Molecular xenomonitoring of diurnally subperiodic Wuchereria bancrofti infection in Aedes (Downsiomyia) niveus (Ludlow, 1903) after nine rounds of Mass Drug Administration in Nancowry Islands, Andaman and Nicobar Islands, India.

A group of four human inhabited Nancowry Islands in Nicobar district in the Andaman and Nicobar Islands, India having a population of 7674 is the lone focus of diurnally sub-periodic Wuchereria bancrofti (DspWB) that is transmitted by Aedes niveus (Ludlow). Microfilaria (Mf) prevalence was above 1% even after nine rounds of Mass Drug Administration (MDA) with DEC and albendazole. Molecular xenomonitoring (MX) was conducted to identify appropriate vector sampling method and assess the impact. BioGents Sentinel traps, gravid traps and human baited double bed nettraps were used in three locations in each village to collect Aedes niveus female mosquitoes. Subsequently daytime man landing collections (MLC) were carried out in all the 25 villages in the islands. Collections were compared in terms of the number of vector mosquitoes captured per trap collection. Females of Ae. niveus were pooled, dried and processed for detecting filarial parasite DNA using RT-PCR assay. Vector infection rate was estimated using PoolScreen software. Only 393 female mosquitoes including 44 Ae. niveus (11.2%) were collected from 459 trap collections using three trapping devices. From 151 MLCs, 2170 Ae. niveus female mosquitoes were collected. The average prevalence of W. bancrofti DNA was 0.43%. Estimated upper 95% CI exceeded the provisional prevalence threshold of 0.1% in all the villages, indicating continued transmission as observed in Mf survey. MLCs could be the choice, for now, to sample Ae. niveus mosquitoes. The PCR assay used in MX for nocturnally periodic bancroftian filariasis could be adopted for DspWB. The vector-parasite MX, can be used to evaluate interventions in this area after further standardization of the protocol.

Introduction

Lymphatic filariasis (LF) is endemic in 72 countries where about 1.39 billion people are at the risk of acquiring infection. Wuchereria bancrofti is the predominant parasite while Brugia malayi and B. timori are restricted in distribution [1]. Global Programme to Eliminate Lymphatic Filariasis (GPELF) launched in 2000 [2] has made a significant impact on infection and disease [3]. Transmission control with mass drug administration (MDA) (with an option of supplementing integrated vector management) and alleviation of sufferings of the diseased with morbidity management and disability prevention (MMDP) are the recommended strategies for achieving the goal of LF elimination. Transmission Assessment Survey (TAS) is the recommended protocol to evaluate the impact of the programme and take a decision on stopping the intervention [4]. Recently, molecular xenomonitoring (MX) has been demonstrated to be a potential surveillance tool to supplement TAS for different parasite and vector combinations [5-7].

In 2004, India, contributing around 44.3% of the global burden [1], launched the National programme to eliminate LF in 256 endemic districts in 21 States and Union Territories with about 610 million running the risk of contracting infection. Administration of MDA with diethylcarbamazine citrate (DEC) and albendazole (ALB) simultaneously is the main strategy to interrupt transmission besides recommending an integrated vector management, wherever feasible. Wuchereria bancrofti is prevalent in all the endemic States while B. malayi is restricted to six States and Union Territories in India. Nocturnally periodic W. bancrofti is prevalent in all the endemic districts. Diurnally sub-periodic W. bancrofti (DspWB), a physiological variant is confined to four (Chowra, Teressa, Kamorta and Nancowry) remotely located group of Nancowry Islands in Nicobar district in the and Nicobar Islands [8, 9]. These islands are known to be endemic for only DspWB. Ever since the incrimination of the day biting Aedes (Downsiomyia) niveus mosquito (earlier known as Downsiomyia nivea, Ochlerotatus (Finlaya) niveus and Aedes (Finlaya) niveus) as the vector of this form of filariasis in these islands [10], there has been significant advancement towards understanding the distribution and bionomics of vector mosquito [11-13]. The vector mosquito prefers to breed primarily in innumerable and inaccessible tree holes spread in the forested tracts of Nancowry islands [10-12].

As in other endemic districts, the National Filariasis Elimination Programme is being implemented in the Nancowry Islands of Nicobar district since 2004. Microfilaria (Mf) survey carried out after six rounds of MDA in 2011 to assess the impact of MDA showed that the overall microfilaria (Mf) prevalence was 3.28% in four islands [14]. As the Mf rate was more than 1%, MDA was continued and nine rounds of MDA were completed by 2014. Even after 9 rounds of Mass Drug Administration, microfilaria prevalence (1.7%) was above the transmission threshold level (1%), indicating continued transmission [15].

Mf survey is operationally feasible in this area since only day blood samples are needed. However, most of the Nicobarese go into the forest daily to collect forest products like firewood and their availability for blood sampling during the daytime remains uncertain. In such situations, monitoring LF infection in the population at risk via mosquitoes (MX) offers a key pathway for illustrating possible transmission as it has been recommended as an instrument for observing the impact of MDA on LF transmission [5, 6, 16–19]. MX protocol has already been developed for W. bancrofti parasite and other vector combinations [16, 17, 19]. Although infection in Ae. niveus was detected earlier using microscopy [11], the scope of MX has not yet been attempted for DspWB and Ae. niveus vector combination. The present study was carried out to evaluate the impact of nine rounds of MDA using MX. However, collection of the vector mosquito has been a challenge though Biogent Sentinel (BGS) has proved useful for other Aedes vector species elsewhere [20]. Therefore, apart from BGS, we used different mosquito sampling devices for collection of the vector species in the islands to compare and identify the most productive one. The study also aimed at assessing the vector infection by molecular assay developed for Culex quinquefasciatus—W. bancrofti combination [19, 21], which could be used as a surveillance tool in evaluating MDA in Ae. niveus transmitted W. bancrofti infection.

Materials and methods

Study area

The Nancowry group comprise seven islands (Chowra, Teressa, Katchal, Kamorta, Nancowry, Trinket & Bompoka) (Fig 1). Out of these seven, Bompoka is not inhabited by humans. Post tsunami, the inhabitants of Trinket Island have been rehabilitated in Kamorta. Katchal is non-endemic for filariasis. Therefore, the study was undertaken in the four human inhabited islands that are endemic for LF (Chowra, Teressa, Kamorta, and Nancowry Islands) between May 2014 and July 2015 [Fig 2A–2D]. The total population of the four inhabited islands is 7674 [22]. The population in these islands ranges from 713 (Nancowry) to 3757 (Kamorta). There are 25 villages in the four Islands and population in these villages ranges from 60 (Kanahinot) to 1759 (Kamorta Hqs). The total area ranges from 5.85 km2 (Chowra) to 131 km2 (Kamorta). The islands are predominantly inhabited by the Nicobarese tribe. The total number of households in these islands is 2026 with the average family size being 4. The Asian tsunami struck the A & N islands on 26th December 2004 and the rehabilitation measures including construction of permanent shelters were accomplished between 2005 and 2006. Briefly, a shelter comprises a living-cum-dining room, two bedrooms and a kitchen. The flooring is cemented concrete. There is a small veranda/porch, which has the entry to the shelter. The plinth area of each shelter measures approximately 450 sq. ft. All the shelters are provided with basic sanitary facilities. These shelters are proximal to the forest (tropical evergreen) and are prone to mosquito menace from the surrounding forest. Other than domestic containers, there are no other breeding habitats within the vicinity of shelters. The tribal community frequent the forest for their livelihood and are at the risk of getting mosquito bites. The islands are accessible only through Govt.-run ferries and all the essential commodities are transported from the mainland routed through the Andaman and Nicobar Administration. All these islands are within the jurisdiction of Nancowry Tehsil (sub-district), an administrative unit of a district.

Fig 1

Map of study area showing the location of Nancowry Islands, Andaman and Nicobar Islands, India.

Fig 2

Map depicting sampling locations by islands (2A: Map of Nancowry, 2B: Map of Chowra, 2C: Map of Kamorta; 2D: Map of Teressa).

Mosquito sampling

Collection sites

Initially, a total of 21 villages (sites) from three islands (Teressa, Nancowry and Kamorta) were selected for mosquito sampling using BioGents Sentinel Traps (BGS, Biogents, AG, Regensburg Germany), human baited double bed net-trap (HBDNT) and gravid traps (GT). None of these devices were productive in terms of collecting adequate number of mosquitoes to reach the sample size. Subsequently, man landing collections (MLCs) were carried out in all the 25 villages in the four islands (Teressa, Nancowry Kamorta and Chowra) from December 2014 to July 2015. Three settings, the domestic (space within the human dwelling and the close surroundings), peri-domiciliary (an area under 10-meter radius from the backyard of the human dwelling) and sylvan (area within 20–25 meter radius from peri-domiciliary) settings were identified.

