Detection of Wuchereria Bancrofti Through Molecular Xenomonitoring of Putative Mosquito Vectors in the Philippines

Introduction

Lymphatic filariasis (LF) is a major public health burden in which approximately 863 million people in 47 countries are still suffering from this neglected tropical disease [1]. LF is a parasitic disease caused by three species of thread- like nematodes: Wuchereria bancrofti, Brugia malayi, and Brugia timori. Bancroftian filariasis is responsible for 90% of infections, while the remaining 10% is attributed to brugian filariasis. These parasites spread from person to person through mosquito bites. Mosquitoes belonging to the genera Aedes, Anopheles, Culex, Mansonia, and Ochlerotatus are incriminated as filaria vectors [2, 3]. LF infection is acquired via repeated bites of infected mosquitoes for a duration of months or years, yet chronic conditions appear several years later such as lymphoedema, abnormal enlargement of body parts, and hydrocoele in men. Infected individuals experience severe pain and permanent physical disability, as well as become victims of social stigma and poverty [1].

In 2000, the World Health Organization (WHO) launched the Global Programme to Eliminate Lymphatic Filariasis (GPELF) as a response to end the escalating LF cases worldwide. GPELF has two key strategies: (1) to interrupt parasite transmission by providing mass drug administration (MDA) in endemic areas; and (2) to prevent disease progression and alleviate suffering of infected individuals through morbidity management and disability prevention (MMDP) [1, 3]. Annual MDA, using a single dose of albendazole in combination with either diethylcarbamazine (DEC) or ivermectin for a span of at least five years, was the primary strategy anticipated to reduce density of circulating filaria parasites below the transmission threshold. Monitoring the success of MDA requires implementation of a surveillance program to confirm the interruption or reestablishment of disease transmission during the post-intervention period [2, 4].

The transmission assessment survey (TAS) was recommended by WHO as a standard methodology for the confirmation of LF elimination in endemic areas. With roughly 24 months apart in implementation, there are three TAS, known as TAS-1, TAS-2, and TAS-3, to be conducted. TAS evaluates if the prevalence of microfilaria in the blood or circulating filarial antigen in infected human populations attained a certain level in which transmission can no longer be sustained, which, in turn, signals when MDA can be stopped. However, there are evidences suggesting inconsistencies in the current TAS guidelines, particularly test sensitivity, parasite level changes in human populations, and size of evaluation units. TAS alone is insufficient in carrying out MDA-related decisions [5, 6, 7].

A non-invasive surveillance tool, known as xenomonitoring, was developed to complement TAS. Xenomonitoring indirectly tracks the progress of LF elimination programs by utilizing infections in wild-caught mosquito vectors to diagnose if disease transmission is presently occurring in humans [2, 7, 8]. Dissection is the inexpensive, gold standard method for filaria parasite detection in mosquitoes, however, efficiency and sensitivity decline when prevalence is below 1%. In large-scale epidemiological surveys, mosquito dissection becomes more costly, labor-intensive, and time-consuming [2]. These limitations led to the development of molecular xenomonitoring (MX) which uses PCR techniques to detect filarial DNA in mosquito vectors [6, 8]. MX has been extensively utilized for years due to its superior testing throughput, high sensitivity and specificity of detection, and species-level parasite identification [5]. Despite these advantages, LF MX has not been globally integrated as a routine monitoring tool for post-MDA surveillance [6, 8]. The implementation of MX as an additional programmatic strategy will strengthen and standardize the current protocol of LF elimination programs [5].

In the Philippines, LF is caused by W. bancrofti and B. malayi parasites endemic in 46 out of 81 provinces in the country [9]. The National Filariasis Elimination Program (NFEP), established by the Department of Health, aims to eliminate LF as a public health problem in the Philippines by utilizing a comprehensive approach and providing universal access to quality health services. About 76% of cases are also evident in poorest municipalities in the country [10]. NFEP initially encountered setbacks particularly in formulating effective MDA coverage plans and in sustaining MMDP activities. By 2015, there were significant improvements in logistic chain management, MDA implementation, and monitoring and surveillance. Regrettably, there are remaining provinces which need to accomplish and pass the evaluations conducted by WHO. Endemic areas declared LF- free are also at risk of potential disease resurgence [9].

