Larvae of Spirocerca lupi and another spirurid species in the same dung beetles: notes on species identification.

Various dung beetle species of the family Scarabaeidae have been reported as intermediate hosts of Spirocerca lupi from different geographical locations, but there has been no data from south-east Asian countries. In the present study in Vietnam, by using morphological and molecular approaches, we identified the third-stage larvae of S. lupi for the first time in two dung beetle species, Catharsius molossus and Copris szechouanicus, adding them as new intermediate hosts of this nematode. In addition, both beetle species were infected with larvae of another spirurid nematode of Physocephalus sp. At large magnifications above 200×, these two spirurid larvae can be differentiated from each other by their body size (1880-2662 vs. 1417-1635 µm) and detailed morphological features of the anterior and posterior ends, such as the length of the buccal cavity, the position of the nerve ring and excretory pore, and tail characters. However, it is difficult to distinguish them at a lower magnification due to their minute size. Moreover, morphometric data of S. lupi larvae vary among reports and the body length may overlap with that of Physocephalus sp., thus, misidentification may occur, indicating the necessity of careful examination for accurate identification. The findings of the present study also supposed that larvae of S. lupi and more spirurid nematodes may be concurrently found in various dung beetle species, including C. molossus and C. szechouanicus, in other south-east Asian countries. Thus, more investigations in various countries should be conducted to identify spirurid larvae from dung beetles in order to fully understand their role as intermediate hosts of S. lupi and other spirurid nematodes.

Molecular phylogenetic relationships of Metastrongylus nematodes with emphasis on specimens from domestic pigs in Vietnam.

Lungworms of the genus Metastrongylus are parasitic nematodes in the respiratory tract of swine. Although they infect both wild boars and domestic pigs, studies on Metastrongylus infections in wild boars in Europe, the Americas and Africa are numerous, while those in domestic pigs are few. There are several studies analysing the molecular phylogenetic relationships of few individual Metastrongylus species with other nematode taxa, but there are no studies on the phylogenetic relationships of species within the genus Metastrongylus. In Southeast Asia, reports on swine lungworms are extremely scarce and do not include any nucleotide sequence data. Therefore, the aim of the present study is to survey Metastrongylus infection in domestic pigs raised in Dien Bien Province, Northern Vietnam, and to analyse the molecular phylogenetic relationships of Metastrongylus species. Based on morphological and molecular data, we identified two species: Metastrongylus apri and Metastrongylus pudendotectus. The prevalence of the former species was found to be significantly higher than the latter one (24.1% vs. 2.3%). We observed pigs exhibiting a coinfection with the two lungworm species or a single infection with only M. apri. However, we did not observe any pigs being infected with just M. pudendotectus. Vietnamese Metastrongylus specimens showed slight morphological and molecular differences compared to those from other countries. The molecular analyses revealed a close genetic relationship between M. apri and Metastrongylus salmi, while both these species were far distant from M. pudendotectus. The present study highlights the needs for further studies to clarify the morphological features and ecological and phylogenetic relationships of Metastrongylus species at the global scale.

Effect of Magnetic Magnetite (Fe3O4) Nanoparticle Size on Arsenic (V) Removal from Water.

Magnetic magnetite (Fe3O4) nanoparticles with average sizes of 5.11, 10.53, and 14.76 nm were synthesized by the chemical co-precipitation method. The surface area of Fe3O4 nanoparticles (average size of 5.11 nm) had the largest value of 167 m²/g. The adsorption capacity for removing arsenic (As(V)) from water at 3 ppm concentration was investigated by atomic absorption spectroscopy. Results showed that the As(V) adsorption capacity of Fe3O4 was dependent on particle size. The maximum absorption efficiency (Hmax) reached 99.02%, the equilibrium time was 30 min; the maximum Langmuir isotherm adsorption capacity was 14.46 mg/g with Fe3O4 nanoparticle an average size of 5 nm. The results indicate that reducing the size of Fe3O4 nanoparticles is a promised way for As(V) ion removal from water and wastewater treatment.

Appearance of US-like porcine epidemic diarrhoea virus (PEDV) strains before US outbreaks and genetic heterogeneity of PEDVs collected in Northern Vietnam during 2012-2015.

