 Ln 4; etc.: The authors could consider changing "indigenoustlyacquired" or "indigenous cases" to "autochthonous" in all locations.
The terminology was revised as suggested.