Pneumococcal Bacteremia Requiring Hospitalization in Rural Thailand: An Update on Incidence,Clinical Characteristics,Serotype Distribution,and Antimicrobial Susceptibility, 2005–2010

Background

Streptococcus pneumoniae is an important cause of morbidity and mortality in Southeast Asia, but regional data is limited. Updated burden estimates are critical as pneumococcal conjugate vaccine (PCV) is highly effective, but not yet included in the Expanded Program on Immunization of Thailand or neighboring countries.

Methods

We implemented automated blood culture systems in two rural Thailand provinces as part of population-based surveillance for bacteremia. Blood cultures were collected from hospitalized patients as clinically indicated.

Results

From May 2005– March 2010, 196 cases of pneumococcal bacteremia were confirmed in hospitalized patients. Of these, 57% had clinical pneumonia, 20% required mechanical ventilation, and 23% (n = 46) died. Antibiotic use before blood culture was confirmed in 25% of those with blood culture. Annual incidence of hospitalized pneumococcal bacteremia was 3.6 per 100,000 person-years; rates were higher among children aged <5 years at 11.7 and adults ≥65 years at 14.2, and highest among infants <1 year at 33.8. The median monthly case count was higher during December–March compared to the rest of the year 6.0 vs. 1.0 (p<0.001). The most common serotypes were 23F (16%) and 14 (14%); 61% (74% in patients <5 years) were serotypes in the 10-valent PCV (PCV 10) and 82% (92% in <5 years) in PCV 13. All isolates were sensitive to penicillin, but non-susceptibility was high for co-trimoxazole (57%), erythromycin (30%), and clindamycin (20%).

Conclusions

We demonstrated a high pneumococcal bacteremia burden, yet underestimated incidence because we captured only hospitalized cases, and because pre-culture antibiotics were frequently used. Our findings together with prior research indicate that PCV would likely have high serotype coverage in Thailand. These findings will complement ongoing cost effectiveness analyses and support vaccine policy evaluation in Thailand and the region.     

Serotonergic activity of HP 184: Does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl)-1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 μM HP 184 enhanced spontaneous release of [³H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30–40% after a 14–21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain: plasma ratio of HP 184 was approximately 2∶1, with brain concentrations of 1.6 μg/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.     

Dihydropyridine sensitive calcium channels in a smooth muscle cell line

The pharmacological properties of voltage sensitive calcium channels (VSCC) were examined in a rat aortic smooth muscle cell line (A10). The inorganic VSCC blockers Co2+ and Cd2+ blocked 45Ca2+ uptake into these cells in both 5 mM K+ and 50 mM K+ (depolarizing) conditions. The organic VSCC antagonists nitrendipine, nimodipine, D-600 and diltiazem also blocked 45Ca2+ uptake at low concentrations. The relative potencies of blockade were similar to those found in intact vascular smooth muscle. The VSCC "agonist" BAY K8644 enhanced 45Ca2+ uptake and this effect could be reversed by nitrendipine. These results indicate that A10 cells possess VSCC and that these VSCC behave similarly to those in authentic smooth muscle.     

Incidence and Epidemiology of Hospitalized Influenza Cases in Rural Thailand during the Influenza A (H1N1)pdm09 Pandemic, 2009–2010

Background

Data on the burden of the 2009 influenza pandemic in Asia are limited. Influenza A(H1N1)pdm09 was first reported in Thailand in May 2009. We assessed incidence and epidemiology of influenza-associated hospitalizations during 2009–2010.

Methods

We conducted active, population-based surveillance for hospitalized cases of acute lower respiratory infection (ALRI) in all 20 hospitals in two rural provinces. ALRI patients were sampled 1∶2 for participation in an etiology study in which nasopharyngeal swabs were collected for influenza virus testing by PCR.

Results

Of 7,207 patients tested, 902 (12.5%) were influenza-positive, including 190 (7.8%) of 2,436 children aged <5 years; 86% were influenza A virus (46% A(H1N1)pdm09, 30% H3N2, 6.5% H1N1, 3.5% not subtyped) and 13% were influenza B virus. Cases of influenza A(H1N1)pdm09 first peaked in August 2009 when 17% of tested patients were positive. Subsequent peaks during 2009 and 2010 represented a mix of influenza A(H1N1)pdm09, H3N2, and influenza B viruses. The estimated annual incidence of hospitalized influenza cases was 136 per 100,000, highest in ages <5 years (477 per 100,000) and >75 years (407 per 100,000). The incidence of influenza A(H1N1)pdm09 was 62 per 100,000 (214 per 100,000 in children <5 years). Eleven influenza-infected patients required mechanical ventilation, and four patients died, all adults with influenza A(H1N1)pdm09 (1) or H3N2 (3).

Conclusions

Influenza-associated hospitalization rates in Thailand during 2009–10 were substantial and exceeded rates described in western countries. Influenza A(H1N1)pdm09 predominated, but H3N2 also caused notable morbidity. Expanded influenza vaccination coverage could have considerable public health impact, especially in young children.     

Studies on pituitary melanotrophs reveal the novel GABAB antagonist CGP 35-348 to be the first such compound effective on endocrine cells.

