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Fulltext Droplet digital PCR application for the detection of SARS-CoV-2 in air sample

Rashid SA, Nazakat R, Muhamad Robat R, Ismail R, Suppiah J, Rajendran K, et al.

Front Public Health, 2023;11:1208348.
PMID: 37965510 DOI: 10.3389/fpubh.2023.1208348

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.

Matched MeSH terms: RNA, Viral/analysis
Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus

Chin KL, Teoh BT, Sam SS, Loong SK, Tan KK, Azizan NS, et al.

Trop Biomed, 2022 Dec 01;39(4):518-523.
PMID: 36602210 DOI: 10.47665/tb.39.4.005

Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.

Matched MeSH terms: RNA, Viral/analysis
Maternal hepatitis C (HCV) infection and Anti-D immunoglobulin therapy: study testing antibodies, RNA and Genotype of HCV in Baghdad

Al-Kubaisy W, Daud S, Al-Kubaisi MW, Al-Kubaisi OW, Abdullah NN

J Matern Fetal Neonatal Med, 2019 Oct;32(20):3464-3469.
PMID: 29656685 DOI: 10.1080/14767058.2018.1465557

Introduction: Hepatitis C virus (HCV) infection is a serious health problem. It is a major contributor to end-stage liver disease. Worldwide, 1-8% of all pregnant women were infected. Women with viral hepatitis may be at an increased risk of pregnancy complications. There are several obstetrics intervention acts as risk factors, which are specific to women pertaining the HCV infection; anti-D immunoglobulin (Ig) therapy may be one of them. Our objectives were to estimate the prevalence of HCV antibodies (anti-HCV), RNA, and genotype distribution among women with anti-D Ig therapy. Materials and methods: A cross sectional study was conducted. A sample of 154 Rhesus negative (Rh - ve) pregnant women regardless of the anti-D Ig therapy was collected. Anti-HCV were tested using third generation enzyme immunoassay (EIA-3) and immunoblot assay (Lia Tek-111), subsequently. In addition, 89 serum samples were subjected to molecular analysis using RT-PCR and DNA enzyme immunoassay (DEIA) method for the detection of HCV-RNA and genotypes. Results: Anti-HCV, and HCV-RNA seroprevalence were significantly higher (17.1, 35.5%) among women with anti-D Ig than their counter group (6.4, 13.16%), p = .038, .018, respectively. Significant direct positive dose response correlation (r = 0.78, p = .005) had been seen between number of anti-D Ig therapy and anti-HCV seropositive rate. Anti-D Ig therapy act as a risk factor (odds ratio (OR) = 3.01, 95%CI: 1.01-8.9) especially from the third dose onward. Women with anti-D Ig therapy were at higher risk (3.6 times more) of positive HCV-RNA (OR =3.6, 95%CI =1.19-10.837). Genotype HCV-1b showed higher prevalent (52.9%) among the recipients of anti-D Ig therapy while genotype HCV-3a (6.6%) was the lowest. Conclusions: Our study showed that Anti-D immunoglobulin therapy acts as a risk factor for acquiring HCV infection. Screening for HCV should be recommended for all recipients of anti-D Ig. Not only HCV antibodies but HCV-RNA detection being recommended for the diagnosis of HCV infection. A brief rational: Pregnant women with HCV infection are at risk of adverse obstetric outcome. Anti-D Ig therapy may be a risk factor for HCV infection. Hence, we conducted a cross sectional study with the objectives to estimate the prevalence of HCV antibodies (anti-HCV), RNA, and genotype distribution among women with anti-D Ig therapy. We found that anti-HCV and HCV-RNA seroprevalence were significantly higher in women with anti-D Ig. In addition, women with anti-D Ig therapy were 3.6 times more at risk of positive HCV-RNA with genotype HCV-1b showed higher prevalence. Therefore, anti-D Ig therapy is a risk factor for acquiring HCV infection and we recommend screening for HCV for all recipients of anti-D Ig. In addition, the diagnosis of HCV infection, should be made with HCV antibodies and HCV-RNA detection.

Matched MeSH terms: RNA, Viral/analysis
Fulltext A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus

Sam SS, Teoh BT, Chee CM, Mohamed-Romai-Noor NA, Abd-Jamil J, Loong SK, et al.

Sci Rep, 2018 12 05;8(1):17632.
PMID: 30518924 DOI: 10.1038/s41598-018-36043-6

Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.

