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Background Given the continued successes of the world’s lymphatic filariasis (LF) elimination programs and the growing successes of many malaria elimination efforts, the necessity of low cost tools and methodologies applicable to long-term disease surveillance is greater than ever before. As many countries reach the end of their LF mass drug administration programs and a growing number of countries realize unprecedented successes in their malaria intervention efforts, the need for practical molecular xenomonitoring (MX), capable of providing surveillance for disease recrudescence in settings of decreased parasite prevalence is increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25 or fewer, making high-throughput examination a challenge. The new method we present here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-of-concept for a practical alternative to traditional methodologies resulting in significant cost and labor savings. Methodology/Principal Findings Excreta/feces of laboratory reared Aedes aegypti or Anopheles stephensi mosquitoes provided with a Brugia malayi microfilaria-positive or Plasmodium vivax-positive blood meal respectively were tested for the presence of parasite DNA using real-time PCR. A titration of samples containing various volumes of B. malayi-negative mosquito feces mixed with positive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR amplification of B. malayi and P. vivax DNA from the excreta/feces of infected mosquitoes was demonstrated, and B. malayi DNA in excreta/feces from one to two mf-positive blood meal-receiving mosquitoes was detected when pooled with volumes of feces from as many as 500 uninfected mosquitoes. Conclusions/Significance While the operationalizing of excreta/feces testing may require the development of new strategies for sample collection, the high-throughput nature of this new methodology has the potential to greatly reduce MX costs. This will prove particularly useful in post-transmission-interruption settings, where this inexpensive approach to long-term surveillance will help to stretch the budgets of LF and malaria elimination programs. Furthermore, as this methodology is adaptable to the detection of both single celled (P. vivax) and multicellular eukaryotic pathogens (B. malayi), exploration of its use for the detection of various other mosquito-borne diseases including viruses should be considered. Additionally, integration strategies utilizing excreta/feces testing for the simultaneous surveillance of multiple diseases should be explored.
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RESEARCH ARTICLE
A Novel Xenomonitoring Technique Using
Mosquito Excreta/Feces for the Detection of
Filarial Parasites and Malaria
Nils Pilotte
1,2
*, Weam I. Zaky
1
, Brian P. Abrams
3
, Dave D. Chadee
4
, Steven A. Williams
1,2
1Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of
America, 2Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst,
Massachusetts, United States of America, 3Department of Science, Groton School, Groton, Massachusetts,
United States of America, 4Department of Life Sciences, University of the West Indies, St Augustine,
Trinidad and Tobago, West Indies
*npilotte@smith.edu
Abstract
Background
Given the continued successes of the worlds lymphatic filariasis (LF) elimination programs
and the growing successes of many malaria elimination efforts, the necessity of low cost
tools and methodologies applicable to long-term disease surveillance is greater than ever
before. As many countries reach the end of their LF mass drug administration programs and
a growing number of countries realize unprecedented successes in their malaria interven-
tion efforts, the need for practical molecular xenomonitoring (MX), capable of providing
surveillance for disease recrudescence in settings of decreased parasite prevalence is
increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25
or fewer, making high-throughput examination a challenge. The new method we present
here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-
of-concept for a practical alternative to traditional methodologies resulting in significant cost
and labor savings.
Methodology/Principal Findings
Excreta/feces of laboratory reared Aedes aegypti or Anopheles stephensi mosquitoes pro-
vided with a Brugia malayi microfilaria-positive or Plasmodium vivax-positive blood meal
respectively were tested for the presence of parasite DNA using real-time PCR. A titration of
samples containing various volumes of B.malayi-negative mosquito feces mixed with posi-
tive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR
amplification of B.malayi and P.vivax DNA from the excreta/feces of infected mosquitoes
was demonstrated, and B.malayi DNA in excreta/feces from one to two mf-positive blood
meal-receiving mosquitoes was detected when pooled with volumes of feces from as many
as 500 uninfected mosquitoes.
PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004641 April 20, 2016 1 / 14
a11111
OPEN ACCESS
Citation: Pilotte N, Zaky WI, Abrams BP, Chadee
DD, Williams SA (2016) A Novel Xenomonitoring
Technique Using Mosquito Excreta/Feces for the
Detection of Filarial Parasites and Malaria. PLoS
Negl Trop Dis 10(4): e0004641. doi:10.1371/journal.
pntd.0004641
Editor: Hans-Peter Fuehrer, Vienna, AUSTRIA
Received: December 17, 2015
Accepted: March 29, 2016
Published: April 20, 2016
Copyright: © 2016 Pilotte et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and/or its Supporting Information
files.
Funding: This work was made possible by a Grand
Challenges Explorations award from The Bill and
Melinda Gates Foundation to NP (award #
OPP1098462). The funders had no role in the study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Conclusions/Significance
While the operationalizing of excreta/feces testing may require the development of new
strategies for sample collection, the high-throughput nature of this new methodology has
the potential to greatly reduce MX costs. This will prove particularly useful in post-transmis-
sion-interruption settings, where this inexpensive approach to long-term surveillance will
help to stretch the budgets of LF and malaria elimination programs. Furthermore, as this
methodology is adaptable to the detection of both single celled (P.vivax) and multicellular
eukaryotic pathogens (B.malayi), exploration of its use for the detection of various other
mosquito-borne diseases including viruses should be considered. Additionally, integration
strategies utilizing excreta/feces testing for the simultaneous surveillance of multiple dis-
eases should be explored.
Author Summary
As a non-invasive method of indirectly monitoring insect-borne disease, molecular xenomo-
nitoring (MX), the molecular testing of insects for the presence of a pathogen, can provide
important information about disease prevalence without the need for human sampling.
However, given the successes of tropical disease elimination programs, including many lym-
phatic filariasis and malaria elimination efforts, parasite levels in many locations are declin-
ing. This decrease in prevalence requires the sampling of increased numbers of vectors for
disease surveillance and recrudescence monitoring. Such increased sampling poses a chal-
lenge since it results in additional costs and labor. In light of these difficulties, high-through-
put methodologies for MX are necessary to provide elimination programs with cost-
reducing alternatives to long-term disease surveillance. Here we demonstrate proof-of-con-
cept for a new method that samples large numbers of mosquitoes using PCR to screen
excreta/feces for filarial or malarial parasites. If operationalized, this approach to MX will
provide a practical first-alertsystem that will enable cost-minimizing surveillance in post-
transmission-interruption settings. Given this potential, the applicability of this approach to
the monitoring of various mosquito-borne diseases should be explored further, as this plat-
form will prove useful for surveillance efforts for a wide variety of pathogens.
Introduction
Spanning 73 countries and territories and placing an estimated 1.39 billion individuals at risk
of infection, lymphatic filariasis (LF) presents a considerable risk to global health [1]. Similarly,
with an estimated 198 million malaria infections and 584,000 malaria-related deaths in 2013,
the global burden of human malaria is staggering [2]. Yet despite the wide ranging impacts of
these diseases, global elimination efforts have made significant strides, spearheaded by mass
drug administration (MDA) programs supported by large pharmaceutical donors [35] and
the widespread use of insecticidal bed nets [69]. As a result, disease prevalence in many loca-
tions has decreased dramatically, enabling a growing number of countries to discontinue their
treatment efforts for LF [5,10] and spurring the creation of an increasing number of malaria
elimination programs [1113]. However, lessons learned as a result of LF elimination efforts
have shown that the cessation of MDA, recommended after the successful passing of a trans-
mission assessment survey [14], results in an additional set of programmatic challenges.
Alternative Xenomonitoring for Filarial Parasites and Malaria
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Foremost in such post-intervention settings is the issue of post-MDA surveillance, as vigilant
monitoring is required to ensure that recrudescence of disease has not occurred [15]. This
monitoring is costly and current efforts for LF are centered upon the periodic sampling of the
human population in order to examine circulating levels of filarial antigen [1617]. While
effective, these efforts require blood sampling of the human population. The invasive nature of
this practice, coupled with the requirement of informed consent, results in participation chal-
lenges [14] that logically increase as populations become further removed from the time of
widespread disease transmission. While still largely of future concern, similar challenges likely
await the malaria community as control efforts continue to reduce the burden of disease, mak-
ing this programmatic obstacle one of utmost global importance.