Porcine epidemic diarrhoea virus (PEDV) is the aetiologic agent of porcine epidemic diarrhoea (PED), a highly contagious enteric disease that is threatening the swine industry globally. Since PED was first reported in Southern Vietnam in 2009, the disease has spread throughout the country and caused substantial economic losses. To identify PEDVs responsible for the recent outbreaks, the full-length spike (S) gene of 25 field PEDV strains collected from seven northern provinces of Vietnam was sequenced and analysed. The sequence analysis revealed that the S genes of Vietnamese PEDVs were heterogeneous and classified into four genotypes, namely North America and Asian non-S INDEL, Asian non-S INDEL, new S INDEL and classical S INDEL. This study reported the pre-existence of US-like PEDV strains in Vietnam. Thirteen Vietnamese variants had a truncated S protein that was 261 amino acids shorter than the normal protein. We also detected one novel variant with an 8-amino acid insertion located in the receptor-binding region for porcine aminopeptidase N. Compared to the commercial vaccine strains, the emerging Vietnamese strains were genetically distant and had various amino acid differences in epitope regions and N-glycosylation sites in the S protein. The development of novel vaccines based on the emerging Vietnamese strains may be contributive to the control of the current PED outbreaks.

Pathological Characterization of an Outbreak of Porcine Reproductive and Respiratory Syndrome in Northern Vietnam.

In 2007, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged in Vietnam and spread to nearly all regions of the country by 2010. Ten representative pigs of different age groups, infected naturally with HP-PRRSV in northern Vietnam in 2010, were used to characterize the pathological features of the infection. Infection was confirmed using reverse transcriptase polymerase chain reaction and viral isolation. The clinical signs and gross findings in these pigs included high fever (>40.2°C), red skin, blue ears, anorexia, respiratory distress, diarrhoea, haemorrhagic pleurisy and lymphadenopathy. Reproductive failure was the main clinical feature in sows. PRRSV infection-associated microscopical lung and lymph node lesions were observed frequently, regardless of age of the animals. Lung lesions were characterized by interstitial pneumonia and were occasionally associated with haemorrhage and fluid exudation following alveolar collapse. Lymph nodes exhibited characteristic haemorrhage and apoptosis, lymphocytic depletion and disorganization secondary to fibrosis and capillary formation. Haematoxylin and eosin staining or caspase-3 immunohistochemistry revealed apoptosis induction in various tissues and organs, particularly the lymph nodes and lungs. Primarily haemorrhagic microscopical lesions were observed commonly in other organs including the spleen, liver, heart and kidney. Immunohistochemical examination revealed HP-PRRS antigen in the lung, lymph node, liver and kidney macrophages, and lung and kidney epithelial cells. Pigs infected naturally with HP-PRRS in the field have multisystemic disease characterized by marked apoptotic cell death.

Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam.

SETTING: The molecular diagnosis of tuberculosis (TB) in Viet Nam is often based on the detection of insertion sequence (IS) 6110 in Mycobacterium tuberculosis. However, 8-11% of M. tuberculosis strains in South-East Asia do not contain this target and this undermines the validity of these molecular tests. OBJECTIVE: We quantified the frequency of M. tuberculosis strains lacking IS6110 in rural Viet Nam and studied their epidemiological and clinical characteristics. DESIGN: Consecutively diagnosed adult TB patients in rural Southern Viet Nam submitted two sputum samples for culture, IS6110 restriction fragment length polymorphism (RFLP) spoligotyping and 15-loci variable number tandem repeat typing. Polymerase chain reaction (PCR) was performed to confirm the absence of IS6110 elements in strains lacking IS6110 hybridisation in RFLP. RESULTS: Among 2664 TB patient isolates examined, 109 (4.1%) had no IS6110 element. Compared to other strains, these no-copy strains were less often resistant to anti-tuberculosis drugs, particularly to streptomycin (adjusted OR 0.2, 95%CI 0.1-0.5), and showed significant geographic variation. No associations with TB history or demographic factors were found. CONCLUSIONS: Strains without the IS6110 target pose a problem in Viet Nam as regards false-negative molecular TB diagnosis in PCR. Compared to other strains circulating in Viet Nam, no-copy strains are more susceptible to anti-tuberculosis drugs.

The ethics of a clinical trial when the protocol clashes with international guidelines.