A permanent house is the collection spot in domestic setting (A permanent shelter/house in the study area typically had walls made up of aerated cement concrete blocks, with thick exteriors, and false ceiling under the corrugated galvanized iron (CGI) roof comprising 9mm × 4mm thick processed bamboo boards supported by steel frame, one or two bedrooms, a dining-cum-living space and a front porch/veranda. The flooring is cemented concrete. Such a residential structure covered by a single roof is known as a domicile. A cluster of such residential structures/permanent shelters, where the villagers live for a larger part of the day, is defined as a domestic setting).

The peri-domiciliary area is an annex of each house, consisting of a yard of about 10-meter radius. This is not connected to the domicile (porch, kitchen, bedrooms etc.), structures like community hall, machan-a raised platform covered on all three sides by tin sheets-structure. People spend their daytime in this area and engage themselves in processing coconut into copra. Pens for poultry like hens and chicken were constructed on wooden poles located near a tree, where chickens sleep and the chickens, which were allowed to wander freely, would nest inside the house, and underneath the foliage of the bushes located at different distances from the home constituting the peri-domiciliary setting.

A sylvan setting was defined as a location with steady forest canopy cover, contiguous to the peri-domiciliary setting. The forest canopy is constituted by tropical evergreen forests, characterized by dense forest with fruiting trees and secondary forest growth.

Sample size

We assumed a Mf prevalence of 1%, which is the threshold recommended for conducting transmission assessment survey (TAS) for arriving at a decision on stopping MDA. Expecting random biting of mosquitoes at this prevalence level in humans, we assumed the prevalence of infection in mosquito to be at 1%. The sample size derived was 1592 mosquitoes from the four islands, with an absolute precision of 0.5% (0.5–1.5%) and a design effect of 1 at 95% confidence level.

Mosquito sampling devices

BGS Trap (with human lure as host seeking attractant, supplied by the manufacturer) and HBDNT (with human volunteer as bait) were used for sampling host seeking (feeding phase) vector mosquitoes and GT was used to trap females attracted for oviposition (oviposition phase) with fusions as attractants. The locations and timings were fixed on the basis of the host seeking and oviposition behaviour and related activities of the vector mosquito. Ae. niveus is a forest dwelling, canopy habitat mosquito [23], frequenting houses in proximity to forest, zoophilic and prefers to feed on primates (primatophilic) [24]. Each of these trapping devices was placed in all the three settings (domestic, peri-domiciliary and sylvan settings).

BG-Sentinel trap (BGS)

Battery operated BGS traps using BG sentinel lure as attractant was deployed for sampling vector mosquitoes between May and October 2014. In the domestic setting, the BGS traps were placed in the porch of the household. In the peri-domiciliary and sylvan settings, the BGS traps were hung from a tree branch 20–30 cm above the ground, as Ae. niveus flies low and has a propensity to bite at the feet. Grease was smeared on the cords used for suspending the trap to prevent predation of collected mosquitoes by ants. The traps were set in the morning (7.00 AM) and removed in the evening (5.00 PM).

Gravid trap (GT)

A modified version of the battery operated CDC GT [19] was used to sample gravid mosquitoes during May-October, 2014. In each village, three traps were set in each setting. In order to maximize the trap collections, two types of infusion were used, using cashew leaves (Anacardium occidentale) or cumin seeds (Cuminum cyminum) as they were reported to have potency in attracting Aedes spp. [25, 26]. The GTs were set in areas which were considered safe and there was no obstruction. Batteries were recharged each day and we did not observe any instances of disruption. A total of 153 trap collections spreading over 51 days were made, spending 918 hrs (Table 1). The traps were set in the forenoon between 6.00 AM and 12.00 noon.

Table 1

Details of different trapping devices, numbers and duration in three different ecological settings for sampling Ae. niveus in Nancowry Islands.

Ecotopes	Teressa1			Nancowry2		Kamorta2
	Type of Traps	Total No. of traps	No. of days	Total No. of traps	No. of days	Total No. of traps	No. of days
Domestic	BGS#	40	8	20	4	5	1
Peri-domestic	BGS#	40	8	20	4	5	1
Sylvan	BGS#	40	8	20	4	45	9
Total		120	24	60	12	55	11
Domestic	GT	24	8	12	4	9	3
Peri-domestic	GT	24	8	12	4	9	3
Sylvan	GT	24	8	12	4	27	9
Total		72	24	36	12	45	15
Domestic	HBDNT@	8	8	4	4	3	3
Peri-domestic	HBDNT@	8	8	4	4	3	3
Sylvan	HBDNT@	8	8	4	4	9	9
Total		24	24	12	12	15	15

# BGS-Lure

@ HBDNT-Human

1 GT with Cumin seed infusion

2 GT with Cashew leaf infusion

Human baited double net trap (HBDNT)

The assembly of the HBDNT comprised two layers of mosquito net. The first layer comprised an inner mosquito net (2.4 m length × 1.60 m breadth × 1.80 m height), which was gracefully stitched to the second layer of a mosquito net (2.9 m length × 2.10 m breadth × 1.80 m height). During the HBDNT catches, an adult volunteer, who acted as bait laid on a strong, flexible and water-resistant tarpaulin sheet, placed inside the inner mosquito net, between 8.00 AM and 1.00 PM. Potable water was always available during this period. It was ensured that the bait had his breakfast, prior to entry into the double bed net. The bait answered a call of nature during the period of collection. The inner mosquito net was neatly tucked underneath the tarpaulin sheet to keep the mosquitoes out. Thus, the bait was fully protected from mosquito bites. The gap between the first and the second mosquito net was 25 cm and was rolled up to 50 cm above the ground to allow the attracted mosquitoes to come close to the first mosquito net. After every 10 minutes, an insect collector caught mosquitoes resting in the outer net. Then, the external mosquito net was rolled up and all the trapped mosquitoes were captured with oral aspirators by insect collector. Thus, mosquitoes were collected at 10 minutes’ interval. HBDNT collections were conducted between July and December 2014.

Man landing collections (MLCs)

Collections were carried out between December 2014 and July 2015 during the peak period of abundance of Ae. niveus [13]. Human volunteers to act as bait were identified in consultation with the village head. Only male volunteers in the age group of 20–30 consenting to the study participated. Collections were conducted in two sittings, one in the morning between 4:00 and 7.00 AM and the other in the evening between 5.00 and 7.00 PM, coinciding with the peak biting activity of the vector species [11]. During the period of study, there were no reported local dengue/chikungunya or zika cases in the Islands. The volunteer was made to sit on a chair, outdoors near a human dwelling, exposing both the arms below elbow and limbs below knees. Mosquitoes that landed and attempted feeding on the exposed parts were collected using oral aspirators by a trained technical staff. Utmost care was taken by not allowing the mosquitoes to bite the human bait. The native Nicobarese spend considerable part of the day working in the forests/coconut plantations for harvesting coconuts and miscellaneous food articles. Ae. niveus is a sylvan mosquito, breeding in tree holes in the forest and resting outdoors [10-12]. It is learnt from the local tribal community that they are bitten by mosquitoes when they frequent the forests. Anticipating that the vector species is an opportunistic biter on the people engaged in the forest, MLCs were conducted in the sylvan settings only. The duration of collection time varied on the days of intermittent rains and depending on the continuous availability of the volunteer.

Identification of mosquitoes and processing for PCR

Mosquitoes from types of collections were transferred into test tubes, labelled with date, place and type of collection and transported alive to the field laboratory of RMRC for further processing. In the laboratory, mosquito samples were anaesthetized with ether and a trained entomologist identified the species using stereomicroscope and standard taxonomic keys [27, 28]. Technicians trained in mosquito taxonomy processed the mosquitoes. The mosquitoes were separated species wise, according to sex and gonotrophic phase (unfed, blood fed, semi-gravid) and recorded. Mosquitoes collected by MLCs in a given location and collection day were pooled separately. Each pool of mosquitoes representing the location and date of collection was considered as a sample for extraction and assay for detecting filarial infection. Female Ae. niveus mosquitoes were pooled in vials each with 10 mosquitoes, dried overnight using dry bath and stored at -20°C. The samples were then transported to Vector Control Research Centre, Puducherry (an Institute under Indian Council of Medical Research and a collaborating institute for the study) for molecular assay to detect filarial parasite DNA.

