Semi-Automated Microfluidic Device Combined with a MiniPCR-Duplex Lateral Flow Dipstick for Screening and Visual Species Identification of Lymphatic Filariae.

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.

A miniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection.

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.

Investigation on the Prevalence of Canine Microfilaremia in Thailand Using a Novel Microfluidic Device in Combination with Real-Time PCR.

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.

Diagnosis of feline filariasis assisted by a novel semi-automated microfluidic device in combination with high resolution melting real-time PCR.

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.

Therapeutic trial of doxycyclin plus ivermectin for the treatment of Brugia malayi naturally infected cats.

Lymphatic filariasis (LF) is one of the neglected tropical diseases which causes permanent and long term disability worldwide. LF is caused by filarial nematode parasites, i.e. Wuchereria bancrofti, Brugia malayi, and B. timori. All available antifilarial drugs currently being used have shown a limited adulticidal activity. Discoveries of endosymbiont rickettsia-like bacterium, Wolbachia in filarial nematodes provided a novel approach for antibiotic use in eradication of filarial diseases. The earlier studies revealed the macrofilaricidal efficacy of doxycycline against filarial nematodes. Chemotherapeutic efficiency of doxycycline has been studied against many filarial parasites, but there are still no therapeutic trials of the drug regimens for B. malayi treatment in naturally infected cats. Thus, this study would be the first attempt to study the efficiency of doxycycline (DOXY) alone or in combination with ivermectin (IVM) for treatment of B. malayi in naturally infected cats. A total of 26 B. malayi-infected cats in the endemic areas were recruited and divided into 3 groups, receiving different treatment regimens; a single dose of ivermectin only (IVM), doxycycline only (DOXY) and a combination of ivermectin and doxycycline (DOXY-IVM). The efficacy of each therapatic regimen was evaluated by detecting the presence of microfilaria using parasitological and molecular techniques monthly up to 2 years after starting the treatment. The IVM treated group had a significant rapid reduction of microfilariae in the first month; however, recurrence of microfilaraemia was observed in some cats. By contrast, the DOXY and DOXY-IVM groups showed a better result with a gradual decrease in microfilariae with no recurrence. These 2 groups were not only virtually deprived of infection but also sustained the sterility of infection through the course of study. These results revealed the advantages of using in B. malayi treatment in cats. Doxycycline showed to have both microfilaricidal and adulticidal effects on lymphatic filariae which maintained the long-term response to control of B. malayi infection in cats.