Electrochemical aptasensor detection of electron transfer flavoprotein subunit beta for leptospirosis diagnosis.

Electron transfer flavoprotein subunit beta (ETFB) of Leptospira interrogans is a biomarker for diagnosing leptospiral infection. Thus, the ETFB-specific nuclease-resistant RNA aptamer ETFB3-63 was developed and used in an electrochemical aptasensor to assay ETFB. Although the majority of reported biosensors detect various genes and antibodies of L. interrogans, this is the first attempt to construct an electrochemical biosensor to detect ETFB protein for the diagnosis of leptospiral infection. The ETFB protein can be detected without any extraction phase. In this assay, a single-stranded DNA probe complementary to the ETFB3-63 sequence was immobilized on a screen-printed carbon electrode (SPCE). The aptamer was then incubated and hybridized with the antisense probe on the SPCE. In the presence of ETFB, the aptamer dissociates from the aptamer/probe complex on the SPCE to bind with the protein. Methylene blue was then added to intercalate with the remaining hybridized aptamers, and its signal was measured using differential pulse voltammetry. The signal arising from the intercalated methylene blue decreased with increasing concentration of ETFB, showing a linear response in the range of 50-500 nM of ETFB and 10 to 109 leptospira cells per mL, respectively. The aptasensor signal was also specific to L. interrogans but not to 12 related bacteria tested. In addition, the aptasensor showed similar performance in detecting ETFB spiked in human serum to that in buffer, indicating that proteins in the serum do not interfere with the assay. Therefore, this assay has great potential to develop into a point-of-care electrochemical device that is accurate, cost-effective, and user-friendly for leptospirosis diagnosis.

Characterization of recombinant flagellin B protein from Leptospira interrogans.

Symptoms of the early phase leptospirosis often are non-specific and can be a major problem in making a diagnosis of febrile illnesses. Rapid diagnosis of leptospirosis is of extreme importance, because antibiotic treatment provides greatest benefit when administered in early stage of the disease. Recombinant flagellin B (FlaB) gene (flaB) of Leptospira interrogans serovar Autumnalis strain Akiyami A was heterologously expressed and purified. The 35 kDa recombinant FlaB was 99% similar to the reference strain in GenBank. Rabbit polyclonal antirecombinant FlaB antibodies recognized using immunoblotting yeilded 35-36 kDa doublet from one saprophytic and eight pathogenic Leptospira serovars. Western blot assay showed that recombinant FlaB could distinguish leptospirosis from non-leptospirosis sera. This recombinant FlaB can be used in serodiagnosis of leptospirosis and identification of Leptospira spp.

Draft genome sequence data of the multidrug-resistant bacterium Staphylococcus haemolyticus 010503B isolated from an aerosol sample in a hospital waiting area in Thailand.

Staphylococcus haemolyticus 010503B is a multidrug-resistant bacterium isolated from an outpatient clinic in a hospital waiting area in Thailand. Here we present the draft genome sequence of S. haemolyticus 010503B. The paired-end reads were generated on the Illumina NextSeq 550 sequencer using genomic DNA from the pure culture of S. haemolyticus 010503B. The draft genome consisted of 114 contigs with a total size of 2,457,654 base pairs, an N50 of 57,312 base pairs and a GC content of 32.60%. The dDDH between 010503B and Staphylococcus haemolyticus SM 131T was 91.9%, identifying the strain as Staphylococcus haemolyticus. The data presented holds promise for bacterial classification, comparative genomics, analysing antimicrobial resistance comprehensively, and assessing bacterial virulence factors of S. haemolyticus. The draft genome sequence data has been deposited at NCBI under Bioproject accession number PRJNA550309.

Comparison of immunogenicity between intradermal and intramuscular injections of repeated annual identical influenza virus strains post-pandemic (2011-2012) in COPD patients.

