First report of Acanthamoebae spp. isolation from a volcanic mud spring in the Philippines.

Acanthamoebae spp. is considered highly adaptive. The present study aims to establish the occurrence of free-living amoebae, particularly Acanthamoebae, to exist in extreme environments such as volcanic mud springs. Fifty surface water samples were collected from mud springs (34 samples), and flat rocks (16 samples) were collected, processed, and cultured. After 14 days of incubation, 32 (64%) plates showed positive amoebic growth. Nineteen (55.8%) of these plates came from the mud spring collection site, while 13 (81.2%) samples are from flat rock sources. DNAs from positive samples were made to react to polymerase chain reaction (PCR) using primer sets JDP1 5'GGCCCAGATCGTTTACCGTGAA-3' and JDP2 5'TCTCACAAGCTGCTAGGGAGTCA-3' for cells that resemble Acanthamoebae. Sequencing and basic local alignment search tool (BLAST) revealed a 99% similarity of isolates to Acanthamoebae spp. Identification of Acanthamoebae spp that can survive in higher temperatures is important public health information. The existence of such isolates in the environment has dire health implications, which suggests revisitation of water treatment protocols. Detection of such organisms in environmental sources used for recreational purposes provides information to local and international tourists who frequent them. This will result in the mitigation of potential future infection.

Detection of Acanthamoeba spp. in groundwater sources in a rural area in the Philippines.

Research on free-living amoebae (FLA) and its public health implication as an etiologic agent of parasitic infection in humans has recently gained traction in the Philippines. This study aimed to identify potential FLAs in collected groundwater samples from Masinloc, Zambales, Philippines. Fifty-four (54) water samples were collected in 250-mL sterile polyethylene containers by purposive sampling from selected groundwater sources in six (6) barangays of Masinloc. The samples were vacuum filtered through a 1.2-µm pore glass microfiber filter, cultured onto non-nutrient agar (NNA) lawned with Escherichia coli, and observed microscopically for amoebic growth for 14 days using light microscopy. Amoebic growth was observed in 11.1% (6/54) of water samples. DNAs from positive samples were extracted and were made to react with polymerase chain reaction using Acanthamoeba-specific JDP1 (5'-GGCCCAGATCGTTTACCGTGAA-3') and JDP2 (5'-TCTCACAAGCTGCTAGGGAGTCA-3') primers, and universal primer Euk A (5'-AACCTGGTTGATCCTGCCAGT-3') and Euk B (5'-TGATCCTTCTGCAGGTTCACCTAC-3'). The presence of Acanthamoeba genotypes T4, T7, and T11 was confirmed using molecular and phylogenetic analysis. Our results confirmed that groundwater sources from two of six sampling sites (33.3%) in Masinloc, Zambales, were contaminated with potentially pathogenic FLAs. Proper identification of risk factors that may cause contamination consequently leads to the implementation of programs that will prevent future infections.

Clinical enzymes as possible immunological biomarkers in the diagnosis of malaria, human African and American trypanosomiasis

@#Peripheral blood smear microscopy still remains the gold standard for diagnosing malaria and trypanosomiasis. Microscopy is a labor-intensive process and requires great amount of skill to accomplish. Even though cheap and easy to perform, it still has several limitations. This hinders the microscopist in identifying protozoan structure or differentiating species from one another. Considering these factors in the performance of microscopic examination, it is crucial to identify new strategies for parasite identification and species differentiation. Innovations in clinical enzymology, immunodiagnostics, and molecular technology would be of help in resolving the problem. This study mainly focused on the possible role of clinical enzymes in malaria and trypanosomiasis diagnosis. Enzymes play a vital role in parasite physiology and metabolism. They enable the parasite to survive inside the living host by initiating different metabolic cycles. These enzymes can either be expressed on the surface of the protozoa or excreted in the extracellular environment. Lactate dehydrogenase, aldolase, and glutamate dehydrogenase were the significant enzymes associated with pathogenic Plasmodium spp. Other malarial enzymes were also identified but further validation is still required to establish their use as diagnostic biomarkers. Whereas, the enzyme matrix metalloproteinase was identified as significant for diagnosing and differentiating Trypanosoma spp. Analysis of these enzymes can be used as alternative means for microscopy in parasite identification and differentiation. Application of these enzymes as immunologic markers in various diagnostic test kits should be further evaluated.

Transform method of processing for speckle strain-rate measurements.

We have developed a highly sensitive method for measuring thermal expansion, mechanical strain, and creep rates. We use the well-known technique of observing laser speckle with a pair of linear array cameras, but we employ a data-processing approach based on a two-dimensional transform of the speckle histories from each camera. This technique can effect large gauge sizes, which are important in the assessment of the spatial statistics of creep. Further, the algorithm provides simultaneous global estimates of the strain rates at both small- and large-scale sizes. This feature may be of value in the investigation of materials with different short- and long-range orders. General advantages of our technique are compact design, modest resolution requirements, insensitivity to slow surface microstructure changes (as seen with oxidation), and insensitivity to zero-mean-noise processes such as turbulence and vibration. Herein we detail the theory of our technique and the results of a number of experiments. Thesetests are intended to demonstrate the performance advantages and limitations of the transform method of processing speckle strain-rate data.

