Prime-boost vaccination using recombinant Mycobacterium bovis BCG and recombinant vaccinia virus DIs harboring HIV-1 CRF01_AE gag in mice: influence of immunization routes.

We have previously reported that live vector-based HIV-1 gag vaccine candidate using BCG as a vector was achievable in BALB/c mice. Although the gag-specific CTL induced by this live candidate vaccine is significantly high, persistence of CTL remains unclear. Thus, efforts were made to explore the potential of recombinant Vaccinia virus DIs strain harboring the same HIV-1 CRF01_AE gag gene (rVaccinia/ HIV-1gagE) present in the BCG construct, using different immunization routes. After one month following a single subcutaneous (s.c.) injection of rBCG/HIV-1gagE, higher CTL responses were recognized against various peptide epitopes along the whole gag protein compared to that by intradermal (i.d.) route. A prime-boost regimen having only rDIs/HIV-1gagE injected i.d. induced very low CTL levels. However, within two months, by priming with rBCG/HIV-1gagE s.c. and boosting with rVaccinia/HIV-1gagE intravenously (i.v.), CTL levels were greater (20-68% specific cell lysis) than those obtained by priming and boosting both i.d. (18-35%). After seven months, both prime-boost immunization with rBCG/HIV-lgagE s.c. and with rVaccinia/HIV-1gagE either i.v. or i.d. sustained similar CTL levels. Our studies exhibit that the prime-boost vaccination of rBCG/HIV-1gagE following by rVaccinia/HIV-1gagE i.d. could be used to elicit prolonged CTL responses as well as memory T-cells in mice, which might be more practical than using i.v. route.

Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Demographic, socio-economic, behavioral and clinical factors predicting virologic failure with generic fixed-dose combination antiretroviral therapy before universal health insurance coverage in northern Thailand.

We conducted a 2-year prospective cohort study to investigate multiple aspects of factors predicting the outcome of fixed-dose combination antiretroviral (ARV) therapy with lamivudine, stavudine, and nevirapine (GPOvir) at a government referral hospital in northern Thailand. At 6 and 24 months after the initiation of GPOvir, viral load (VL) was measured to determine virologic failure (>400 RNA copies/ml) and demographic, socio-economic, behavioral and clinical data were collected. From 10 April 2002 to 31 January 2004, 409 patients participated in this study: 64/364 (17.0%) at 6 months and 55/345 (15%) at 24 months virologically failed treatment. On univariate analysis, besides ARV experience [odds ratio (OR), 3.08, 95% confidence interval (CI), 1.71 -5.57] and the frequency of delayed doses (OR, 2.97; 95% CI, 1.47-6.00), we identified one socioeconomic factor significantly associated with virologic failure: "not having child" (OR, 1.85; 95% CI, 1.03 - 3.34). Although the association with "not having child" became marginal on multivariate analysis, results of in-depth interviews and group discussions indicated that having a child was a strong motivating factor for good treatment compliance. We suggest that patients without children may need more attention. Further investigation of socio-economic factors is warranted.

Enhancement of cell-mediated immune response in mice by whole HIV-1 gag in Mycobacterium bovis BCG as a live vaccine candidate.

In this study, we employed a recombinant Mycobacterium bovis Bacille Calmette-Guerin (BCG) harboring whole HIV-1 CRF01_AE gag DNA as a candidate vaccine to investigate specific cell-mediated immunity in BALB/c mice. Construction of the stable expression recombinant BCG was achieved by demonstrating by Western blot detection of protein of approximately 55 kDa. By a single injection of 0.1 mg of the recombinant HIV-1 gag protein expressing BCG subcutaneously into mice, after 2 weeks various specific cytotoxic T-lymphocyte (CTL) responses were exhibited against a single gag epitope of amino acid positions 294-304, and also against various peptide regions along the entire gag protein with moderate CTL activities (10-35% specific cell lysis), which increased to relatively high levels (50-68%) after one month. However, after two months activities were 3-3.7 fold lower. On the other hand, gag-specific lymphocyte proliferation was detected 9.3 fold higher than that of non-immunized mouse spleen cells. Our results indicate that in mice, BCG can be used as a recombinant live vector to induce cellular immune responses to HIV-1 gag antigen.

The epidemiological relationship between Salmonella enterica serovar typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from humans and swine in Thailand.