We compared the antibody responses and persistence of the reduced-dose, 9 µg hemagglutinin (HA)/strain intradermal (ID) injection via the Mantoux technique and the 15 µg HA/strain intramuscular (IM) injection of the repeated annual identical trivalent, inactivated, split-virion vaccine 2011-2012 in chronic obstructive pulmonary disease (COPD) patients. Eighty patients were randomized to ID (n = 41) and IM (n = 39) groups. Four weeks post-vaccination, the antibody responses of the two groups were similar; those for influenza A(H1N1)pdm09 and influenza A(H3N2)-but not influenza B-met the criteria of the Committee for Proprietary Medicinal Products (CPMP). The antibody responses for influenza A(H1N1)pdm09 rapidly declined in both groups, especially with the ID injection, whereas those for influenza A(H3N2) maintained above the CPMP criteria throughout 12 months post-vaccination. The geometric mean titres for influenza A(H1N1)pdm09 persisted above the protective threshold (≥ 40) until 6 months post-vaccination in both the ID and IM groups. The seroprotection rates of the ID and IM groups were above 60% until 3 months and 6 months post-vaccination, respectively. In conclusion, the 9 µg HA/strain ID injection of vaccine 2011-2012 elicited antibody responses similar to the standard dose of 15 µg of the HA/strain IM injection at 4 weeks post-vaccination. However, the antibody responses for influenza A(H1N1)pdm09 rapidly declined, especially in the case of the ID injection, whereas they were comparable for influenza A(H3N2). Additional strategies for increasing vaccine durability should be considered, especially for new pandemic strains affecting elderly COPD patients.

Analysis of differential proteomes in pathogenic and non-pathogenic Leptospira: potential pathogenic and virulence factors.

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel-based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non-pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non-pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.

Detection and differentiation between pathogenic and saprophytic Leptospira spp. by multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) was developed for diagnosing leptospirosis and differentiating pathogenic and saprophytic leptospires. Specific primers were designed to amplify 23S rDNA from pathogenic Leptospira and saprophytic Leptospira spp. PCR products from 27 pathogenic and 5 (including 1 intermediate) saprophytic serovars were 615 and 316 base pairs (bp), respectively. After the restriction enzyme's digestion of PCR products, the fragments by SacI of pathogenic serovars and by PstI of saprophytic serovars were 339 and 276 bp and 202 and 114 bp, respectively. The PCR primers enabled amplification of DNA from L. meyeri serovar Ranarum as a pathogenic Leptospira spp. The PCR assay could detect 1 to 2 cells of leptospires and not amplify DNA from other 18 bacterial species. The sensitivity and specificity of this PCR in rat kidney, using isolation as gold standard, were 98.6% and 100%, respectively. The most appropriate sample preparation of blood for detecting DNA was buffy coat. Among the sample preparations from 7 laboratory-confirmed leptospirosis cases, leptospiral DNA was detected in all 7 buffy coat preparations, whereas leptospiral DNA was detected in only 3 plasma or serum samples. The PCR assay may be useful as a diagnostic tool for leptospirosis.

Assessment of Southern blot ribotyping for differentiation of Leptospira strains isolated from field rats.

A Southern blot ribotyping based on EcoRV and HindIII digestion with two 16S and 23S rDNA probes for differentiating 27 Leptospira serovars was developed. The results between ribotyping and serotyping among 40 leptospiral strains isolated from field rats trapped in the northeastern region of Thailand during 1999-2000, were compared. A combination of Southern blot ribotyping, using EcoRV or HindIII digestion with both 16S and 23S rDNA as the probes, successfully typed 27 Leptospira serovars into 24 ribotypes with the discriminatory index (D) values of 0.99. The 16S- and 23S-EcoRV ribopatterns produced 17 and 9 profiles, respectively, with D values of 0.95 and 0.63, respectively. Ribopatterns of HindIII from both specific probes yielded 17 patterns. The D values of 16S- and 23S-HindIII ribopatterns were 0.94 and 0.93, respectively. With EcoRV digestion, the 16S rDNA probe was more discriminative than the 23S rDNA probe for differentiating Leptospira serovars. Moreover, the 16S-EcoRV (11 profiles), 16S-HindIII (11 profiles), and 23S-HindIII (10 profiles) ribopatterns produced higher numbers of distinct and unique profiles than the 23S-EcoRV (5 profiles). The results showed 100% concordance between ribotyping and serotyping, leading to all 40 isolates being successfully typed. The current study revealed that ribotyping as a quick and powerful tool for differentiating Leptospira serovars, has potential value in epidemiological studies.