Molecular xenomonitoring (MX), the testing of vectors for the presence of parasite genetic
material, has been proposed as a non-invasive means of conducting post-MDA surveillance
for LF [14,1718]. Although precise correlations between levels of parasite within the vector
population and levels within the human population have not been conclusively established, par-
asite presence within the vector population is indicative of the potential for disease transmission.
Furthermore, when monitoring for LF in locations endemic for the Wuchereria bancrofti para-
site, a pathogen without a known zoonotic host [19], presence is directly indicative of active
human infection. Yet despite its many advantages, MX is costly and when used for monitoring
in a post-MDA setting, typically requires the collection and sampling of many thousands of
mosquitoes [18,2021]. Therefore, as a growing number of countries continue to enter the sur-
veillance phases of their LF eradication programs, alternative methodologies for streamlining,
simplifying, and reducing the costs associated with post-MDA monitoring will be required.
As an alternative to traditional approaches to MX, excreta and feces produced by mosqui-
toes potentially harboring parasites can be tested for the presence of pathogen DNA. Previous
work has demonstrated that vector feces-monitoring for the PCR-based detection of Trypano-
soma cruzi can be used as a means of surveying insect host infection status [22]. Similarly, it
has been shown that genetic material from the Brugia malayi parasite can be successfully
detected in the excreta and feces collected from individual mosquitoes [23]. Building upon
these findings, we describe methodological proof-of-principle for the real-time PCR-based
monitoring for B.malayi parasite DNA in pools of mosquito excreta/feces as a platform for the
surveillance of large numbers of insects. While unconventional, excreta/feces monitoring has
the potential to provide significant time, cost, and labor savings over traditional MX methodol-
ogies due to its exceptionally high-throughput nature. Furthermore, as excreta/feces collection
would likely prove readily adaptable to a variety of both passive and active trapping practices
and platforms, its potential feasibility as an exceedingly low cost, long-term surveillance tool is
great. Equally promising, initial experiments have demonstrated that this approach to MX can
be applied to the detection of Plasmodium vivax DNA, indicating its possible usefulness for the
monitoring of both unicellular and multicellular eukaryotic pathogens. Given these encourag-
ing findings, the further exploration of mosquito excreta/feces testing as a new method for dis-
ease surveillance purposes is warranted and efforts to adapt this alternative MX approach to
other mosquito-borne illnesses should be pursued.
Materials and Methods
Mosquito Rearing for the Accumulation of Excreta/Feces
Accumulation of excreta/feces from mosquitoes potentially infected with B.malayi.
Mosquito cartons containing the excreta/feces from female Aedes aegypti mosquitoes potentially
infected with the B.malayi parasite were received from the Filarial Research Reagent Resource
Center (FR3) located at the University of Georgia, College of Veterinary Medicine, Athens, GA.
Alternative Xenomonitoring for Filarial Parasites and Malaria
PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004641 April 20, 2016 3 / 14
Rearing and infection of mosquitoes occurred in accordance with SOP Number 8.3available
on the FR3 website (http://www.filariasiscenter.org/parasites-resources/Protocols/materials-1).
Accumulation of excreta/feces from mosquitoes potentially infected with P.vivax.Mos-
quito cartons containing the excreta/feces from female Anopheles stephensi mosquitoes poten-
tially infected with the P.vivax parasite were received from the Centers for Disease Control and
Prevention, Atlanta, GA, USA. Rearing of mosquitoes occurred in accordance with the protocols
described in chapter 2.4 of the Methods in Anopheles Researchmanual [24] available on the
BEI Resources website (https://www.beiresources.org/Catalog/VectorResources.aspx). Infection
of mosquitoes occurred in accordance with previously described methodologies [25].
Accumulation of feces from uninfected mosquitoes. Moist filter paper rafts containing
Culex quinquefasciatus eggs were received from BEI Resources (www.beiresources.org). Eggs
were rinsed into open-topped plastic vessels containing approximately 1 L of tap water at a
depth of approximately 5 cm and a small volume of standard flake-based fish food was added
to the water in each container. Upon maturation into pupae, 50 mosquitoes were transferred
into plastic containers, approximately 5 cm in diameter, containing 1 cm of tap water. These
containers were then placed into waxed cardboard cartons (approximately 18 cm in diameter
by 14.5 cm in height). Cartons were covered with standard mesh tulle and mosquitoes were
allowed to emerge as adults. Upon emergence, a cotton ball soaked in 10% sucrose was placed
on top of each carton and this solution was refreshed daily. Mosquitoes remained within the
cartons producing feces until they expired naturally (1020 days). At this time, the expired
mosquitoes were removed and the cartons were collected, flattened, and stored at 4°C.
Extraction of DNA from Mosquito Excreta/Feces
Preliminary experiments were designed to determine the effectiveness/efficiency of extracting
DNA from the excreta/feces of mosquitoes potentially infected with the B.malayi parasite. To
make this determination, various extraction protocols and techniques were tested in order to eval-
uate their efficiency (Table 1). Because the FR3-derived mosquito cartons containing excreta/feces
from potentially infected insects were non-waxed, initial samples were either scraped off of the
cartons using a metal spatula, or strips of the carton material (hereafter referred to as carton strips)
were directly used as the starting material for the extraction procedure. The amplification of B.
malayi parasite DNA from all extracts was evaluated using the previously described real-time PCR
primer-probe pairing [26]. Results demonstrated that DNA extractions performed using the
QIAamp DNA Micro Kit (Qiagen, Valencia, CA) provided the most consistent and effective
detection of parasite DNA. For this reason, this kit was used in all subsequent experiments.
To adapt the Qiagen protocol for use with the bulky, brittle mosquito carton material,
minor modifications were made to the manufacturers suggested instructions for DNA extrac-
tion from bloodspots. Briefly, carton strips were soaked in 360 μl of Buffer ATL for 1 hour
prior to incubation with Proteinase K at 56°C. Additionally, following incubation at 70°C, sam-
ples were centrifuged at maximum speed for 5 min and supernatants were transferred to new
1.7 ml microcentrifuge tubes. Tubes were centrifuged for an additional 5 min at maximum
speed to pellet residual debris and the supernatants were transferred to QIAamp MinElute col-
umns. Lastly, all samples were incubated in Buffer AE at room temperature for 5 min prior to
the elution of samples from the columns.
Evaluation of Positivity of Excreta/Feces from Mosquitoes Potentially
Infected with B.malayi
Although preliminary experiments demonstrated that excreta/feces derived from vector mos-
quitoes fed on B.malayi microfilaria (mf)-positive blood resulted in the amplification of
Alternative Xenomonitoring for Filarial Parasites and Malaria
PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004641 April 20, 2016 4 / 14
parasite DNA, the availability of mf-containing blood does not guarantee that all mosquitoes
will feed or ingest parasites while feeding. Additionally, as the FR3s standard operating proce-
dure (SOP 8.3) requires that mosquitoes spend three to five days as adults prior to the time an
infective blood meal is introduced, a substantial volume of parasite-negative feces was pro-
duced and deposited into mosquito cartons prior to blood feeding. Furthermore, as mosquitoes
are known to excrete while taking a blood meal [27], it is likely that excreta would be deposited
before parasite DNA had reached/been incorporated into the voided material. Therefore, a por-
tion of the voided material collected from mosquitoes provided with mf-positive blood would
likely not contain parasites and would therefore not result in a positive PCR. For this reason, a
large panel of potentially positive excreta/feces samples was tested in order to estimate the rates
of sample positivity. In total, 59 independent samples were tested, with each sample consisting
of a 0.48 cm
2
carton strip. Based upon observations of the volume of excreta/feces produced by
single mosquitoes housed in 50 ml conical tubes, it was estimated that the volume of excreta/
feces on each carton strip was equivalent to the average volume produced by one to two mos-
quitoes over a 24 hour period. Negative control extractions were performed on similar volumes
of mosquito feces collected from uninfected C.quinquefasciatus. All samples underwent DNA
extraction using the modified Qiagen procedure described above and were analyzed by 45
cycles of real-time PCR using the published reagent concentrations and cycling protocol [26].
2μl aliquots of each DNA extract were tested in triplicate and samples returning two or more
positive results were considered positive for B.malayi parasite DNA.
