Background The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. Methods The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were ‘spiked’ with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. Results In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi , 15 as Dirofilaria immitis and 7 as| D. repens . Conclusions We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

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We conducted a survey of canine microfilaraemia in 768 dogs in Chanthaburi, Samut Sakhon, and Narathiwat provinces of Thailand using a novel semi-automated, microfluidic device that is easy and rapid to perform. Microfilariae species were identified using High Resolution Melting real-time PCR (HRM real-time PCR). The prevalence of canine microfilaremia was 16.2% (45/278) in Chanthaburi and 5.5% (12/217) in Samut Sakhon. The prevalence of canine microfilaremia in Narathiwat was 22.7% (67/273). Brugia pahangi and Dirofilaria immitis were the predominant species of filariae found in the infected dogs from Chanthaburi and Narathiwat, respectively. The low prevalence of canine microfilaremia of Samut Sakhon may reflect the success of the Soi Dog foundation’s efforts and the establishment of veterinary control programs. An effective disease control and prevention strategies is needed in Chanthaburi and Narathiwat to reduce the risks of zoonotic transmission of the parasites. An appropriate drug treatment should be given to infected dogs and prophylactic drugs are suggested to be given to dogs age ≤1-year-old to prevent filarial infection. The novel microfluidic device could be implemented for surveillance of filariae infection in other animals.

Lymphatic filariasis (LF) is a leading cause of permanent disability worldwide that has been listed as a neglected tropical disease by the World Health Organization. Significant progress made by the Global Program to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decline in the population of the worm that causes LF infection. Diagnostic assays capable of detecting low levels of parasite presence are needed to diagnose LF. There is also a need for new tools that can be used in areas where multiple filarial species are coendemic and for mass screening or for use in a point-of-care setting. In the present study, we applied our previously developed semi-automated microfluidic device in combination with our recently developed mini polymerase chain reaction (miniPCR) with a duplex lateral flow dipstick (DLFD) (miniPCR-DLFD) for rapid mass screening and visual species identification of lymphatic filariae in human blood. The study samples comprised 20 Brugia malayi microfilariae (mf) positive human blood samples, 14 Wuchereria bancrofti mf positive human blood samples and 100 mf negative human blood samples. Microfilariae detection and visual species identification was performed using the microfluidic device. To identify the species of the mf trapped in the microfluidic chips, DNA of the trapped mf was extracted for miniPCR amplification of W. bancrofti and B. malayi DNA followed by DLFD. Thick blood smear staining for microfilariae detection was used as the gold standard technique. Microfilariae screening and visual species identification using our microfluidic device plus miniPCR-DLFD platform yielded results concordant with those of the gold standard thick blood smear technique. The microfluidic device, the miniPCR and the DLFD are all portable and do not require additional equipment. Use of this screening and visual species identification platform will facilitate reliable, cost-effective, and rapid surveillance for the presence of LF infection in resource-poor settings.

Background: Lymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematode parasites which is transmitted by the bites of infective mosquitoes. The diagnosis of LF traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears which has several limitations. Materials and methods: In the present study, we developed a lateral flow (LF) strip to be used in combination with a novel microfluidic device and polymerase chain reaction (PCR) for a rapid and visual detection of lymphatic filariae, Brugia malayi/ Wuchereria bancrofti infection in human blood samples. Results: The assay targets B. malayi HhaI gene and W. bancrofti Ssp gene. Prior to perform the assay, no DNA product clean up step required, thus, it can shorten time and reduce cost. The LF strip can detect as low as 10 pg of DNA product and no cross-reactivity with DNA of other parasites such as Gnathostoma spinigerum, cysticercisis soliumnor with DNA of other filariae i.e. Brugia pahangi, Dirofilaria immitis andD. repens. Conclusions: The developed LF strip shows high sensitivity and specificity, in combination with a novel microfluidic device and PCR, can be used as an alternative tool for the diagnosis of lymphatic filariasis.

Background: Lymphatic filariasis is a major neglected tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. Accordingly, access is now needed to a cost-efficient, non-laborious diagnostic assay(s) capable of detecting low levels of microfilariae and without the need for infrastructure and expensive equipment.Methods: We developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD) assay. The design of the PCR primers was guided by sequence alignments of the HhaI and SspI genes of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection, accuracy, and cross-reactivity of the assay were evaluated, after which the assay was deployed to assess infection using blood samples obtained from Myanmar study participants living along the Thailand-Myanmar border, a region in Tak province endemic W. bancrofti. In addition, blood samples were provided by from Thais in Narathiwat province residing in an area endemic for brugian filariasis. We also combined a previously reported semi-automated microfluidic device with the miniPCR-DLFD to facilitate rapid detection and species identification of microfilariae in human blood.Results: The miniPCR-DLFD assay exhibited a detection limit of two microfilariae per mililiter blood sample, and cross-amplification was not observed with from other parasites. For field validation, microfilariae and DNA of W. bancrofti were detected from two (0.6%) and five (1.5%) out of 328 (100%) blood samples, respectively. For the Narathiwat samples, microfilariae and DNA of B. malayi were detected from one (0.46%) and two (0.9%) out of 216 (100%) blood samples, respectively. A rapid and visual of species identification was accurately obtained in all microfilariae entrapped by our previously developed microfluidic device.Conclusion: A miniPCR-DLFD platform alone or coupled with the microfluidic device provided rapid and visual diagnosis of lymphatic filariasis. The microfluidic device, the miniPCR, and the DLFD are all portable. Coupled with a portable microcentrifuge for DNA extraction, this is a promising platform for the diagnosis of lymphatic filariasis in point-of-collection settings with a modest cost (~USD $ 5) per sample.

Lymphatic filariasis (LF) is an important tropical disease that affects over a billion people in more than 80 countries and approximately 40 million people are currently suffering from severe disfigurement and disability. A diagnostic tool is the principal impact factor to determine the infection status of lymphatic filariasis. The purpose of the present study was to investigate nucleic acid of Wuchereria bancrofti as well as antifilarial IgG4 in a Myanmar immigrant community living along the Moei River, a natural border between Mae Sot, Tak province Thailand and Myawaddy, Myanmar which is an endemic area of bancroftian filariasis. Blood was collected from 300 Myanmar immigrants in Mae Sot district, Tak Province. The nucleic acid of W. bancrofti was assessed in the study population using our recent published miniPCR-Duplex Lateral Flow dipstick (DLFD) platform as well as the standard PCR technique. The antifilarial IgG4 was detected in the study population using the developed ELISA which used BmSxp protein as antigen. The miniPCR-DLFD method delivered results comparable to the standard PCR technique and it enables convenient and rapid visual detection of the parasite nucleic acid. Furthermore, the ELISA using BmSxp antigen demonstrated a sensitivity, specificity, and positive and negative predictive values of 98.1%, 98.9%, 96.3%, and 99.4% respectively. The W. bancrofti nucleic acid and antifilarial IgG4 were detected in 1.6% (5/300), and 2% (6/300) of the study population, accordingly. The results of this study also revealed important epidemiological data about LF on the Thai–Myanmar border.