Leptospirosis in Takeo Province, Kingdom of Cambodia, 2003.

BACKGROUND: In Cambodia, epidemiology and disease burden of leptospirosis were not addressed as they do not have an existing surveillance system and have limitations on their laboratory diagnosis. OBJECTIVE: Define the existence of leptospirosis and determine the antibodies to serovars of leptospires in Cambodia. MATERIAL AND METHOD: One hundred and twenty-one suspected cases of leptospirosis were enrolled in this cross-sectional study, between September 8 and November 30, 2003 from Takeo Provincial Hospital in Doun Keo District, Cambodia. RESULTS: Common clinical manifestations were fever (96%), headache (92%), and myalgia (87%). Common risk behaviors were throwing garbage on the ground (84%), pulling out sprouts (77%), fertilizing (49%), and plowing (47%). Microscopic agglutination test result confirmed four cases and polymerase chain reaction test result confirmed seven cases. Two cases each showed antibodies to serovars Javanica and Australis. An estimated annual incidence of leptospirosis in Takeo province was 7.65 per 100,000 populations. Further studies to define epidemiology and burden of disease are needed. CONCLUSION: Increasing awareness and knowledge on leptospirosis among people are necessary to decrease the impact of leptospirosis in Cambodia.

Trends of HIV seropositivity at Siriraj Hospital: 13 year's observation from 1992-2004.

OBJECTIVE: The aim of the present report was to observe the trend of seroprevalence rates of HIV seropositivity for routine services at Siriraj Hospital for 13 years. MATERIAL AND METHOD: The prevalence rate of HIV seropositivity was analyzed in three groups of subjects: 1) patients who attended the hospital with HIV related diseases; 2) pregnant women at first visit to the antenatal care clinic; 3) emigrating workers who have applied for employment in foreign countries. RESULTS: Of the 13 year-observation, HIV seroprevalence rates in the groups of patients, pregnant women and emigrating workers was 10.6% (95%CI 8.9-12.3%), 2.0% (95%CI 1.8-2.2%) and 0.6% (95%CI 0.4-0.8%), respectively. CONCLUSION: The low prevalence of HIV seropositivity in the group of emigrating workers may be due to self selection, whereas the prevalence in pregnant women, which was rather consistent at about 2.0%, may represent the infection rate in the general population. The seroprevalence rate measured in the group of pregnant women demonstrates that Thailand should increase efforts to confine the spread of HIV infection in the community.

The immunogenicity of the intradermal injection of seasonal trivalent influenza vaccine containing influenza A(H1N1)pdm09 in COPD patients soon after a pandemic.

The antibody responses of a reduced-dose intradermal seasonal influenza vaccination have never been studied in COPD patients soon after a pandemic. A total of 149 COPD patients (60 y of age or older) were randomized to receive trivalent influenza vaccine (Sanofi-Pasteur, France) either 9 µg of hemagglutinin (HA) per strain split into 2-site intradermal (ID) injections via the Mantoux technique or one intramuscular (IM) injection of 15 µg of HA per strain. The geometric mean titers, seroconversion factors, seroconversion rates and seroprotection rates for influenza A(H3N2) and B administered through the ID injection (n = 75) were similar to those obtained with the IM injection (n = 74) 4 weeks post-vaccination. The antibody responses for influenza A(H1N1)pdm09 administered through the ID injection were lower than those obtained with the IM injection, but all of these responses met the 3 criteria proposed by the Committee for Proprietary Medicinal Products (CPMP) for annual re-licensure. The seroprotection rates 4 weeks post-vaccination for influenza A(H1N1)pdm09 were 64.0% (95%CI 52.7-74.0%) in the ID group vs. 78.4% (95% CI 67.6-86.3%) in the IM group (p = 0.053). Influenza-related acute respiratory illness (ARI), diagnosed as a 4-fold rise in HI titers with a convalescent titer > 1:40, and/or the RT-PCR between the ID group (5.3%) and the IM group (8.1%) were not significantly different. The reduced-dose intradermal influenza vaccine may expand vaccine coverage in cases of vaccine shortage.

