Mongkol Pongsuchart, Amornpun Sereemaspun and Kiat Ruxrungtham
Background: Nucleic acid lateral flow (NALF) strip test is currently a promising method in biomedical applications for point-of-care DNA detection. However, sensitivity of NALF is a major limitation when tested without amplification of the DNA sample.
Objective: This study introduces UV crosslink as an additional step to enhance sensitivity of the test strip. Methods: By applying UV exposure to the NALF platform with different irradiation energies and times, specificity test with target DNA and non-target DNA.
Results: the results revealed increasing signals of approximately 40% from all test strips compared to those without UV exposure. Furthermore, the sensitivity enhancement by UV crosslink of NALF dipstick has been shown to be independent from DNA sequences, hybridization specificity and target DNA length.
Conclusion: Data presents a new step to improve the sensitivity of NALF assay. It allows immediate visualization and quantification. There may be a potential application in other NALF platform products.
Kiat Ruxrungtham, Stephen Kerr, Anchalee Avihingsanon, Nittaya Phanuphak, Pariya Ruxrungtham and Praphan Phanuphak
Prevalence estimates of human immunodeficiency virus (HIV) infection in Asia are diverse, ranging from <0.1% to 1.1%. In Asia, approximately 4.7 million people are living with HIV and 310,000 are newly infected each year. The fastest growth of the epidemic throughout Asia is among men who have sex with men (MSM). Drug resistance varies from 4%-5% in an Asian cohort to 10% in an acutely infected MSM cohort in Bangkok. A rise (4%-14%) in the prevalence of syphilis is associated with the HIV epidemic in Asia. Tuberculosis is among the most common coinfections; however, HIV testing of tuberculosis patients is not routine. Coinfection with HBV, HCV, and triple coinfection varies from 8.7%-11%, 5%-18%, and 0.4%-3% respectively. Although the World Health Organization 2013 guidelines to start antiretroviral therapy (ART) early are implemented, most patients present late, with low CD4 counts, resulting in a high mortality during the first year of ART. While integrase inhibitor-based treatments are preferred first-line treatments in high income countries, efavirenz-based treatments remain preferred treatments in resource-limited countries. HIV-1 viral load testing is not available in most of these countries, making low-cost point-of-care testing accessibility an urgent priority. Effective ART coverage to prevent new HIV infections among children in Asia remains low. To provide life-long ART for children, better use of current first-line regimens and access to pediatric second-line formulations are important. To end AIDS in Asia by 2030, committed policy with innovative strategies to enhance “reach recruit, treat and retain” combined with effective prevention strategies are required.
In Thailand, more than 150,000 patients are currently treated with antiretroviral drugs under the support of the National AIDS Program (NAP). The appointed Adults and Adolescents Committee consisted of 28 members who are experts in HIV research, patient care or health care policy. Relevant published literature, guidelines, and the most recent relevant clinical trials presented internationally were reviewed. Several peer review and clinical studies conducted in Thailand were included in the review process. Special considerations for patients with co-infection of tuberculosis or hepatitis B were incorporated. Appropriate cut-off of CD4+ T-cell counts when to commence ART among Thai patients have been considered. It is now recommended to start ART at CD4+ T-cell count <350 cells/mm3. For treatment-naive patients, the preferred initial therapy is a nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimen containing lamivudine plus zidovudine or tenofovir. Stavudine will be phased out in a two-year plan at the national program level. Viral load and CD4+ T-cell counts should be monitored at least once and twice a year. To achieve long-term treatment success, enhancing adherence together with the proper management of antiretroviral-related toxicity is critical. In summary, the major changes from the Thai 2008 guidelines include commencing ART earlier. ART is recommended regardless of CD4+ T cell count if patients have an indication to treat their HBV co-infection. Preferred first regimen uses AZT or TDF, not d4T as the NRTI-backbone. Furthermore, efavirenz is now considered a preferred NNRTI, along with nevirapine.
Hepatitis B virus (HBV) has been the most prevalent blood-borne pathogen wherein utero transmission has still not been properly managed. Recent practice guidelines suggested that an antiviral drug should be administered to third-trimester pregnancies with significant viremia (>2 × 105 IU/mL).
To develop a novel turbidity-based loop-mediated isothermal amplification (LAMP) coupled with heat treatment DNA extraction method that is a rapid, cost-effective, and feasible viral load assessment and could be applied to antenatal screening.
Primers and reagents were designed, turbidity-based platform and heat treatment method were added, and evaluated for optimal efficiency. Assay sensitivity was tested from serially diluted standard HBV DNA. Assay specificity was tested with six standard viral DNAs. Clinical samples were analyzed and the results were compared with those of quantitative polymerase chain reaction (qPCR) diagnostic records.
The optimized condition was 60°C with no betaine, 1.4 mM deoxyribonucleotide triphosphates (dNTPs) and 6 mM of MgSO4 for 60 min. The assay accurately detected samples with standard HBV DNA at >2 × 105 IU/mL in both distilled water and spiked serum. Results can be interpreted within 31.48 ± 1.41 min in real-time turbidimeter. The amplification is exclusively specific to HBV, but not with the other six human-specific viruses. Moreover, the assay showed comparable performance within 95% confidence interval to the previously developed HBV LAMP toward clinical specimens.
This newly developed method was accurate, affordable, and flexible to further implementation to large-scale third-trimester pregnancy screening.