Rapid genotyping of Plasmodium vivax Pvs25 and Pv38 genes by using mismatch specific endonuclease.

Mismatch specific endonuclease (MSE) method was used to detect natural polymorphisms in Pvs25 and Pv38 genes of Plasmodium vivax. Eighty seven patients with P. vivax were recruited in the Republic of Korea (ROK). Pvs25 and Pv38 genes were amplified by polymerase chain reaction (PCR), and the PCR amplicons were mixed with reference DNA sequences. Following the denaturation and gradual annealing, the product mixtures were cleaved by the MSE. Heteroduplex types were readily detected by gel electrophoresis, where extra bands with shorter sizes would appear from the cleavage. After MSE cleavage of 657- bp product from Pvs25 mixtures, three genotypes were detected, while Pv38 mixtures with 1220-bp products presented two genotypes in ROK isolates. After the MSE cleavage, the mismatched samples of Pvs25 and Pv38 were completely sequenced, and the results were in complete agreement with the MSE analyses. In conclusion, genotyping of Pvs25 and Pv38 with MSE cleavage could be a potential method for the high-throughput screening of the large field samples.

Rapid genotyping of Plasmodium vivax Pvs25 and Pv38 genes by using mismatch specific endonuclease

Mismatch specific endonuclease (MSE) method was used to detect natural polymorphisms in Pvs25 and Pv38 genes of Plasmodium vivax. Eighty seven patients with P. vivax were recruited in the Republic of Korea (ROK). Pvs25 and Pv38 genes were amplified by polymerase chain reaction (PCR), and the PCR amplicons were mixed with reference DNA sequences. Following the denaturation and gradual annealing, the product mixtures were cleaved by the MSE. Heteroduplex types were readily detected by gel electrophoresis, where extra bands with shorter sizes would appear from the cleavage. After MSE cleavage of 657- bp product from Pvs25 mixtures, three genotypes were detected, while Pv38 mixtures with 1220-bp products presented two genotypes in ROK isolates. After the MSE cleavage, the mismatched samples of Pvs25 and Pv38 were completely sequenced, and the results were in complete agreement with the MSE analyses. In conclusion, genotyping of Pvs25 and Pv38 with MSE cleavage could be a potential method for the high-throughput screening of the large field samples.

Autocalibrating parallel imaging of in vivo trabecular bone microarchitecture at 3 Tesla.

In this work the generalized autocalibrating partially parallel acquisition (GRAPPA) technique was implemented with modified reconstruction and applied to in vivo high-resolution (HR) magnetic resonance imaging (MRI) of the trabecular bone microarchitecture at 3 Tesla (T) with a multiple-acquisition balanced steady-state free precession (b-SSFP) sequence. Trabecular bone is made up of a network of microstructures (80-140 microm), and its structural deterioration is associated with the skeletal metabolic disorder osteoporosis. HR-MRI is a promising noninvasive tool for assessing the trabecular microarchitecture in vivo, but it involves long acquisition times. Using partially parallel imaging (PPI) to accelerate the acquisition may help mitigate this shortcoming and allow more flexibility in protocol design. In this study the effects of GRAPPA-based reconstruction on image characteristics and the measurement of trabecular bone structural parameters were evaluated. Initial studies showed that image quality and depiction of microstructure were preserved in the GRAPPA-based reconstruction, indicating the feasibility of PPI in HR-MRI of trabecular bone. The results also demonstrated the potential of PPI for increasing the signal-to-noise ratio (SNR) efficiency of multiple-acquisition b-SSFP imaging protocols.

Fully balanced steady-state 3D-spin-echo (bSSSE) imaging at 3 Tesla.

In this work a 3D fully balanced steady-state spin-echo sequence (bSSSE) was designed and implemented on a 3-Tesla whole-body MR scanner. In contrast to previously introduced nonbalanced SSSE (nbSSSE) sequences, bSSSE features fully rewound imaging gradients. Additionally, all phase encodings and the readout prephasing are performed after the refocusing pulse. Its performance was compared with previously used gradient-echo (GE) and nbSSSE sequences. The signal response of each sequence was simulated by numerical solution of the Bloch equation. All sequences were then optimized based on these simulations for high-resolution (HR) imaging of tissues with short relaxation times and applied to in vivo HR-MRI of trabecular bone. bSSSE outperformed nbSSSE sequences in terms of SNR and SNR efficiency. Measurements of structural bone parameters revealed thinner trabeculae and a lower bone/bone-marrow fraction for the bSSSE sequence compared to the nbSSSE sequence. By applying a parallel imaging technique based on generalized autocalibrating partially parallel acquisition (GRAPPA) with a reduction factor of 2, we were able to maintain a clinically feasible scan time using bSSSE. Comparisons of structural bone parameters revealed a difference of less than 3% for all structural parameters between parallel and conventional imaging.