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

The efficacy and effectiveness of influenza vaccination among Thai elderly persons living in the community.

OBJECTIVE: To determine the efficacy and cost-effectiveness of influenza vaccination in the Thai elderly living in an urban community. MATERIAL AND METHOD: The study design was a stratified, randomized, double blind, placebo-controlled trial. A total of 635 participants aged 60 years and older living in an urban community was randomized to receive an influenza vaccine or tetanus toxoid as a placebo injection. All participants were followed up 4-6 weeks in the community for influenza-like illness and treatment received, hospitalization and death for one year. A hemagglutination inhibition (HI) test for influenza virus antibody of all participants was done on the day of vaccination as well as 1 month, 5 months, and 12 months after the vaccination. Main outcome measures were immune response rate and protective titer, influenza-like illness, serological influenza, treatment received for influenza-like illness and their expenses, hospitalization and death during the study period. RESULTS: The immune response rate of vaccinations was 97.1% and protective titer for A (H1N1) and A (H3N2) strains were 96.4 and 98.6%, respectively. The incidence of influenza-like illness was 4.83% in the vaccine group compared with 10.88% in the placebo group. The relative risk reduction was 56% (95% CI = 14 to 77%). The survival analysis also showed that vaccinations significantly reduced the incidence of influenza (p = 0. 009). The number needed to prevent one episode was 17 persons (95% CI = 9 to 71 persons). The adverse reactions of vaccinations were mild and tolerable. However, the number of treatments received for influenza-like illness and their cost were not significantly different between the two groups. None of the subjects had pneumonia nor needed hospitalization during the study period. Seven participants died during the year of follow up, but not from influenza. CONCLUSION: In Thai elderly living in the community, influenza vaccination reduced the incidence of influenza-like illness by half, but not the number of treatments received for influenza-like illness, their cost, and its serious complications. In the year of the study, considering the cost of vaccines and the numbers needed to prevent one episode of infection from the provider's viewpoint, it may not be cost-effective to recommend that all Thai older persons living in the community should receive influenza vaccination annually. Vaccination recommendation for the elderly should be promptly implemented in expectation of a severe epidemic in Thailand.

Acute respiratory illness in patients with COPD and the effectiveness of influenza vaccination: a randomized controlled study.

STUDY OBJECTIVES: To determine the effectiveness of influenza vaccination on influenza-related acute respiratory illness (ARI) and overall ARI in patients with COPD, and its relationship to the degree of airflow obstruction. DESIGN: Stratified, randomized, double-blind, placebo-controlled trial. SETTING: From June 1997 to November 1998 at a single university hospital. PATIENTS AND INTERVENTIONS: One hundred twenty-five patients with COPD were stratified based on their FEV(1) as having mild, moderate, and severe COPD. Within each group, they were randomized to the vaccine group (62 patients who received purified, trivalent, split-virus vaccine) or the placebo group (63 patients). MEASUREMENTS: The number of episodes and severity of total ARI, classified as outpatient treatment, hospitalization, and requirement of mechanical ventilation; and the number of episodes and severity of influenza-related ARI. RESULTS: The incidence of influenza-related ARI was 28.1 per 100 person-years and 6.8 per 100 person-years in the placebo group and vaccine group, respectively (relative risk [RR], 0.24 [p = 0.005]; vaccine effectiveness, 76%). The incidences were 28.2, 23.8, and 31.2 per 100 person-years in the patients with mild, moderate, and severe COPD, respectively, in the placebo group, and 4.5, 13.2, and 4.6 per 100 person-years in the patients with mild, moderate, and severe COPD, respectively, in the vaccine group (RR, 0.16 [p = 0.06]; vaccine effectiveness, 84%; RR, 0.55 [p = 0.5]; vaccine effectiveness, 45%; and RR, 0.15 [p = 0.04]; vaccine effectiveness, 85%, in the patients with mild, moderate, and severe COPD, respectively). Bivariate analysis revealed that the effectiveness of influenza vaccination was not modified by the severity of COPD, comorbid diseases, age, gender, or current smoking status. There was no difference in the incidence or severity of total ARI between the placebo group and the vaccine group. CONCLUSIONS: Influenza vaccination is highly effective in the prevention of influenza-related ARI regardless of the severity of COPD. Influenza vaccination does not prevent other ARIs unrelated to influenza. The effectiveness of influenza vaccination in the prevention of overall ARI in patients with COPD will depend on how much the proportion of influenza-related ARI contributes to the incidence of total ARI. Influenza vaccination should be recommended to all patients with COPD.