Due to their nature and complexity, clinical trials often take some time to launch after the protocol has been designed and ethics approval obtained. During this time, there may be changes in international treatment guidelines and recommendations that result in a conflict between study protocol and recommended international best practice. Here, we describe the situation that arose in a pharmacokinetic study on the use of two different doses of rifabutin in patients with human immunodeficiency virus-associated tuberculosis who initiated antiretroviral therapy (ART) with a lopinavir-ritonavir-based regimen in South Africa and Viet Nam. The study protocol specified that ART should be started 10 weeks after the start of anti-tuberculosis treatment. The study in South Africa was approved in June 2008, went ahead as scheduled and was completed in August 2010. The study in Viet Nam was approved in October 2008 and was started in June 2010. A few weeks later, the World Health Organization released their 2010 guidelines for adult ART; one of its strong recommendations (with moderate quality of evidence) was that ART should be started 2-8 weeks after the start of anti-tuberculosis treatment. Emerging scientific evidence also supported this recommendation. The investigators felt that the Viet Nam study protocol was in conflict with recommended international best practice, and the trial was stopped in October 2010. An amended study protocol in which ART was started at 2 weeks was developed and implemented. The ethics issues around this decision and the need to change the study protocol are discussed in this article.

Du fait de la nature et de la complexité des études cliniques, leur mise en œuvre est souvent longue après l'élaboration du protocole et son approbation éthique. Pendant cette période, il peut y avoir un changement des lignes directrices internationales de traitement et des recommandations qui provoquent un conflit entre le protocole et les meilleures pratiques recommandées internationalement. Nous décrivons ici la situation apparue dans une étude pharmacocinétique portant sur l'utilisation de deux doses différentes de rifabutin chez des patients atteints de tuberculose (TB) associée au virus de l'immunodéficience humaine et commençant un traitement antirétroviral (ART) à base de lopinavir-ritonavir en Afrique du Sud et au Viet Nam. Le protocole de l'étude spécifiait de commencer l'ART 10 semaines après le début de la thérapie antituberculeuse. En Afrique du Sud, l'étude a été approuvée en juin 2008, s'est déroulée comme prévu et a été achevée en juin 2010. Au Viet Nam, l'étude a été approuvée en octobre 2008 et a démarré en juin 2010. Quelques semaines après, l'Organisation Mondiale de la Santé a publié ses lignes directrices de 2010 pour l'utilisation de l'ART chez les adultes, dont l'une des vives recommandations (basée sur des données de qualité modérée) était de commencer l'ART entre 2 et 8 semaines après le début du traitement de la TB. L'arrivée de nouvelles preuves scientifiques est aussi venue à l'appui de cette recommandation. Les investigateurs ont eu le sentiment que le protocole d'étude au Viet Nam était en conflit avec les meilleures pratiques internationales et l'étude a été arrêtée en octobre 2010. Un protocole d'étude amendé a été développé et mis en œuvre. Les problèmes éthiques entourant cette décision et la nécessité de changer le protocole sont discutés dans ce papier.

Los ensayos clínicos, dadas sus características y su complejidad, suelen exigir mucho tiempo desde la elaboración del protocolo y la aprobación por parte del comité de ética hasta su realización. Durante este lapso, pueden surgir modificaciones en las recomendaciones y las directrices terapéuticas internacionales, lo cual genera un conflicto entre el protocolo del estudio y las prácticas óptimas recomendadas. A continuación se describe la situación que se presentó en Suráfrica y Viet Nam durante un estudio de farmacocinética sobre el uso de dos dosificaciones diferentes de rifabutina, en pacientes aquejados de tuberculosis (TB) asociada con la infección por el virus de la inmunodeficiencia humana (HIV), quienes habían comenzado el tratamiento antirretrovírico (ART) con un régimen basado en la asociación lopinavir y ritonavir. El protocolo del estudio precisaba que el ART se debía comenzar 10 semanas después de haber iniciado el tratamiento antituberculoso. En Suráfrica, el estudio recibió la aprobación en junio del 2008, comenzó en el tiempo previsto y se completó en agosto del 2010. En Viet Nam, se obtuvo la aprobación del estudio en octubre del 2008 y se comenzó en junio del 2010. A las pocas semanas, la Organización Mundial de Salud publicó sus directrices del ART en los adultos del 2010, una de cuyas recomendaciones más firmes consistía en que el ART se debía iniciar entre 2 y 8 semanas después de haber comenzado el tratamiento antituberculoso (con una calidad probatoria moderada). Algunos resultados científicos de aparición reciente respaldaban igualmente esta recomendación. Los investigadores consideraron que el protocolo del estudio en Viet Nam entraba en conflicto con las prácticas óptimas internacionales recomendadas e interrumpieron su realización en octubre del 2010. Se introdujeron modificaciones al protocolo, según las cuales el ART se comenzaría a las 2 semanas y se puso en práctica el estudio. En el presente artículo se analizan los aspectos éticos en torno a esta decisión y a la necesidad de modificar el protocolo del estudio.