Assay Sensitivity Testing
In order to determine detection limits for the presence of B.malayi-infected excreta/feces in
large pools of uninfected mosquito feces, a titration of samples was created, with each sample
containing a 0.48 cm
2
strip from a carton used to house mosquitoes provided with a B.malayi-
positive blood meal mixed with various volumes of uninfected mosquito feces. Feces from
uninfected C.quinquefasciatus mosquitoes were removed from cartons using a cotton swab,
Table 1. Evaluation of extraction methods for the isolation of DNA from mosquito excreta/feces.
DNA Extraction Method Quantity of Excreta/Feces
(Mosquito Excreta/Feces/Days)*
# of Samples (# of
Positives)
Qiagen DNeasy Blood and Tissue
(Qiagen, Valencia, CA)
62.5 2 (0)
Overnight Soak in 1 x PBS 62.5 8 (0)
Overnight Soak in 1 x TE 62.5 8 (0)
Phire Plant Direct PCR Kit (Thermo Fisher
Scientic, Vantaa, Finland)
62.5 8 (0)
Published Insect Feces Extraction
[22]
12 5 (0)
Published Insect Feces Extraction
[22]
+ Phenol/Chisam Purication
12 5 (0)
QIAamp DNA Micro Kit Extraction
(Qiagen, Valencia, CA)
12 5 (5)
Nucleospin Blood DNA Kit (Macherey-
Nagel, Bethlehem, PA)
12 5 (0)
Nucleospin Blood DNA Kit (Macherey-
Nagel, Bethlehem, PA)
12 5 (0)
*Mosquito Excreta/Feces/Days are dened as the estimated quantity of excreta/feces produced by a
single mosquito over a 24 hour period.
doi:10.1371/journal.pntd.0004641.t001
Alternative Xenomonitoring for Filarial Parasites and Malaria
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and the feces-covered cotton was added to each sample. As 50 uninfected mosquitoes were
raised in each carton, and adult mosquitoes were observed to survive for a minimum of 10 days
(with most surviving considerably longer), it was conservatively estimated that each carton
contained a minimum of 500 mosquito feces/days (i.e. the amount of feces produced by 500
mosquitoes in one 24 hour period, or the amount of feces produced by a single mosquito over
a 500 day period). While the distribution of feces within cartons was not precisely uniform, by
sectioning cartons based upon total internal surface area (approximately 1,050 cm
2
), it was
possible to roughly estimate the number of mosquito feces/days being added to each sample.
Samples estimated to contain approximately 62.5, 125, 250, and 500 feces/days were prepared.
Negative control extractions were also prepared using mosquito feces collected from uninfected
C.quinquefasciatus. All samples were extracted and tested in duplicate reactions using the
same extraction and detection methods as described above for the evaluation of PCR positivity
testing.
Adaptation of Excreta/Feces Testing to the Detection of P.vivax DNA
To test whether the detection of mosquito-borne pathogen DNA from mosquito excreta/feces
was possible for species other than the B.malayi parasite, a set of samples was created using
mosquito excreta/feces produced by carton-raised A.stephensi that had been fed on P.vivax-
positive blood. As was done for B.malayi detection, samples were prepared by excising 0.48
cm
2
carton strips containing potentially positive excreta/feces. To establish proof-of-principle,
20 samples were prepared and DNA was extracted using the modified Qiagen protocol
described above. DNA extracts from each sample were tested using a previously described
primer-probe set for the universal detection of Plasmodium species [28] with reaction recipes
and cycling conditions remaining consistent with the authorspublished protocol.
Results
Evaluation of PCR Positivity of Excreta/Feces from Mosquitoes
Potentially Infected with B.malayi
Carton strips were excised from containers used to house A.aegypti mosquitoes provided with
B.malayi mf-containing blood and testing was conducted to determine the percentage of
excreta/feces samples containing B.malayi DNA. Such testing was necessary since the produc-
tion of feces can occur prior to the provision of an infective blood meal or before the ingestion
of a blood meal. Furthermore, the availability of infective blood does not guarantee that each
individual mosquito will feed and, dependent upon the mosquito species, localization of para-
site material to voided excreta/feces may take time following blood meal ingestion. Accord-
ingly, DNA was extracted from 59 independent samples, each consisting of a carton strip
measuring 0.48 cm
2
and containing excreta/feces from one to two mosquitoes over a 24 hour
period (i.e. one to two mosquito feces/days). Real-time PCR testing, using 2 μl of template
DNA resulted in positive detection for 21 out of 59 samples tested (35.6%). For positive sam-
ples, mean Ct values ranged from 26.62 (+/- 0.24) to 41.98 (+/- 0.03) (Table 2). Because only a
fraction of the deposited mosquito excreta/feces would contain parasite DNA, 35.6% may be a
true indication of the frequency of positive samples.
Assay Sensitivity Testing
A titration of samples containing potentially positive 0.48 cm
2
carton strips mixed with varying
amounts of uninfected mosquito feces was prepared in order to estimate the limits of detection
for B. malayi-based excreta/feces testing. In total, five samples containing an estimated 62.5
Alternative Xenomonitoring for Filarial Parasites and Malaria
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mosquito feces/days, six samples containing an estimated 125 mosquito feces/days, six samples
containing an estimated 250 mosquito feces/days, and two samples containing an estimated
500 mosquito feces/days were assayed. As expected, due to the uncertainty of which samples
actually contained B.malayi DNA, a fraction of the samples failed to give positive PCR detec-
tion of B.malayi DNA. However, detection of parasite DNA proved possible at all tested levels
of sensitivity (Table 3).
Adaptation of Excreta/Feces Testing to the Detection of Plasmodium
vivax DNA
To explore whether excreta/feces testing would efficiently detect pathogen DNA from species
other than B.malayi, testing for the presence of the human malaria-causing parasite P.vivax
was performed. To demonstrate proof-of-concept, 20 samples were prepared and tested by
PCR. Each sample was comprised of a 0.48 cm
2
carton strip excised from a mosquito container
Table 2. PCR positivity of excreta/feces from mosquitoes potentially infected with B.malayi.
Sample # Ct Value (Std. Dev.) Sample # Ct Value (Std. Dev.)
1 Negative 32 Negative
2 27.75 (+/- 0.15) 33 Negative
3 28.67 (+/- 0.17) 34 Negative
4 Negative 35 Negative
5 38.25 (+/- 2.79) 36 Negative
6 Negative 37 Negative
7 Negative 38 Negative
8 36.53 (+/- 2.33) 39 39.07 (+/- 1.07)
9 40.49 (+/- 0.12) 40 Negative
10 Negative 41 Negative
11 34.80 (+/- 0.70) 42 Negative
12 37.55 (+/- 0.91) 43 Negative
13 41.96 (+/- 2.67) 44 40.74 (+/- 2.17)
14 40.54 (+/- 1.26) 45 Negative
15 Negative 46 31.69 (+/- 0.50)
16 Negative 47 Negative
17 Negative 48 38.88 (+/- 0.49)
18 Negative 49 37.69 (+/- 0.69)
19 Negative 50 Negative
20 Negative 51 40.32 (+/- 0.43)
21 Negative 52 Negative
22 Negative 53 37.49 (+/- 1.53)
23 Negative 54 Negative
24 Negative 55 39.48 (+/- 2.37)
25 41.44 (+/- 0.95) 56 26.62 (+/- 0.24)
26 27.91 (+/- 0.54) 57 Negative
27 Negative 58 Negative
28 41.98 (+/- 0.03) 59 Negative
29 Negative Negative Extract #1 Negative
30 Negative Negative Extract #2 Negative
31 Negative
doi:10.1371/journal.pntd.0004641.t002
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having housed A.stephensi female mosquitoes provided with Plasmodium-positive blood.
Real-time PCR testing of DNA extracted from each sample clearly demonstrated the adaptabil-
ity of excreta/feces testing to the detection of P.vivax since all samples were positive with Ct
values ranging from 26.82 (+/- 0.26) to 29.21 (+/- 0.80) (Table 4).
Table 3. Limits for the detection of B.malayi DNA in mosquito excreta/feces samples.
Sample ID Quantity of Potentially Positive Excreta/Feces (Mosquito
Excreta/Feces/Days)*
Quantity of Negative Excreta/Feces (Mosquito
Excreta/Feces/Days)*
Ct Value (Std.
Dev.)