A total of 138 isolates of S. Typhimurium and S. 4,[5],12:i:- from humans and swine in Thailand during 2003-2006, were evaluated for antimicrobial sensitivity by the disk diffusion method against 10 antimicrobial drugs and pulsed-field gel electrophoresis (PFGE) with endonuclease Xbal to investigate the epidemiological relationship among isolates. It was found that all isolates were classified into 27 antimicrobial resistance patterns, and 80% of S. Typhimurium and 95.4% of S. 4,[5],12:i:- isolates were resistant to three or more antimicrobial agents. By PFGE testing, the 84 PFGE patterns were categorized into A to Z patterns. Eighty percent of S. Typhimurium and 71.3% of S. 4,[5],12:i:- isolates in 7 major PFGE patterns had close clonal relationships (_85% similarity). Our studies indicate the spread of genetically identical clones of S. Typhimurium and S. 4,[5],12:i:- in humans and swine in Thailand.

Safety and efficacy of hyptis suaveolens extract for therapy of influenza in healthy adults.

Objective: To determine the safety and efficacy of H.suaveolens extract for therapy of influenza in healthy adults. Methods: The study was randomized double blind placebo controlled study conducted in 15 community and general hospitals from May to August 2006. The study subjects were healthy adults who had influenza-like symptoms and positive preliminary diagnostic test for influenza A or B from respiratory secretions. They were randomized to receive H.suaveolens extract 500 mg 3 times daily or the placebo 3 times daily for 7 days. The subjects were evaluated for the severity of symptoms related to influenza, adverse effects of the medications and the presence of influenza viruses from respiratory secretions at entry, day 4 and day 7 after treatment. Results: There were 39 subjects in the placebo group and 46 in the H.suaveolens group. There was a significant improvement in symptoms of the patients in both groups on day 4 and day 7 when compared with that at entry. However, the average duration of fever of the patients in both groups was not significantly different (3.1 days in the placebo group vs. 3 days in the H.suaveolens group, p=0.749). The recovery rates of influenza A and influenza B viruses from respiratory secretions of the subjects on day 4 and day 7 after treatment in both groups were not significantly different. A trend of less positive culture for influenza A virus in the patients receiving H.suaveolens extract (32.5%) compared with those receiving a placebo (47.1%) was observed. The compliance to medications was satisfactory. No serious adverse effects due to study medications were observed. Conclusion: H.suaveolens extract 1.5 grams per day for 7 days is safe but it is not effective in relieving influenza-related symptoms in adults with influenza. The lack of efficacy of H.suaveolens extract might be due to an insufficient dosage of the extract.

Typing of Thai clinical isolates of Mycobacterium leprae and analysis of leprosy transmission by polymorphism of tandem repeats.

Mycobacterium leprae isolates from Thai leprosy patients were typed for strain differentiation and analysis of leprosy transmission using the six base tandem repeat, GACATC, in rpoT gene and TTC repeat as genetic markers. M. leprae DNA was isolated from skin biopsies of new untreated leprosy patients living in remote areas or in suburban regions of Thailand where leprosy is in low prevalence. In M. leprae strains of 100 patients, TTC alleles exhibited variations in length with 10 to 30, 33 and 35 repeats, the most common alleles being 15, 16, 17 and 19 repeats. All isolates contained three copies of the six base repeat in rpoT gene. Application of TTC repeats in tracking leprosy transmission in two families with multi-cases identified a single (but different) strain of M. leprae in each family.

Identification of clinical isolates of Leptospira spp by pulsed field gel-electrophoresis and microscopic agglutination test.

Twenty-five clinical isolates of Leptospira spp were characterized by microscopic agglutination test (MAT) and pulsed field gel-electrophoresis (PFGE) in comparison with 23 reference Leptospira serovars and with the saprophytic L. biflexa serovar Patoc. PFGE DNA profiling was more specific and reliable than MAT.

Mycobacterium avium and Burkholderia pseudomallei (Melioidosis) coinfection in an HIV-positive patient.

A 29 year old HIV positive Thai female with CD4 count of 10 cells/mm3 presented with chronic diffuse abdominal pain, fever, weight loss, anemia and leucopenia. Ultrasonography demonstrated diffuse upper abdominal lymphadenopathy with ascites. Microbiological and molecular work up of the specimen obtained by ultrasound-guided lymph node aspiration revealed co-infection with Burkholderia pseudomallei and Mycobacterium avium. Indirect hemagglutination, IgM-indirect fluorescent antibody, and IgG-indirect fluorescent antibody to Burkholderia pseudomallei were < 1:20, < 1:50 and < 1:50, respectively, at nine months, four months before the culture diagnosis and two months, eight months after the culture diagnosis of Burkholderia pseudomallei infection. The patient was treated initially with two weeks of intravenous ceftazidime, followed by oral cotrimoxazole, doxycycline and chloramphenicol. Clarithromycin and ofloxacin were added after the identification of Mycobacterium avium and its susceptibility test. The patients demonstrated clinical improvement with decreasing abdominal pain and resolution of fever.