Matched MeSH terms: RNA, Viral/analysis
Fulltext Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma

Higuchi H, Yamakawa N, Imadome KI, Yahata T, Kotaki R, Ogata J, et al.

Blood, 2018 06 07;131(23):2552-2567.
PMID: 29685921 DOI: 10.1182/blood-2017-07-794529

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Matched MeSH terms: RNA, Viral/analysis
Characterization of significant molecular determinants of virulence of Enterovirus 71 sub-genotype B4 in Rhabdomyosarcoma cells

Yee PTI, Mohamed RAH, Ong SK, Tan KO, Poh CL

Virus Res, 2017 06 15;238:243-252.
PMID: 28705680 DOI: 10.1016/j.virusres.2017.07.010

One of the leading causes of the hand, foot and mouth disease (HFMD) is Enterovirus 71 (EV-A71), displaying symptoms such as fever and ulcers in children but some strains can produce cardiopulmonary oedema which leads to death. There is no FDA-approved vaccine for prevention of severe HFMD. The molecular determinants of virulence for EV-A71 are unclear. It could be a single or a combination of amino acids that determines virulence in different EV-A71 genotype/sub-genotypes. Several EV-A71 strains bearing single nucleotide (nt) mutations were constructed and the contribution of each mutation to virulence was evaluated. The nt(s) that contributed to significant reduction in virulence in vitro were selected and each mutation was introduced separately into the genome to construct the multiply mutated EV-A71 strain (MMS) which carried six substitutions of nt(s) at the 5'-NTR (U700C), VP1-145 (E to G), VP1-98E, VP1-244K and G64R in the vaccine seed strain that had a partial deletion within the 5'-NTR region (nt. 475-485) of Δ11bp. In comparison to the wild type strain, the MMS showed low virulence as it produced very low RNA copy number, plaque count, VP1 and had 105-fold higher TCID50, indicative of a promising LAV candidate that should be further evaluated in vivo.

Matched MeSH terms: RNA, Viral/analysis
Fulltext Loss in lung volume and changes in the immune response demonstrate disease progression in African green monkeys infected by small-particle aerosol and intratracheal exposure to Nipah virus

Cong Y, Lentz MR, Lara A, Alexander I, Bartos C, Bohannon JK, et al.

PLoS Negl Trop Dis, 2017 04;11(4):e0005532.
PMID: 28388650 DOI: 10.1371/journal.pntd.0005532

Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that emerged in the late 1990s in Malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in Bangladesh and India. NiV infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of NiV contaminated foods. In the work presented here, the development of disease was investigated in the African Green Monkey (AGM) model following intratracheal (IT) and, for the first time, small-particle aerosol administration of NiV. This study utilized computed tomography (CT) and magnetic resonance imaging (MRI) to temporally assess disease progression. The host immune response and changes in immune cell populations over the course of disease were also evaluated. This study found that IT and small-particle administration of NiV caused similar disease progression, but that IT inoculation induced significant congestion in the lungs while disease following small-particle aerosol inoculation was largely confined to the lower respiratory tract. Quantitative assessment of changes in lung volume found up to a 45% loss in IT inoculated animals. None of the subjects in this study developed overt neurological disease, a finding that was supported by MRI analysis. The development of neutralizing antibodies was not apparent over the 8-10 day course of disease, but changes in cytokine response in all animals and activated CD8+ T cell numbers suggest the onset of cell-mediated immunity. These studies demonstrate that IT and small-particle aerosol infection with NiV in the AGM model leads to a severe respiratory disease devoid of neurological indications. This work also suggests that extending the disease course or minimizing the impact of the respiratory component is critical to developing a model that has a neurological component and more accurately reflects the human condition.

Matched MeSH terms: RNA, Viral/analysis
Fulltext The Nature of Exposure Drives Transmission of Nipah Viruses from Malaysia and Bangladesh in Ferrets

Clayton BA, Middleton D, Arkinstall R, Frazer L, Wang LF, Marsh GA

PLoS Negl Trop Dis, 2016 06;10(6):e0004775.
PMID: 27341030 DOI: 10.1371/journal.pntd.0004775

Person-to-person transmission is a key feature of human Nipah virus outbreaks in Bangladesh. In contrast, in an outbreak of Nipah virus in Malaysia, people acquired infections from pigs. It is not known whether this important epidemiological difference is driven primarily by differences between NiV Bangladesh (NiV-BD) and Malaysia (NiV-MY) at a virus level, or by environmental or host factors. In a time course study, ferrets were oronasally exposed to equivalent doses of NiV-BD or NiV-MY. More rapid onset of productive infection and higher levels of virus replication in respiratory tract tissues were seen for NiV-BD compared to NiV-MY, corroborating our previous report of increased oral shedding of NiV-BD in ferrets and suggesting a contributory mechanism for increased NiV-BD transmission between people compared to NiV-MY. However, we recognize that transmission occurs within a social and environmental framework that may have an important and differentiating role in NiV transmission rates. With this in mind, ferret-to-ferret transmission of NiV-BD and NiV-MY was assessed under differing viral exposure conditions. Transmission was not identified for either virus when naïve ferrets were cohoused with experimentally-infected animals. In contrast, all naïve ferrets developed acute infection following assisted and direct exposure to oronasal fluid from animals that were shedding either NiV-BD or NiV-MY. Our findings for ferrets indicate that, although NiV-BD may be shed at higher levels than NiV-MY, transmission risk may be equivalently low under exposure conditions provided by cohabitation alone. In contrast, active transfer of infected bodily fluids consistently results in transmission, regardless of the virus strain. These observations suggest that the risk of NiV transmission is underpinned by social and environmental factors, and will have practical implications for managing transmission risk during outbreaks of human disease.

Matched MeSH terms: RNA, Viral/analysis
Three different patterns of CD4 recovery in a cohort of Chinese HIV patients following antiretroviral therapy - a five-year observational study

Naftalin CM, Wong NS, Chan DP, Wong KH, Reidpath DD, Lee SS

Int J STD AIDS, 2015 Oct;26(11):803-9.
PMID: 25281539 DOI: 10.1177/0956462414553826

To explore the heterogeneity of CD4 responses following highly active antiretroviral therapy, the patterns of CD4 recovery of HIV-1-infected Chinese patients who have been on their first antiretroviral regimen for ≥5 years were analysed. The CD4 trajectories were traced, smoothed and differentiated into three defined profiles. Half (56.3%) were 'satisfactory responders', with CD4 gain of >100 cells/μL and a peak of >350 cells/μL, plateauing before the end of Year 5. Thirty-three (24.4%) were 'continuing responders' whose CD4 rise persisted at Year 4-5. The remaining 26 (19.3%) were 'poor responders'. Presentation with AIDS before therapy was common not just among 'poor' but also paradoxically the 'continuing' responders. While a majority had responded well to antiretroviral therapy, older patients and those with AIDS diagnosis before initiation of therapy may never achieve a satisfactory level even with effective treatment. Categorization of HIV patients by their CD4 trajectory may support the prediction of immunological outcome over time, and ultimately inform treatment choices.

Matched MeSH terms: RNA, Viral/analysis*
Fulltext A Malaysia 97 monovalent foot-and-mouth disease vaccine (>6PD50/dose) protects pigs against challenge with a variant FMDV A SEA-97 lineage virus, 4 and 7 days post vaccination

Nagendrakumar SB, Hong NT, Geoffrey FT, Jacqueline MM, Andrew D, Michelle G, et al.

Vaccine, 2015 Aug 26;33(36):4513-9.
PMID: 26192355 DOI: 10.1016/j.vaccine.2015.07.014

Pigs play a significant role during outbreaks of foot-and-mouth disease (FMD) due to their ability to amplify the virus. It is therefore essential to determine what role vaccination could play to prevent clinical disease and lower virus excretion into the environment. In this study we investigated the efficacy of the double oil emulsion A Malaysia 97 vaccine (>6PD50/dose) against heterologous challenge with an isolate belonging to the A SEA-97 lineage at 4 and 7 days post vaccination (dpv). In addition, we determined whether physical separation of pigs in the same room could prevent virus transmission. Statistically there was no difference in the level of protection offered by 4 and 7 dpv. However, no clinical disease or viral RNA was detected in the blood of pigs challenged 4 dpv, although three of the pigs had antibodies to the non-structural proteins (NSPs), indicating viral replication. Viral RNA was also detected in nasal and saliva swabs, but on very few occasions. Two of the pigs vaccinated seven days prior to challenge had vesicles distal from the injection site, but on the inoculated foot, and two pigs had viral RNA detected in the blood. One pig sero-converted to the NSPs. In contrast, all unvaccinated and inoculated pigs had evidence of infection. No infection occurred in any of the susceptible pigs in the same room, but separated from the infected pigs, indicating that strict biosecurity measures were sufficient under these experimental conditions to prevent virus transmission. However, viral RNA was detected in the nasal swabs of one group of pigs, but apparently not at sufficient levels to cause clinical disease. Vaccination led to a significant decrease in viral RNA in vaccinated pigs compared to unvaccinated and infected pigs, even with this heterologous challenge, and could therefore be considered as a control option during outbreaks.

Matched MeSH terms: RNA, Viral/analysis
Reversible thermo-pneumatic valves on centrifugal microfluidic platforms

Aeinehvand MM, Ibrahim F, Harun SW, Kazemzadeh A, Rothan HA, Yusof R, et al.

Lab Chip, 2015 Aug 21;15(16):3358-69.
PMID: 26158597 DOI: 10.1039/c5lc00634a

Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel biochemical assays on a single microfluidic disk. The integration of complex, sequential microfluidic procedures on these platforms relies on robust valving techniques that allow for the precise control and manipulation of fluid flow. The ability of valves to consistently return to their former conditions after each actuation plays a significant role in the real-time manipulation of fluidic operations. In this paper, we introduce an active valving technique that operates based on the deflection of a latex film with the potential for real-time flow manipulation in a wide range of operational spinning speeds. The reversible thermo-pneumatic valve (RTPV) seals or reopens an inlet when a trapped air volume is heated or cooled, respectively. The RTPV is a gas-impermeable valve composed of an air chamber enclosed by a latex membrane and a specially designed liquid transition chamber that enables the efficient usage of the applied thermal energy. Inputting thermo-pneumatic (TP) energy into the air chamber deflects the membrane into the liquid transition chamber against an inlet, sealing it and thus preventing fluid flow. From this point, a centrifugal pressure higher than the induced TP pressure in the air chamber reopens the fluid pathway. The behaviour of this newly introduced reversible valving system on a microfluidic disk is studied experimentally and theoretically over a range of rotational frequencies from 700 RPM to 2500 RPM. Furthermore, adding a physical component (e.g., a hemispherical rubber element) to induce initial flow resistance shifts the operational range of rotational frequencies of the RTPV to more than 6000 RPM. An analytical solution for the cooling of a heated RTPV on a spinning disk is also presented, which highlights the need for the future development of time-programmable RTPVs. Moreover, the reversibility and gas impermeability of the RTPV in the microfluidic networks are validated on a microfluidic disk designed for performing liquid circulation. Finally, an array of RTPVs is integrated into a microfluidic cartridge to enable sequential aliquoting for the conversion of dengue virus RNA to cDNA and the preparation of PCR reaction mixtures.

Matched MeSH terms: RNA, Viral/analysis
Fulltext Sustained Virologic Response to a Dual Peginterferon alfa-2a and Ribavirin in Treating Chronic hepatitis C Infection: A Retrospective Cohort Study

Naing C, Sitt T, Aung AT, Aung K

Medicine (Baltimore), 2015 Jul;94(30):e1234.
PMID: 26222859 DOI: 10.1097/MD.0000000000001234

In Myanmar, hepatitis C virus (HCV) infection prevalence is 2%. A combination therapy of pegylated interferon alfa-2a and ribavirin (PEG-IFNa/RBV) is a standard treatment, but the effect of this antiviral therapy needs evaluation as to determine the efficacy and safety of dual PEG-IFNa/RBV therapy in treating patients infected with HCV in Myanmar.This was a retrospective analysis of data from a single clinic exclusively for gastrointestinal diseases in Yangon, Myanmar. We assessed treatment responses at the defined time points and stratified by genotypes of HCV. We also determined incidences of adverse events (AEs). We investigated independent predictors of sustained virologic response (SVR) in the participants.A total of 362 HCV-infected cases were included in this study. The majority were females (51.7%) with mean age of 47.12 years (±11.6) and noncirrhosis patients (82%). Rapid virologic response (RVR), early virologic response (EVR), end of treatment response (ETR), and SVR 24 weeks after completion of the dual treatment were 50.3% (178/362), 88% (314/357), 80.1% (286/357), and 85.6% (167/195), respectively. The most frequently reported AEs were nausea/anorexia (72.8%) and flu-like symptoms (62.4%). In multivariate analysis, 4 factors were independently associated with SVR; SVR to genotype 3 (odds ratio [OR] 2.4, 95% CI: 1.24-4.62), EVR (OR 0.54, 95% CI: 0.3-0.95), and duration of treatment (OR 1.52, 95% CI: 1.18-1.98). Study limitations were acknowledged.The efficacy and safety of the dual therapy in treating HCV-infected patient in Myanmar was acceptable. We recommend a prospective randomized control trial looking at duration of therapy and rates of achieving SVR, which could significantly impact the care of HCV-infected patients in Myanmar and perhaps other countries as well.

Matched MeSH terms: RNA, Viral/analysis*
Fulltext Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification

Teoh BT, Sam SS, Tan KK, Danlami MB, Shu MH, Johari J, et al.

J Clin Microbiol, 2015 Mar;53(3):830-7.
PMID: 25568438 DOI: 10.1128/JCM.02648-14

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.

Matched MeSH terms: RNA, Viral/analysis
Fulltext First round of external quality assessment of dengue diagnostics in the WHO Western Pacific Region, 2013

Pok KY, Squires RC, Tan LK, Takasaki T, Abubakar S, Hasebe F, et al.

Western Pac Surveill Response J, 2015 Jun 30;6(2):73-81.
PMID: 26306220 DOI: 10.5365/WPSAR.2015.6.1.017

Accurate laboratory testing is a critical component of dengue surveillance and control. The objective of this programme was to assess dengue diagnostic proficiency among national-level public health laboratories in the World Health Organization (WHO) Western Pacific Region.

Matched MeSH terms: RNA, Viral/analysis
Eco-virological survey of Aedes mosquito larvae in selected dengue outbreak areas in Malaysia

Rohani A, Aidil Azahary AR, Malinda M, Zurainee MN, Rozilawati H, Wan Najdah WM, et al.

J Vector Borne Dis, 2014 Dec;51(4):327-32.
PMID: 25540966

BACKGROUND & OBJECTIVESI: Transovarial transmission of dengue virus in the Aedes vectors is now a well-documented phenomenon reported from many parts of the endemic areas in the world, which played an important role in initiating and maintaining the outbreak in human populations. This study investigated the factors affecting breeding habitats and the relationship with transovarial dengue virus in larvae of Aedes aegypti and Ae. albopictus.

Matched MeSH terms: RNA, Viral/analysis
Fulltext The assessment of risk factors for the Central/East African Genotype of chikungunya virus infections in the state of Kelantan: a case control study in Malaysia

Yusoff AF, Mustafa AN, Husaain HM, Hamzah WM, Yusof AM, Harun R, et al.

BMC Infect Dis, 2013 May 08;13:211.
PMID: 23656634 DOI: 10.1186/1471-2334-13-211

BACKGROUND: The aims of the study were to assess the risk factors in relation to cross border activities, exposure to mosquito bite and preventive measures taken.An outbreak of chikungunya virus (CHIKV) infection in Malaysia has been reported in Klang, Selangor (1998) and Bagan Panchor, Perak (2006). In 2009, CHIKV infection re-emerged in some states in Malaysia. It raises the possibilities that re-emergence is part of the epidemics in neighbouring countries or the disease is endemic in Malaysia. For this reason, A community-based case control study was carried out in the state of Kelantan.
METHODS: Prospective case finding was performed from June to December 2009. Those who presented with signs and symptoms of CHIKV infection were investigated. We designed a case control study to assess the risk factors. Assessment consisted of answering questions, undergoing a medical examination, and being tested for the presence of IgM antibodies to CHIKV. Descriptive epidemiological studies were conducted by reviewing both the national surveillance and laboratory data. Multivariable logistic regression analysis was performed to determine risk factors contributing to the illness. Cases were determined by positive to RT-PCR or serological for antibodies by IgM. CHIKV specificity was confirmed by DNA sequencing.
RESULTS: There were 129 suspected cases and 176 controls. Among suspected cases, 54.4% were diagnosed to have CHIKV infection. Among the controls, 30.1% were found to be positive to serology for antibodies [IgM, 14.2% and IgG, 15.9%]. For analytic study and based on laboratory case definition, 95 were considered as cases and 123 as controls. Those who were positive to IgG were excluded. CHIKV infection affected all ages and mostly between 50-59 years old. Staying together in the same house with infected patients and working as rubber tappers were at a higher risk of infection. The usage of Mosquito coil insecticide had shown to be a significant protective factor. Most cases were treated as outpatient, only 7.5% needed hospitalization. The CHIKV infection was attributable to central/east African genotype CHIKV.
CONCLUSIONS: In this study, cross border activity was not a significant risk factor although Thailand and Malaysia shared the same CHIKV genotype during the episode of infections.

Matched MeSH terms: RNA, Viral/analysis
Genome sequence of a novel HIV-1 circulating recombinant form 54_01B from Malaysia

Ng KT, Ong LY, Takebe Y, Kamarulzaman A, Tee KK

J Virol, 2012 Oct;86(20):11405-6.
PMID: 22997423

We report here the first novel HIV-1 circulating recombinant form (CRF) 54_01B (CRF54_01B) isolated from three epidemiologically unlinked subjects of different risk groups in Malaysia. These recently sampled recombinants showed a complex genome organization composed of parental subtype B' and CRF01_AE, with identical recombination breakpoints observed in the gag, pol, and vif genes. Such a discovery highlights the ongoing active generation and spread of intersubtype recombinants involving the subtype B' and CRF01_AE lineages and indicates the potential of the new CRF in bridging HIV-1 transmission among different risk groups in Southeast Asia.

Matched MeSH terms: RNA, Viral/analysis
Genome sequences of a novel HIV-1 CRF53_01B identified in Malaysia

Chow WZ, Al-Darraji H, Lee YM, Takebe Y, Kamarulzaman A, Tee KK

J Virol, 2012 Oct;86(20):11398-9.
PMID: 22997419

A novel HIV-1 genotype designated CRF53_01B was recently characterized from three epidemiologically unrelated persons in Malaysia. Here we announced three recently isolated full-length genomes of CRF53_01B, which is likely to be phylogenetically linked to CRF33_01B, circulating widely in Southeast Asia. The genome sequences may contribute to HIV-1 molecular surveillance and future vaccine development in the region.

Matched MeSH terms: RNA, Viral/analysis
Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA

Wong KT, Ng KY, Ong KC, Ng WF, Shankar SK, Mahadevan A, et al.

Neuropathol. Appl. Neurobiol., 2012 Aug;38(5):443-53.
PMID: 22236252 DOI: 10.1111/j.1365-2990.2011.01247.x

To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished.

Matched MeSH terms: RNA, Viral/analysis*
Fulltext Impact of antiretroviral therapy on tuberculosis incidence among HIV-positive patients in high-income countries

del Amo J, Moreno S, Bucher HC, Furrer H, Logan R, Sterne J, et al.

Clin Infect Dis, 2012 May;54(9):1364-72.
PMID: 22460971 DOI: 10.1093/cid/cis203

BACKGROUND: The lower tuberculosis incidence reported in human immunodeficiency virus (HIV)-positive individuals receiving combined antiretroviral therapy (cART) is difficult to interpret causally. Furthermore, the role of unmasking immune reconstitution inflammatory syndrome (IRIS) is unclear. We aim to estimate the effect of cART on tuberculosis incidence in HIV-positive individuals in high-income countries.
METHODS: The HIV-CAUSAL Collaboration consisted of 12 cohorts from the United States and Europe of HIV-positive, ART-naive, AIDS-free individuals aged ≥18 years with baseline CD4 cell count and HIV RNA levels followed up from 1996 through 2007. We estimated hazard ratios (HRs) for cART versus no cART, adjusted for time-varying CD4 cell count and HIV RNA level via inverse probability weighting.
RESULTS: Of 65 121 individuals, 712 developed tuberculosis over 28 months of median follow-up (incidence, 3.0 cases per 1000 person-years). The HR for tuberculosis for cART versus no cART was 0.56 (95% confidence interval [CI], 0.44-0.72) overall, 1.04 (95% CI, 0.64-1.68) for individuals aged >50 years, and 1.46 (95% CI, 0.70-3.04) for people with a CD4 cell count of <50 cells/μL. Compared with people who had not started cART, HRs differed by time since cART initiation: 1.36 (95% CI, 0.98-1.89) for initiation <3 months ago and 0.44 (95% CI, 0.34-0.58) for initiation ≥3 months ago. Compared with people who had not initiated cART, HRs <3 months after cART initiation were 0.67 (95% CI, 0.38-1.18), 1.51 (95% CI, 0.98-2.31), and 3.20 (95% CI, 1.34-7.60) for people <35, 35-50, and >50 years old, respectively, and 2.30 (95% CI, 1.03-5.14) for people with a CD4 cell count of <50 cells/μL.
CONCLUSIONS: Tuberculosis incidence decreased after cART initiation but not among people >50 years old or with CD4 cell counts of <50 cells/μL. Despite an overall decrease in tuberculosis incidence, the increased rate during 3 months of ART suggests unmasking IRIS.

Matched MeSH terms: RNA, Viral/analysis

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