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.

Cellular requirements for lipopolysaccharide adjuvanticity. A role for both T lymphocytes and macrophages for in vitro responses to particulate antigens.

By employing primary cultures of purified spleen cells from lipopolysaccharide (LPS) responder (C3H/HeN or C57BL/10Sn) or nonresponder (C3H/HeJ or C57BL/10ScN) mice incubated with particulate antigen and LPS prepared by phenol-water extraction (Ph), we have presented evidence that both T cells and macrophages (MO) are required for LPS-induced adjuvanticity. First, MO derived from C3H/HeN spleen cells, when mixed with responder, C3H/HeN lymphocytes and Ph-LPS, elicited enhanced antibody responses to sheep erythrocytes (SRC) antigen, whereas lymphocytes from the nonresponder, C3H/HeJ mouse strain did not evoke this response. Similarly, purified T cells from C3H/HeN spleens, when cultured with responder, nu/nu spleen cells, and Ph-LPS yielded enhanced anti-TNP PFC responses; whereas, C3H/HeJ T cells did not potentiate immune responses when mixed with optimal concentrations of Ph-LPS. LPS prepared by butanol-water extraction elicited significant adjuvant effects with all cell combinations. Finally, purified responder T cells and MO enabled either responder or nonresponder B cells to elicit LPS potentiation. These data indicate that T cells and MO are controlling LPS-induced augmentation of B-cell responses.

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.

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.

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.

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.

Clinical and neurophysiological study of the effects of multiple doses of artemisinin on brain-stem function in Vietnamese patients.

The qinghaosu (artemisinin) group of drugs is the most important new class of antimalarials developed in the last fifty years. Although there has been no clinical evidence of neurotoxicity, an unusual pattern of damage to specific brain-stem nuclei has been reported in experimental animals receiving high doses of arteether or artemether. Detailed clinical examinations, audiometry, and brain stem auditory evoked potentials (BSAEPs) were assessed in 242 Vietnamese subjects who had previously received up to 21 antimalarial treatment courses of artemisinin or artesunate alone and 108 controls from the same location who had not received these drugs. There was no evidence of a drug effect on the clinical or neurophysiological parameters assessed. In this population there was no clinical or neurophysiological evidence of brain-stem toxicity that could be attributed to exposure to artemisinin or artesunate.

Isoniazid resistance, mycobacterial genotype and outcome in Vietnamese adults with tuberculous meningitis.

SETTING: Centre for Tropical Diseases, a 500-bed hospital for infectious diseases in Ho Chi Minh City, Vietnam. OBJECTIVE: The factors that determine outcome in adults with tuberculous meningitis are poorly understood. The objective of the study was to investigate the relationship between admission clinical features, HIV infection, drug resistance, mycobacterial genotype and outcome in adults with tuberculous meningitis. DESIGN: Clinical and laboratory data were recorded prospectively for 56 Vietnamese adults with tuberculous meningitis confirmed by culture of cerebrospinal fluid. Variables associated with in-hospital mortality, IV infection, drug resistance and microbial genotype were assessed by univariate and multivariate analysis. RESULTS: Admission coma score independently predicted death in hospital (OR 0.73, 95%CI 0.61-0.87, P = 0.001). HIV-infected adults with tuberculous meningitis were more likely to be infected with Mycobacterium tuberculosis resistant to isoniazid (P = 0.011) and streptomycin (P = 0.002). Isoniazid resistance, streptomycin resistance, HIV infection and microbial genotype were not associated with increased in-hospital mortality. CONCLUSION: Treatment of tuberculous meningitis before the onset of coma saves lives. Resistance to isoniazid and/or streptomycin does not appear to affect outcome.

Risk factors for typhoid fever in the Mekong delta, southern Viet Nam: a case-control study.

In order to identify risk factors for typhoid fever in a highly endemic area, we undertook a case-control study in the Mekong delta, Viet Nam. Cases were 144 consecutive patients admitted to hospital with blood culture-confirmed typhoid fever. Two controls (1 in the hospital and 1 in the community) were chosen for each case. Standardized interviews were conducted with questions regarding recent contact with a typhoid fever patient, eating habits, hygiene and socio-economic level. Cases were more likely to have been in contact with a patient with typhoid fever than hospital controls (adjusted odds ratio (OR) = 5.2, 95% confidence interval (95% CI) 1.7-15.9) or community controls (adjusted OR = 11.9, 95% CI 2.3-60.7); 11% and 14% of typhoid fever cases (compared to hospital or community controls, respectively) were attributable to recent contact with a patient with this disease. These findings suggest that strategies directed towards the persons in contact with a patient might reduce the incidence of secondary cases of typhoid fever.

Influence of Soil Saturation and Temperature on Erwinia chrysanthemi Soft Rot of Carrot.

In 1998, soft rot caused by Erwinia chrysanthemi resulted in an estimated loss of 1,800 tons of carrots in California. The disease appeared to be related to unusually high temperatures and excessive irrigation. To determine the optimum conditions for development of soft rot under controlled conditions, pots of carrots inoculated with E. chrysanthemi were saturated with water and incubated at 20, 25, 30, or 35°C. Plants were harvested and examined for disease 12, 24, 36, 48, 72, and 96 h after inoculation. Negligible disease occurred after 12 h. Disease severity and incidence increased with increasing temperature and duration of saturation from 24 to 96 h. In a second experiment, carrot disks were inoculated with three isolates each of E. chrysanthemi and E. carotovora subsp. carotovora and incubated at 15, 20, 25, 30, and 35°C. After 48 h, the disks were washed to remove rotted tissue and reweighed. Neither bacterium reduced carrot disk weight at 15°C. In general, moderate weight reduction occurred at 20 and 25°C. The greatest degree of soft rot was caused by E. chrysanthemi at 30 and 35°C. E. carotovora subsp. carotovora isolates were relatively less virulent than E. chrysanthemi at 30°C and none of the E. carotovora subsp. carotovora isolates reduced carrot disk weight at 35°C. This is the first report of E. chrysanthemi causing soft rot of carrot in California. Based on these results, growers should limit the length of time carrot roots are exposed to saturated soil, especially at high soil temperatures.

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.

First Report of Verticillium dahliae on Stevia (Stevia rebaudiana) in North America.

Stevia (Stevia rebaudiana Bertoni, Asteraceae) is the source of stevioside, a sweet, low-calorie sugar substitute. Acreage of stevia in California has been increasing in recent years. In October 1999, stevia plants in a commercial field exhibited stunting, leaf necrosis, and vascular discoloration. Verticillium dahliae was consistently isolated from diseased root and stem pieces plated on water agar and acidified potato dextrose agar (APDA). Colonies became dark with age on APDA, formed single celled conidia on hyaline verticillate condiophores, and produced dark microsclerotia. Stevia (cv. R-set 1) plants were grown from seed in soilless potting mix in 20-cm-diameter pots. Roots of eight 4-week-old stevia plants were submerged in a 106 conidia per ml suspension of one of the isolates for 2 min. Eight control plants were dipped in sterile water. The plants were then repotted in soilless potting mix in 10-cm-diameter pots. Four weeks later, inoculated plants exhibited stunting, necrotic lower leaves, and discolored vascular systems. Control plants remained healthy. V. dahliae was reisolated on water agar from diseased plants. The experiment was conducted twice and the results were identical. This is the first report of V. dahliae on stevia in North America.

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.

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.