Oriental Mindoro was declared LF-free last 2013 after successful results from TAS-1. However, the province failed TAS-2 in 2015 which prompted recommendation from WHO to conduct TAS strengthening by implementing two more rounds of MDA (2017-2018). Sultan Kudarat has been implementing MDA from 2011 to 2017, but high burden municipalities in the province are still not able to reach the threshold level (<1%). Sultan Kudarat will continue with MDA until the threshold is achieved, followed by the initiation of TAS-1.

As evidence to support TAS, this study was conducted to detect the presence of filarial parasites in mosquitoes from two endemic provinces, Oriental Mindoro (Region 4B) and Sultan Kudarat (Region 12), in the Philippines through molecular xenomonitoring.

Discussion

The significant vectors of W. bancrofti parasites are (1) culicines in most urban and semi-urban areas; (2) anophelines in more rural areas of Africa; and (3) aedines in the Pacific Islands [21]. In the Philippines, there are five incriminated mosquito vectors of LF. W. bancrofti is transmitted by Ae. poicilius, Cx. quinquefasciatus, and An. flavirostris mosquitoes. Contrarily, Ma. uniformis and Ma. bonneae mosquitoes are responsible for the transmission of another filarial parasite, B. malayi [9].

This study collected four filaria mosquito vectors including aedines, anophelines, culicines, and mansonioides. W. bancrofti was detected in Ae. poicilius in Brgy. Sta. Clara, Kalamansig, Sultan Kudarat even though there were only 14 households. Cx. quinquefasciatus was collected in all sampling sites for both provinces, but W. bancrofti was detected in just two barangays – Brgy. Poblacion II, Victoria, Oriental Mindoro and Brgy. Sta. Maria, Kalamansig, Sultan Kudarat.

Collected mosquito vectors were pooled prior to real-time PCR detection. This hindered the identification of specific households positive for filarial parasites. Nevertheless, a specific real-time PCR assay is capable of detecting LF parasites in pools of 50 to 100 mosquitoes even if ingestion of microfilaria-positive blood occurred for more than two weeks [22]. Detection of W. bancrofti in primary and secondary mosquito vectors in Oriental Mindoro and Sultan Kudarat could be indicative of ongoing LF transmission. As indirect indicators of LF infection, PCR-positive mosquitoes in the sampling sites revealed that LF-infected individuals are or were recently nearby [23]. The existence of filarial parasites in mosquitoes denotes microfilaria reservoir in the local human population [24] since humans are the only recognized definitive host of W. bancrofti [25]. Furthermore, the use of heads and thoraxes of mosquitoes potentially improved the chances of detecting third-stage infective larvae (L3), although for this study, the real-time PCR protocol used only detected filarial DNA. Further studies should be conducted on the correlation of mosquito vectors and LF prevalence surveys in humans to solidify evidence of ongoing disease transmission. Molecular protocol specific for filarial L3 detection should be utilized to incriminate other probable mosquito vectors collected within the study sites.

BGS traps were placed near previously or currently known LF case/s and presumed mosquito breeding sites. Both mosquito breeding sites and flight range are important factors to be recognized when conducting vector surveillance. Aedes mosquitoes usually breed in clean, stagnant water- holding natural and/or artificial containers [21]. The primary filaria vector Ae. poicilius is known to oviposit in water-filled leaf axils of abaca (Musa textilis), banana (Saba variety, Musa sapientum), screwpine (Pandanus), and taro (Colocasia esculenta) plants which are found near human habitations [9, 26]. Most of the barangays with collected Ae. poicilius were noted to have at least one of these plants which serve as breeding sites of these mosquitoes. On the contrary, Cx. quinquefasciatus, a secondary filaria vector in the Philippines, is ubiquitous in nature because they can breed in a wide range of bodies of water, particularly in polluted and stagnant waters. These include canals, cesspits, drains, marshy swamps, septic tanks, and empty containers or tree holes filled with rainwater [21]. Cx. quinquefasciatus increases in numbers during the rainy season, and in areas with open sewers, poor sanitation, and untreated waste waters [27]. The sampling sites in both provinces displayed the above- mentioned breeding sites which support the abundance of Cx. quinquefasciatus in the mosquito collections.