Extraction and detection of W. bancrofti parasite DNA

Extraction of W. bancrofti parasite DNA from the pooled mosquito samples was performed following the manufacturer’s instructions using “DNA extraction Solution Kit Genie” (Genie-Bangalore). Real-time polymerase chain reaction (RT- PCR) assay was carried out following the technique described earlier [29] with 12.5 μl of FastStart Essential DNA probes Master (Roche Diagnostics, Germany) along with 450 nmol/L of each primer: LDR1-5’ATTTTGATCATCTGGGAACGTTAATA-3’;LDR2-5’CGACTGTCTAATCCATTCAGAGTGA-3’ and 125 nmol/L probe (6 FAM-ATCTGCCCATAGAAATAACTACGGTGGATCTG-TAMRA) in a final volume of 25μl in 96-well MicroAmp optical plates (Roche Diagnostics, Germany). One microliter of the extracted DNA was used as a template in RT- PCR along with 1 ng, 100 pg and 10 pg of purified genomic DNA samples as positive controls and water negative controls. All RT-PCR reactions were run in duplicates. Cycle of quantification (Cq) values for each sample is thus a single value reflecting the cycle number used for quantification. Thermal cycling parameters used were 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1min. Thermal cycling and data analysis were done with Light Cycler® 96 (Roche, Germany) instrument using the sequence detection system (SDS) software (Applied Biosystems). Cq values of samples ranging from 1.0–39.0 were considered positive, and samples that failed to reach the fluorescence threshold beyond 39 were considered indeterminate and repeated to confirm the negativity or positivity of those samples following standard procedures.

Data analysis

The density of man landing vector mosquito per hour was calculated by dividing the number of Ae. niveus collected by the number of hours spent. The numbers of Ae. niveus collected in different traps were too small to compare the trap densities by statistical analysis. Therefore, the actual numbers are presented in the results section. Kruskal-Wallis one-way analysis of variance (ANOVA) was used to compare the difference in density of Ae. niveus in the MLCs between the villages and islands. The heterogeneity chi-square test was used to compare the pool positivity rates among islands. P value less than 0.05 was considered as significant. All statistical analyses were carried out with STATA version 14.0. The prevalence of W. bancrofti DNA in Ae. niveus (vector infection rate) was estimated using the PoolScreen software (v. 2.02) software [30, 31] from the data generated from qPCR (quantitative PCR) assays. The PoolScreen software calculates the maximum likelihood estimate of the prevalence and its 95% confidence interval.

Institutional Human Ethics Committee Clearance

The study protocol was approved by the Institutional Human Ethics Committee of the RMRC, Port Blair. It was assured that all the necessary precautions would be followed to collect mosquitoes before probing and biting the volunteer. Written informed consent was obtained from each of the adult male volunteers who were trained for participation to act as bait.

Results

The types of trapping devices used, their numbers and hours used in different settings for sampling Ae. niveus in the Nancowry Islands is furnished in Table 1. Overall, in 21 villages, a total of 235 BGS trap collections involving a total of 2350 hours (Hrs) were completed. The total number of GTs used in all the settings and villages was 153 for 918 hours. The number of traps used varied between the settings and villages and the difference was due to the availability of suitable spots in the respective settings. A total of 51 HBDNT collections were completed, spending a total of 255 hours.

Species composition

The numbers of female mosquito species collected from BGS, GT and HBDNT (combining three ecological settings) and from MLCs are depicted in the Fig 3. The BGS trap sampled six species of mosquitoes (n = 220). Ae. albopictus was relatively more in number forming 40.0% of the total collected followed by Ae. aegypti (32.7%). Only 24 female Ae. niveus were collected by BGS traps, which constituted 10.9% of all the mosquitoes collected by various methods. Other species were Ae. edwardsi (10.5%), Ae. malayensis (5.0%) and Armigeres subalbatus (0.9%). A total of 73 female mosquitoes belonging to seven species of mosquitoes were trapped in the GT. Ae. albopictus (41.0%) and Ae. aegypti (34.3%) were the dominant species. Only 11 female Ae. niveus were collected. In total, 100 female mosquitoes belonging to 6 species were sampled through HBDNT. Ae. aegypti (44.0%) and Ae. albopictus (, 34.0%) were the dominant species. Only 11 female Ae. niveus were collected. Only a total of 2170 female Ae. niveus were collected from MLCs.

Fig 3

The numbers of female mosquitoes captured by different traps by species.

MLC

As many as 151 MLCs were completed and a total of 2170 female mosquitoes of Ae. niveus were collected. The number of mosquitoes collected ranged from 20 to 210 in 77 sampling spots (Table 2). The density of man landing vector mosquito ranged from 1.0 to 8.0 per hour in different sites and from 1.77 to 5.68 per hour in different islands. Kruskal-Wallis one-way ANOVA showed that while the density did not differ significantly among sites (χ2 = 23.4, D.F. = 24, P = 0.49), it differed significantly between islands (χ2 = 18.3, D.F. = 3, P = 0.0004).

Table 2

Number of Ae. niveus collected through man landing collections (MLCs).

Island	Villages	Period	No. MLCs	No. sampling spots	Total man hours spent	No. Ae. niveus collected	Man landing rate@	Man biting rate#
Teressa	Bengali	April-May, 2015	6	3	30	120	4.00	48.00
Teressa	Aloorang	April-May, 2015	3	3	15	60	4.00	48.00
Teressa	Kalasi	April-May, 2015	7	6	35	130	3.71	44.57
Teressa	Enam	April-May, 2015	3	3	15	50	3.33	40.00
Teressa	Minyuk	April, 2015	3	3	15	70	4.67	56.00
Teressa	Luxi	April,2015	2	1	10	50	5.00	60.00
Teressa	Chukmachi	April, 2015	2	2	10	60	6.00	72.00
SUB TOTAL			26	19	130	540	4.15	49.85
Nancowry	Champin	Dec 2014, Jan-Feb, 2015	12	3	60	140	2.33	28.00
Nancowry	Balu Basthi	Dec 2014, Jan-Feb, 2015	17	4	85	170	2.00	24.00
Nancowry	Tapong	Jan-Feb, 2015	7	2	35	150	4.29	51.43
Nancowry	Hitui	January, 2015	9	4	45	90	2.00	24.00
SUB TOTAL			45	13	225	550	2.44	29.33
Kamorta	Head Quarters	Dec 2014, Jan-Feb, 2015	12	4	60	120	2.00	24.00
Kamorta	Chota Enak	Dec 2014, Jan-Feb, 2015	6	2	30	40	1.33	16.00
Kamorta	Bada Enak	December, 2014	3	2	15	30	2.00	24.00
Kamorta	Vikas Nagar	Feb-Mar, 2015	7	4	35	60	1.71	20.57
Kamorta	Dering	Feb-Mar, 2015	7	4	35	70	2.00	24.00
Kamorta	Kakana	Feb-Mar, 2015	4	2	20	20	1.00	12.00
Kamorta	Pillpillow	Feb-Mar, 2015	4	2	20	40	2.00	24.00
Kamorta	Munak	March, 2015	12	6	60	120	2.00	24.00
Kamorta	Changuah	March, 2015	6	3	30	40	1.33	16.00
SUB TOTAL			61	29	305	540	1.77	21.25
Chowra	Raihon	June-July, 2015	8	5	40	210	5.25	63.00
Chowra	Kuitasuk	June-July, 2015	3	3	15	120	8.00	96.00
Chowra	Tae-ela	June-July, 2015	3	3	15	50	3.33	40.00
Chowra	Chongkamong	June-July, 2015	3	3	15	90	6.00	72.00
Chowra	Al-hiat	June-July, 2015	2	2	10	70	7.00	84.00
SUB TOTAL			19	16	95	540	5.68	68.21
Overall			151	77	755	2170	2.87	34.49

@ No. of Ae. niveus collected ÷ total man hours spent

# No. of Ae. niveus collected × 12 hours

Detection of W. bancrofti DNA in Ae. niveus

Vector mosquitoes collected from MLCs alone were processed for assessing vector infection as the number collected from trap collections were fewer. Of the 217 pools of the vector mosquito, two got damaged and 215 pools were processed by RT-PCR; 9 pools were found positive for W. bancrofti DNA. Pool positivity was 4.2%. Maximum pool positivity was found in Chowra (7.6%, n = 53), followed by Kamorta and Nancowry (3.70%, n = 54), while the least was in Teressa (1.9%, n = 54). The pool positivity rates were not significantly different between islands (χ2 = 2.28, P = 0.52). The pool screening calculation indicated a maximum likelihood estimate (MLE) of infection of 0.77% (95% CI: 0.25–1.86%) in Chowra, followed by 0.37% (95% CI: 0.07–1.22%) in Nancowry. The infection rate in Ae. niveus was the least in Teressa (0.20%, 95% CI: 0.01–0.90) (Table 3).

Table 3

PoolScreen estimation of W. bancrofti in Ae. niveus mosquitoes after nine rounds of mass drug administration (DEC + albendazole) in Nancowry islands, India, 2014–15.