Flexibility in the administration schedule of varicella vaccination in healthy adolescents and young adults.

A clinical trial to assess the immunogenicity and reactogenicity of two doses of varicella vaccine (live attenuated Oka-strain, GlaxoSmithKline Biologicals), when either given 8 or 4 weeks apart in healthy seronegative adolescents and young adults, was conducted in Khon Kaen and Bangkok, Thailand. Contrary to seroconversion rates generally reported for this age group, in our study all subjects were already seropositive after the first vaccine dose. After the first vaccine dose, geometric mean titers (GMTs) for anti-varicella antibodies were 78.4 (median 64) for the adolescent group and 136.5 (median 128) for the young adult group. Six weeks after administration of the second dose, anti-varicella GMTs reached 331.7 (median 256) and 636.9 (median 512) for the adolescent and young adult groups, respectively, with a 4.2-4.7-fold increase from pre-vaccination titers. The difference in GMTs between post-dose I and dose II was statistically significant for each group. The reactogenicity after the first and second doses of vaccination was low: no varicella rash was seen, in either the shorter or longer schedule. GlaxoSmithKline Biologicals varicella vaccine (Varilix) offered a high flexibility, administration possible at either 4 or 8 weeks interval, whilst eliciting good immunogenicity and good tolerability.

Prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in low and high endemic areas in Thailand.

OBJECTIVE: To investigate the prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in urban and rural areas in low and high endemic areas in Thailand. MATERIAL AND METHOD: A total of 1,664 serum samples were collected from rodents and shrews in areas of low and high endemicity for leptospirosis. Four areas classified by case rates (CR) per 100,000 population of leptospirosis were urban Area I Bangkok (CR = 0.07), rural Area II (CR = 0.24), rural Area III (CR = 1.97) and rural Area IV (CR = 48.20). All serum samples were investigated for antibodies to leptospires by microscopic agglutination test (MAT) using antigens from each of the 22 pathogenic serovars of Leptospira interrrogans: australis, autumnalis, ballum, bangkok, bataviae, bratislava, canicola, celledoni, copenhageni, djasiman, grippotyphosa, hardjo, hebdomadis, icterohaemorrhagiae, javanica, pomona, pyrogenes, rachmati, saigon, sejroe, tarassovi and wolffi and one non-pathogenic strain of L. biflexa serovar patoc. RESULTS: Ninety-four (5.6%) serum samples were positive for Leptospira antibodies. The most commonly detected antibodies were to serovars pyrogenes (39.1%), sejroe (19.1%), bataviae (10.0%), pomona (6.4%), autumnalis (5.5%), copenhageni (3.6%) and javanica (3.6%). The positive rates in Area I, II, III and IV were 7.6 per cent, 2.9 per cent, 4.6 per cent and 7.1 per cent, respectively. The seroprevalence in rural areas tended to increase significantly with high endemicity for leptospirosis (Chi-square for trend, p = 0.04). The seropositive rates by animal species were 39/496 (7.9%), 22/322 (6.8%), 23/492 (4.7%), 6/170 (3.5%), 4/175 (2.3%), 0/4 (0%) and 0/5 (0%) in Rattus norvegicus, Rattus exulans, Rattus rattus, Bandicota indica, Bandicota savilei, Mus musculus and Suncus murinus, respectively. There was a statistical trend between seropositive rates in R. exulans and endemicity for leptospirosis (Chi-square for trend, p = 0.04). CONCLUSION: The 5.6 per cent of rodents and shrews trapped in urban and rural areas in Thailand were reservoirs of leptospires. The results of high seroprevalence in rats also indicate the high endemicity for leptospirosis.