Association of streptomycin resistance mutations with level of drug resistance and Mycobacterium tuberculosis genotypes.

OBJECTIVES: To determine 1) the relationship between specific streptomycin (SM) resistance mutations and the minimum inhibitory concentration (MIC), and 2) whether these mutations are preferentially associated with the Beijing genotype in Viet Nam. METHODS: A total of 131 consecutive Mycobacterium tuberculosis isolates resistant to either isoniazid (INH) or rifampicin (RMP), collected previously, were tested for SM resistance, spoligotyped and sequenced in the rpsL, rrs and gidB genes. The MIC for 50 mutants was also determined. RESULTS: Overall, 116/131 isolates were SM-resistant. The three most frequently occurring mutation sites in rpsL and rrs were at codon 43 of rpsL (72/116, 62.1%), rpsL88 (22/116, 18.9%) and rrs514 (8/116, 6.9%). Mutations in the rrs910 region were found in two isolates (1.7%), and three isolates had mutations in both rpsL and rrs (2.6%). gidB mutations were found in both resistant and susceptible strains. Among SM-resistant isolates resistant to INH/RMP, the Beijing genotype was strongly associated with rpsL43 mutation (aOR 23.6, 95%CI 2.9-193.4, P = 0.002). The median MIC for each mutation was as follows: rpsL43 = 256 µg/ml, rpsL88 = 16 µg/ml, 515 loop = 4 µg/ml, 910 region = 8 µg/ml, and double mutation = 256 µg/ml. We found a strong association between rpsL43 and high drug resistance levels, with all rpsL43 mutants having an MIC >256 µg/ml (P < 0.001).

Epiregulin (EREG) variation is associated with susceptibility to tuberculosis.

Although host genetics influences susceptibility to Mycobacterium tuberculosis, the human genes regulating pathogenesis remain largely unknown. We used M. tuberculosis-stimulated macrophage gene expression profiling in conjunction with a case-control genetic association study to discover epiregulin (EREG), as a novel candidate tuberculosis (TB) susceptibility gene. Using a genome-wide association study dataset, we found that among the 21 genes with greater than 50-fold induction, EREG had the most polymorphisms associated with TB. We genotyped haplotype-tagging polymorphisms in discovery (N = 337 cases, N = 380 controls) and validation (N = 332 cases) datasets and an EREG polymorphism (rs7675690) was associated with susceptibility to TB (genotypic comparison; corrected P = 0.00007). rs7675690 was also associated more strongly with infections caused by the Beijing lineage of M. tuberculosis when compared with non-Beijing strains (controls vs Beijing, OR 7.81, P = 8.7 × 10(-5); non-Beijing, OR 3.13, P = 0.074). Furthermore, EREG expression was induced in monocytes and peripheral blood mononuclear cells stimulated with M. tuberculosis as well as TLR4 and TLR2/1/6 ligands. In murine macrophages, EREG expression induced by M. tuberculosis was MYD88- and TLR2-dependent. Together, these data provide the first evidence for an important role for EREG as a susceptibility gene for human TB.

Improving the diagnosis of pulmonary tuberculosis in HIV-infected individuals in Ho Chi Minh City, Viet Nam.

SETTING: District 6, An Hoa Clinic in Ho Chi Minh City (HCMC), Viet Nam. OBJECTIVE: To evaluate the performance of various algorithms in tuberculosis (TB) screening and diagnosis in a human immunodeficiency virus (HIV) infected population in HCMC, Viet Nam. DESIGN: A cross-sectional study of 397 consecutive HIV-infected patients seeking care at the An Hoa Clinic from August 2009 to June 2010. Data on participant demographics, clinical status, chest radiography (CXR) and laboratory results were collected. A multiple logistic regression model was developed to assess the association of covariates and pulmonary TB (PTB). RESULTS: The prevalence of sputum culture-confirmed PTB, acid-fast bacilli (AFB) positive TB, and multidrugresistant TB among the 397 HIV-infected patients was respectively 7%, 2%, and 0.3%. Adjusted odds ratios for low CD4+ cell count, positive sputum smear, and CXR to positive sputum culture were respectively 3.17, 32.04 and 4.28. Clinical findings alone had poor sensitivity, but combining CD4+ cell count, AFB sputum smear and CXR had a more accurate diagnostic performance. CONCLUSION: Results suggest that symptom screening had poor clinical performance, and support the routine use of sputum culture to improve the detection of TB disease in HIV-infected individuals in Viet Nam. However, when routine sputum culture is not available, an algorithm combining CD4+ cell count, AFB sputum smear and CXR is recommended for diagnosing PTB.

