Suboptimal Exposure to Anti-TB Drugs in a TBM/HIV+ Population Is Not Related to Antiretroviral Therapy.

A placebo-controlled trial that compares the outcomes of immediate vs. deferred initiation of antiretroviral therapy in HIV +ve tuberculous meningitis (TBM) patients was conducted in Vietnam in 2011. Here, the pharmacokinetics of rifampicin, isoniazid, pyrazinamide, and ethambutol were investigated in the presence and absence of anti-HIV treatment in 85 patients. Pharmacokinetic analyses show that HIV therapy has no significant impact on the pharmacokinetics of TB drugs in this cohort. The same population, however, displayed generally low cerebrospinal fluid (CSF) and systemic exposures to rifampicin compared to previously reported HIV -ve cohorts. Elevated CSF concentrations of pyrazinamide, on the other hand, were strongly and independently correlated with increased mortality and neurological toxicity. The findings suggest that the current standard dosing regimens may put the patient at risk of treatment failure from suboptimal rifampicin exposure, and potentially increasing the risk of adverse central nervous system events that are independently correlated with pyrazinamide CSF exposure.

Measuring IPM Impacts in California and Arizona.

Integrated pest management (IPM) is a method of reducing economic, human health, and environmental risks from pests and pest management strategies. There are questions about the long-term success of IPM programs in relation to continued use of pesticides in agriculture. Total pounds of pesticides applied is a mis-measure of the impact of IPM in agriculture. A more complete measurement of the long-term impact of IPM includes consideration of changes in agricultural production practices and productivity, toxicity of the pesticides used, risks from human exposure to pesticides, and environmental sampling for pesticides in air and water resources. In recent decades, agricultural IPM programs have evolved to address invasive pests, shifts in endemic pest pressures, reductions in pest damage tolerance in markets, and increases in crop yields. Additionally, pesticide use data from Arizona and California revealed reduced use of pesticides in some toxicity categories but increased use of pesticides in a couple of categories. Data from federal and California programs that monitored pesticide residue on food have documented low pesticide risk to consumers. Environmental monitoring programs documented decreased pesticide levels in surface water resources in agricultural watersheds in the western United States and low levels of pesticides in air resources in agricultural areas in California. The focus of IPM assessment should be on reducing economic, human health, and environmental risks, not on pounds of pesticides applied. More broadly, IPM programs have evolved to address changes in pests and agricultural production systems while continuing to reduce human health and environmental risk from pesticides.

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.

What is the evidence of a role for host genetics in susceptibility to influenza A/H5N1?

The apparent family clustering of avian influenza A/H5N1 has led several groups to postulate the existence of a host genetic influence on susceptibility to A/H5N1, yet the role of host factors on the risk of A/H5N1 disease has received remarkably little attention compared to the efforts focused on viral factors. We examined the epidemiological patterns of human A/H5N1 cases, their possible explanations, and the plausibility of a host genetic effect on susceptibility to A/H5N1 infection. The preponderance of familial clustering of cases and the relative lack of non-familial clusters, the occurrence of related cases separated by time and place, and the paucity of cases in some highly exposed groups such as poultry cullers, are consistent with a host genetic effect. Animal models support the biological plausibility of genetic susceptibility to A/H5N1. Although the evidence is circumstantial, host genetic factors are a parsimonious explanation for the unusual epidemiology of human A/H5N1 cases and warrant further investigation.

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.

Effect of magnesium sulphate on urinary catecholamine excretion in severe tetanus.

Severe tetanus is characterised by muscle spasms and autonomic dysfunction. We recently reported the results of a randomised placebo controlled trial of magnesium sulphate infusions for the treatment of severe tetanus which showed magnesium was associated with improved muscle spasm and cardiovascular control. We hypothesised that magnesium controlled autonomic dysfunction by reducing catecholamine release and thus urinary excretion. Urinary adrenaline and noradrenaline concentrations were measured during the first 24 h of therapy in 180 adults with severe tetanus randomised to treatment with magnesium (n = 92) or placebo (n = 88). Magnesium therapy was associated with lower urinary adrenaline excretion and higher urinary noradrenaline excretion. High urinary adrenaline concentrations were associated with documented autonomic dysfunction. Patients given magnesium had significantly less autonomic dysfunction, required less cardiovascular stabilising drugs, and had lower urinary concentrations of adrenaline. These findings suggest adrenaline is important in the pathophysiology of severe tetanus and magnesium controls autonomic dysfunction by reducing adrenaline release.

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.

Magnesium sulphate for treatment of severe tetanus: a randomised controlled trial.

BACKGROUND: The most common cause of death in individuals with severe tetanus in the absence of mechanical ventilation is spasm-related respiratory failure, whereas in ventilated patients it is tetanus-associated autonomic dysfunction. Our aim was to determine whether continuous magnesium sulphate infusion reduces the need for mechanical ventilation and improves control of muscle spasms and autonomic instability. METHODS: We did a randomised, double blind, placebo controlled trial in 256 Vietnamese patients over age 15 years with severe tetanus admitted to the Hospital for Tropical Medicine, Ho Chi Minh City, Vietnam. Participants were randomly assigned magnesium sulphate (n=97) or placebo solution (n=98) intravenously for 7 days. The primary outcomes were requirement of assisted ventilation and of drugs to control muscle spasms and cardiovascular instability within the 7-day study period. Analyses were done by intention to treat. This trial is registered as an International Standard Randomised Clinical Trial, number ISRCTN74651862. FINDINGS: No patients were lost to follow-up. There was no difference in requirement for mechanical ventilation between individuals treated with magnesium and those receiving placebo (odds ratio 0.71, 95% CI 0.36-1.40; p=0.324); survival was also much the same in the two groups. However, compared with the placebo group, patients receiving magnesium required significantly less midazolam (7.1 mg/kg per day [0.1-47.9] vs 1.4 mg/kg per day [0.0-17.3]; p=0.026) and pipecuronium (2.3 mg/kg per day [0.0-33.0] vs 0.0 mg/kg per day [0.0-14.8]; p=0.005) to control muscle spasms and associated tachycardia. Individuals receiving magnesium were 4.7 (1.4-15.9) times less likely to require verapamil to treat cardiovascular instability than those in the placebo group. The incidence of adverse events was not different between the groups. INTERPRETATION: Magnesium infusion does not reduce the need for mechanical ventilation in adults with severe tetanus but does reduce the requirement for other drugs to control muscle spasms and cardiovascular instability.

Polymorphisms of the gene coding for copper/zinc superoxide dismutase (SOD1) in patients with Japanese encephalitis.

Japanese encephalitis is the commonest form of encephalitis globally. Most cases develop characteristic encephalitis but some also present with flaccid paralysis. The paralysis is secondary to damage at the alpha motor neurone, the site that is also damaged in amyotrophic lateral sclerosis (ALS). The gene coding for superoxide dismutase 1 (SOD1) is thought to be involved in ALS and may also be linked to susceptibility to Japanese encephalitis. To investigate this possibility, polymorphisms in the SOD1 gene were investigated, in 61 cases of Japanese encephalitis, 61 matched controls and 171 population controls, in Vietnam. Novel polymorphisms, found only in three of the cases and one of the population controls, may be involved with susceptibility to Japanese encephalitis and potentially to other flavivirus infections that lead to damage to the cells of the anterior horn. Further research on this possible association is required.

Urinary catecholamine excretion in tetanus.

Imperfect understanding of the pathophysiology of tetanus has limited therapeutic advances. Autonomic disturbance is a major cause of mortality and is believed to be associated with catecholamine release, predominantly norepinephrine. We measured epinephrine and norepinephrine concentrations in 24-h urine collections from tetanus and critically ill patients suffering from other severe diseases. In patients with severe tetanus, mean (SD) epinephrine was 164.18 (129.37) nmol x day(-1) compared with 45.18 (37.74) nmol x day(-1) in mild-moderate disease (p = 0.008). In the severe group, mean (SD) norepinephrine was 411.64 (208.5), and 121.00 (81.81) nmol x day(-1) in moderately ill patients (p < 0.001). Compared with critically ill control patients, median epinephrine was 331.77 in tetanus patients and 89.70 nmol x day(-1) in controls (p < 0.001). Median norepinephrine concentration was 788.02 nmol x day(-1) in tetanus and 300.05 nmol x day(-1) in control patients, p = 0.006. The study finds a novel result of increased epinephrine excretion in tetanus and confirms that catecholamine excretion in tetanus exceeds that in other critically ill patients. These results should be considered in designing more effective therapeutic strategies.

Predicting the clinical outcome of tetanus: the tetanus severity score.

OBJECTIVES: To create a new tetanus score and compare it with the Phillips and Dakar scores. METHODS: We used prospectively acquired data from consecutive patients admitted to the Hospital for Tropical Diseases, Ho Chi Minh City, to create the Tetanus Severity Score (TSS) with multivariate logistic regression. We compared the new score with Phillips and Dakar scores by means of resubstituted and prospective data, assessing performance in terms of sensitivity, specificity and area under receiver operator characteristic curves. RESULTS: Resubstitution testing yielded a sensitivity of 77% (298/385) and a specificity of 82% (1,183/1,437) for the TSS; 89% (342/385) and 20% (281/1,437) for the Phillips score; and 13% (49/385) and 98% (1,415/1,437) for the Dakar score. The TSS showed greatest discrimination with 0.89 area under the receiver operator characteristic curve (95% CI 0.88-0.90); this was 0.74 for the Dakar score and (95% CI 0.71-0.77) and 0.66 for the Phillips score (95% CI 0.63-0.70; P values <0.001). Prospective testing showed 65% (13/20) sensitivity and 91% (210/230) specificity for the TSS; 80% (16/20) and 51% (118/230) for the Phillips score; and 25% (5/20) and 96% (221/230) for the Dakar score. The TSS achieved the greatest area under TSS of 0.89 (95% CI 0.82-0.96), significantly greater than the Phillips score [0.74 (0.6-0.88), P = 0.049] but not the Dakar score [0.80, (0.71-0.90), P = 0.090]. CONCLUSIONS: The TSS is the first prospectively developed classification scheme for tetanus and should be adopted to aid clinical triage and management and as a basis for clinical research.

Fake antimalarials in Southeast Asia are a major impediment to malaria control: multinational cross-sectional survey on the prevalence of fake antimalarials.

OBJECTIVE: To assess the prevalence of counterfeit antimalarial drugs in Southeast (SE) Asia. DESIGN: Cross-sectional survey. SETTING: Pharmacies and shops selling antimalarial drugs in Myanmar (Burma), Lao PDR, Vietnam, Cambodia and Thailand. MAIN OUTCOME MEASURES: Proportion of artemisinin derivatives or mefloquine containing drugs of substandard quality. RESULTS: Of the 188 tablet packs purchased which were labelled as 'artesunate' 53% did not contain any artesunate. All counterfeit artesunate tablets were labelled as manufactured by 'Guilin Pharma', and refinements of the fake blisterpacks made them often hard to distinguish from their genuine counterparts. No other artemisinin derivatives were found to be counterfeited. Of the 44 mefloquine samples, 9% contained <10% of the expected amount of active ingredient. CONCLUSIONS: An alarmingly high proportion of antimalarial drugs bought in pharmacies and shops in mainland SE Asia are counterfeit, and the problem has increased significantly compared with our previous survey in 1999-2000. This is a serious threat to public health in the region.

The role of the polymerase chain reaction in the diagnosis of bacterial meningitis in Vietnam.

Bacterial meningitis remains an important cause of morbidity and mortality in Vietnam. Diagnosis is hampered by the ready availability of antibiotics in the community, leading to late presentation, masked clinical signs, and poor organism detection during the microscopical examination and culture of cerebrospinal fluid (CSF). In order to improve organism detection at the Hospital for Tropical Diseases in Ho Chi Minh City, a diagnostic PCR-based protocol was developed. This protocol was followed in the investigation of CSF samples from 36 patients with clinical signs of bacterial meningitis. Each sample was first tested in a semi-nested PCR using primers for the 16sRNA gene common to all bacteria. The products of this reaction were then amplified using a 16sru8 primer and primers specific for Neisseria meningitidis, Haemophilus influenzae or Streptococcus spp. The samples found positive for Streptococcus were further investigated in a nested PCR using primers specific for the pneumolysin gene of S. pneumoniae. The sensitivity of detection was increased from 36% with culture to 44% with PCR. Although the sample size was small, the results indicate that PCR would be a feasible and useful adjunct in the diagnosis of bacterial meningitis, particularly in areas where community antibiotic use is common.

First Report of Powdery Mildew on Potato Caused by Golovinomyces cichoracearum in California.

In October 2003, potato plants in three fields (cv. Desiree, Satina, Midas, and Mondial) in Lancaster, California exhibited symptoms and signs of powdery mildew. Disease symptoms were most severe on cvs. Desiree and Santina. Disease expression was greater along sprinkler lines and in localized areas from which the disease spread to surrounding plants. Severely affected plants began collapsing just prior to water cutoff. Early symptoms comprise small dark areas on the adaxial surface of leaves, along the veins, and at the petioles. Dark lesions consisting of mycelia and conidiophores were also visible on the main stems of affected plants. As the disease progressed, leaves were covered by a gray powdery fungal mass, and older leaves became necrotic. Conidial chains arising from the hyaline, epiphytic mycelia consisted of two to eight conidia. The cylindric to doliform conidia measured 16.8 to 22.8 µm wide (mean = 19.2, standard error = 0.36, N = 30) × 28.8 to 45.6 µm long (mean = 32.4, standard error = 0.75, N = 30). No cleistothecia were observed. Identification of the causal agent as Golovinomyces cichoracearum (synonyms G. orontii and Erysiphe cichoracearum) based on morphology was confirmed by internal transcribed spacer (ITS)-polymerase chain reaction (PCR). Conidia were washed off the affected leaves, concentrated by filtration and centrifugation, and sonicated to release genomic DNA. PCR was performed on the sonicated conidia with primers ITS4 and ITS5 (2), and the resulting amplicon was purified and sequenced. BLAST analysis of the ITS sequence revealed a 99% homology to E. cichoracearum from an Ambrosia sp. (GenBank Accession No. AF011292). Pathogenicity was confirmed on potato seedlings cv. Red La Soda. Inoculations were performed twice on six plants (three pots) each time. A sterile brush was used to transfer conidia from the affected leaves to seedlings consisting of two to three fully expanded leaves. A plastic bag was placed around each pot containing two seedlings for 1 to 2 days and then removed. Noninoculated controls were stroked with a sterile brush, placed in a plastic bag for 1 to 2 days, and kept in the greenhouse on a separate bench. Two control plants were included for each inoculation. Plants were maintained in a greenhouse at approximately 25 to 28°C and 40 to 60% relative humidity. After 7 days, dark spots were visible on the leaves of all inoculated plants, and conidiophores with conidia identical to those of the isolate used as the inoculum source were apparent after 10 days. The controls showed no disease symptoms or signs. To our knowledge, this is the first report of powdery mildew caused by G. cichoracearum on potato in California. The first field report of the disease was from Washington in 1950 (1), with subsequent reports from Utah and Ohio. References: (1) J. D. Menzies. Plant Dis. Rep. 34:140, 1950. (2) T. J. White et al. PCR Protocols. Academic Press, New York, 1990.

Characterization of rotaviruses causing diarrhoea in Vietnamese children.

Faecal samples from 123 children admitted to the Centre for Tropical Diseases in Ho Chi Minh city, Vietnam, with acute watery diarrhoea were screened by negative-stain electron microscopy for viral enteropathogens. In addition to the 48 children who were found to be infected with rotavirus only, one had both rotavirus and astrovirus, two had adenovirus 40/41, and one had astrovirus only. The rotaviruses were subjected to molecular analysis by electropherotyping, G- and P-genotyping (by reverse-transcriptase PCR), and amplicon sequencing. By use of newly designed PCR primers, all 49 isolates could be G-genotyped and all but one P-genotyped. Novel variants of G1-G1*--were the most commonly detected G-genotype and such variants of P[8]-P[8*]--were the second commonest P-genotype. The P[8*] and G1* amplicons were, respectively, only 92%-93.4% and 88.1%-89% similar to the corresponding sequences from the prototype P[8] G1 rotavirus, Wa. Several unusual P- and G-genotype combinations were detected. Four (8%) of the children investigated were each found to be co-infected with two different rotaviruses. These data add to our knowledge of the continuing evolution and diversity of human rotaviruses, and should help in the rational design of vaccines.