Comparison of leptospiral serovars identification by serology and cultivation in northeastern region, Thailand.

Blood from patients suspected of leptospirosis 148 specimens were cultured for leptospira. Twenty two specimens were positive (15%). The isolated leptospira were tested against the 24 serovars of standard antisera by Microscopic Agglutination Test (MAT). It was found that all 22 leptospira isolates reacted strongly against L. autumnalis, except 1 isolate that also reacted against serovar djasiman. The patient's sera were collected from only 14 cases. When the sera of the 14 patients were tested with the 24 reference serovars by MAT it was found that sera reacted the most against L. australis and in decreasing order against L. bratislava, L. autumnalis, L. rachmati, L. copenhageni, L. javanica. There had some cross reactions against several serovars in a single patient. The present study showed inconsistency between culture results and serum assays. Since sera showed cross reactivities against several serovars, it was not possible to determine which serovar was etiologic. Therefore, the isolation of leptospira though time consuming is specific in the identification of the serovar.

HIV infection among substance abusers in Thanyarak Institute On Drug Abuse, Thailand, 1987-2002.

A study of HIV infection among substance abusers from 1987 to 2002 was performed in Thanyarak Institute on Drug Abuse (TIDA). From 118,676 anti HIV tests (6,076 - 10,626 tests each year) of TIDA inpatients, 17,526 tests were positive (474 - 2,041 tests each year) In 71,403 new substance abuse cases (3,724 - 6,184 cases each year), 12,401 cases were positive (17.4%), highest in 1990 28.3% and then decreased to 8% in the last three years. In injecting drug users (IDU), the mean anti HIV positivity rate was 21.7% (range 19.2 - 29.4%) higher than those among non IDU about 8 times 2.8% (range 1.9 - 3.6%). The HIV incidence rates were from inpatients that were previously anti-HIV negative, the authors found the mean incidence rate was 7.3% per year. Anti HIV positive cases were highest in injecting heroin users (IHU) about 36.8% (range 31.5 - 46.1%). Although numbers of IHU in Thailand have decreased in the last 6 years because of changing to use methamphetamine by smoking and dying from HIV subtype B'. The authors must continue the effective preventive programs of both avoid sharing injecting equipments and promoting 100% condom program to control this reservoir of HIV infection.

Early containment of severe acute respiratory syndrome (SARS); experience from Bamrasnaradura Institute, Thailand.

BACKGROUND: On March 11, 2003, a World Health Organization (WHO) physician was admitted to Bamrasnaradura Institute, after alerting the world to the dangers of severe acute respiratory syndrome (SARS) in Vietnam and developing a fever himself. Specimens from the first day of his admission were among the first to demonstrate the novel coronavirus, by culture, reverse transcription-polymerase chain reaction (RT-PCR), and rising of specific antibody, but proper protective measures remained unknown. The authors instituted airborne, droplet and contact precautions from the time of admission, and reviewed the efficacy of these measures. MATERIAL AND METHOD: A specific unit was set up to care for the physician, beginning by roping off an isolated room and using a window fan to create negative pressure, and later by constructing a glass-walled antechamber, designated changing and decontamination areas, and adding high-efficiency particulate air (HEPA) filters. The use of personal protective equipment (PPE) was consistently enforced by nurse managers for all the staff and visitors, including a minimum of N95 respirators, goggles or face shields, double gowns, double gloves, full head and shoe covering, and full Powered Air Purifying Respirator (PAPR) for intubation. To assess the adherence to PPE and the possibility of transmission to exposed staff a structured questionnaire was administered and serum samples tested for SARS coronavirus by enzyme-linked immunosorbent assay (ELISA). Exposure was defined as presence on the SARS ward or contact with laboratory specimens, and close contact was presence in the patient's room. RESULTS: The WHO physician died from respiratory failure on day 19. 112 of 129 exposed staff completed questionnaires, and the 70 who entered the patient's room reported a mean of 42 minutes of exposure (range 6 minutes-23.5 hours). 100% reported consistent handwashing after exposure, 95% consistently used a fit-tested N95 or greater respirator, and 80% were fully compliant with strict institutional PPE protocol. No staff developed an illness consistent with SARS. Serum samples from 35 close contacts obtained after day 28 had a negative result for SARS coronavirus antibody. CONCLUSIONS: Hospitalization of one of the earliest SARS patients with documented coronavirus shedding provided multiple opportunities for spread to the hospital staff, but strict enforcement of conservative infection control recommendations throughout the hospitalization was associated with no transmission.