Mixed infections with Opisthorchis viverrini and intestinal flukes in residents of Vientiane Municipality and Saravane Province in Laos.

Faecal examinations for helminth eggs were performed on 1869 people from two riverside localities, Vientiane Municipality and Saravane Province, along the Mekong River, Laos. To obtain adult flukes, 42 people positive for small trematode eggs (Opisthorchis viverrini, heterophyid, or lecithodendriid eggs) were treated with a 20-30 mg kg(-1) single dose of praziquantel and purged. Diarrhoeic stools were then collected from 36 people (18 in each area) and searched for helminth parasites using stereomicroscopes. Faecal examinations revealed positive rates for small trematode eggs of 53.3% and 70.8% (average 65.2%) in Vientiane and Saravane Province, respectively. Infections with O. viverrini and six species of intestinal flukes were found, namely, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus caninus, Prosthodendrium molenkampi, and Phaneropsolus bonnei. The total number of flukes collected and the proportion of fluke species recovered were markedly different in the two localities; in Vientiane, 1041 O. viverrini (57.8 per person) and 615 others (34.2 per person), whereas in Saravane, 395 O. viverrini (21.9 per person) and 155207 others (8622.6 per person). Five people from Saravane harboured no O. viverrini but numerous heterophyid and/or lecithodendriid flukes. The results indicate that O. viverrini and several species of heterophyid and lecithodendriid flukes are endemic in these two riverside localities, and suggest that the intensity of infection and the relative proportion of fluke species vary by locality along the Mekong River basin.

Feasibility of in vivo structural analysis of high-resolution magnetic resonance images of the proximal femur.

Previously, high resolution MRI to assess bone structure of deep-seated regions of the skeleton such as the proximal femur was substantially limited by signal-to-noise ratio (SNR). With the advent of new optimized pulse sequences in MRI at 1.5 T and 3 T, it may now be possible to depict and quantify the trabecular microarchitecture in the proximal femur. The purpose of this study was to investigate the feasibility of assessing trabecular microstructure of the human proximal femur in vivo with MR imaging at 1.5 T and 3 T. MR images of six young, healthy male and female subjects were acquired using standard clinical 1.5-T and high-field 3-T whole-body MR scanners. Using a T2/T1-weighted 3D FIESTA sequence (and a 3D FIESTA-C sequence at 3 T to avoid susceptibility artifacts) a resolution of 0.234 x 0.234 x 1.5 mm(3) was achieved in vivo. Structural parameters analogous to standard bone histomorphometry were determined in femoral head and trochanter regions of interest. Bone mineral density (BMD) measurements were also obtained using dual-energy X-ray absorptiometry (DXA) for the femoral trochanter in the same subjects. The bone structure of the proximal femur is substantially better depicted at 3 T than at 1.5 T. Correlation between the structural parameters obtained at both field strengths was up to R =0.86 for both the femoral head and the trochanteric region. However, the resolution of the images limits the application of 3D structural analysis, making the assessment more akin to 2D textural measures, which may be correlated to histomorphometric but are not identical measures. This feasibility study establishes the potential of MRI as a means of imaging proximal femur structure, and improvements in technique and resolution enhancements are warranted.

Suppressed cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with Toxoplasma gondii tachyzoites.

Mechanisms of host immunosuppression after infection with Toxoplasma gondii are unclear. This study was performed to observe cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with live, nonreplicating (irradiated) RH tachyzoites on stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS). For lymphocyte cultivation, 3 groups were prepared: coculture with live nonirradiated tachyzoites separated by a transwell (group T), live irradiated tachyzoites without a transwell (group R), and no tachyzoites (group C). Compared with group T, groups R and C, on stimulation with Con A, revealed significantly (P < 0.05) lower levels of interleukin-2 (IL-2) and IFN-gamma, but not IL-10. The levels of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were also significantly (P < 0.05) lower in groups R and C than in group T after stimulation with LPS. The results suggest that intracellular infection of murine splenic lymphocytes with T. gondii tachyzoites could impair their capacity to produce cytokine and immunoglobulin secretions.