Villages	Island	Mf rate (%)$	Mosquitoes collected	Total pools tested	No of pools positive for parasite DNA	% pools positive for parasite DNA	Prevalence of W. bancrofti DNA in Ae. niveus [95%CI]#
Bengali	TERESSA	3.34	120	12	0	0	0.0 [0,2.74]
Aloorang	TERESSA	0	60	6	1	16.67	1.66 [0.10,8.18]
Kalasi	TERESSA	2.6	130	13	0	0	0.0 [0.0,2.56]
Enam	TERESSA	0.57	50	5	0	0	0.0 [0.0,0.54]
Minyuk	TERESSA	4.59	70	7	0	0	0.0 [0.0,4.28]
Luxi	TERESSA	2	50	5	0	0	0.0 [0.0,5.540
Chukmachi	TERESSA	9.27	60	6	0	0	0.0 [0.0,4.83]
	Sub Total	3.04	540	54	1	1.85	0.19[0.01,0.9]
Champin	NANCOWRY	0	140	14	0	0	0.0 [0.0, 2.40]
Balu Basthi	NANCOWRY	0	170	17	1	5.88	0.59 [0.03,2.85]
Tapong	NANCOWRY	0.69	140	14	0	0	0.00 [0.0,2.40]
Hitui	NANCOWRY	0	90	9	1	11.11	1.11[0.07,5.41]
	Sub Total	0.19	540	54	2	3.7	0.37[0.07,1.22]
Head Quarters	KAMORTA	0.21	120	12	0	0	0.0 [0.0,2.74]
Chota Enak	KAMORTA	0	40	4	0	0	0.0 [0.0,6.51]
Bada Enak	KAMORTA	0	30	6	0	0	0.0 [0.0,7.91]
Vikas Nagar	KAMORTA	0	60	6	0	0	0.0 [0.0,4.83]
Dering	KAMORTA	5.06	70	7	1	14.29	1.42[0.09,6.98]
Kakana	KAMORTA	0.68	20	2	0	0	0.0 [0.0,10.16]
Pillpillow	KAMORTA	0	40	4	1	25	2.47 [0.15,12.52]
Munak	KAMORTA	0	120	12	0	0	0.0 [0.0,2.74]
Changuah	KAMORTA	5.56	40	4	0	0	0.0 [0.0,6.51]
	Sub Total	0.56	540	54	2	3.7	0.37[0.07,1.22]
Raihon	CHOWRA	2.09	210	21	1	4.76	0.48 [0.03,2.31]
Kuitasuk	CHOWRA	5.48	120	12	1	8.33	0.83[0.05,4.04]
Tae-ela	CHOWRA	1.96	50	5	1	20	1.99[0.12,9.88]
Chongkamong	CHOWRA	2.91	80	8	1	12.5	1.25[0.07,6.09]
Al-hiat	CHOWRA	3.45	70	7	0	0	0.0 [0.0,4.28]
	Sub Total	2.92	530	53	4	7.55	0.77[0.25,1.86]

# Maximum likelihood estimate using PoolScreen

$ Shriram et al. 2020 microfilaraemia by village

Vector infection was recorded only in 9 villages. The pool positivity varied between 4.8% (Raihon) and 25% (Pillpillow) in different villages sampled in the four islands. The overall infection in the vector mosquito was 0.43% (95% CI: 0.21–0.78). Villages with no Mf carriers contain infected mosquitoes, with PoolScreen estimation, sometimes exceeding the 0.1% provisional threshold (Table 3) Also, the upper confidence limit of the vector infection exceeded the 1.0% provisional threshold in all the screened villages.

Discussion

Although Nicobar district in the Andaman and Nicobar Islands is endemic for nocturnally periodic Wuchereria bancrofti, the four Nancowry Islands in the district are endemic only for DspWB with a population of 7674 at risk. Assessment by the Directorate of Health Services, Andaman & Nicobar administration under the aegis of National Vector Borne Diseases Control Programme (NVBDCP), following nine rounds of MDA indicated that the Mf prevalence was >1% in sentinel/spot check sites in Nicobar district (Personal Communication, NVBDCP data, Andaman & Nicobar Islands). Since Mf prevalence was above the pre-TAS benchmark of <1%, MDA was being continued in the district. Additionally, studies carried out in the Nancowry Islands in 2014 also showed that the microfilaria prevalence was >1% [15]. This study carried out MX as a supplementary measure to assess the impact of MDA.

Mf prevalence <1% would provide better yield for surveillance by methods like MX when it is difficult to sample human blood [5, 7, 18]. There are two components of MX, collection of vector mosquitoes and performing molecular assay. Collection of vector mosquitoes is a major challenge, particularly with species of Anopheles and Aedes. Therefore, this study assessed the efficiency of four different collection methods (three trap types and MLCs) in collecting adequate numbers to secure the sample size of Ae. niveus for MX. The number of Ae. niveus females collected from all the three devices (BGS, GT and HBDNT) was very small. GT, which is being used for collection of Cx. quinquefasciatus did not seem to adapt to Ae. niveus, the tree-hole breeding mosquitoes. BGS-lure and HBDNT set at daytime, signalled the presence of human blood meal to the host seeking mosquitoes. Such trapping methods attracted predominantly Ae. albopictus and Ae. aegypti and very few Ae. niveus. Thus, the category of lures did not adapt to Ae. niveus. The HBDNT used human bait for attraction of mosquitoes but the bait is secured from landing and biting. Two nets as a physical barrier could have limited the entry of mosquitoes by reducing the human signal from the human bait inside the inner net and diverting to other available hosts in the proximity.

In MLCs, the mosquitoes aggressively pursue the host. Therefore, the yields are more than the HBDNT and other passive collections. The MLCs yielded collections from all the sites and in a total of 257 hours of collections, it was possible to achieve more than the minimum sample size required. However, comparison of results of MLC with those of other collections hasthe limitation that collections were conducted at different time periods and using different collecting methods. In Samoa, studies of the sampling of Ae. polynesiensis (diurnal) and Ae. samoanus (nocturnal) vector mosquitoes showed that BGS traps with any category of lure captured a greater number of mosquitoes in comparison to both CDC traps and the MLC [32]. Our study showed that MLC was better than the trap collections. This method, however, has operational and ethical issues and can be used until more productive devise is available as an alternative method.

The RT-PCR assay developed for periodic W. bancrofti could detect the DNA of DspWB. Lack of parallel data on vector infection assessed by dissection and microscopy limits us from commenting on the false positives. The method used in this study for DNA extraction is cheaper than the other extraction methods [21] and hence should be technically and operationally feasible. Our earlier study of vector infection based on dissection and microscopy prior to LF elimination programme in the Nancowry group of islands showed 2.65% and 0.5% infection and infectivity respectively in Ae. niveus mosquitoes [11]. No data on vector infection was however available during or following MDA. The present MX study showed relatively low vector infection level (0.43%) after nine rounds of MDA but still higher than the provisional threshold of 0.1% derived for Aedes transmitted filariasis [16, 19]. The assay detected vector infection in nine villages from all the four islands and in all these villages was more than 0.1%. The Mf prevalence in the islands ranged between 0% and 9.3% in different villages with an overall Mf prevalence of 1.7% after nine rounds of MDA [15]. In Samoa, MX conducted after seven rounds of MDA showed 4.7% vector infection in Ae. polynesiensis when Mf prevalence in human was as low as 0.6% [32].

In the mainland, Cx. quinquefasciatus is the vector of widely prevalent nocturnally periodic Wuchereria bancrofti and three species of Mansonia are the vectors of Brugian filariasis confined to certain pockets. In these islands, Cx. quinquefasciatus was the other vector species collected. But we did not consider this mosquito as it is a nocturnal biting mosquito and hence not appropriate for testing filarial infection. Cx. quinquefasciatus, the omnipresent and ubiquitous vector of nocturnal periodic form of Wuchereria bancrofti was less abundant (1.7% of the total mosquitoes collected) in our earlier study in the sites selected for the study. None of the Cx. quinquefasciatus dissected for filarial infection was found positive [11]. In the present study we could collect only 12 female mosquitoes of Cx. quinquefasciatus from gravid traps and HBDNT collections and hence were not included for molecular assay.

MX has been observed to be an indicator of human filarial infection transmitted by different mosquito vectors in wide-ranging ecological situations in American Samoa [7, 20], French Polynesia [33], Egypt [16, 34], Sri Lanka [6], Sierra Leone [35], Ghana [36] and India [5]. In American Samoa, MX results showed evidence of ongoing transmission of W. bancrofti by Ae. polynesiensis after two successful TASs, which was confirmed by TAS3 [37].