Mortality analysis of HIV-1 infected patients for prioritizing antiretroviral drug therapy.

Mortality data of patients, classified according to their clinical status and CD4+ cell count status, would be very useful to guide clinicians to prioritizing patients who need antiretroviral drug therapy. In the current study, the authors re-analyzed data derived from a previously published retrospective study of HIV-1-infected individuals at Lampang Hospital in northern Thailand. According to the Cox proportional hazard model, compared to asymptomatic patients with a high CD4+ cell count (> 200 cell/microl), the mortality rate of asymptomatic patients with a medium CD4+ cell count (100-199 cell/microl) did not significantly differ. However, the mortality rate of patients with a CD4+ cell count below 100 cell/microl was at least 16 times higher, regardless of the presence of clinical symptoms. Based on these results, the authors produced a Lampang Hospital guideline of antiretroviral drug use; priority of antiretroviral therapy should, therefore, be given to patients with CD4+ cell count < 100 cell/microl.

Isolation and identification of influenza virus strains circulating in Thailand in 2001.

Local influenza surveillance plays an important role in preparing for, and responding to, epidemics and pandemics. Between January and December 2001, the National Institute of Health of Thailand collected a total of 711 throat swab specimens from outpatients affected with acute respiratory symptoms from several centers throughout Thailand, of which 374 were virus-positive. Of these, 338 (90.4%) were positive for influenza virus by immunofluorescence testing. By hemagglutination-inhibition (HI) testing, 155 of the type A viruses were found to be subtype H1N1 strains closely related to A/New Caledonia/20/99, and 70 were subtype H3N2 A/Moscow/ 10/99-like viruses. For type B, the isolates were antigenically B/Sichuan/379/9-like by HI, although a number of the strains could be shown to be more closely related to earlier influenza B strains by genetic analysis. The strains circulating in Thailand were antigenically similar to strains isolated worldwide during the same period and to strains recommended by the WHO for inclusion in the vaccines for use in 2001-2002.

Characteristic of HIV-1 in V3 loop region based on seroreactivity and amino acid sequences in Thailand.

The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.

Co-existence of HIV-1 subtypes B' and E infections among Thai injecting drug users.

Subtypes B' and E are the two major subtypes of HIV-1 among injecting drug users (IDU) in Thailand. However, there are not many reports on subtype distribution during the early epidemic. Random blood specimens collected during 1994-2000 from 3,286 IDU at the Thanyarak Hospital were tested for HIV antibody and subtyped by using peptide binding enzyme immunoassay. The prevalence rate of HIV infection was 36.8%. All HIV-seropositive IDU were ascertained for "year of first HIV seropositivity" from their medical records. Of 1,512 HIV-seropositive samples, 1,408 (93.1%) were typeable. During 1987-1988, the proportion of subtype B' was as high as 80% but decreased rapidly to 27.6% during 1999-2000. At the same time, the proportions of subtype E increased correspondingly (Chi-square test for the trend, p < 0.05). The relatively high proportion of subtype E among IDU since an early stage of the epidemic suggests early co-existence of both subtypes and needs further investigation.

The development of situation assessment and trend monitoring model of cancers in Thailand.

This study is aimed to develop appropriate models for situation assessment and trend monitoring of cancers in Thailand. This study used system approach and was carried out during 1991-1993. It is composed of two parts : Part I was a review of cancer surveillance systems of Thailand and other countries. Results of the first part were used as direct inputs for Part-II study. Pats II was solicitation of experts’ opinions. Experts included public health specialists, cancer specialists, and to solicit their ideas on how the cancer surveillance system should look like. The questionnaire survey was followed by a brain-storming session where the results of the survey were presented and shaped to reach a consensus of opinion that past experiences of cancer surveillance systems faced several difficulties including duplicate reporting, inaccurate/insufficient diagnoses, unknown or unfamiliar abbreviations used in writing the surveillance report cards, and clinical diagnoses inconsistent with and/or lacking supports of pathological findings. Experts’ conclusion was that cancer registry. In particular, cancer registry should start as hospital-based to be later expanded to population-based registry. In addition, development and research activities are needed to obtain supplementary information on risk factors and risk groups for cancers and to develop appropriate indicators for evaluation.