Effects of arterial compliance and non-Newtonian rheology on correlations between intimal thickness and wall shear.

A minimally diseased (mean intimal thickness = 56 microns) human aortic bifurcation was replicated in rigid and compliant flow-through casts. Both casts were perfused with physiological flow waves having the same Reynolds and unsteadiness numbers; the pulse pressure in the compliant cast produced radial strains similar to those expected from post-mortem measurements of the compliance of the original tissue. The compliant cast was perfused with a Newtonian fluid and one whose rheology was closer to that of blood. Wall shear rate histories were estimated from near-wall velocities obtained by laser Doppler velocimetry at identical sites in both casts. Intimal thickness was measured at corresponding sites in the original vessel and linear regressions were performed between these thicknesses and several normalized shear rate measures obtained from the histories. The correlations showed a positive slope--that is, the intima was thicker at sites exposed to higher shear rates--consistent with earlier results for relatively healthy vessels, but their significance was often poor. There was no significant effect of either model compliance or fluid rheology on the slopes of the correlations of intimal thickness against any normalized shear rate measure.

The effect of compliance on wall shear in casts of a human aortic bifurcation.

Rigid and compliant casts of a human aortic bifurcation were subjected to physiologically realistic pulsatile fluid flows. At a number of sites near the wall in the approximate median plane of the bifurcation of these models, fluid velocity was measured with a laser Doppler velocimeter, and wall motion (in the case of the compliant cast) was determined with a Reticon linescan camera. The velocity and wall motion data were combined to estimate the instantaneous shear rates at the cast wall. Analysis showed that at the outer walls the cast compliance reduced shear rates, while at the walls of the flow divider the shear rate was increased.

Variations in geometry and shear rate distribution in casts of human aortic bifurcations.

Shear rates measured at sites along the walls of ten casts of human aortic bifurcations are presented to provide an indication of the variability in the shear rate distribution that can occur among individuals. The geometric characteristics and flow parameters for each of the casts are also given. Much of the variability in wall shear distribution seen here results from variations in arterial geometry; variations in flow parameters can widen further the normal range of shear distributions.

Effect of flow partition on wall shear in a cast of a human coronary artery.

The effect of flow partition on wall shear was determined in a cast of a human coronary artery. A laser Doppler anemometer was used to measure the time varying velocities at selected sites close to the walls of the cast. Shear rates were derived by dividing the measured velocities at each site by the perpendicular distance from measurement site to the wall. At the bifurcation of the left main coronary artery into its anterior descending and circumflex branches the shear at sites near the outer walls of the two branches was more strongly affected by the flow partition than at sites on the flow divider and near the walls of the left main coronary artery.

Correlation between wall shear and intimal thickness at a coronary artery branch.

Pulsatile velocities were measured by laser Doppler anemometry at fourteen sites near the walls of a cast of a minimally diseased human left coronary artery bifurcation. The flow wave used in the experiments was physiologically realistic. The sites selected for hemodynamic measurement were at the outer walls of the left main artery and its anterior descending and circumflex branches, and along the flow divider. The intimal and medial thicknesses at corresponding sites in the original branch were also measured. Wall shear rates were derived from the velocity data. The correlations between time-average or maximum instantaneous wall shear rate and intimal thickness had negative slopes (P less than 0.005); that is, the intima was generally thicker at sites exposed to lower shears. These results are consistent with those obtained earlier using other human arterial bifurcations.

Measurement of wall motion and wall shear in a compliant arterial cast.

Initial measurements of the time-varying wall shear rate at two sites in a compliant cast of a human aortic bifurcation are presented. The shear rates were derived from flow velocities measured by laser Doppler velocimetry (LDV) near the moving walls of the cast. To derive these shear rate values, the distance from the velocimeter sampling volume to the cast wall must be known. The time variation of this distance was obtained from LDV measurements of the velocity of the wall itself.

Shear-dependent thickening of the human arterial intima.

Hemodynamic data were obtained by laser Doppler anemometry at 163 sites in 10 flow-through casts of minimally diseased human aortic bifurcations, and the intimal thickness at each of these sites in the original vessels was measured (mean = 208 micron, range = 10-967 micron). Analysis of the results suggests that the intimal thickness at sites exposed to high shear stresses increases quickly to a modest value, growing slowly thereafter, while the thickness at sites exposed to low shears rises more slowly but, after time, reaches higher values. Thus the intima of younger vessels is thicker where shear is high, and that of older vessels is thicker where shear is low. This behavior is rationalized by a parsimonious model in which a substance from the lumen enters the wall and is catabolized or otherwise removed. The intimal permeability and removal rate both increase as the shear stress is raised. Intimal thickness is related to the amount of the substance in the wall. This model fits the experimental data with an overall standard error of estimate of 105 micron. Although the model is an extreme simplification of the actual thickening process, it shows that the observed results can be the consequence of competing shear-dependent processes.