In view of the ongoing transmission, MDA was continued in Nicobar district including the four Islands with additional inputs as per the national accelerated plan of LF elimination [38]. A pilot study with mass distribution of DEC fortified salt as a supplementary measure to the ongoing MDA was implemented in two islands in 2016 and after one year of intervention total interruption of transmission was achieved [15]. MX was not carried out after DEC salt distribution and the impact was assessed only from infection in human. DEC salt intervention is now being implemented in the remaining two islands. In this context, MX can be a useful supplementary surveillance tool to TAS for evaluation of the impact as well as post-MDA monitoring. Exploring an alternative method of mosquito sampling to MLC will make MX operationally more feasible.

Conclusions

MLC is productive in sampling day biting and forest dwelling Ae. niveus transmitting DspWB and other trapping devices were not efficient. However, there is a need to identify an alternative vector sampling method and standardised protocol in view of ethical concerns about involving human bait in the field. RT-PCR assay developed for nocturnally periodic WB can be used for detecting DspWB DNA. MX in Ae. niveus can be a supplementary to TAS and post-MDA period for early detection of risk of transmission.

30 Sep 2019

Dear Dr. SHRIRAM:

Thank you very much for submitting your manuscript "Molecular xenomonitoring of diurnally subperiodic Wuchereria bancrofti infection in Downsiomyia nivea (L) after nine rounds of Mass Drug Administration in Nancowry islands, Andaman and Nicobar Islands, India" (#PNTD-D-19-01106) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

When you are ready to resubmit, please be prepared to upload the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file); the other a clean version (uploaded as the article file).

(3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type.

Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) If applicable, we encourage you to add a list of accession numbers/ID numbers for genes and proteins mentioned in the text (these should be listed as a paragraph at the end of the manuscript). You can supply accession numbers for any database, so long as the database is publicly accessible and stable. Examples include LocusLink and SwissProt.

(5) To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

We hope to receive your revised manuscript by Nov 29 2019 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit a revision, go to https://www.editorialmanager.com/pntd/ and log in as an Author. You will see a menu item call Submission Needing Revision. You will find your submission record there.

Sincerely,

Audrey Lenhart

Associate Editor

PLOS Neglected Tropical Diseases

Michael French

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

In addition to the addressing the comments from the reviewers, please note that Downsiomyia nivea is no longer the correct species name. The authors are referred to Wilkerson et al. (2015) and are requested to use Aedes niveus.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Objectives need to be more clearly defined

Reviewer #2: Objectives of the study were obscured when the authors failed to clearly visualize the context. Are they evaluating molecular xenomonitoring for infection status or intervention or both? The type of intervention they mentioned here is not discussed anywhere in the manuscript. There a number of trapping method analysis for the specific mosquito species which could also come in to the objective if they say something in the background. The sample size is not satisfactory because the prevalence they assumed is not correct. Also some analysis are not supported by the sample size. Not proper tools are followed for data analysis as these are count and possibly over dispersed. Proper definitions are necessary for the densities and rates. Proper management of volunteer engagement is not mentioned could be an ethical issue.

Reviewer #3: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

→ Issues are raised with the stated objectives in the Peer Review comments document; authors may want to consider adding (acknowledging) what is believed to be a secondary objective

-Is the study design appropriate to address the stated objectives?

→ Study design is not clearly stated but readers can understand what the authors did, and for the stated purposes their design seems fine but more details are definitely needed

-Is the population clearly described and appropriate for the hypothesis being tested?

→ Population is described but could be more clear (though from tables it is possible to gain additional details)

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

→ Unclear, especially as all assumptions are not detailed; suggest this paper go to statistical review once it is in better shape (including because it is not clear if all necessary adjustments are made or if authors could have done different calculations based on their existing data – hopefully next version will clarify this to the point this can be better assessed).

-Were correct statistical analysis used to support conclusions?

→Unclear, see above (eg they need to add more details)

-Are there concerns about ethical or regulatory requirements being met?

→Possibly – though as this was funded and performed by Indian government agencies it could be tempting to assume ethical clearance was fine. Still, authors need to add more details, including the actual clearance numbers, and especially because they used human landing catches and human baited double trap nets – neither of which are sufficiently described in detail (including the steps taken to ensure each were done with full ethical consideration and to minimize human risk/harm).

Additional Comments on Methods (from Reviewer Document to be Uploaded):

METHODS

Overall: This section needs better organization, including that some information that applies to all collected mosquitoes (e.g., processing, pooling) is currently described in one sub-section (e.g., gravid traps) and that some information in this section actually belongs in other sections (e.g., Results). ALSO please review PLoS NTD guidance on section headings (line 116 does not conform).

Study area:

1) What were the dates of this study?

2) Line 120: Here it is clear the 4 islands in the study are endemic for only DspWB, but would be helpful to state this earlier (e.g., lines 82-90, above)

3) Line 124: Please spell out or denote abbreviations or acronyms before their use (e.g., line 28)

4) What is ‘Nancowry Tehsil’? If not an important detail, can be removed (or should be defined)

5) 125-8 and 134-135: a) These seem to present similar information; suggest presenting in one section only; b) what is ‘post-tsunami’ (please state year); c) are there any other important features (e.g., standard numbers of residents, number of houses per shelter, sanitation features) that may relate to mosquito parameters?

6) Territorial area and number of houses are provided; would be helpful to have total population or perhaps better yet population density.

Sites for mosquito sampling:

1) Line 131: It appears ‘i.e.’ is used incorrectly

2) Line 132: BGS not defined yet

3) See q5, above

Mosquito sampling:

1) Some info on assumption of 1% mf prevalence (when last survey showed above 3%) would be helpful

2) Does author have a source for sample size calculation?

3) Line 140: three of ‘five’ islands or ‘four’ per elsewhere?

4) Lines 148-9: Were traps rotated? How often were nets etc. collected?

5) Line 157-63: Can authors explain when (hours set/collected), for how long (how many days), and locations (more specifics on placement)?

6) Line 172: Were gravid traps battery operated?

7) Line 174-5: How did authors check for differences in mosquito abundances as collected by the 2 different infusions (just says ‘were compared’)?

8) Lines 176-8: Can authors give more detail on where traps were set, and what does ‘closer’ mean (closer than what? Than the location of porches used for BGS?)

9) Authors state gravid traps left for 3 days; were BGS traps also left for 3 days, and if so authors should state (in any case authors should provide congruent details on each trap method)

10) Line 183: Can authors explain why gravid traps were set between 6-12 whereas HBDNT were conducted 8am – 1pm (slight difference), could have affected results if adjusted (authors should provide timings re: BGS traps as well)

11) Lines 184-7: Several things: a) Do these methods apply for mosquito processing for all trapping method (i.e., should they be in a separate section, and not only under ‘gravid traps’ as they currently are? b) Can authors provide more detail on how species/sex were identified (e.g., by a taxonomist? entomologist? trained field staff?) [Lines 204-5 mentions stereomicroscope and taxonomic keys – is that the same for this section?] Authors could organize these sections better

12) Line 194: Who was the ‘native Nicobarese’- was it only one person, or several people, and who was the person(s)? It is assumed a child was not used so can authors state more about the ‘bait’?

13) Line 196-7: What does this mean: ‘…attended to his individual needs…’? (eating? urinating? what? And could any of this ‘attending to needs’ have affected mosquito attraction?

14) Can authors please explain the choices of collections / trap days / hours: Lines 165 (BGS): 235 collections / 47 trap days / 2350 hours; Line 178 (Gravid): 154 collections / 51 days / 918 hours; Line 206-7: 51 HBDNT collections / 255 hours.

15) If some traps were left several days (e.g. gravid traps for 3 days, maybe BGS if authors clarify) what methods did authors take to avoid dessication? (and, in Results, what proportion of mosquitoes was unable to be processed via PCR?)

16) Table 1: Please edit; inconsistent capitalization; abbreviations not defined (‘Gr.’) and acronyms not used (HBDNT); it’s leaF not leaVE; ‘Sylvan’ used in text ‘Sylvatic’ used in table, ‘Hours’ v ‘Hrs’ etc. etc. – this needs much cleaning up! It is authors responsibility to clean these tables, PLoS NTDs or most other journals will not copy edit tables

17) Line 210-11: These are results, should not be here; MLC info should be new (its own) paragraph –in fact lines 210-25 need seriously editing/organization to remove off-topic issues and keep on track with MLC info.

18) Line 212-15: Is this the study period for all 4 collection types, or only MLCs? Best to clearly state when the studies took place, and state this early on in the Methods section.

19) Line 220-21: What is ‘necessary care’ – authors could include information they used to convince ethics committee; as written this is very thin on details to convey that MLC was done with appropriate consideration/caution.