Role of intestinal goblet cells in the expulsion of Gymnophalloides seoi from mice.

Role of intestinal goblet cells (GCs) in the expulsion of Gymnophalloides seoi was studied using 4 strains of mice, ICR, C3H/ HeN, BALB/c, and C57BL/6, after infection with 200 metacercariae isolated from oysters. On day 7 postinfection (PI), significantly higher (P < 0.05) worm recovery rates (WRRs) were observed in ICR (29.5 +/- 12.0%) and C3H/HeN (14.8 +/- 8.2%) than in BALB/c (5.7 +/- 5.3%) and C57BL/6 (0.8 +/- 1.1%) mice. Alteration of the GC mucins was marked in C57BL/6 mice. On day 14 PI, 5.2 +/- 5.2% and 0.6 +/- 0.7% of worms were recovered only from ICR and C3H/HeN mice. When C57BL/6 mice were immunosuppressed with prednisolone, WRR on day 7 PI increased to 11.7 +/- 13.9%, whereas the GC hyperplasia and mucin alteration diminished significantly. The results suggest that expulsion of G. seoi from the intestine is dependent on immune responses of the host, and GCs may be an important effector.

Recovery, growth, and development of Acanthoparyphium tyosenense (Digenea: Echinostomatidae) in experimental chicks.

Chicks were experimentally infected with Acanthoparyphium tyosenense (Digenea: Echinostomatidae) metacercariae per os, and the growth and development of worms in this host were observed from days I to 38 postinfection (PI). The worms grew rapidly and matured sexually in the small intestine (chiefly in the jejunum) of chicks by day 5 PI. and survived at least up to day 38 Pi, although worm recovery decreased after day 5 PI. Both parenchymal and reproductive organs increased greatly in size from day 2 to day 10 PI and then continued to increase gradually in size up to day 38 PI. The number of uterine eggs reached a peak on days 10 and 15 PI and then decreased gradually. The results suggest that chicks are a fairly suitable definitive host for experimental infection with A. tyosenense.

Surface ultrastructure of the advanced third-stage larvae of Gnathostoma nipponicum.

The surface ultrastructure of advanced third-stage larvae (AL3) of Gnathostoma nipponicum was studied using scanning electron microscopy. The larvae were recovered from the grass snake Rhabdophis tigrina in the Republic of Korea. Parasites had a globular head bulb with a pair of lips at the anterior end and 2 labial papillae and an amphid on each lip. The head bulb was characteristically armed with 3 transverse rows of hooklets, averaging 36, 38, and 43 in number, increasing posteriorly. A total of 213-232 minute unidentate cuticular spines were present along the entire length of the larvae, forming the transverse striations. Two pairs of cervical papillae were located between the 8th and 12th transverse striations, and a pair of body papillae was seen laterally on the posterior third of the body. A pair of caudal phasmids was recognized near the posterior extremity. The surface ultrastructure of AL3 of G. nipponicum is unique compared with that of other species.

Stictodora lari (Digenea: Heterophyidae): the discovery of the first human infections.

Stictodora lari Yamaguti, 1939 (Digenea: Heterophyidae) was originally reported as an avian intestinal parasite and is here reported in humans. The patients were adults, aged 46-69 (2 men and 4 women), who resided in coastal villages in Sachon-gun, Kyongsangnam-do, or Shinan-gun, Chollanam-do. in the Republic of Korea. The worms were recovered after praziquantel treatment and purgation with magnesium salts. A total of 15 S. lari specimens (1-10 per individual) was collected, together with 5 other species of intestinal flukes. The flukes were morphologically characterized by a small body size (0.70-0.86 mm long and 0.27-0.36 mm wide), a ventrogenital sac, and a gonotyl armed with 70-80 spines in the form of a comma or reversed comma lying along their lateral margin. The patients had eaten the raw flesh of mullets and gobies that had been caught in an estuary near their villages. To the best of our knowledge, this is the first record of a human S. lari infection.

High prevalence and seasonality of cryptosporidiosis in a small rural village occupied predominantly by aged people in the Republic of Korea.