A12 62.5 Negative
B12 62.5 Negative
C12 62.5 Negative
D12 62.5 37.89 (+/- 2.31)
E12 62.5 38.77 (+/- 0.20)
F12 125 30.98 (+/- 0.20)
G12 125 Negative
H12 125 Negative
I12 125 Negative
J12 125 Negative
K12 125 Negative
L12 250 29.56 (+/- 0.01)
M12 250 35.88 (+/- 0.04)
N12 250 Negative
O12 250 38.73 (+/- 0.91)
P12 250 Negative
Q12 250 Negative
R12 500 Negative
S12 500 38.40 (+/- 1.03)
Negative
#1
N/A 62.5 Negative
Negative
#2
N/A 62.5 Negative
Negative
#3
N/A 62.5 Negative
*Mosquito Excreta/Feces/Days are dened as the estimated quantity of excreta/feces produced by a single mosquito over a 24 hour period.
doi:10.1371/journal.pntd.0004641.t003
Table 4. PCR positivity of excreta/feces from mosquitoes infected with P.vivax.
Sample # Ct Value (std. dev.) Sample # Ct Value (std. dev.)
1 28.23 (+/- 0.58) 12 28.77 (+/- 0.09)
2 27.84 (+/- 0.05) 13 28.83 (+/- 0.49)
3 27.85 (+/- 0.15) 14 27.88 (+/- 0.19)
4 27.85 (+/- 0.29) 15 27.36 (+/- 0.14)
5 26.82 (+/- 0.26) 16 26.82 (+/- 0.10)
6 27.98 (+/- 0.28) 17 28.00 (+/- 0.27)
7 26.94 (+/- 0.20) 18 28.48 (+/- 0.29)
8 27.26 (+/- 0.32) 19 28.40 (+/- 0.26)
9 27.19 (+/- 0.32) 20 28.07 (+/- 0.12)
10 28.37 (+/- 0.49) Negative Extract #1 Negative
11 29.21 (+/- 0.80) Negative Extract #2 Negative
doi:10.1371/journal.pntd.0004641.t004
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Discussion
While sensitive and less intrusive to the local population than human sampling, the number of
studies implementing current MX practices for the surveillance of LF or malaria has been lim-
ited. Although such efforts provide valuable data [10,1718,21] the routine use of MX for
post-MDA LF surveillance or long-term recrudescence monitoring is not yet standard proce-
dure. Despite the existence of effective molecular tools [2829], vector monitoring for malaria
is even more uncommon and World Health Organization recommendations for infection
monitoring and prevalence estimation rely solely on human sampling [2]. Limited implemen-
tation has occurred for multiple reasons, including the need to process and test large numbers
of mosquitoes from areas suspected of having low parasite density within the vector population
[10,18,21]. Difficulties in establishing a concrete correlation between vector-parasite levels
and human prevalence have further restricted MX implementation [21]. Yet despite these
shortcomings, MX continues to receive attention as the need for post-intervention disease sur-
veillance continues to grow and mosquito trap designs continue to improve [3034]. Accord-
ingly, methodologies capable of harnessing the advantageous aspects of MX while making its
practice more practical and inexpensive would be of great benefit to global LF and malaria
elimination efforts, as well as to monitoring efforts for other vector-borne diseases.
The work presented here provides methodological proof-of-concept for a novel approach to
MX with the potential to greatly reduce the cost, time, and labor associated with large-scale sur-
veillance efforts. The successful amplification of parasite DNA from pooled mosquito excreta/
feces containing B.malayi genetic material has demonstrated that high-throughput MX for LF
is feasible. In the past, real-time PCR-based MX for the presence of the filariasis-causing para-
sites has been restricted to the testing of pools of 25 or fewer mosquitoes. This is because the
biological mass of mosquitoes and high yields of mosquito DNA associated with pools of large
size results in the inability to detect the presence of small quantities of parasite DNA [35].
However, excreta/feces testing enables the sampling of material obtained from vast numbers of
mosquitoes, while simultaneously limiting the biological mass associated with each sample. As
we have demonstrated, it is possible to detect trace amounts of parasite DNA in pools contain-
ing the voided material from as many as 500 uninfected mosquitoes. Future studies implement-
ing this approach will benefit from the drastic reduction in cost of DNA extractions and PCR
(approximately 20-fold). Furthermore, as it has been shown that non-vector mosquitoes rid
themselves of parasite material more rapidly than vector species (as indicated by a shortened
period of time during which parasite detection is possible within non-vectors [23]), one would
expect to find greater quantities of parasite DNA within the excreta/feces of non-vector mos-
quitoes. Therefore, the testing of mixed pools of vector and non-vector excreta/feces should be
possible. While such testing will result in reduced ability to directly correlate the presence of
parasite with individual vector species, it will likely increase the sensitivity of detection when
surveying for the presence of parasite in post-transmission-interruption settings as both vector
and non-vector mosquitoes potentially harboring parasite material will be screened. In addi-
tion, it is likely that excreta/feces testing will eliminate the need for the labor intensive and time
consuming species-sorting efforts which are commonplace in current MX work [10,1718,21,
36]. By drastically reducing the numbers of pools that must be screened and by eliminating the
need for sorting mosquitoes by species, labor-related time and costs are dramatically reduced.
While operationalizing this alternative approach to MX presents some implementation hur-
dles, adaptation of current passive and active trapping methods to the collection of mosquito
excreta/feces is possible. Such adaptation could occur by transferring live mosquitoes from a
trap to a holding carton, in which they would be sugar fed using a cotton ball, thereby encour-
aging the voiding of waste material. Expired mosquitoes would then be removed and additional
Alternative Xenomonitoring for Filarial Parasites and Malaria
PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004641 April 20, 2016 9 / 14
mosquitoes could be added following further collection from the trap. Periodic testing of the
accumulated excreta/feces would enable the high-throughput screening of the voided material
from a series of such traps. Any trap with the capacity to maintain live mosquitoes could be
used for this purpose including the CDC Gravid Trap, the Ifakara tent trap and others [30,37].
Alternatively, collection of excreta/feces could occur directly within traps of various designs.
One such design proving readily adaptable to excreta/feces collection in preliminary experi-
ments is the Large Passive Box Trapdeveloped by Ritchie, et al [38]. While work aimed at
evaluating the adaptability of this trap to the collection of various species of mosquitoes is cur-
rently ongoing, and further efforts to optimize this trap for the purpose of excreta/feces collec-
tion will be required, simply lining the internal surfaces of this passive trap with waxed paper
provides an uncomplicated method for collecting the accumulated material voided by trapped
mosquitoes (S1 Fig). Swabbing the excreta/feces from the waxed paper then enables the PCR
analysis of pooled material.
Additional testing will be required to determine the stability of parasite DNA in mosquito
excreta/feces over time and under field conditions. However, in the proof-of-concept experi-
ments described in this paper, mosquito excreta/feces containing parasite DNA was allowed
to accumulate for 1416 days prior to transfer to cold storage. In this setting, parasite DNA
remained stable and detectable (Table 3). While further validation under conditions mimicking
tropical temperatures and humidity will be required, these results are encouraging, as DNA sta-
bility within tropical and sub-tropical climates could present another hurdle when operationa-
lizing this method in the field.
Since production of feces can occur prior to the provision of a parasite-positive blood meal
and since this provision does not ensure that all mosquitoes will ingest and/or metabolize a
parasite, a percentage of the excreta/feces samples collected will likely test negative for parasite
DNA. It is therefore difficult using blood-fed mosquitoes to definitively assess the consistency
of detection of parasite DNA in excreta/feces. During initial testing, we demonstrated that 21
out of 59 samples comprised of 0.48 cm
2
carton strips derived from containers used to rear
mosquitoes with a B.malayi-positive blood source were positive (Table 2). However, although
sufficient to fulfill our primary aim of providing methodological proof-of-concept, it cannot be
conclusively determined whether the remaining 38 samples were all truly negative for parasite
DNA. While spiking uninfected excreta/feces samples with extracted B.malayi genomic DNA
would provide clear positive and negative samples, this approach is extremely artificial and has
decreased biological relevance since it eliminates any possible effects of mosquito metabolism
on the integrity of parasite DNA. Since the major uses of excreta/feces testing will likely center
on mapping and long-term, low-cost, post-transmission-interruption recrudescence monitor-
ing, marginally reduced consistency of detection has diminished significance as continuous,
sustainable, high-throughput surveillance would enable detection of even low-levels of parasite
prevalence. The high-throughput nature of this testing was clearly demonstrated by the posi-
tive detection of parasite DNA derived from pools containing various volumes of negative
feces up to 500 mosquito feces/days (Table 3). Detection proved possible at all tested sensitivity
levels and with overall sample positivity rates similar to those obtained when testing potentially
positive excreta/feces samples without the addition of negative feces (36.8% vs. 35.6% respec-
tively). Thus, the inclusion of large amounts of negative feces does not appear to alter detection
efficiency. Given these findings, sustainable, high-throughput surveillance efforts using
excreta/feces screening could serve as a first-alertplatform, with positive detection serving as
ared flagfor recrudescence in settings of known transmission interruption. In such a sce-
nario, detection would spur the implementation of more traditional surveillance and monitor-
ing studies.