20) Lines 221-25: Again, does this pertain to how all mosquitoes were processed, pooled, etc., or just the ones from MLC? There should be a section on how mosquitoes were preserved, pooled, etc. that is comprehensive or provides same information for each of the 4 types of collection methods (2 traps, HBDNT, MLC). Please see comments above re: providing more detail on how mosquitoes were handled, speciated, staged, pooled, stored, and prepared for molecular analysis especially who did these steps.

21) Lines 229-30: Isn’t the VCRC part of the ICMR? Please sort out acronyms.

22) Line 229 and re: RT-PCR: Two things: a) Here it’s finally stated (that real-time PCR is being used); please state this in Abstract and other places requested above; b) Please also consider using acronyms consistently – RT-PCR defined line 229 then acronym is dropped by line 232.

23) Lines 253-55: What kind of ANOVA – can authors be specific? How did authors account for potential effects of using different infusions for gravid traps (and any other parameters that were different within and between methods – e.g. time of day, I if more than one ‘bait’ was used, etc.)?

24) Line 260: define qPCR; check all acronyms in text.

25) Lines 264-66: Some of this information should be included above in Methods, including re:MLCs and HBDNT (re: informed consent, no children). The ethics section is very limited, and normally includes a study protocol/ethical approval number – can authors provide this (should be able to if funded by and conducted in conjunction with the Indian government, as it seems).

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Analysis of the Results need reorganisation

Reviewer #2: Data analysis is not sufficient and as mentioned earlier proper tools are not used. Results are not presented clearly and not described properly. The authors have to remove one table.

Reviewer #3: -Does the analysis presented match the analysis plan?

→Analysis plan not clearly or comprehensively stated; thus it is hard to assess. More details need to be added, and Methods and Results sections should be reorganized – this may help clarify on next draft.

-Are the results clearly and completely presented?

→ Results section should be reorganized – This includes that some Results (at least one line) were presented in Methods section.

-Are the figures (Tables, Images) of sufficient quality for clarity?

→ Several issues exist with Tables, mainly copy-editing and editing. Some of the tables seem to be excessive or provide detail that is unnecessary to understanding the paper. Figures and Tables were added into the main text, not always put immediately after first mention, and then added at the end of the document again - which is NOT following the submission rules of PLoS NTDs (strange the journal accepted that and sent out to Peer Reviewers in this form).

Additional Comments on Results (from Reviewer Document to be Uploaded):

RESULTS

Editing / copy-editing required: Recommend this is edited (organization) copy-edited (readability, grammar) as clearer writing could frame the results better.

1) Line 277: Ae. edwardsi actually has comparable proportion to Do. nivea, the vector of interest – could authors comment on this or provide more information here or in Discussion?

2) Line 278: First mention of ‘Ar’ without defining (spelling out) this mosquito.

3) Lines 278-9: What does this mean - ‘were comparable’ – could authors provide more information?

4) Table 2: Please clean tables carefully- a)What is DBNT? (text refers to this throughout as HBDNT); b) how is table organized? Mosquitoes aren’t alphabetical, or by species; please consider rearranging, even putting Do. nivea first as it’s the stated mosquito of interest.

5) Line 291: Is that significant? If authors are not considering p>0.05 then can authors clearly state this in Methods?

6) Text presents information on 6 mosquitoes but under the heading of BGS, with the following table (Table 2) showing 6 species collected via all trapping methods save MLCs. By the next text referring to 3 mosquitoes and the following table (Table 3), only 3 mosquitoes are presented via BGS. Why do authors provide further info on these 3 mosquitoes?

7) Line 300: Figure missing number; also figure if published goes immediately after paragraph where first mentioned (not at end of page). Still/again, please note submission is not supposed to include figures inside main manuscript.

8) Check consistency in Tables and Figures; most use acronym DBT whereas text (including above in line 303 as opposed to figure in line 309) uses HBDNT.

9) Just checking – is it correct that the authors did not collect information on physiological status (e.g., unfed, bloodfed, semi-gravid, gravid)? This information should be stated, including if the pools were comprised of a mix of all females of all physiological status or if the pools included specific types (e.g., more bloodfed).

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: Conclusions are supported by the data in the manuscript and are of pubic health relevant. However, it could be written in a better way. Limitations are not discussed. This article would add another evidence of the importance of molecular xenomonitoring.

Reviewer #3: -Are the conclusions supported by the data presented?

There are significant issues with the Discussion section (per PLoS NTDs section title rules, this section is called Discussion not Conclusions). This includes the very last part the authors include, called ‘Conclusions’ There is a serious lack of detail, reflection (in relation to their own work as well as contrasted by others), and discussion of limitations. These points are further flagged in the Peer Review document to be uploaded next but many are also copied below.

-Are the limitations of analysis clearly described?

Not sufficiently –see above and below

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Not enough – this paper needs a more ‘global’ view on their work, including in relation to sites with other vector/parasite combinations.

-Is public health relevance addressed?

Kind of, but to be honest this seems to be more of scientific interest (rare parasite/vector combination) than of great public health interest, though the 10,000 or so people who live in the study sites could benefit greatly from better understanding of this vector/parasite combination.

Additional Comments on Discussion (from Reviewer Document to be Uploaded):

DISCUSSION

Editing / copy-editing required: Recommend this is edited (organization) copy-edited (readability, grammar) as clearer writing could frame the results better.

1) Line 351: What is an ‘appraisal’ – what test was used? What method? Who did this?

2) Line 352: a) What are sentinel/spot check islands? B) What is NVBDCP?

3) Line 354-5: What does this mean – ‘mf’ and ‘day samples’? If sampling in daytime is ideal, isn’t it best to detect antigen (e.g., via ICT as in TAS, with operational (daytime) feasibility being one reason WHO recommends ICT for TAS) as opposed to mf (e.g., thick blood film, filtration, etc.), which requires a night blood draw due to nocturnal periodicity for W. bancrofti? If authors suggest DspWB can be subject to daytime blood draws wouldn’t they be missing out on the rest of transmission if in A & N they also have (nocturnal periodic) W. bancrofti? if Nicobarese are not available in the day (assuming authors meant ideally they’d test for antigen via ICT) then couldn’t authors use parasitological methods such as TBFs? The arguments here are not clearly stated

4) Line 353-57: this is a non-traditional argument for MX; many often focus on the operational infeasibility of surveying humans as MDA drives prevalence (mf, antigen) levels down; it seems here authors are arguing for MX in relation to the inability to contact some Nicobarese during the daytime (i.e. practical issues) as opposed to traditional arguments of diagnostic limitations after MDA and that sampling requires more and more humans as population infection prevalence decreases.

5) Lines 359-362: Check acronyms – MX? XM? And naming conventions - Aedes

6) Lines 362-64: Please clarify what this means – ‘achieve the sample size’ In the end, authors did compare collection methods and commented several times on productivity. Per earlier comments authors may choose to make this a secondary objective or comment more on the findings related to their discoveries of trap type. Also here and above authors mention 3 trapping devices but could frame this as 4 different collection methods (3 trap types and MLCs)

7) Line 367: This is the first time authors mention Do. nivea is forest dwelling, despite commenting a lot on forests above (e.g., Methods and how sampling sites were chosen); authors could mention this earlier (e.g., Introduction).

8) Lines 368-70: Please explain this logic (describe how former sentence means latter) and edit the sentences for readability.

9) Lines 373-75: These concepts are not introduced until now and should be included in Methods.

10) Line 377: What are ‘more efforts’ – please clarify, cite.

11) Line 378: Please cite some evidence/examples of the ‘matter of fact.’

12) Lines 380-83: Why is this evidence presented (presumably as a contrast to study findings) when the authors do not discuss their own findings in relation, in particular to BGS trap productivity?

13) Lines 372-94: Please improve organization –including because concepts are unevenly and confusingly divided between paragraphs, repeated (lines 380-83 and 390-92), and presented without sufficient contrast to or reflection on relation to authors’ own results (e.g., Samoa data).

14) Line 396: What assay was developed? Do authors mean the assay they used (also please cite) and is this the first time the assay was used for DspWB (if so, authors could state this)?

15) Lines 396-411: Would help to frame threshold and mf prevalences on same scale or help the reader interpret these; also line 402 is first mention of ‘infectivity’ so would be

16) Line 401: What is the RMRC? Please define all acronyms

17) Lines 404-6: This seems like a strong statement – what about any issues with the PCR including false negatives? There seems to be no discussion of difficulties of diagnostics, including reliability of PCR methods (including that authors seemed to use a protocol developed for nocturnally periodic W. bancfrofti but yet use it for DspWB, even if this same protocol was used in the previous study). Authors could expand on other limitations surrounding molecular analysis, including if any existed related to the speciation, pooling, kits, or any other steps or parts of the molecular analysis.

