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

Loymek, Sumas; Phuakrod, Achinya; Zaelai, Kati; Sripumkhai, Witsaroot; Vongjaroensanti, Prapakorn; Wongkamchai, Sirichit

doi:10.3390/vetsci8030039

Cited by 9 publications

(6 citation statements)

References 17 publications

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“…We also applied our developed microfluidic device for investigation on the prevalence of canine microfilaremia [19]. However, our microfluidic device requires an expensive specialized instrument (i.e., Light Cycler LC480 to perform a high-resolution melt (HRM) real-time PCR) for species identification of the trapped mf [12,19].…”

Section: Discussionmentioning

confidence: 99%

“…Thick blood smear staining for mf detection was performed using ethylenediaminetetraacetic acid (EDTA) blood obtained from study samples according to the standard World Health Organization (WHO) procedure [19]. Briefly, 50 µL of EDTA blood was deposited onto a clean glass slide and then the blood was spread to create a circular smear.…”

Section: Thick Blood Smear Staining For Microfilariae Detectionmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Thick Blood Smear Staining For Microfilariae Detectionmentioning

confidence: 99%

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

et al. 2022

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“…Outside Europe, dirofilariosis was detected in Thailand [35,36], French Guyana [37], Nepal [38], Iran [39], and the USA [8,40].…”

Section: Discussionmentioning

confidence: 99%

Identity of Microfilariae Circulating in Dogs from Western and South-Western Romania in the Last Decade

et al. 2021

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Dirofilaria infections in dogs are recognized as widespread mosquito-borne diseases with zoonotic potential, that are caused by the filarioid nematode (Onchocercidae) species Dirofilaria immitis and Dirofilaria repens. The long-term survey conducted in western and south-western Romania was undertaken in order to provide valuable data on the occurrence of Dirofilaria infections in dogs. Blood samples from 1088 dogs, originating from 73 localities of 11 western and south-western counties in Romania, were collected and examined using the modified Knott test. Subsequently, all of the microscopically positive samples were subjected to molecular analysis for confirmation. Altogether, the data obtained showed the percentage of dogs with circulating microfilariae to be 21.42% (233/1088) of dogs tested in the investigated region. The identified species, in cases of monoinfections, were D. immitis, D. repens, and Acanthocheilonema reconditum in 106 (9.74%) samples, 102 (9.38%) samples, and 1 (0.09%) sample, respectively. Twenty-four (2.21%) samples were simultaneously positive for D. immitis and D. repens. There was no association (p > 0.05) between infection status and breed; however, sex and lifestyle were positively associated (p < 0.05) with the percentage of dogs with circulating microfilariae and might be regarded as risk factors for infection. The results of the present investigation indicate potential zoonotic risks for humans living in the screened area and support the imperative to increase awareness among both veterinarians and physicians, regarding the continuous spread of these zoonotic filariae.

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“…We next ]performed a real time PCR with high resolution melting analysis (HRM-real time PCR) for species identi cation of the trapped mf [29]. The e ciency of the device was evaluated in laboratory and it was applied to study the prevalence of canine and feline micro laremia [29,37]. In this study, we applied the micro uidic device to detect micro lariae of B. malayi and W. bancrofti in human blood.…”

Section: Discussionmentioning

confidence: 99%

A miniPCR-duplex Lateral Flow Dipstick Platform in Combination with the Microfluidic Device for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

Phuakrod

Sripumkhai

Jeamsaksiri

et al. 2021

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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.

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

Cited by 9 publications

References 17 publications

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

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

Identity of Microfilariae Circulating in Dogs from Western and South-Western Romania in the Last Decade

A miniPCR-duplex Lateral Flow Dipstick Platform in Combination with the Microfluidic Device for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

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