One obstacle to understanding the action and physiological significance of the responsiveness of various endocrine cells to gamma-aminobutyric acid (GABA) has been that previously available substances, all active as GABAB antagonists in the nervous system, are ineffective on endocrine cells. The introduction of a potent new member of this class, CGP 35-348, of very different chemical structure, encouraged us to examine its effect on endocrine cells. For this purpose, we studied melanotroph secretion from pituitary neurointermediate lobes. We found that CGP 35-348, in contrast to previously available members of this class, suppressed completely, in rat and toad, secretory responses to baclofen, the classic GABAB agonist. Analysis, in toad, showed CGP 35-348 did not affect responses to GABAA agonists (muscimol; isoguvacine), dopamine, or neuropeptide Y. When tested against GABA, the physiological ligand present in the innervation of melanotrophs (along with dopamine and neuropeptide Y), CGP 35-348 completely suppressed the secretory response, which, in toad, is purely inhibitory and unaffected by bicuculline, the specific GABAA antagonist. In addition, CGP 35-348 unmasked a stimulant effect that bicuculline blocked. In CGP 35-348, we thus have a new tool with which to analyse responses to GABA and their physiological involvement in endocrine cells.     

Measurement of the cytosolic sodium ion concentration in rat brain synaptosomes by a fluorescence method

A method for the measurement of the cytosolic Na⁺ concentration in intact synaptosomes is described. This method makes use of a pH sensitive dye (BCECF) that can be loaded into the cytosol and a relatively specific ionophore (monensin) that can exchange Na⁺ for H⁺ across the synaptosomal membrane. By setting conditions such that there is no electrochemical potential difference for H⁺ across the membrane (no membrane potential and pH_i = pH_o), addition of ionophore would induce a H⁺ flux only if there is a concentration difference for Na⁺. Thus, when there is no fluorescence change (no cytosolic pH change) extracellular [Na⁺] equals intrasynaptosomal [Na⁺. The intrasynaptosomal [Na⁺] concentration was determined to be 7 ± 3 mM (n = 5; mean ± S.E.). The results obtained with this fluorescence method are compared with estimates obtained by atomic absorption spectrometry. Limitations and applications of the method are discussed.     

The effects of dihydropyridines on neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of (3H) norepinephrine and (3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of (3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.     

Characterization of voltage-sensitive calcium channels in a clonal pituitary cell line

We have pharmacologically characterized voltage sensitive calcium channels (VSCCs) in GH3 cells, an anterior pituitary clonal cell line known to secrete prolactin and growth hormone. Raising the medium K+ concentration from 5 to 50 mM caused an immediate increase in net 45Ca2+ uptake which remained apparent over a 15 minute time course. 45Ca2+ uptake was maximally stimulated nearly 10-fold over basal levels. This K+-induced stimulation of Ca2+ uptake was not prevented by 10-5M tetrodotoxin or by replacing sodium with choline in the assay medium. Ca2+ uptake was, however, inhibited by several VSCC antagonists: nitrendipine, D-600, diltiazem and Cd2+. Further, the novel dihydropyridine VSCC agonists, BAY K8644 and CGP 28392, enhanced 50 mM K+-stimulated 45Ca2+ uptake and these effects were blocked by nitrendipine.     

Maitotoxin-Induced Release of γ-[3H]Aminobutyric Acid from Cultures of Striatal Neurons

The potent marine toxin, maitotoxin, induced the release of gamma-[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose-dependent manner. Maitotoxin-induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin-induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D-600 at concentrations of 10(-6) M did not block maitotoxin-induced or 70 mM K+-induced release. High concentrations of D-600 (10(-4) M) partially blocked both maitotoxin- and 70 mM K+-induced release. The dihydropyridine calcium agonist BAY K8644 (10(-6) M) did not enhance maitotoxin-induced or 70 mM K+-induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage-sensitive calcium channels.     

HP 749 enhances calcium-independent release of [3H]norepinephrine from rat cortical slices and synaptosomes

Previous studies have shown that, at concentrations of 1 M and 10 M, HP 749 increased electrically-stimulated release of [³H]norepinephrine (NE) from rat cortical slices. These effects were Ca²⁺-dependent, indicating an effect on release from vesicular stores. At 100 M, HP 749 had two effects. In addition to enhancing the Ca²⁺-dependent electrically-evoked release, it also induced a rise in the basal efflux (spontaneous release) of [³H]NE, which was observed in both cortical slices and synaptosomes. The spontaneous release effect was (1) not blocked by the reuptake inhibitor nomifensine, (2) not affected by removal of external calcium, (3) not blocked by vesicular depletion with reserpine, and (4) not inhibited by the sodium channel blocker tetrodotoxin (TTX). As would be expected, the spontaneous [³H]NE release induced by the cytoplasmic releaser tyramine and the sodium channel activator veratridine were blocked by nomifensine and TTX, respectively. Notably, however, the Ca²⁺-independent veratridine-induced release was completely blocked by 100 M HP 749. The mechanism of spontaneous release of [³H]NE caused by 100 M HP 749 is unresolved at present; however, the data are consistent with this release originating from a cytoplasmic source.