Alternative Xenomonitoring for Filarial Parasites and Malaria
PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004641 April 20, 2016 10 / 14
By successfully detecting P.vivax DNA in pools of excreta/feces produced by Plasmodium-
positive-blood fed A.stephensi, we have provided proof-of-principle for the application of this
platform to the detection of malaria parasites. Furthermore, the increased rates of sample posi-
tivity and decreased Ct values seen when assaying for P.vivax are not entirely surprising and
indicate this system may work even better for malaria than LF. Estimates have suggested that
the ratio of Plasmodium merozoites to gametocytes within the peripheral blood is as great as
156:1 [3940]. Given this ratio, the vast number Plasmodium merozoites ingested during a
blood meal (up to 32 per infected erythrocyte [41]), and knowledge that merozoites obtained
during blood feeding are unable to undergo further development within the mosquito host
(only gametocytes undergo further development [42]), the great majority of ingested parasites
are simply metabolized and/or eliminated by the mosquito. In contrast, while mosquito hosts
possess measures that provide partial protection against filarial infection [4344], and environ-
mental conditions are thought to impact rates of parasite survival [45], all filarial parasites
taken up as part of a blood meal are of the correct lifecycle stage (mf) to potentially undergo
further development within the vector host. Therefore, due to the varying natures of their life-
cycles, it follows that a greater percentage of filarids ingested during a blood meal are able to
successfully develop within the mosquito host as compared to Plasmodium. Since successful
parasite development would likely mean the absence of parasite DNA in mosquito excreta/
feces, the lower levels of sample positivity and the more modest Ct values observed during B.
malayi testing compared to P.vivax testing seem logical.
With its adaptability to both B.malayi and P.vivax, MX of mosquito excreta/feces for vari-
ous other mosquito-borne pathogens should be explored. Given the successes realized with the
detection of these parasites, it is extremely likely that similar detection will prove possible for
W.bancrofti and other malaria species. However, the applicability of this new platform to
other types of pathogens should also be examined, since improved high-throughput screening
for RNA viruses such as Dengue, Chikungunya, and Zika would be welcomed programmatic
tools. Furthermore, since all species of biting insects draw from the same reservoir of blood
within a target host, the possibility of cross-vector monitoring should also be considered. For
example, excreta/feces samples collected from mosquitoes could be monitored for the presence
of disease-causing agents having unrelated insect hosts (such as Leishmania ssp. or Loa loa).
Adaptability to various pathogens and the possibility of cross-vector monitoring could also
make excreta/feces sampling an attractive strategy for tropical disease integration efforts. In
light of these factors, and the potential time, cost, and labor savings associated with such appli-
cations, we believe that this proof-of-concept study suggests that further evaluation of this new
method is warranted.
Supporting Information
S1 Fig. Mosquito excreta/feces collection in the modified Large Passive Box Trap.Voided
material from mosquitoes entering the passive trap collects on wax paper lining the trap sur-
faces below. Outlined in red is one such mosquito and a series of excreta/feces spotswhich
has accumulated.
(TIF)
Acknowledgments
The authors wish to thank Molly Riggs, Chris Evans, Dr. Mike Dzimianski, Dr. Prasit Supa-
korndej, and Dr. Andy Moorehead (Filarial Research Reagent Resource Center, University
of Georgia College of Veterinary Medicine, Athens, GA) for providing B.malayi-positive
Alternative Xenomonitoring for Filarial Parasites and Malaria
PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004641 April 20, 2016 11 / 14
mosquito excreta/feces, and Dr. Paul Howell (Centers for Disease Control and Prevention,
Atlanta, GA) for the provision of Plasmodium-positive material. We would also like to extend
our sincere appreciation to Dr. Eric Ottesen and Dr. Patrick Lammie (Task Force for Global
Health, Decatur, GA), as well as Dr. Sandra Laney (Vulcan, Inc., Seattle, WA) for their contin-
ued support, encouragement, and advice.
Author Contributions
Conceived and designed the experiments: NP WIZ DDC SAW. Performed the experiments:
NP WIZ BPA. Analyzed the data: NP WIZ SAW. Contributed reagents/materials/analysis
tools: NP SAW. Wrote the paper: NP. Contributed to the editing of the manuscript: WIZ BPA
SAW.
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Supplementary resource (1)

... A proof-of-concept high-throughput method for collecting excreta/faeces from large numbers of mosquitoes in a superhydrophobic cone, then screening samples for filarial and malarial DNA, was developed [50] and tested in two rural communities of Ghana [51]. Mosquitoes (greater than 95% Anophleles spp., mostly An. gambiae s.l.) were collected indoors by aspirators and outdoors by box GTs and BGS traps. ...
... The high-throughput method for collecting excreta from a large numbers of mosquitoes then screening samples for arboviral, filarial and malarial DNA is promising even if it requires the development of new strategies for sample collection [50,67]. Techniques for testing mosquito saliva using honeybaited nucleic acid preservation cards or sugar bait stations for arboviruses provide evidence of pathogen transmission, not possible by processing pools, and are an alternate and sensitive method to mitigate against the problems of low prevalence of infection in mosquitoes, expensive labour costs and the need for specialized equipment [64,68]. ...
Article
The scientific community recognizes that molecular xenomonitoring (MX) can allow infected mosquitoes to serve as a proxy for human infection in vector-borne disease surveillance, but developing reliable MX systems for programmatic use has been challenging. The primary aim of this article is to examine the available evidence to recommend how MX can best be used for various purposes. Although much of the literature published within the last 20 years focuses on using MX for lymphatic filariasis elimination, a growing body of evidence supports its use in early warning systems for emerging infectious diseases (EIDs). An MX system design must consider the goal and target (e.g. diseases targeted for elimination versus EIDs), mosquito and pathogen characteristics, and context (e.g. setting and health system). MX is currently used as a ‘supplement’ to human surveillance and will not be considered as a ‘replacement’ until the correlation between pathogen-infection rates in human and mosquito populations is better understood. Establishing such relationships may not be feasible in elimination scenarios, due to increasingly dwindling human infection prevalence after successful control, but may still be possible for EIDs and in integrated disease surveillance systems. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases'.
... Additionally, MX of E/F has the potential to substantially increase the throughput of mosquito processing while maintaining sensitivity. In laboratory studies this technique has been shown to successfully amplify pathogen DNA in the E/F from one positive mosquito within an E/F pool from up to 500 negative mosquitoes [22]. The use of mosquito E/ F for MX purposes has recently been demonstrated for a variety of arboviruses [23][24][25], showing that viral RNA can also be detected in the field [26]. ...
... While we have demonstrated proof-of-concept for detection from field-collected mosquitoes and taking into account that E/F screening can be done in larger pool sizes [22], additional work needs to be done to evaluate the cost-effectiveness of using E/F compared to carcasses. ...
Article
Full-text available
We recently developed a superhydrophobic cone-based method for the collection of mosquito excreta/feces (E/F) for the molecular xenomonitoring of vector-borne parasites showing higher throughput compared to the traditional approach. To test its field applicability, we used this platform to detect the presence of filarial and malaria parasites in two villages of Ghana and compared results to those for detection in mosquito carcasses and human blood. We compared the molecular detection of three parasites (Wuchereria bancrofti, Plas-modium falciparum and Mansonella perstans) in mosquito E/F, mosquito carcasses and human blood collected from the same households in two villages in the Savannah Region of the country. We successfully detected the parasite DNA in mosquito E/F from indoor resting mosquitoes, including W. bancrofti which had a very low community prevalence (2.5-3.8%). Detection in the E/F samples was concordant with detection in insect whole carcasses and human blood, and a parasite not vectored by mosquitoes was detected as well.Our approach to collect and test mosquito E/F successfully detected a variety of parasites at varying prevalence in the human population under field conditions, including a pathogen (M. perstans) which is not transmitted by mosquitoes. The method shows promise for further development and applicability for the early detection and surveillance of a variety of pathogens carried in human blood.