Multiplex allele-specific polymerase chain reaction for detection of isoniazid resistance in Mycobacterium tuberculosis.

SETTING: Pham Ngoc Thach Tuberculosis Reference Hospital, Ho Chi Minh City, Viet Nam. DESIGN: A multiplex allele-specific polymerase chain reaction (MAS-PCR) was developed to detect mutations at the two most common sites responsible for isoniazid (INH) resistance in Mycobacterium tuberculosis: katG315 and inhA-15. The MAS-PCR is able to detect rare mutations at katG315, in addition to katG S315T. Conventional phenotypic proportion drug susceptibility testing on Löwenstein-Jensen media was used as a gold standard to compare the sensitivity and specificity of the commercial MTBDRplus line-probe assay and the MAS-PCR in 100 INH-resistant and 50 INH-susceptible isolates collected consecutively at Pham Ngoc Thach Hospital reference laboratory. RESULTS: The sensitivity and specificity on culture isolates were 90% (n = 90/100, 95%CI 0.83-0.94) and 100% (n = 50/50, 95%CI 0.93-1.0), respectively, for the MAS-PCR and the MTBDRplus assay. CONCLUSION: The MAS-PCR described here represents an alternative method for rapid screening for INH resistance in M. tuberculosis isolates.

Decrease in risk of tuberculosis infection despite increase in tuberculosis among young adults in urban Vietnam.

OBJECTIVE: To assess whether the increase in tuberculosis (TB) notification rates among young adults in Vietnam reflects increased transmission in the population at large. METHOD: Trends of case notification rates of new smear-positive TB were calculated from routinely reported data of district TB units over the period 1996-2005. Results from repeated tuberculin surveys among children aged 6-9 years were obtained to calculate the trend in annual risk of TB infection (ARTI). FINDINGS: From 1996 to 2006, notification rates in the age group 15-24 years increased by 4.3% per year, and more so in highly urbanised (6.7%) than in rural districts (1.7%). The ARTI in urban districts declined from 2.4% in 1992 to 1.2% in 1998 and 0.9% in 2005. In rural districts, the ARTI increased from 0.7% in 1991 to 1.2% in 1997, and then declined to 0.9% in 2006. CONCLUSION: The increase in TB notification rates among young adults in Ho Chi Minh Province is accompanied by a decrease in ARTI in children. This suggests that the trend in TB notification among young adults reflects increased rates of progression from infection to disease and/or increased transmission within this age group, rather than increased transmission in the population at large.

Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology.

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.

The Beijing genotype is associated with young age and multidrug-resistant tuberculosis in rural Vietnam.

BACKGROUND: Associations between multidrug resistance and the Mycobacterium tuberculosis Beijing genotype have been described mainly in populations with poor tuberculosis (TB) control such as prisons and inner cities, and may reflect shared risk factors rather than a biological association. OBJECTIVE: To study the association between genotype and drug resistance among TB patients in a population with adequate TB control. SETTING: Three rural districts in Vietnam. The study was performed at the Pham Ngoc Thach Tuberculosis and Lung Disease Hospital, Ho Chi Minh City, and the Tien Giang Provincial Tuberculosis and Lung Disease Hospital, My Tho, Vietnam. METHODS: Pretreatment sputum specimens were collected for culture, drug susceptibility testing and spoligotyping of all sputum smear-positive pulmonary TB patients consecutively diagnosed over a 3-year period. RESULTS: Beijing genotype infections were observed in 614 of 1744 (35%) patients. Beijing strains were more common among female (adjusted odds ratio [aOR] 1.4, P = 0.005), young (aOR 2.8, P < 0.001) and previously treated patients (aOR 2.4, P < 0.001). The Beijing genotype was associated with any resistance (aOR 3.7, P < 0.001) and multidrug resistance (aOR 6.8, P < 0.001) among new patients, and with any resistance (aOR 2.7, P = 0.005) but not with multidrug resistance (aOR 1.4, P = 0.545) among previously treated patients. CONCLUSION: In Vietnam, Beijing genotype is associated with young age and in new patients with multidrug resistance despite adequate TB control, suggesting a biological association. This potentially undermines the effectiveness of TB control in countries where Beijing genotype infections are common.