Distribution of the geometric parameters of human aortic bifurcations.

The distribution of flow divider offset, branch angle, and angular asymmetry in a collection of 70 human aortic bifurcations are reported. A computer algorithm was used to determine these parameters objectively. The mean values with standard deviations and ranges are: 1) magnitude of flow divider offset = 1.4 +/- 1.4 mm (range = 0.04 to 8.1 mm); 2) branch angle = 35.0 degrees +/- 11.1 degrees (range = 10.4 degrees to 61.3 degrees); and 3) angular asymmetry = 15.3 degrees +/- 12.9 degrees (range 0.2 degrees to 56.1 degrees). Since these parameters have been shown to have a significant influence on hemodynamic shear, their variability suggests a corresponding variability in the fluid dynamic stresses to which the bifurcations were exposed in life.

Nonquasi-steady character of pulsatile flow in human coronary arteries.

This experiment was conducted to determine if the pulsatile flow through the proximal portion of the left coronary artery system in man exhibits quasi-steady characteristics. Steady and pulsatile flows were passed through an idealized model whose dimensions were based on a vascular cast. The mean Reynolds number was 180 and the unsteadiness number was 2.7. Velocity profiles were measured by laser Doppler anemometry at several locations along diameters in the parent and both daughter channels in the neighborhood of the "left main" bifurcation. Analysis of the results along one diameter in the "left main" channel shows that unsteady flow in the larger coronary arteries may not be simulated by a series of steady flow experiments.

Comparison of steady and pulsatile flow near the ventral and dorsal walls of casts of human aortic bifurcations.

Steady and pulsatile flows were passed through casts of human aortic bifurcations and, by means of a laser Doppler anemometer, fluid velocities were measured at selected sites near the ventral and dorsal walls. At these sites, in the vicinity of the bifurcation, the influence of secondary flow is significant and therefore an appreciation of the phasic variation of secondary flow patterns is important. Results are presented comparing the flow direction in both steady and pulsatile flow at sites in three casts. The common features of the flow at these sites were the persistence of the flow direction during the accelerating and decelerating phases of the pulsatile cycle, and the consistently smaller angle (measured from the inlet centerline) of the pulsatile flow direction as compared to the angle of the flow direction in steady flow.

Correlation of human arterial morphology with hemodynamic measurements in arterial casts.

A realistic pulsatile flow was passed through a cast of the aortic bifurcation of a 63-yr-old male with mild atherosclerosis, and a laser Doppler anemometer was used to measure fluid velocities in the cast at 15 selected sites near the lateral and medial walls. Intimal, medial and adventitial thicknesses were measured and sudanophilia was scored at corresponding sites in the vessel from which the cast had been made. A negative correlation was found between intimal thickness (IT) and wall shear rate. The strongest negative correlation (p less than 0.005) was between IT and "pulse shear rate" (PSR), defined by analogy with pulse pressure. Sudanophilia also correlated negatively with PSR (p less than 0.01). Medial thickness correlated positively with shear rate, and most strongly with the mean (i.e., time-average) rate (p less than 0.005). From an analysis of the fluid mechanical data, it appears possible to separate the effects of bifurcation geometry and the shape of the arterial cross section on interfacial shear.

Correlation between intimal thickness and fluid shear in human arteries.

A realistic pulsatile flow was passed through a cast of the aortic bifurcation of a 63-year-old male with mild atherosclerosis, and a laser Doppler anemometer was used to measure fluid velocities in the cast at 15 selected sites near the lateral and medial walls. Intimal, medial and adventitial thickness were measured, and sudanophilia was scored, at corresponding sites in the vessel from which the cast had been made. A negative correlation was found between intimal thickness (IT) and wall shear rate (i.e., the velocity gradient at the blood-artery interface). The strongest negative correlation (P less than 0.005) was between IT and "pulse shear rate" (PSR), defined by analogy with pulse pressure. Sudanophilia also correlated negatively with PSR (P less than 0.01). These results suggest that large excursions of interfacial shear, at levels too low to cause damage, may inhibit intimal thickening.

Hemodynamic measurements in human arterial casts, and their correlation with histology and luminal area.

A new approach is presented for studying the vascular response to hemodynamic stress. A laser doppler anemometer is used to make velocity measurements very near the walls of human arterial casts; these measurements are then correlated with the histology of the artery from which the cast was made. Several illustrative results are given which suggest that the velocity profiles along the outer walls of aortic bifurcations may be significantly determined by the longitudinal variation of cross-sectional area. The shapes of these profiles were qualitatively different for each cast studied. In one specimen, the location of initial lipid deposits appeared to correlate with flow acceleration.