... More sensitive and rapid tools are needed emergently for schistosomiasis surveillance and quick response [20,21]. Molecular xenomonitoring is an alternative approach to detect pathogens in their vectors, which had been reported for monitoring the transmission of parasitic diseases, such as malaria, filariasis, sleeping sickness as well as schistosomiasis, etc. [22][23][24]. Polymerase-chain reaction (PCR), nested-PCR, quantitative PCR (q-PCR) and isothermal amplification techniques, etc. were widely used as the tools of molecular xenomonitoring, presenting advantages over traditional parasitological methods to detect early and light infections in vectors. Loopmediated isothermal amplification (LAMP), a novel isothermal amplification method developed by Notomi in 2000 [25], is considered to have great potential to be used for field settings due to its various advantages such as high sensitivity, rapidity and ease of use in laboratories without expensive equipment. ...
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Background Due to sustained control activities, the prevalence of Schistosoma japonicum infection in humans, livestock and snails has decreased significantly in P. R. China, and the target has shifted from control to elimination according to the Outline of Healthy China 2030 Plan. Applying highly sensitive methods to explore the presence of S. japonicum infection in its intermediate host will benefit to assess the endemicity or verify the transmission interruption of schistosomiasis accurately. The aim of this study was to access the presence of S. japonicum infection by a loop-mediated isothermal amplification (LAMP) method through a 5-year longitudinal study in five lake provinces along the Yangtze River. Methods Based on previous epidemiological data, about 260 villages with potential transmission risk of schistosomiasis were selected from endemic counties in five lake provinces along the Yangtze River annually from 2015 to 2019. Snail surveys were conducted in selected villages by systematic sampling method and/or environmental sampling method each year. All live snails collected from field were detected by microscopic dissection method, and then about one third of them were detected by LAMP method to assess the presence of S. japonicum infection with a single blind manner. The infection rate and nucleic acid positive rate of schistosomes in snails, as well as the indicators reflecting the snails’ distribution were calculated and analyzed. Fisher's exact test was used to examine any change of positive rate of schistosomes in snails over time. Results The 5-year survey covered 94,241 ha of environment with 33,897 ha of snail habitats detected accumulatively. Totally 145.3 ha new snail habitats and 524.4 ha re-emergent snail habitats were found during 2015–2019. The percentage of frames with snails decreased from 5.93% [45,152/761,492, 95% confidence intervals ( CI ): 5.88–5.98%] in 2015 to 5.25% (30,947/589,583, 95% CI : 5.19–5.31%) in 2019, while the mean density of living snails fluctuated but presented a downward trend generally from 0.20 snails/frame (155,622/761,492, 95% CI : 0.17–0.37) in 2015 to 0.13 snails/frame (76,144/589,583, 95% CI : 0.11–0.39) in 2019. A total of 555,393 live snails were collected, none of them was positive by dissection method. Totally 17 pooling snail samples were determined as positives by LAMP method among 8716 pooling samples with 174,822 of living snails, distributed in 12 villages of Hubei, Hunan, Jiangxi and Anhui provinces. The annual average positive rate was 0.41% (95% CI : 0.13–0.69%) in 2015, 0% in 2016, 0.36% (95% CI : 0.09–0.63%) in 2017, 0.05% (95% CI : 0–0.16%) in 2018, 0.05% (95% CI : 0–0.15%) in 2019, respectively, presenting a downward trend from 2015 to 2019 with statistical significance ( χ ² = 11.64, P < 0.05). Conclusions The results suggest that S. japonicum infection still persisted in nature along the Yangtze River and traditional techniques might underestimate the prevalence of schistosomiasis in its intermediate hosts. Exploring and integrating molecular techniques into national surveillance programme could improve the sensitivity of surveillance system and provide guidance on taking actions against schistosomiasis. Graphical Abstract
... Non-invasive field sampling techniques such as the collection of feces, hair, footprints, and urine samples can provide useful information about wildlife, without needing to see or interact with the animal [2,6]. For many species, scats are easy to find in the environment and can provide in-depth information about diet [7], stress and reproductive hormones [8,9], sex [10], and parasite load [11]. However, extracting this information from scat can be time-consuming, expensive, and require multiple procedures [12]. ...
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The ability to measure and monitor wildlife populations is important for species management and conservation. The use of near-infrared spectroscopy (NIRS) to rapidly detect physiological traits from wildlife scat and other body materials could play an important role in the conservation of species. Previous research has demonstrated the potential for NIRS to detect diseases such as the novel COVID-19 from saliva, parasites from feces, and numerous other traits from animal skin, hair, and scat, such as cortisol metabolites, diet quality, sex, and reproductive status, that may be useful for population monitoring. Models developed from NIRS data use light reflected from a sample to relate the variation in the sample’s spectra to variation in a trait, which can then be used to predict that trait in unknown samples based on their spectra. The modelling process involves calibration, validation, and evaluation. Data sampling, pre-treatments, and the selection of training and testing datasets can impact model performance. We review the use of NIRS for measuring physiological traits in animals that may be useful for wildlife management and conservation and suggest future research to advance the application of NIRS for this purpose.
... Molecular xenomonitoring surveillance techniques are often associated with parasites transmitted by hematophagous insects, such as lymphatic filaria in mosquito vectors [20,[34][35][36] and trypanosomes in tsetse flies [18,19]. However, several assays have been developed for detecting trematode species in freshwater snails, including Fasciola spp. ...
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Schistosomiasis, a neglected tropical disease of medical and veterinary importance, transmitted through specific freshwater snail intermediate hosts, is targeted for elimination in several endemic regions in sub-Saharan Africa. Multidisciplinary methods are required for both human and environmental diagnostics to certify schistosomiasis elimination when eventually reached. Molecular xenomonitoring protocols, a DNA-based detection method for screening disease vectors, have been developed and trialed for parasites transmitted by hematophagous insects, such as filarial worms and trypanosomes, yet few have been extensively trialed or proven reliable for the intermediate host snails transmitting schistosomes. Here, previously published universal and Schistosoma-specific internal transcribed spacer (ITS) rDNA primers were adapted into a triplex PCR primer assay that allowed for simple, robust, and rapid detection of Schistosoma haematobium and Schistosoma bovis in Bulinus snails. We showed this two-step protocol could sensitively detect DNA of a single larval schistosome from experimentally infected snails and demonstrate its functionality for detecting S. haematobium infections in wild-caught snails from Zanzibar. Such surveillance tools are a necessity for succeeding in and certifying the 2030 control and elimination goals set by the World Health Organization.
... PCR-based assays also enable the testing of sample types other than blood. Most importantly, in the context of LF and malaria, this capacity allows researchers to indirectly and non-invasively sample a population using unique methods such as molecular xenomonitoring (MX): the testing of hematophagous arthropods for the presence of parasite-derived genetic material [15][16][17][18]. ...
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Background Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both next-generation sequencing (NGS) technologies and bioinformatics-based analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent. Methodology/Principal findings Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome’s most abundant tandem repeat. While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing. Conclusions/Significance Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements. While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study’s objectives.
... PCR-based assays also enable the testing of sample types other than blood. Most importantly, in the context of LF and malaria, this capacity allows researchers to indirectly and non-invasively sample a population using unique methods such as molecular xenomonitoring (MX): the testing of hematophagous arthropods for the presence of parasite-derived genetic material [15][16][17][18]. ...
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Background Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both next-generation sequencing (NGS) technologies and bioinformatics-based analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent.Methodology/Principal Findings Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome’s most abundant tandem repeat. While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing. Conclusions/Significance Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements. While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study’s objectives.