Validation of the line-probe assay for rapid detection of rifampicin-resistant Mycobacterium tuberculosis in Vietnam.

BACKGROUND: Delays in identifying multidrug-resistant tuberculosis (MDR-TB) contribute to higher TB morbidity and mortality, and ongoing transmission. The line-probe assay (LiPA) is a rapid, commercially available polymerase chain reaction based assay that detects most mutations in the rpoB gene for rifampicin (RMP) resistance. We validated and compared this assay with conventional drug susceptibility testing (DST). METHODS: We re-cultured a random sample of stored isolates known to be either RMP-resistant or RMP-susceptible according to DST (proportion method). We performed a blinded comparison between LiPA and conventional DST. Genetic sequencing of the rpoB gene was performed on RMP-resistant isolates and discordant results. RESULTS: We tested 79 RMP-resistant and 64 RMP-susceptible strains. Concordance of LiPA with DST was 94%. For detecting RMP resistance, LiPA sensitivity was 90% and specificity was 100%. Molecular analysis of possible false-negative isolates by LiPA revealed an absence of mutations in the rpoB gene. RMP resistance was a good proxy for MDR-TB, as 66 (93%) of 71 RMP-resistant isolates were also isoniazid-resistant. CONCLUSION: The LiPA provided rapid results that were highly predictive of RMP resistance and MDR-TB. False-negatives occurred, but only among isolates with mutations in regions not assessed by LiPA. Performance and cost-effectiveness should be evaluated in patients during routine program conditions.

Comparison of MAS-PCR and GenoType MTBDR assay for the detection of rifampicin-resistant Mycobacterium tuberculosis.

SETTING: Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, the tertiary referral hospital for tuberculosis (TB) in Southern Vietnam. OBJECTIVE: To develop and evaluate a simple, rapid and accurate multiplex allele specific polymerase chain reaction (MAS-PCR) test to detect rifampicin (RMP) resistance point mutations at codons 516, 526 or 531 in the rpoB gene of Mycobacterium tuberculosis. DESIGN: The novel MAS-PCR was compared with the commercial M. tuberculosis Drug Resistance (MTBDR) test in 104 RMP-resistant and 50 RMP-susceptible routine isolates, defined by conventional 1% phenotypic susceptibility testing. RESULTS: The sensitivity of the MAS-PCR and MTBDR tests was respectively 83.7% (95%CI 75.1-90.2) and 93.3% (95%CI 86.6-97.3). Both tests were 100% specific. The negative predictive value was 74.6% (95%CI 65.3-83.1) for the MAS-PCR and 87.7% (95%CI 80.0-93.6) for the MTBDR test. CONCLUSION: The MTBDR test, although more sensitive, is currently prohibitively expensive in resource-poor, high-burden settings. The MAS-PCR described here presents a less laborious economic alternative. A susceptible result returned by either test cannot be used to exclude multidrug-resistant TB.

Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR.

SETTING: Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, Ho Chi Minh City, Vietnam. OBJECTIVE: Fluoroquinolones (FQs) are increasingly used in the treatment of tuberculosis (TB) and are the second-line drugs of choice for treatment of multidrug-resistant TB. We aimed to set up a polymerase chain reaction (PCR) based assay to detect the most common FQ-resistance-associated mutations in gyrase A (gyrA) of Mycobacterium tuberculosis. DESIGN: A total of 42 FQ-resistant and 40 FQ-susceptible isolates were collected in 2005-2006 and sequenced in gyrA. Using sequencing results as gold standard, a real-time PCR using three locked nucleic acid probes (LNA-PCR) was designed to detect mutations at positions 90, 91 and 94 (97% of gyrA FQ-resistance-associated mutations) and evaluated. RESULTS: Sequencing of 42 FQ-resistant isolates revealed no gyrA mutations in 10 isolates, 20 isolates had a single mutation and 12 isolates showed double peaks at resistance-associated alleles, suggesting a heterogeneous population. With LNA-PCR, all wild-type and 19/20 mutant isolates were correctly identified. Eleven of 12 heterogeneous isolates were correctly identified as resistant mutants. Overall, 71% ([19 + 11]/42) of phenotypically FQ-resistant isolates were detected. Specificity was 100% on 40 FQ-susceptible isolates. CONCLUSION: This assay provides a simple and rapid means to reliably detect FQ-resistance-associated gyrA mutations in M. tuberculosis.