Typically, the maximum mosquito flight distance spans from 50 meters to 50 kilometers depending on the species. The most common type of mosquito dispersal is associated with shorter daily flights in relation to the mosquitoes’ behavioral response for survival. This non-oriented dispersal is intentional and integral for their mating, host- seeking, biting, resting, and egg-laying behavior [28]. A study of Bockarie reported two contradicting scenarios: (1) filaria-positive mosquitoes were detected in households with microfilaria-negative residents; and (2) filaria-negative mosquitoes were detected in households with microfilaria- positive residents [24]. This may be connected to the flight capacity, availability of suitable oviposition sites, and host preference of mosquito vectors.

Ae. poicilius is acknowledged as an anthropophilic, night- biting species suggesting preference for human hosts over animals. Previous findings concluded approximately 70% human blood index for this species. People who have outdoor activities at night are more at risk of acquiring LF because Ae. poicilius also shows activeness during the first few hours of the night. Furthermore, Ae. poicilius has prominent rate of survival in nature and favorably permissive for development of third-stage infective larvae [29, 30]. Cx. quinquefasciatus is also identified as night-biter which favors both avian and mammalian hosts [27]. Cx. quinquefasciatus was formerly characterized as a weak LF mosquito vector in the Philippines because the microfilaria experimentally fed to these mosquitoes showed comparatively poorer development than in Ae. poicilius [26]. W. bancrofti, the predominant filarial parasite in the Philippines, exclusively infects humans which indicates lack of other epidemiologically significant non- human host. Additionally, W. bancrofti exhibits nocturnally periodic form synchronous with its competent night-biting mosquito vectors Ae. poicilius and Cx. quinquefasciatus [31, 32].

With reference to the GPELF launched by WHO, endemic countries were committed to eliminate LF as a public health problem by 2020. MDA was the main strategy which focused on limiting the number of circulating filarial parasites in endemic areas. Monitoring LF infection in human populations through TAS and in mosquito vectors using MX were undertaken after stopping MDA [2]. In TAS, blood from infected individuals is tested for the presence of microfilaria or anti-filarial IgG4 using microscopic or serologic techniques, respectively. Challenges were observed during the implementation of TAS. Individuals became reluctant to submit to regular blood collections over time. Assessment of infections in humans is deemed as a “lagging indicator” because of the prepatent period which could extend for months or years. The prepatent period, defined as the period from infection to manifestation of the parasite in the host’s body, should be substantially considered [33]. Due to these concerns, assessment of infections in mosquito vectors provides a more favorable approach. The primary basis of MX is the capability of mosquito vectors to competently take up filarial parasites via ingestion of infected human blood. MX is non-invasive and only involves mosquitoes [6] which makes it ideal to use as indirect guide of the progress of LF control and elimination programs [2, 8]. MX, being highly sensitive and specific, is an effective complement to human-based TAS in quantifying persistent low levels of LF infection in endemic areas which require further treatment with MDA and follow- up intervention [2, 4, 8]. A previous study in American Samoa mapped LF after MDA and reported that DNA of W. bancrofti was still detected in pools of mosquitoes using a highly sensitive real-time PCR assay. In Sri Lanka, a comprehensive LF assessment showed that pools of Cx. quinquefasciatus were positive for W. bancrofti six years after MDA was stopped [4]. Household-based MX surveys are beneficial in conjunction with TAS for programmatic decision-making of stopping MDA

and post-MDA surveillance as evident in another study in Sri Lanka in which MX identified more areas with low levels of transmission than TAS. This is relevant in endemic areas which passed TAS but the number of LF-positive individuals is almost at the critical threshold value [8].