18) Lines 417-19: This sentence makes little sense in context here – why do authors present examples of general/other MX sites, discuss their results, and then discuss American Samoa (and with no citation) without contrasting to their own results?

19) Lines 419-20: How does the earlier part of sentence support the conclusion after ‘thus’ – this is a very confusing few sentences – the first part of this sentence relates to the previous sentence, the second part seems to potentially relate to the first part but the first part of this sentence but is not the sole justification for authors to conclude the second part. Also – potentially due to verb tense –as written it seems that authors use the American Samoa study results to indicate MX can be considered supplementary (in general). Suggest complete revision of this paragraph.

20) Lines 421-26: Please revise, including for writing, punctuation, and (suggested) selection of themes presented in the final paragraph of Discussion before ‘Conclusions’

21) Line 422: Here is the first time authors mention 11 rounds of MDA; please clarify here and elsewhere, including per earlier comments (also, see title).

22) Lines 421-26: The first parts of the paragraph present different info on MDA – including introducing more round than previously stated and unpublished data on DEC salt, both of which seem important for the final conclusion about MX being a potential tool to assess transmission but neither of which are discussed in sufficient detail to support or further explain this conclusion. Also, authors may want to consider framing this idea (as they have previously) that MX can be used as a supportive or additional tool to assess transmission but still cannot be recommended as the only tool due to a variety of issues (including, but not limited to, difficulties related to diagnostics in the mosquito – something which, again, authors may want to further discuss in this section).

23) ‘Conclusions:’ Authors have previously stated their aim was not to test different collection methods, but 50% of the sentences in their conclusions focus on this, suggest reconsider framing if this was part of the aim and if so rebalance the paper or devote more time to discussion on collection methods (including limitations of each, in Discussion section).

24) Lines 434-36: Again, authors may want to take care using the term ‘alternative’ rather than ‘supplementary’ or ‘additional’ as MX literature in general, and this paper in particular (and for this vector/parasite combination) do not present sufficient evidence to support that MX can be used as the only (i.e., ‘alternative’) tool to assess if transmission is ongoing.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: The authors need to re-analysis the sample size and data analysis specified in the comments. Proper description of density/rates are lacking.

Reviewer #3: Please see my comments above and in the document I am going to upload. The presentation of data in the tables could be improved, as well as the presentation of data (including if enough data was presented) in the Results section.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: Molecular xenomonitoring is a surveillance strategy to detect circulating filarial parasites in an endemic population which is either under intervention or post-intervention. It is an important surveillance tool when human sample collection is difficult. This manuscript provided data from an endemic area where parasite load is high even after many rounds of MDA. The parasite is diurnal in that area where most of the people are outside of their house so blood collection for diagnosis is difficult. Also they undertook many sampling strategies for the specific vector which will be an important add to the scientific literature and benefit scientists and researchers of similar interest. However, the manuscript has failed to clearly portrait its objective. Their assumption for sample collection is incorrect and lacks proper analysis. Some of the analysis lacks enough data. Description could be made in a better way as the article needed to be improved by rewording.

Reviewer #3: Additional Comments - Overall, on Abstract, Author Summary, Introduction as these were not listed above as well as Final comments (from Reviewer Document to be Uploaded):

NOTE: This manuscript should be edited for English, scientific writing, and organization, as well as copy-edited for typos and grammatical errors.

Overall Comments: The paper presents interesting information, aiming to provide evidence on a parasite-vector combination that is infrequently reported in the literature as well as in a setting (islands) where elimination may be achievable (theoretically) faster than elsewhere.

First, the entire paper should be edited and copy edited for grammar (e.g., punctuation, standard norms in capitalization, subject/tense agreement, abbreviations/acronyms defined before first use) and readability (both scientific and standard English). Better organization – between and within sections – could help the reader understand key concepts, especially in the Methods (what authors did) and Discussion (what their findings mean), that are not necessarily clear as the paper currently stands. Some small editing points include that words like viz.’ (used 7 times) and i.e., are used incorrectly; larger issues include problems with commas and other punctuation, as well as many problems with abbreviations and acronyms (incorrect, undefined before first use, or sometimes used once without being spelled out elsewhere).

Next, authors have not submitted the manuscript per instructions for PLoS NTDs - Tables and Figures are supposed to be separate/at the end? If the journal has sent paper like this to reviewers combining all elements then please note there are several mistakes: e.g., line 309 (no Figure number, figure titles should be at the bottom as opposed to table titles on top). Please check the guidance and revise / sort out.

Some important thoughts – particularly those related to mosquitoes and MX – are not carried through completely to their logical conclusions. This paper needs much better organization to help the reader follow each thought, including with the work of these authors as well as in relation or with consideration of that of other colleagues/study sites. The title of the paper is MX for post MDA surveillance, so the authors should focus on MX and the entomology but relate their findings more clearly to what this means for MDA, and if MDA can be stopped after what is currently presented as a confusing (or conflicting) number of rounds of MDA. Still, if the authors are able to address many of the comments below (as this reviewer hopes) it may strengthen the paper to warrant a recommendation of acceptance.

************************************************************************************

FOCUSED Comments:

************************************************************************************

ABSTRACT

1) BACKGROUND (lines 33-4): What does this sentence mean – could it be reworded? Isn’t MX conducted to provide follow up (to baseline, which is assumed to have provided evidence of pre-MDA infection levels in or before 2004 when MDA commenced) data on infection prevalence in humans? That is, isn’t this entire study being done in order to assess the impact of MDA in the form of DEC-fortified salt (comparing previous baseline data with this follow-up data)?

2) METHODS: Could benefit from more info on design and molecular methods (e.g. pooling details, PCR type stated clearly). Did authors test for sign cant differences in abundance and infection between islands and within villages (and if so, how)?

3) RESULTS: (line 42): ‘productive’ is a subjective term; could authors state numbers of females captured per trap type? Also, could they indicate if there were significant differences in infection between the 4 islands, or within the 25 villages (each)?

4) CONCLUSIONS: (lines 48-99): Based on the 2 sentences in these conclusions and evidence from the study there is no compelling link or justification for this sentence to be the main conclusion. Suggest authors rephrase this section. Also, if the stated study aim was to assess progress of MDA after 12 cycles via screening for infection in mosquitoes, shouldn’t the authors present some conclusions related to what their results mean in relation to MDA based on mosquito infection – i.e., something programmatic? As written, MDA progress itself (as evaluated by MX) isn’t addressed.

5) KEY WORDS: Are some cut off and/or do authors want to consider some more (e.g., vector species, MDA, DEC-fortified salt, etc.) to help those searching key words to find their manuscript?

6) Abstract would benefit from copy-editing (some missing punctuation, subject/verb/tense agreement, and perhaps streamlined wording).

AUTHOR SUMMARY

1) Recommend editing – among other things, several sentences don’t make grammatical sense as written (e.g., first sentence, lines 63-64)

2) It seems ‘viz.’ is not always used correctly, including author summary (and is overused throughout this manuscript)

3) Wuchereria bancrofti could be abbreviated in second use; conversely vector name is not fully written out in first use, nor is MDA (abbreviations should be written out in here, like in Abstract, and then again redefined in first use in main text)

4) Lines 64-8: could be presented more basically (e.g., ‘filarial infection in mosquitoes was above the acceptable threshold, indicating XXX; ‘filarial in infection in humans was also above the acceptable threshold, indicating YYY’) – ideally the authors would plainly state what infection in humans and mosquitoes meant about filarial transmission in terms that the lay person would understand, rather than stating exact numbers without sufficient interpretation.’

5) Similar to comments on Abstract, authors could draw a better line between study conclusions and why MX is supportive tool for assessing MDA impact.

INTRODUCTION

Overall a good, succinct Introduction but a few suggested modifications:

1) Please check punctuation (commas and parentheses are especially misused, including those that are extra or lacking), grammar, and phrasing.

2) Lines 74-5: Please capitalize GPELF words accordingly.

3) Lines 75-6: GPELF has 2 ‘pillars:’ 1) interrupting transmission (MDA and IVC/IVM) and 2) managing morbidity; please note ‘pillar 1’ is not just MDA. It seems especially important that a paper on MX highlights the importance of the vector (IVM/IVC for interrupting transmission as well for surveillance (MX) to detect if transmission has been interrupted).

4) Lines 79-80: Is India only using MDA for ‘pillar 1’? Please clarify per point (3) above.