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Background: Results from an increasing number of studies suggest that mosquito excreta/feces (E/F) testing has considerable potential to serve as a supplement for traditional molecular xenomonitoring techniques. However, as the catalogue of possible use-cases for this methodology expands, and the list of amenable pathogens grows, a number of fundamental methods-based questions remain. Answering these questions is critical to maximizing the utility of this approach and to facilitating its successful implementation as an effective tool for molecular xenomonitoring. Methods: Utilizing E/F produced by mosquitoes or tsetse flies experimentally exposed to Brugia malayi , Plasmodium falciparum , or Trypanosoma brucei brucei , factors such as limits of detection, throughput of testing, adaptability to use with competent and incompetent vector species, and effects of additional blood feedings post parasite-exposure were evaluated. Two platforms for the detection of pathogen signal (quantitative real-time PCR and digital PCR (dPCR)) were also compared, with strengths and weaknesses examined for each. Results: Experimental results indicated that high throughput testing is possible when evaluating mosquito E/F for the presence of either B. malayi or P. falciparum from both competent and incompetent vector mosquito species. Furthermore, following exposure to pathogen, providing mosquitoes with a second, uninfected bloodmeal did not expand the temporal window for E/F collection during which pathogen detection was possible. However, this collection window did appear longer in E/F collected from tsetse flies following exposure to T. b. brucei . Testing also suggested that dPCR may facilitate detection through its increased sensitivity. Unfortunately, logistical obstacles will likely make the large-scale use of dPCR impractical for this purpose. Conclusions: By examining many E/F testing variables, expansion of this technology to a field-ready platform has become increasingly feasible. However, translation of this methodology from the lab to the field will first require field-based pilot studies aimed at assessing the efficacy of E/F screening.
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Background: Results from an increasing number of studies suggest that mosquito excreta/feces (E/F) testing has considerable potential to serve as a supplement for traditional molecular xenomonitoring techniques. However, as the catalogue of possible use-cases for this methodology expands, and the list of amenable pathogens grows, a number of fundamental methods-based questions remain. Answering these questions is critical to maximizing the utility of this approach and to facilitating its successful implementation as an effective tool for molecular xenomonitoring. Methods: Utilizing E/F produced by mosquitoes or tsetse flies experimentally exposed to Brugia malayi , Plasmodium falciparum , or Trypanosoma brucei brucei , factors such as limits of detection, throughput of testing, adaptability to use with competent- and incompetent-vector species, and effects of additional blood feedings post parasite-exposure were evaluated. Two platforms for the detection of pathogen signal (quantitative real-time PCR and digital PCR [dPCR]) were also compared, with strengths and weaknesses examined for each. Results: Experimental results indicated that high throughput testing is possible when evaluating mosquito E/F for the presence of either B. malayi or P. falciparum from both competent- and incompetent-vector mosquito species. Furthermore, following exposure to pathogen, providing mosquitoes with a second, uninfected bloodmeal did not expand the temporal window for E/F collection during which pathogen detection was possible. However, this collection window did appear longer in E/F collected from tsetse flies following exposure to T. b. brucei . Testing also suggested that dPCR may facilitate detection through its increased sensitivity. Unfortunately, logistical obstacles will likely make the large-scale use of dPCR impractical for this purpose. Conclusions: By examining many E/F testing variables, expansion of this technology to a field-ready platform has become increasingly feasible. However, translation of this methodology from the lab to the field will first require the completion of field-based pilot studies aimed at assessing the efficacy of E/F screening.
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Background The escalating level of mosquito resistance to pyrethroid insecticides threatens the effectiveness of insecticide-treated nets (ITNs) for malaria control in Malawi. An evaluation of the effectiveness of ITNs for preventing malaria in children aged 6–59 months old, after 1 year of mass distribution of LLINs was conducted in Machinga District, Malawi, an area of moderate pyrethroid resistance. Methods A facility-based, case–control study among children 6–59 months was conducted in an area of pyrethroid resistance between March and September 2013 in Machinga District. Cases and controls were children with fever who sought care from the same hospital and tested positive and negative, respectively, for malaria parasites by microscopy. Results A high proportion of both cases (354 of 404 or 87.6 %) and controls (660 of 778 or 84.8 %) slept under an ITN the night before the survey. In univariable logistic regression, older age (24–59 months versus 6–23 months, p < 0.001), sleeping on the floor versus a mattress (p < 0.001), and open versus closed house eaves (p = 0.001) were associated with increased odds of malaria, whilst secondary education of the caretaker, having windows on multiple walls, and being in the least poor wealth quintile (p < 0.001 for each) reduced the odds of malaria; ITN use was not associated with malaria (p = 0.181). In multivariable analysis, older age (p < 0.001) and secondary education of the caregiver (p = 0.011) were the only factors significantly associated with malaria. Conclusion This study did not find a significant personal protective effect of ITNs. However, high use of ITNs in the community and recent findings of lower malaria incidence in ITN users compared to bed net non-users from a cohort study in the same area suggest that ITNs provide community protection to both users and non-users alike in this area.
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Preventing malaria during pregnancy is important for the health of mothers and newborns. Interventions, which include distribution of bed nets and administration of intermittent preventive treatment (IPT), typically occur at the first antenatal visit, usually in the second or third trimester of pregnancy. In 2012, during the course of ongoing clinical studies of malaria among pregnant women in Malawi, a universal bed net campaign was implemented by the Government. This study tested the hypothesis that a universal bed net campaign would decrease the prevalence of malaria among pregnant women at their first antenatal visit. Some 1661 women were recruited for two studies from 2009 to 2014. Quantitative PCR (qPCR) was conducted from dried blood spots collected at the first antenatal care visit (prior to administration of IPT or any study interventions) from women who were in their first or second pregnancy and less than 28 weeks gestation by clinical assessment. Overall, 320 of 1629 (19.6 %) women tested for malaria at their first antenatal visit were infected. Malaria infection rates declined from 28.4 % before the universal bed net campaign, to 18.5 % in 2012, to 15.0 % in the years following the universal bed net campaign. The odds of malaria infection at the time of first antenatal visit in 2012 and the years following the bed net campaign were significantly lower than in the years prior to the intervention (OR 0.6, 95 % CI 0.4–0.8; and OR 0.4, 95 % CI 0.3–0.6, respectively). A similar pattern was observed for the prevalence of clinical malaria. The inverse trend was observed for reported bed net use. However bed net use and malaria infection were not significantly associated on the individual level. Malaria infection in pregnant women is common even after a bed net campaign in Malawi, though prevalence rates declined. These early infections may cause maternal anaemia and placental malaria resulting in adverse maternal and fetal outcomes. Infection early in pregnancy may also contribute to malaria transmission as pregnant women represent a significant untreated reservoir of parasites. Universal bed net distribution appears to have moderate success in preventing malaria early in pregnancy and these findings support continued efforts to target women early in pregnancy and all women of childbearing age.
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Objective To investigate if the first national insecticide-treated bed-net campaign in Burkina Faso, done in 2010, was followed by a decrease in childhood malaria in a district with high baseline transmission of the disease. Methods We obtained data on the prevalence of Plasmodium falciparum parasitaemia in children aged 2 weeks to 36 months from malaria surveys in 2009 and 2011. We assessed morbidity in children younger than 5 years by comparing data from the Nouna health district’s health management information system before and after the campaign in 2010. We analysed mortality data from 2008 to 2012 from Nouna’s health and demographic surveillance system. Findings The bed-net campaign was associated with an increase in the reported use of insecticide-treated nets. In 2009, 73% (630/869) of children reportedly slept under nets. In 2011, 92% (449/487) did. The campaign had no effect on the proportion of young children with P. falciparum parasitaemia after the rainy season; 52% (442/858) in 2009 and 53% (263/499) in 2011. Cases of malaria increased markedly after the campaign, as did the number of children presenting with other diseases. The campaign was not associated with any changes in child mortality. Conclusion The 2010 insecticide-treated net campaign in Burkina Faso was not associated with a decrease in care-seeking for malaria or all-cause mortality in children younger than 5 years. The most likely explanation is the high coverage of nets in the study area before the campaign which could have had an effect on mosquito vectors, limiting the campaign’s impact.
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In Hainan Province, China, great achievements in elimination of falciparum malaria have been made since 2010. There have been no locally acquired falciparum malaria cases since that time. The cost-effectiveness of elimination of falciparum malaria has been analyzed in Hainan Province. There were 4,422 falciparum malaria cases reported from 2002 to 2012, more cases occurred in males than in females. From 2002 to 2012, a total of 98.5 disability-adjusted life years (DALYs) were reported because of falciparum malaria. Populations in the age ranges of 15-25 and 30-44 years had higher incidences and DALYs than other age groups. From 2002 to 2012, malaria-related costs for salaries of staff, funds from the provincial government, national government, and the Global Fund were US$3.02, US$2.24, US$1.44, and US$5.08 million, respectively. An estimated 9,504 falciparum malaria cases were averted during the period 2003-2012. The estimated cost per falciparum malaria case averted was US$116.5. The falciparum malaria elimination program in Hainan was highly effective and successful. However, funding for maintenance is still needed because of imported cases.