Occurrence and protective level of influenza infections using serology in patients with COPD in vaccination study.

OBJECTIVE: To investigate the prevalence, occurrence and protective level of influenza infections using serology in patients with chronic obstructive pulmonary disease (COPD) during a one-year influenza vaccination study. MATERIAL AND METHOD: A total of 123 patients with COPD were enrolled during the period of 1997 to 1998. There were 61 patients in the vaccine group and 62 patients in the placebo group with a mean age +/- SD of 67.6 +/- 8.0 and 69.1 +/- 7.5, respectively. The vaccine was composed of influenza A/Texas/36/91 (H1N1), A/Nanchang/933/95 (H3N2) and B/Harbin/07/94 strains. Antibodies to influenza viruses were detected by hemagglutination inhibition (HI) test using antigens of vaccine strains. RESULTS: The incidence of influenza proven by serological examination was 22/123 (17.9%) cases. Among 17/62 (27.4%) influenza cases in the placebo group representing natural infections, 3 (17.6%) were diagnosed as A (H1N1), 8 (47.1%) as A (H3N2), 3 (17.6%) as type A, 1 (5.9%) as type B and 2 (11.8%) as untypeable viruses. The 8.2% of influenza cases found in the vaccine group was significantly lower than 27.4% of that in the placebo group (Chi-square test, p = 0.01). The protection rate of influenza vaccination was 71%. Among 23 acute blood samples from 22 influenza cases, the titers ranged from < 10 to 20 corresponding to its type/subtype. In the vaccine group, 5 influenza cases occurred at 7, 7, 10, 11 and 11 months after vaccination. The HI antibodies to influenza A (H1N1), A (H3N2) and B viruses at titers of > or = 10 vs > or = 40 were 50.4% vs 21.9%, 54.5% vs 28.5% and 17.9% vs 4.1%, respectively. CONCLUSION: The findings indicated that from 1997 to 1998, the occurrence of influenza as natural infection was 27.4%. Influenza A (H3N2) was more frequently prevalent than A (H1N1) and B viruses. The influenza vaccination in COPD patients was effective. The protective HI antibody titers were > or = 40. The patients without protective HI antibody to A (H1N1), A (H3N2) and B viruses were 78.1%, 71.5% and 95.9%, respectively. Such patients were considered to be at high-risk for influenza and recommended to have vaccination.

Kinetics of the antibody response to seasonal influenza vaccination among the elderly.

Influenza vaccination, which has been targeted to the elderly and those at serious risk of complications, is recommended. The purpose of this study was to determine antibody responses after influenza vaccination among Thai elderly persons living in the community. A total of 591 subjects consisting of 308 vaccinees and 283 non-vaccinees were enrolled in the study. Antibodies to H1N1, H3N2, and B viruses were detected by hemagglutination inhibition (HI) testing. The numbers of subjects who had protective antibody titers ≥40 and geometric mean titers (GMTs) of antibodies against A(H1N1), A(H3N2), and B viruses prior to vaccination were similar for the vaccine and placebo groups. The seroprotection rates and GMTs for influenza virus A(H1N1), A(H3N2), and B strains after influenza vaccination at 1, 5, and 12 mo in the vaccine group were significantly higher than those in the placebo group. The seroprotection rates for the A(H1N1) and A(H3N2) strains, but not the B strain, met Committee for Proprietary Medicinal Products (CPMP) criteria (>60%). GMTs and seroprotection rates against influenza B strain in the vaccinees at all time points were <40% and <60%, respectively, and significant differences between the vaccinees and the placebo controls were observed. The GMTs and seroprotection rates for influenza strains in those with pre-existing antibody titers ≥40 were significantly higher than those in the group with pre-existing antibody titers <40. These findings demonstrated that the elderly living in the community developed adequate antibody responses with sustainable titers throughout the 12-month study period after influenza vaccine immunization. Moreover, the presence of pre-existing antibody at a titer ≥40 prior to vaccination strongly affected the antibody response to influenza vaccination.