5) Lines 82-5: It is unclear if B. malayi and/or nocturnally periodic W. bancrofti also exist in A&N; seems DspWB exists but at this point in paper it is not clear if this is the only parasite present. [ed: line 120 states only DspWB exists in study sites but still not clear if this is only for study site (those 4 islands) – i.e. if A & N have more than 1 parasite]

6) Lines 85-90: It is unclear if other vectors exist in Nicobar (e.g. Culex, Mansonia); per this paragraph paper focuses on Do. nivea and Ae. niveus (and per title the former). Authors could present more context – e.g., if other vector/parasite combinations then what proportion of transmission is estimated attributable to those vs. Do. Nivea / DspWB.

7) Lines 92-113: Appears that both nocturnally periodic W. bancrofti and DspWB exist, but no mention if other vectors transmit these parasites and if so if the national program targets them as well (assuming they target vector per q3, above, not just parasite via MDA)

8) Lines 93-5: Authors could briefly explain TAS (or at least that it’s WHO mandated in certain circumstances) and why this site was not eligible (e.g., include recommended thresholds) for readers who may be unfamiliar (e.g., those focused on mosquito rather than human surveillance).

9) What were the dates of this study, and of MDA? In Abstract, Methods, and Results this is not provided; from Introduction, if MDA began in 2004 and 9 annual round were given, does this mean MDA finished in 2013 or 2014?

10) Lines 99-100: Are some words missing?

11) Lines 104-8: This sentence is confusing (perhaps also due to commas placement) – a) what is ‘the statistically robust method’ b) suggest authors do not use ‘this form’ of filariasis but rather DspWB if that’s what they mean.

12) Lines 108-10: Several things – a) this sentence is confusing, including phrases such as ‘…interventions such as mass DEC-fortified salt evaluations…’ (evaluations are not interventions); b) suggest authors consider softening this phrase by replacing ‘will’ with e.g., ‘may’ or ‘could’; c) here or elsewhere suggest authors include the idea that MX alone is not sufficient to indicate if transmission has been interrupted; human surveillance is still required.

13) Lines 110-11: Authors state the aim was ‘to assess vector infection…’ but title specifically states that the parasite of interest was DspWB and the vector of interest was Do. nivea – so suggest authors restate this sentence. However based on the last sentence here, as well as the Methods and Results sections, it seems that a secondary aim was to determine the best mosquito collection method); if this interpretation is correct perhaps authors could rephrase to make the aim more comprehensive.

14) Side note/small point: Authors use ‘MX’ on pages 1, 7, 8, and 9 (all pre manuscript if manuscript technically starts with ‘Introduction’) and then not again until the Discussion. They then switch to ‘xenomonitoring’ and don’t take advantage of abbreviation again until pages 31 and 32. Similarly, authors use LF in 5 instances all before page 5, and then not again. Suggest authors consider if they want to use ‘MX’ and ‘LF’ (and perhaps other acronyms) more extensively in paper after first defining them in Introduction.

************************************************************************************

FINAL Comments:

************************************************************************************

This paper needs better organization, editing (for balance – e.g., minimizing some things or removing some details or even tables, while adding other more important details in other sections), and copy editing. In particular, the paper needs more detail in the Methods and Discussion sections. Finally, the title is about MX but the authors do not focus on MX as much as they could – including general issues (including with diagnostic limitations, findings from other settings, their methods, and their study’s limitations). They also do not identify or accept that they spend significant time discussing collection methods; it may strengthen the paper to add this as a secondary aim. Still, once this is completed, the authors will b able to provide important information for a rarely studied/reported vector/parasite combination so this work would certainly add to LF knowledge in general. Also, this work is interesting for several reasons (including the use of HBDNT and MLCs that are infrequently used these days) and the fact that the work occurs on islands, which are good indications or seemingly easier places to achieve elimination than some other land-locked sites. So, this reviewer wants to convey that - despite number and detail of the above comments - the work of these authors can contribute many useful points of knowledge to the LF community and so this reviewer thanks the authors for their submission and wishes them good luck in addressing these and other peer review comments.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Hoda A. Farid

Reviewer #2: No

Reviewer #3: No

Submitted filename: Reviewer 393.pdf

Click here for additional data file.

Submitted filename: Reviewer Comments.docx

Click here for additional data file.

Submitted filename: PNTD-D-19-01106_Premkumar_LF_India_R1_Sept_2019.pdf

Click here for additional data file.

5 May 2020

Submitted filename: Reviewer Comments.docx

Click here for additional data file.

17 Aug 2020

Dear Dr. SHRIRAM,

Thank you very much for submitting your manuscript "Molecular xenomonitoring of diurnally subperiodic Wuchereriabancrofti infection in Aedes (Downsiomyia) niveus (Ludlow, 1903) after nine rounds of Mass Drug Administration in Nancowry islands, Andaman and Nicobar Islands, India" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Many thanks for addressing the comments from the reviewers. The incorporation of reviewer feedback has very much strengthened this manuscript. There remain a few outstanding points that should be addressed:

1. Please include a description of the timing of the MLCs per village. For example, an additional column in Table 2 to say which month(s) the MLCs occurred in each village.

2. Please explain why MLCs were only conducted in sylvan areas

3. There could have been bias in assessing how effective the traps were compared to MLCs because the traps were mostly set during different months of the year (described in the text as not corresponding to the peak times for Ae. niveus) and also at different times of the day (also described in the text as not corresponding to the peak biting times of Ae. niveus). This should be mentioned/explored in the discussion.

4. Mosquitoes were clearly pooled by location, but were they also pooled based on collection day?

5. In line 422, the authors mention that Cx. quinquefasciatus were not analysed because they are day biting. Especially in this scenario where W. bancrofti is diurnally sub-periodic, it would stand to reason that day-biting Culex could also be an important vector. The authors should mention why this is not apparently the case--has it already been studies on these islands?

6. Lines 432-433 specifically mention that 2 islands that had received DEC salt achieved total disruption of transmission. Did the MX data arising from this study corroborate this finding?

7. Figure 3 (which is currently mis-labeled as Figure 1): please adjust the y-axis to better accommodate the 2170 Ae. niveus captured by MLC; as it appears now, it is confusing to have 2 separate y-axes. To avoid loss of resolution when visualizing the other values, the authors may consider adding a 'break' on the y-axis to demonstrate that the uppermost value is not in scale with the others.

8. Please edit the full manuscript for English grammar and readability.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Audrey Lenhart

Associate Editor

PLOS Neglected Tropical Diseases

Michael French

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Many thanks for addressing the comments from the reviewers. The incorporation of reviewer feedback has very much strengthened this manuscript. There remain a few outstanding points that should be addressed:

1. Please include a description of the timing of the MLCs per village. For example, an additional column in Table 2 to say which month(s) the MLCs occurred in each village.

2. Please explain why MLCs were only conducted in sylvan areas

3. There could have been bias in assessing how effective the traps were compared to MLCs because the traps were mostly set during different months of the year (described in the text as not corresponding to the peak times for Ae. niveus) and also at different times of the day (also described in the text as not corresponding to the peak biting times of Ae. niveus). This should be mentioned/explored in the discussion.

4. Mosquitoes were clearly pooled by location, but were they also pooled based on collection day?

5. In line 422, the authors mention that Cx. quinquefasciatus were not analysed because they are day biting. Especially in this scenario where W. bancrofti is diurnally sub-periodic, it would stand to reason that day-biting Culex could also be an important vector. The authors should mention why this is not apparently the case--has it already been studies on these islands?

6. Lines 432-433 specifically mention that 2 islands that had received DEC salt achieved total disruption of transmission. Did the MX data arising from this study corroborate this finding?

7. Figure 3 (which is currently mis-labeled as Figure 1): please adjust the y-axis to better accommodate the 2170 Ae. niveus captured by MLC; as it appears now, it is confusing to have 2 separate y-axes. To avoid loss of resolution when visualizing the other values, the authors may consider adding a 'break' on the y-axis to demonstrate that the uppermost value is not in scale with the others.

8. Please edit the full manuscript for English grammar and readability.

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

31 Aug 2020

Submitted filename: Response_outstanding issues.docx

Click here for additional data file.

1 Sep 2020

Dear Dr. SHRIRAM,

We are pleased to inform you that your manuscript 'Molecular xenomonitoring of diurnally subperiodic Wuchereriabancrofti infection in Aedes (Downsiomyia) niveus (Ludlow, 1903) after nine rounds of Mass Drug Administration in Nancowry islands, Andaman and Nicobar Islands, India' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Audrey Lenhart

Associate Editor

PLOS Neglected Tropical Diseases

Michael French

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

14 Oct 2020

Dear Dr. SHRIRAM,

We are delighted to inform you that your manuscript, "Molecular xenomonitoring of diurnally subperiodic Wuchereriabancrofti infection in Aedes (Downsiomyia) niveus (Ludlow, 1903) after nine rounds of Mass Drug Administration in Nancowry islands, Andaman and Nicobar Islands, India," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases