Supat Chamnanchanunt

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus.

Downregulation of plasma miR-451 and miR-16 in Plasmodium vivax infection

Malaria is a common parasitic disease in tropical countries, causing one to two million deaths every year. To establish the new biomarker, we analyzed plasma miRNAs obtained from 19 malaria patients and 19 normal subjects, using reverse transcription-based quantitative polymerase chain reaction (RT-qPCR). The average levels of plasma miR-451 and miR-16 were significantly lower in malaria patients, (8.9-fold; p <0.001 and 10.4-fold; p = 0.01, respectively). The levels of other abundant miRNAs in plasma (miR-223, miR-226-3p) did not change significantly in malaria patients. Our data suggest that plasma miR-451 and miR-16 are relevant biomarkers for malaria infection.

Safety and immune responses following administration of H1N1 live attenuated influenza vaccine in Thais

BACKGROUND Emergence and rapid spread of influenza H1N1 virus prompted health authorities to develop a safe and effective influenza vaccine for domestic use. The Thai Government Pharmaceutical Organization (GPO) with technical support from Russia through WHO had prepared a pandemic live attenuated vaccine (PLAIV) using ca-ts attenuated candidate strain A/17/CA/2009/38 (H1N1) for Thais. METHODS Each participant received two doses of intranasal H1N1 vaccine or placebo 21 days apart. All were followed up at 7, 21, 42 and 60 days after first immunization. Blood was drawn for hemagglutination inhibition (HAI) assay from all participants at days 1, 21, 42, and 60 after first immunization. A subset of 40 participants aged 19-49 years was randomly selected for nasal washing at days 1, 21, 42, and 60 to assess IgA using direct enzyme-linked immunosorbent assay (ELISA) along with serum HAI and microneutralization (MN) assay determination. RESULTS A total of 363 subjects aged 12-75 years were randomized into 2 groups (271 vaccinees:92 placebos). Almost all AEs were mild to moderate. Local reactions were stuffy nose (22.3%), runny nose (25.1%), scratchy throat (27.2%) and sore throat (19.3%). Systemic reactions included headache (21.7%), myalgia (13.8%), fatigue (16.8%) and postnasal drip (19.9%). On day 60, HAI seroconversion rates for vaccine:placebo group were 30.3:6.0 for ITT and 29.4:5.1 for PP analysis. Children showed highest seroconversion rate at 44, but it decreased to 39.4 when all 3 assays (HAI, MN assay and ELISA) from subgroup analysis were considered. CONCLUSION The vaccine candidate is safe. The use of more than one assay may be needed for evaluation of immune response because live attenuated vaccines could effectively induce different kinds of responses. Different individuals could also mount different kinds of immune response, even to the same antigen.

Clinical factors for severity of Plasmodium falciparum malaria in hospitalized adults in Thailand.

Plasmodium falciparum is a major cause of severe malaria in Southeast Asia, however, there is limited information regarding clinical factors associated with the severity of falciparum malaria from this region. We performed a retrospective case-control study to compare clinical factors and outcomes between patients with severe and non-severe malaria, and to identify clinical factors associated with the requirement for intensive care unit (ICU) admission of patients with severe falciparum malaria among hospitalized adults in Southeast Asia. A total of 255 patients with falciparum malaria in the Hospital for Tropical Diseases in Bangkok, Thailand between 2006 and 2012 were included. We identified 104 patients with severe malaria (cases) and 151 patients with non-severe malaria (controls). Patients with falciparum malaria with following clinical and laboratory characteristics on admission (1) referrals, (2) no prior history of malaria, (3) body temperature of >38.5°C, (4) white blood cell counts >10×109/µL, (5) presence of schizonts in peripheral blood smears, and (6) albumin concentrations of <3.5 g/dL, were more likely to develop severe malaria (P<0.05). Among patients with severe malaria, patients who met ≥3 of the 2010 WHO criteria had sensitivity of 79.2% and specificity of 81.8% for requiring ICU admission. Multivariate analysis identified the following as independent associated factors for severe malaria requiring ICU admission; (1) ethnicity of Thai [odds ratio (OR) = 3.601, 95% confidence interval (CI) = 1.011–12.822] or Myanmar [OR = 3.610, 95% CI = 1.138–11.445]; (2) referrals [OR = 3.571, 95% CI = 1.306–9.762]; (3) no prior history of malaria [OR = 5.887, 95% CI = 1.354–25.594]; and (4) albumin concentrations of <3.5 g/dL [OR = 7.200, 95% CI = 1.802–28.759]. Our findings are important for the clinical management of patients with malaria because it can help early identification of patients that could develop severe malaria and require ICU admission. Early identification and the timely initiation of appropriate treatments may well improve the outcomes and reduce the mortality of these patients.

Safety and immunogenicity of a live attenuated influenza H5 candidate vaccine strain A/17/turkey/Turkey/05/133 H5N2 and its priming effects for potential pre-pandemic use: a randomised, double-blind, placebo-controlled trial

Summary Background The emergence of highly pathogenic avian influenza H5N1 viruses has raised concerns about their pandemic potential. Vaccination is the most effective way of preventing influenza. In this study, we investigated the safety and immunogenicity of an avian H5N2 live attenuated influenza vaccine (LAIV H5N2) in healthy Thai adults and its priming immune responses with an H5N1 inactivated vaccine boost. Methods This study was done at the Vaccine Trial Centre at Mahidol University, Bangkok, Thailand and was divided into two parts. Part 1 consisted of a randomised, double-blind, placebo-controlled trial done over 18 months. We randomly assigned (2:1) healthy Thai adults aged 18–49 years with a computer generated randomisation sequence (blocks of six) to receive either two intranasal doses (0·25 mL per nostril) of LAIV H5N2 (101 participants) or placebo (51 participants) 21 days apart. For part 2, an open-label trial was done in which previously vaccinated participants (40 from LAIV H5N2 group and 20 placebo) were given one intramuscular dose (0·5 mL) of H5N1 booster vaccine. Participants, investigators, and site-study workers were blinded from randomisation. Immune responses after subsequent immunisation were evaluated using haemagglutination-inhibition and microneutralisation assays and circulating follicular T-helper cells and plasmablast cells were measured in serum and whole blood. The trials are registered with ClinicalTrials.gov, numbers NCT01841918 and NCT02229357. Findings Between Feb 4, 2013, and Feb 28, 2013, 256 individuals were screened, of whom 152 participants were enrolled in part 1 of this study. LAIV H5N2 vaccine was well tolerated. Viral shedding was detected in only six (6%) of 101 participants in the vaccine group 1 day after the first vaccination and in and two (2%) of 98 participants in the group after the second vaccination. There was no serious adverse event in both groups. 51 (50%) of 101 participants in the vaccine group and 28 (55%) of 51 in the placebo group reported at least one adverse event. 80 (84%) of 95 events in the vaccine group and 32 (78%) of 43 events in the placebo groups were reportedly suspected adverse events, probably related to the vaccine; however, most were mild in nature. After two doses of vaccine, 13 (13%) of 100 participants in the vaccine group had an increase in haemagglutination-inhibition titre of more than four-fold and four (4%) of 100 vaccinees developed a rise in neutralisng antibody titre of more than four-fold. 1 year later, after a booster with an inactivated H5N1 vaccine (part 2), 39 (98%) of 40 participants who had previously been vaccinated with LAIV H5N2 had an increase in haemagglutination-inhibition titre of greater than four-fold as early as day 7 compared with three (15%) of 20 participants in the placebo group. Peak geometric mean titre (GMT) for haemagglutination-inhibition antibodies in the previously LAIV H5N2 vaccinated group (566·89 [95% CI 436·97–735·44]) were significantly higher than among those who previously received placebo (25·49 [11·82–54·96]; p<0·0001). The peak GMT by neutralising antibody assay in the H5N2 vaccinated group (1395·85 [1040·79–1872·03]) was also significantly higher than that observed in the placebo group (17·41 [9·05–33·48]; p<0·0001). Importantly, higher cross-reactive haemagglutination-inhibition antibody titres against H5N1 (clades 1, 2.1.3.2, and 2.3.4) were detected in the LAIV H5N2 experienced group than the naive group (p<0·0001). Interpretation Our data suggest that LAIV vaccination induces long-lasting memory immune responses. The limitation of this study was that part 2 was designed as a proof-of-concept study by contrast with part 1. Funding WHO.

Circulating microRNAs in malaria infection: bench to bedside

Severe malaria has a poor prognosis with a morbidity rate of 80% in tropical areas. The early parasite detection is one of the effective means to prevent severe malaria of which specific treatment strategies are limited. Many clinical characteristics and laboratory testings have been used for the early diagnosis and prediction of severe disease. However, a few of these factors could be applied to clinical practice. MicroRNAs (miRNAs) were demonstrated as useful biomarkers in many diseases such as malignant diseases and cardiovascular diseases. Recently it was found that plasma miR-451 and miR-16 were downregulated in malaria infection at parasitic stages or with multi-organ failure involvement. MiR-125b, -27a, -23a, -150, 17–92 and -24 are deregulated in malaria patients with multiple organ failures. Here, the current findings of miRNAs were reviewed in relation to clinical severity of malaria infection and emphasized that miRNAs are potential biomarkers for severe malaria infection.

Clinical Trial of an Oral Live Shigella sonnei Vaccine Candidate, WRSS1, in Thai Adults

ABSTRACT Live attenuated Shigella sonnei vaccine candidate WRSS1, previously tested in U.S. and Israeli volunteers, was evaluated in a population of adult Thai volunteers in which the organism is endemic. In a randomized placebo-controlled, double-blind design, inpatient participants received a single oral dose of 1.6 × 104 CFU of WRSS1. The vaccine was generally well tolerated, with equal numbers of vaccinees and placebo controls showing mild symptoms. Only 3 of 13 vaccinees (23%) had culture-positive stools, while a total of 9 vaccinees were positive by PCR. Lack of vaccine shedding in volunteers correlated with lack of clinical symptoms and immune responses, just as the duration of fecal shedding correlated directly with stronger immune responses. Two months following immunization, 10 vaccinees and 10 newly recruited naive controls received a challenge dose of 1,670 CFU of virulent S. sonnei strain 53G. This dose had previously demonstrated a 75% attack rate for dysentery in Thai volunteers. However, in this study the attack rate for dysentery in naive controls after challenge was 20%. Based on clinical record summaries, 3 vaccinees and 5 naive controls experienced clinically relevant illness (diarrhea/dysentery/fever/shigellosis), and a 40% vaccine efficacy was calculated. When these data are compared to those for the performance of this vaccine candidate in more naive populations, it is clear that a single oral dose of WRSS1 at 104 CFU failed to achieve its full potential in a population in which the organism is endemic. Higher doses and/or repeated immunizations may contribute to improved vaccine shedding and consequent elevation of protective immune responses in a population in which the organism is endemic. (The study has been registered at ClinicalTrials.gov under registration no. NCT01080716.)

Dynamic Measurement of Hemodynamic Parameters and Cardiac Preload in Adults with Dengue: A Prospective Observational Study.

Few previous studies have monitored hemodynamic parameters to determine the physiological process of dengue or examined inferior vena cava (IVC) parameters to assess cardiac preload during the clinical phase of dengue. From January 2013 to July 2015, we prospectively studied 162 hospitalized adults with confirmed dengue viral infection using non-invasive cardiac output monitoring and bedside ultrasonography to determine changes in hemodynamic and IVC parameters and identify the types of circulatory shock that occur in patients with dengue. Of 162 patients with dengue, 17 (10.5%) experienced dengue shock and 145 (89.5%) did not. In patients with shock, the mean arterial pressure was significantly lower on day 6 after fever onset (P = 0.045) and the pulse pressure was significantly lower between days 4 and 7 (P<0.05). The stroke volume index and cardiac index were significantly decreased between days 4 and 15 and between days 5 and 8 after fever onset (P<0.05), respectively. A significant proportion of patients with dengue shock had an IVC diameter <1.5 cm and IVC collapsibility index >50% between days 4 and 5 (P<0.05). Hypovolemic shock was observed in 9 (52.9%) patients and cardiogenic shock in 8 (47.1%), with a median (interquartile range) time to shock onset of 6.0 (5.0–6.5) days after fever onset, which was the median day of defervescence. Intravascular hypovolemia occurred before defervescence, whereas myocardial dysfunction occurred on the day of defervescence until 2 weeks after fever onset. Hypovolemic shock and cardiogenic shock each occurred in approximately half of the patients with dengue shock. Therefore, dynamic measures to estimate changes in hemodynamic parameters and preload should be monitored to ensure adequate fluid therapy among patients with dengue, particularly patients with dengue shock.

Predictive Factors for Death After Snake Envenomation in Myanmar

INTRODUCTION Factors predictive for death from snake envenomation vary between studies, possibly due to variation in host genetic factors and venom composition. This study aimed to evaluate predictive factors for death from snake envenomation in Myanmar. METHODS A prospective study was performed among adult patients with snakebite admitted to tertiary hospitals in Yangon, Myanmar, from May 2015 to August 2016. Data including clinical variables and laboratory parameters, management, and outcomes were evaluated. Multivariate regression analysis was performed to evaluate factors predictive for death at the time of presentation to the hospital. RESULTS Of the 246 patients with snake envenomation recruited into the study, 225 (92%) survived and 21 (8%) died during hospitalization. The snake species responsible for a bite was identified in 74 (30%) of the patients; the majority of bites were from Russells vipers (63 patients, 85%). The independent factors predictive for death included 1) duration from bite to arrival at the hospital >1 h (odds ratio [OR]: 9.0, 95% confidence interval [CI]: 1.1-75.2; P=0.04); 2) white blood cell counts >20 ×103 cells·μL-1 (OR: 8.9, 95% CI: 2.3-33.7; P=0.001); and 3) the presence of capillary leakage (OR: 3.7, 95% CI: 1.2-11.2; P=0.02). A delay in antivenom administration >4 h increases risk of death (11/21 deaths). CONCLUSIONS Patients who present with these independent predictive factors should be recognized and provided with early appropriate intervention to reduce the mortality rate among adults with snake envenomation in Myanmar.

A Randomized Multicenter Trial Comparing Low-Dose Prednisolone Versus Observation for Prevention of Recurrences in Adult Immune Thrombocytopenia:

Adult immune thrombocytopenia (ITP) commonly relapses after stopping treatments. This may be preventable by low-dose steroids. In this multicenter study, adult patients with ITP who had been responding to corticosteroids were randomized with the 2 strata of newly diagnosed and relapsed ITP to prednisolone 7.5 mg/d or observation for 6 months. Relapses were defined by a platelet count below 30 × 109/L and/or clinical bleeding. There were 75 patients evaluable for the efficacy and 77 for safety. The recurrent ITP comprised 57.3%. During the median follow-up of 42 weeks, there were 20.5% (8/39) and 25% (9/36) of recurrences in the prednisolone and control groups (P = .643), with the hazard ratio (HR) of 0.75 (P = .549). The significant factor that could predict recurrences was relapsed ITP with the HR of 2.79 (95% confidence interval, 1.02-7.64, P = .037). Prednisolone showed a trend toward a benefit in the relapsed subgroup (P = .070). Adverse events were not different (P = .540) and mostly mild. In conclusion, prednisolone maintenance could not prolong relapse-free survival. Relapsed patients deserve further investigations for preventive measures.