Rickettsia, Ehrlichia, Anaplasma, and Bartonella in ticks and fleas from dogs and cats in Bangkok.

Flea and tick specimens (5-10 fleas or ticks) on dogs and cats from various sites in Bangkok were tested by polymerase chain reaction and DNA sequencing to detect DNA of bacteria Rickettsia (gltA and 17 kDa genes), Anaplasmataceae (16S rRNA gene), and Bartonella (pap31 and its genes). We confirmed that Rickettsia sp. related to Rickettsia felis was detected in 66 of 98 (67.4%) flea specimens from dogs, whereas 8 Bartonella henselae and 2 Bartonella clarridgeiae were detected in 10 of 54 (18.5%) flea specimens from cats. Further, this work provides the first evidence of 10 Ehrlichia canis (3.3%), 7 Anaplasma platys (2.3%), and 2 Wolbachia spp. (0.66%) in 304 Rhipicephalus sanguineus tick specimens in Thailand.

Serological response to the 2009 pandemic influenza A (H1N1) virus for disease diagnosis and estimating the infection rate in Thai population.

BACKGROUND: Individuals infected with the 2009 pandemic virus A(H1N1) developed serological response which can be measured by hemagglutination-inhibition (HI) and microneutralization (microNT) assays. METHODOLOGY/PRINCIPAL FINDINGS: MicroNT and HI assays for specific antibody to the 2009 pandemic virus were conducted in serum samples collected at the end of the first epidemic wave from various groups of Thai people: laboratory confirmed cases, blood donors and health care workers (HCW) in Bangkok and neighboring province, general population in the North and the South, as well as archival sera collected at pre- and post-vaccination from vaccinees who received influenza vaccine of the 2006 season. This study demonstrated that goose erythrocytes yielded comparable HI antibody titer as compared to turkey erythrocytes. In contrast to the standard protocol, our investigation found out the necessity to eliminate nonspecific inhibitor present in the test sera by receptor destroying enzyme (RDE) prior to performing microNT assay. The investigation in pre-pandemic serum samples showed that HI antibody was more specific to the 2009 pandemic virus than NT antibody. Based on data from pre-pandemic sera together with those from the laboratory confirmed cases, HI antibody titers ≥ 40 for adults and ≥ 20 for children could be used as the cut-off level to differentiate between the individuals with or without past infection by the 2009 pandemic virus. CONCLUSIONS/SIGNIFICANCE: Based on the cut-off criteria, the infection rates of 7 and 12.8% were estimated in blood donors and HCW, respectively after the first wave of the 2009 influenza pandemic. Among general population, the infection rate of 58.6% was found in children versus 3.1% in adults.

The immunogenicity of intradermal influenza vaccination in COPD patients.

We evaluated the immunogenicity of a reduced-dose intradermal trivalent, inactivated, split-virion seasonal influenza vaccine compared to that of a conventional intramuscular vaccination in chronic obstructive pulmonary disease (COPD) patients. One hundred and fifty-six COPD patients randomly received either 0.2 ml (6 microg hemagglutinin (HA) per strain) split into two-site intradermal (ID) injections or a single 0.5 ml (15 microg HA per strain) intramuscular (IM) injection. Geometric mean titers, seroconversion factors, seroconversion rates and seroprotection rates at 4 weeks post-vaccination in the ID group were less than those in the IM group. Only the seroconversion factor to influenza B in the ID group was statistically less than in the IM group (18.8 in the ID group, n=81 versus 37.3 in the IM group, n=75, p=0.045). Nevertheless, each strain of the ID vaccination met all the Committee for Proprietary Medicinal Products (CPMP) criteria. Seroprotection rates were above 60% throughout the year in influenza A (H3N2), for at least 6 months in influenza A (H1N1) and at least 4 weeks in influenza B in both ID and IM groups. The reduced-dose intradermal vaccination may be considered for use in COPD patients in a vaccine shortage situation.