A polymorphism in human TLR2 is associated with increased susceptibility to tuberculous meningitis.

Tuberculous meningitis (TBM) results from the haematogenous dissemination of Mycobacterium tuberculosis from the lung to the brain. Dissemination is believed to occur early during infection, before the development of adaptive immunity. Toll-like receptor 2 (TLR2) mediates recognition of M. tuberculosis and initiates the innate immune response to infection. We hypothesized that polymorphisms in the TLR2 gene influence bacterial dissemination and the development of TBM. A case-control study was designed to test the hypothesis. Cases of bacteriologically confirmed pulmonary tuberculosis (TB) (n=183) and TBM (n=175), and cord blood controls (n=389) were enrolled in Vietnam. TLR2 genotype 597CC was associated with susceptibility to TB (odds ratio (OR)=2.22, 95% confidence interval (CI): 1.23-3.99). The association was found with meningeal rather than pulmonary TB (TBM vs control, OR=3.26, 95% CI: 1.72-6.18), and was strongest when miliary TB was found on chest radiography (controls vs TBM with miliary TB, OR=5.28, 95% CI: 2.20-12.65). Furthermore, the association increased with the severity of neurologic symptoms (grade I TBM, OR=1.93, 95% CI: 0.54-6.92; grade II, OR=3.32, 95% CI: 0.84-13.2; and grade III, OR=5.70, 95% CI: 1.81-18.0). These results demonstrate a strong association of TLR2 SNP T597C with the development of TBM and miliary TB and indicate that TLR2 influences the dissemination of M. tuberculosis.

Molecular analysis of Mycobacterium tuberculosis causing multidrug-resistant tuberculosis meningitis.

SETTING: Tertiary referral hospitals in southern Vietnam. OBJECTIVE: Molecular characterisation of multidrug-resistant (MDR) tuberculous meningitis (TBM). DESIGN: Mycobacterium tuberculosis isolates from the cerebrospinal fluid (CSF) of 198 Vietnamese adults were compared with 237 isolates from patients with pulmonary tuberculosis (PTB) matched for age, sex and residential district. Isolates resistant to isoniazid or rifampicin (RMP) were sequenced in the rpoB and katG genes, inhA promoter and oxyR-ahpC intergenic regions. RESULTS: While drug resistance rates were lower in the CSF (2.5% MDR) than pulmonary isolates (5.9% MDR), the difference was not significant. The most commonly mutated codons were 531, 526 and 516 in rpoB and 315 in katG. Four novel triple mutants in rpoB were identified. CONCLUSION: RMP resistance is a good surrogate marker for MDR-TBM in this setting. However, probes directed against these three codons would have a maximum sensitivity of only 65%. A rapid phenotypic detection test may be more applicable for the diagnosis of MDR-TBM.

Stability of canine distemper virus (CDV) after 20 passages in Vero-DST cells expressing the receptor protein for CDV.

Isolates 007Lm, S124C and Ac96I and a Vero cell-adapted Onderstepoort strain of canine distemper viruses (CDV) were examined for stability after passages in Vero cells expressing the canine signaling lymphocyte activation molecule (dogSLAM, the intrinsic receptor to CDV). These viruses passage once in Vero cells expressing dogSLAM (Vero-DST) cells (original) and after 20 passages (20p) were compared by using sequence analyses and growth characteristics. All four strains of 20p grew well and were slightly better than their originals. The 20p viruses developed a cytopathic effect slightly lower than the original strains. A few changes in amino acids in the H gene were between the 20p and the original viruses, but the sites of changes were not specific. Fragments of P, M and L genes of all strains showed no nucleotide changes after the passages. These results showed that: (1) passages of CDVs in Vero-DST cells induced amino acid changes only in the H gene, not in the P, M and L genes, unlike in a previous study with Vero cells; (2) passages did not markedly affect the growth characteristics of every viral strain. These results indicate that Vero cells expressing canine SLAM allow the isolation and passaging of CDV without major changes in viral genes.