Lau, et al. (2016) mentioned several disadvantages and flaws of MX. MX demands entomological expertise in mosquito collection and identification, proficiency in molecular techniques, and well-equipped molecular diagnostic laboratory. There are also lapses in sampling strategies, interpretation of MX into operational policies, and unclear guidelines regarding critical thresholds to indicate ongoing transmissions [23].

As of 2021, 44 out of 46 endemic provinces in the country were already declared as LF-free. Sultan Kudarat and Zamboanga del Norte were the remaining provinces with ongoing LF transmission. The NFEP is keen and ambitious in preventing resurgence of the disease in LF- free areas by tracking down and immediately administering treatment to infected individuals, as well as implementing vector control strategies to impede transmission [9]. The role of mosquito surveillance in evaluating ongoing disease transmission should essentially be considered. Detection of filarial parasites in mosquito vectors functions as marker of efficacy of MDA in LF elimination or prognosis of pending LF resurgence.

Succeeding research related to this study should improve the adult mosquito collection and real-time PCR detection of filarial parasites. Longer collection time with more sampling days and/ or repeated collection for several months may be conducted to obtain more diverse collection of mosquitoes. Dorkenoo, et al. (2018) proposed increasing the frequency of monthly mosquito collections and additional sampling sites to achieve an extensive sample size [34]. Different adult collection techniques may be evaluated to establish the most suitable trap for each targeted vector species. For the real-time PCR detection, collected mosquito vectors may be tested individually instead of pooling to easily identify the household/s positive for filarial parasites. Adoption of a duplex real-time PCR protocol targeting L3 of both W. bancrofti and B. malayi yields more comprehensive and extensive data for LF elimination. Vector abundance and disease transmission dynamics are constantly changing which may bring in new vector species [8, 34]. Testing non- vector mosquito species may provide further evidence for vector incrimination (with L3 detection adopted).

Conclusion

This is the first study in the Philippines utilizing molecular xenomonitoring to detect the presence of W.

bancrofti filarial DNA in mosquitoes. Four mosquito vector species were collected from the provinces of Oriental Mindoro (Region 4B) and Sultan Kudarat (Region 12) namely, Ae. poicilius, Cx. quinquefasciatus, An. flavirostris, and Ma. uniformis. W. bancrofti parasites were only detected in Ae. poicilius (Brgy. Sta. Clara, Kalamansig, Sultan Kudarat) and Cx. quinquefasciatus (Brgy. Poblacion II, Victoria, Oriental Mindoro and Brgy. Sta. Maria, Kalamansig, Sultan Kudarat) mosquitoes. This result supports TAS findings of ongoing LF transmission in the two endemic provinces. Further research regarding diverse aspects of this disease are needed to accurately characterize LF transmission in the Philippines.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgement

The authors would like to thank the following: (1) Ms. Katherine Gass, Ms. Julie Kenyon, and colleagues from Task Force for Global Health and Neglected Tropical Diseases Support Center (NTD-SC) for their valuable involvement and advice throughout this study; (2) Mr. Nils Pilotte, Ms. Jessica Grant, Ms. Susan Haynes, and researchers from Smith College who provided the protocols and positive control of Wuchereria bancrofti parasites; (3) Dr. Leda Hernandez from Department of Health - Philippines, as well as Regional Entomologists Mr. Richard Delos Reyes (Region 4B) and Mr. Royfrextopher Boholst (Region 12) for providing status and insights of LF in the sampling sites; (4) local government units of Oriental Mindoro and Sultan Kudarat for their consistent participation and support; and (5) residents of selected sampling sites for allowing us to conduct adult mosquito collection in their household premises. We also acknowledge the contributions of the field and laboratory staff namely, Mr. Rommuel Gutierrez, Mr. Roland Parreno, and Ms. Jean Angela Ariles, of the Department of Medical Entomology, Research Institute for Tropical Medicine.

Funding

This study was funded by the Department of Health – Philippines through their Disease Prevention and Control Bureau (Sub-allotment Advice Numbers 2017-06-400 and 2017-12-2208).