Implementation of LED Fluorescence Microscopy for Diagnosis of Pulmonary and HIV-Associated Tuberculosis in a Hospital Setting in Indonesia

Background

Fluorescence microscopy (FM) has not been implemented widely in TB endemic settings and little evaluation has been done in HIV-infected patients. We evaluated diagnostic performance, time and costs of FM with light-emitting diodes technology (LED-FM), compared with conventional (Zieh-Neelsen) microscopy in a hospital in Indonesia which acts as referral centre for HIV-infected patients.

Method

We included pulmonary tuberculosis suspects from the outpatient and HIV clinic. Direct and concentrated sputum smears were examined using LED-FM and ZN microscopy by two technicians who were blinded for the HIV-status and the result of the comparative test. Mean reading time per slide was recorded and cost of each slide was calculated. Mycobacteria culture served as the reference standard.

Results

Among 404 tuberculosis suspects from the outpatient clinic and 256 from the HIV clinic, mycobacteria culture was positive in 12.6% and 27%, respectively. The optimal sensitivity of LED-FM was achieved by using a threshold of ≥2 AFB/length. LED-FM had a higher sensitivity (75.5% vs. 54.9%, P<0.01) but lower specificity (90.0% vs 96.6%, P<0.01) compared to ZN microscopy. HIV was associated with a lower sensitivity but similar specificity. The average reading time using LED-FM was significantly shorter (2.23±0.78 vs 5.82±1.60 minutes, P<0.01), while costs per slide were similar.

Conclusion

High sensitivity of LED-FM combined with shorter reading time of sputum smear slides make this method a potential alternative to ZN microscopy. Additional data on specificity are needed for effective implementation of this technique in high burden TB laboratories.     

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.     

Complexes of salicylaldehyde acylhydrazones: cytotoxicity, QSAR and crystal structure of the sterically hindered t-butyl dimer

A series of acylhydrazones of salicylaldehyde and their transition metal complexes, predominantly copper(II), have been prepared and characterized. The crystal structure of the Cu(II) complex of the sterically hindered t-butyl derivative contains a phenolato bridged dimer with the ligand coordinated as a tridentate moiety. QSAR analyses of the cytotoxicity of the chelators and their Cu(II) complexes reveals that solubility is the dominant factor for activity. Compounds display a maximum with respect to lipophilicity, allowing optimization of the bioactivity for both the ligands and their complexes. Copper complexes are significantly more cytotoxic than the metal-free ligands and complexes of other metals: Cu > Ni > Zn = Mn > Fe = Cr > Cr > Co.     

Efficient synthesis of the GABAA receptor agonist trans-4-aminocrotonic acid (TACA)

Investigations into the pharmacology of different types of cys-loop GABA receptor have relied for years on the chemical modification of GABA-like compounds. The GABA metabolite GABOB is an attractive molecule to modify due to its convenient chemical structure. In the process of developing new GABA-mimic compounds from GABOB as a starting compound three small molecule GABA derivatives were synthesized using a variety of chemical transformations. Amongst these, a new and reliable method to synthesize TACA (trans-4-aminocrotonic acid) is reported.     

Steroidal saponin production in callus cultures of Solanum aculeatissimum Jacq.

 Callus was induced from the epicotyl of S. aculeatissimum, and the relation between culture conditions and the production of steroidal saponins in the callus was studied. The results indicated that the callus produced the steroidal saponins aculeatiside A and B. The highest production of steroidal saponins occurred at the middle of the log phase. Optimal conditions for the production of steroidal saponins were culturing on MS basal medium supplemented with the combination of 0.05 ppm or 0.1 ppm NAA and 10 ppm BA, and fructose as a carbon source, in the dark at 25  °C. Under these optimal conditions, the callus produced 0.8% (per dry weight) steroidal saponins, or 0.32% aculeatiside A and 0.48% aculeatiside B. Received: 24 December 1999 / Revision received: 21 February 2000 / Accepted: 16 May 2000     

Analysis of the 220-MHz,P.M.R. spectra of some products of the amadori and heyns rearrangements

In order to establish whether p.m.r. spectroscopy is useful for identifying Amadori- and Heyns-rearrangement products, the p.m.r. spectra at 220 MHz of 16 rearrangement products derived from d-glucose or d-fructose and amino acids have been investigated. At pH 3, the protons of the NCH₂ group of N-substituted 1-amino-1-deoxy-d-fructose (Amadori-rearrangement products) resonate at δ 3.25–3.60 in D₂O and are shifted upfield by 0.3–0.6 p.p.m. at pH 9. These protons exchange with deuterium. Also, in D₂O there is an equilibrium of the acyclic, furanose, and pyranose structures, the last being favoured. At pH ? 7, the equilibrium is completely shifted to the β-pyranose form, which adopts exclusively the ²C₅ conformation. At pH 3, the equilibrium favours the β-furanose form. At pH 3, H-1e and H-1a of N-substituted 2-amino-2-deoxy-d-glucoses (Heyns-rearrangement products) resonate at δ 5.55 and 5.04, respectively. At pH 9, the signal for H-2 is shifted upfield by 0.2–0.7 p.p.m. In D₂O solution, these compounds exist as an equilibrium of α- and β-pyranose forms in the ⁴C₁ conformation. The α anomer is stabilised by the amino acid group at position 2. At pH 3, the αβ-ratio is 2–4:1, and, at pH 9, 1.0–1.1:1.     

Environmental and temporal patterns in bioturbation in the Cambrian–Ordovician of Western Newfoundland

The early Paleozoic emergence of bioturbating (sediment-dwelling and -mixing) animals has long been assumed to have led to substantial changes in marine biogeochemistry, seafloor ecology, and the preservation potential of both sedimentary and fossil archives. However, the timing of the rise of bioturbation and environmental patterns in its expansion have long been subjects of debate—resolution of which has been hampered, in part, by a paucity of high-resolution bioturbation data or of systematic investigations of facies trends in lower Paleozoic bioturbation. To address these issues, we conducted an integrated sedimentological and ichnological characterization of the Cambrian–Ordovician Port au Port succession and Cow Head Group of western Newfoundland, encompassing over 350 meters of stratigraphy logged at the centimeter to decimeter scale. We find that, across a wide range of marine facies, bioturbation does not on average exceed moderate intensities—corroborating observations from other lower Paleozoic successions indicating that the early Paleozoic development of bioturbation was a protracted process. Moreover, bioturbation intensities in the Port au Port succession and Cow Head Group are commonly characterized by considerable variability at even fine scales of stratigraphic resolution and changes in bioturbation intensity correlate strongly with variability in sedimentary facies. We observe that facies recording nearshore depositional environments and carbonate-rich lithologies are each characterized by the highest intensities of both burrowing and sediment mixing. These data highlight the need for a high-resolution and facies-specific approach to reconstructing the evolutionary history of bioturbation and suggest that average levels of bioturbation, although relatively low throughout this interval, increased notably earlier in nearshore marine settings.