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Background: The decision to stop mass drug administration (MDA) and monitor recrudescence has to be made when endpoints for elimination of lymphatic filariasis (LF) have been achieved. Highly sensitive and specific diagnostic tools are required to do this. The main objective of this study was to determine most effective diagnostic tools for assessing interruption of LF transmission. Methods: The presence of filarial infection in blood and mosquito samples was determined using five diagnostic tools: Brugia malayi-14 (BM14) antibody detection ELISA, Onchocerca gibsoni antigen (Og4C3) based ELISA, PCR, immunochromatography (ICT) card test and blood smear. The study was carried out in two communities in the Central Region of Ghana. Results: OG4C3 was found to be the most sensitive test but ICT, the second most sensitive, was the most field applicable. PCR was found to be the most specific. Thirteen out of 30 pools of anopheles mosquitoes tested positive for the DNA of Wuchereria bancrofti. Conclusions: Very low antigen prevalence in primary school children indicates that MDA is working, so children born since the intervention was put in place are not getting infected. Inclusion of xenomonitoring in monitoring the effectiveness of MDA will give a better indication as to when transmission has been interrupted especially in areas where microfilaria prevalence is lower than 1%.
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Effective program management is essential for successful elimination of malaria. In this perspective article, evidence surrounding malaria program management is reviewed by management science and malaria experts through a literature search of published and unpublished gray documents and key informant interviews. Program management in a malaria elimination setting differs from that in a malaria control setting in a number of ways, although knowledge and understanding of these distinctions are lacking. Several core features of successful health program management are critical to achieve elimination, including effective leadership and supervision at all levels, sustained political and financial commitment, reliable supply and control of physical resources, effective management of data and information, appropriate incentives, and consistent accountability. Adding to the complexity, the requirements of an elimination program may conflict with those of a control regimen. Thus, an additional challenge is successfully managing program transitions along the continuum from control to elimination to prevention of reintroduction. This article identifies potential solutions to these challenges by exploring managerial approaches that are flexible, relevant, and sustainable in various cultural and health system contexts. © The American Society of Tropical Medicine and Hygiene.
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Lymphatic filariasis (LF) is caused by filarial worms that live in the lymphatic system and commonly lead to lymphoedema, elephantiasis, and hydrocele. LF is recognized as endemic in 73 countries and territories; an estimated 1.39 billion (thousand million) people live in areas where filariasis has been endemic and is now targeted for treatment [1]. Global momentum to eliminate LF has developed over the past 15 years as a result not only of research demonstrating the value of single-dose treatment strategies and point-of-care diagnostic tools, but also of both the generous donations of medicines from the following committed pharmaceutical companies: GlaxoSmithKline (albendazole), Merck (ivermectin), and Eisai (diethylcarbamazine; DEC), and the essential financial support for programme implementation from the donor community [2]. During 2011, more than 50 countries carried out LF elimination programmes, and more than 500 million people received mass treatment [1]. A principal reason for the programme's dramatic expansion and success to date has been the galvanizing of efforts of all key partners around a common policy framework created and coordinated through the World Health Organization's Global Programme to Eliminate Lymphatic Filariasis (GPELF). This report, rather than highlighting the very considerable contributions of each individual partner or even chronicling most of the specific achievements of the GPELF, instead focuses on the details of the underlying processes themselves and their importance in determining programme success.
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A Global Programme to Eliminate Lymphatic Filariasis was launched in 2000, with mass drug administration (MDA) as the core strategy of the programme. After completing 13 years of operations through 2012 and with MDA in place in 55 of 73 endemic countries, the impact of the MDA programme on microfilaraemia, hydrocele and lymphedema is in need of being assessed. During 2000-2012, the MDA programme made remarkable achievements - a total of 6.37 billion treatments were offered and an estimated 4.45 billion treatments were consumed by the population living in endemic areas. Using a model based on empirical observations of the effects of treatment on clinical manifestations, it is estimated that 96.71 million LF cases, including 79.20 million microfilaria carriers, 18.73 million hydrocele cases and a minimum of 5.49 million lymphedema cases have been prevented or cured during this period. Consequently, the global prevalence of LF is calculated to have fallen by 59%, from 3.55% to 1.47%. The fall was highest for microfilaraemia prevalence (68%), followed by 49% in hydrocele prevalence and 25% in lymphedema prevalence. It is estimated that, currently, i.e. after 13 years of the MDA programme, there are still an estimated 67.88 million LF cases that include 36.45 million microfilaria carriers, 19.43 million hydrocele cases and 16.68 million lymphedema cases. The MDA programme has resulted in significant reduction of the LF burden. Extension of MDA to all at-risk countries and to all regions within those countries where MDA has not yet reached 100% geographic coverage is imperative to further reduce the number of microfilaraemia and chronic disease cases and to reach the global target of interrupting transmission of LF by 2020.
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Background: The Sri Lankan Anti-Filariasis Campaign conducted 5 rounds of mass drug administration (MDA) with diethycarbamazine plus albendazole between 2002 and 2006. We now report results of a comprehensive surveillance program that assessed the lymphatic filariasis (LF) situation in Sri Lanka 6 years after cessation of MDA. Methodology and principal findings: Transmission assessment surveys (TAS) were performed per WHO guidelines in primary school children in 11 evaluation units (EUs) in all 8 formerly endemic districts. All EUs easily satisfied WHO criteria for stopping MDA. Comprehensive surveillance was performed in 19 Public Health Inspector (PHI) areas (subdistrict health administrative units). The surveillance package included cross-sectional community surveys for microfilaremia (Mf) and circulating filarial antigenemia (CFA), school surveys for CFA and anti-filarial antibodies, and collection of Culex mosquitoes with gravid traps for detection of filarial DNA (molecular xenomonitoring, MX). Provisional target rates for interruption of LF transmission were community CFA <2%, antibody in school children <2%, and filarial DNA in mosquitoes <0.25%. Community Mf and CFA prevalence rates ranged from 0-0.9% and 0-3.4%, respectively. Infection rates were significantly higher in males and lower in people who denied prior treatment. Antibody rates in school children exceeded 2% in 10 study sites; the area that had the highest community and school CFA rates also had the highest school antibody rate (6.9%). Filarial DNA rates in mosquitoes exceeded 0.25% in 10 PHI areas. Conclusions: Comprehensive surveillance is feasible for some national filariasis elimination programs. Low-level persistence of LF was present in all study sites; several sites failed to meet provisional endpoint criteria for LF elimination, and follow-up testing will be needed in these areas. TAS was not sensitive for detecting low-level persistence of filariasis in Sri Lanka. We recommend use of antibody and MX testing as tools to complement TAS for post-MDA surveillance.
Chapter
Despite over 100 years of discovery supporting the value of nonhuman primate models for malaria research, use of these models has been slow and sporadic and has clearly fallen short of capitalizing on the availability of a wealth of opportunities and possibilities for advancing knowledge on malaria in humans. This chapter provides knowledge relevant for the future use of these models, recognizing that they in many respects have advantages over the use of clinical samples, rodent malaria models, or in vitro cultures. The introduction provides a basic understanding of malaria as a widespread global disease of devastating proportions, the malaria life cycle in humans and nonhuman primates, and the basic uses of these models for research. The history of the discovery and use of dozens of human and simian malaria models in a variety of nonhuman primate hosts is detailed. Basic fundamental information about the species, strains, and their biology and infection characteristics are presented in a succinct and organized manner, making this work a relevant reference for both established scientists and newcomers planning experimental work using these systems. With the recent and ongoing publications of both nonhuman primate and Plasmodium parasite genomes and many functional genomic and systems biology approaches now at the forefront of science, this is an opportune time for this field. Maintenance of the most appropriate nonhuman primate colonies, including Aotus, Saimiri, and macaque species, is critical, along with training of experts to be intimately familiar with the nuances of the various parasite–host interactions and relationships.