The prevalence of Cryptosporidium parvum infection and its seasonality were studied in a small rural village occupied predominantly by aged people in Hwasun-gun, Chollanam-do, Republic of Korea. Fecal samples were collected monthly from November 1996 to October 1997 and examined for enteric parasites. To detect oocysts of C. parvum, the modified acid-fast stain was applied. To determine effects of climatic factors, local weather reports were collected. The overall oocyst prevalence during 1 year was 57.0% (77 of 135), and was significantly higher (P < 0.0001) in aged people--those aged 50-59 years (80.0%) and those aged 60-69 years (69.0%)--than in younger groups. No significant difference (P > 0.05) was noted between men and boys (53.6%) and women and girls (59.5%), but significant (P < 0.05) household clustering was recognized. People aged > 50 years tended to be more repeatedly positive than people in the younger group. The rate varied monthly from 7.8 to 20.0% (average, 12.6%), with significantly higher (P < 0.05) rates in spring than in summer and winter months. This seasonality had little relation with temperature (r = 0.04), but a weak correlation was found with rainfall (r = 0.4). It is of special interest that cryptosporidiosis is highly endemic among the aged population.

Acanthoparyphium tyosenense: the discovery of human infection and identification of its source.

Acanthoparyphium tyosenense Yamaguti, 1939 (Digenea: Echinostomatidae), was originally reported as an avian intestinal parasite; here, its presence is reported in 10 humans in the Republic of Korea. The patients were 9 adults aged 35-66 yr (males and females) and a young girl aged 7 yr residing in 2 coastal villages in Puan-gun, Chollabuk-do. The worms were recovered after treatment with praziquantel and purgation with magnesium salts. A total of 158 specimens (1-107 specimens/individual) was collected, together with varying numbers of other intestinal flukes. The patients had eaten various kinds of brackish water mollusks caught in an estuary near their villages. Five bivalves and a gastropod species suspected as sources of human infection were collected and examined. Two bivalves (Mactra veneriformis and Solen grandis) and the gastropod (Neverita bicolor) were found to be infected with the metacercariae of A. tyosenense; adult flukes were confirmed after the experimental infection of chicks. The results show that A. tyosenense infects humans and that brackish water mollusks are the source of human infection.

A nationwide survey of the prevalence of human Gymnophalloides seoi infection on western and southern coastal islands in the Republic of Korea.

A nationwide survey was performed to know the distribution and prevalence of human Gymnophalloides seoi infection on western and southern coastal islands in the Republic of Korea. A total of 4,178 fecal specimens were collected from residents on 45 (24 western and 21 southern) islands, and examined by Kato-Katz and formalin-ether sedimentation techniques. Eggs of G. seoi were detected from 160 (3.8%) people living on 22 (13 western and 9 southern) islands. The prevalence varied by the location of islands; higher on western islands than on southern islands. The highest prevalence was found on Amtaedo (25.3%), followed by Cheungdo (25.0%), and Anchwado (20.9%) (Shinan-gun). A little lower prevalence was observed on Munyodo (13.3%), Shinshido (12.9%), and Sonyudo (10.3%) (Kunsan-shi). Of the remaining islands, the regions showing the prevalence greater than 5% included Kohado, Dallido (Mokpo-shi), Pyeongildo, Kogumdo (Wando-gun), and Keogumdo (Kohung-gun). A strong age predilection was noted (p < 0.05); 95% of the infected people were over 40 years old. Females showed a little higher prevalence than males. The results indicate that human G. seoi infection is more widely distributed than previously considered. Nine of 11 islands (excluding the 2 known areas Munyodo and Sunyudo) that showed greater prevalence than 5% are regarded as new endemic foci of G. seoi.

Diphyllobothrium latum infection after eating domestic salmon flesh.

Diphyllobothrium latum infection in human is not common in Korea and only thirty seven cases have been reported since 1921. We report two cases of fish tapeworm infection after ingestion of raw cherry salmon (Oncorhynchus masou) caught in the domestic river. Among four family members who ate together raw salmon flesh six months ago, just two, mother and daughter, were infected. It is our expectation that the salmon associated tapeworm infections would be enlisted as one of the major parasitic problems with the growing consumption of salmon in Korea.

Genetic difference in susceptibility and fatality of three strains of mice experimentally infected with Neodiplostomum seoulense.

The genetic influence on host susceptibility to Neodiplostomum seoulense infection and fatality of the host was studied in 3 inbred strains of mice (BALB/c [H-2d], C3H/He [H-2k], and C57BL/6 [H-2b]). The survival of the mice, worm expulsion kinetics, worm size, number of eggs produced per day (EPD), and number of uterine eggs were observed from day 1 to day 40 postinfection (PI) with 100 or 200 metacercariae per mouse. Infection with N. seoulense was highly lethal to all 3 strains of mice, but the lethality was dose-dependent and varied according to the genetic backgrounds of the mice. The C3H/He mice exhibited the highest mortality, the lowest worm burdens and EPD, and the quickest expulsion of worms. It is suggested that different genetic backgrounds of mice appear to affect the host's capacity to expel N. seoulense and the fatality of the hosts themselves.

Molecular differentiation of three species of Metagonimus by simple sequence repeat anchored polymerase chain reaction (SSR-PCR) amplification.

Genomic DNAs from 3 species of Metagonimus in the Republic of Korea, M. yokogawai, M. miyatai, and M. takahashii, were amplified by simple sequence repeat anchored polymerase chain reaction. Primers of 3' or 5' termini of the (CA)n repeats were used; 3 kinds of 3' anchored primers (CA)8RG, (CA)4RG, and (CA)8RY, and 2 kinds of 5' anchored primers BDB(CA)7C and DBDA(CA)7. The results showed distinguishable banding patterns among the 3 species of Metagonimus, suggesting that they have different genotypes.

Persistent endemicity of Gymnophalloides seoi infection in a southwestern coastal village of Korea with special reference to its egg laying capacity in the human host.

Follow-up studies have been conducted every three years on the endemicity of Gymnophalloides seoi infection in a small coastal village of Chollanam-do (Province), Korea, since it was first known as an endemic area in 1994. Special attention was given to its egg laying capacity in the human host. In fecal examinations, the overall helminth egg and/or cyst positive rate was 78.7% (74/94) in 1997 and 76.6% (82/107) in 2000. Among them G. seoi eggs showed the highest rate; 71.3% (67/94) in 1997 and 72.0% (77/107) in 2000. The average number of eggs per gram of feces (EPG) was 1,015 in 1997, while a reduced rate of 353 was observed in 2000. In 1997, total of 320,677 adult flukes of G. seoi (av. 10,344/person, 94-69,125 in range) were collected from the diarrheic stools of 31 treated patients. The EPG/worm obtained from 21 cases ranged from 0.04 to 0.77 (av. 0.23), suggesting density-dependent constraints on the worm fecundity. The relationship between the worm burden (X) and EPG/worm (Y) can be expressed as Y = 0.42.e-1.2 chi (r = 0.49). The results showed that G. seoi infection is persistently endemic in this village.

Surface ultrastructure of Metagonimus takahashii metacercariae and adults.

A scanning electron microscopic study was performed on the surface ultrastructure of metacercariae and adults of Metagonimus takahashii. Metacercariae were collected from the scale of crucian carp (Carassius auratus), and adult flukes were harvested 1-4 weeks after infection to rats. In excysted metacercariae, the oral sucker had type I (numerous) and type II (seven in total) sensory papillae. Tegumental spines were dense and digitated into 5-7 points on the surface anterior to the ventral sucker, but became sparse and less digitated posteriorly toward the end of the body. In adults, seven type II sensory papillae were characteristically arranged around the lip of the oral sucker, and on the inner side of the lip four small and two large type I sensory papillae were symmetrically seen on each side (12 in total). Tegumental spines on anterior two-thirds of the body, were digitated with 9-12 tips ventrally and 8-13 tips dorsally. Sperms entering into the Laurer's canal were observed. The results show that the surface ultrastructure of M. takahashii is generally similar to those of M. yokogawai and M. miyatai except for the digitation of tegumental spines.

High endemicity of Metagonimus yokogawai infection among residents of Samchok-shi, Kangwon-do.

A small-scale epidemiological survey was undertaken during 1997-1998 on the residents along the Osib-chon (Stream), Samchok-shi (City), Kangwon-do (Province), to evaluate the status of Metagonimus yokogawai infection. A total of 165 fecal samples was collected and examined by cellophane thick smear and formalin-ether sedimentation techniques. The egg positive rate of M. yokogawai was 29.7%, showing a remarkable difference between males (46.6%) and females (16.3%). To obtain the adult flukes of M. yokogawai, 11 egg positive persons were treated with praziquantel and purged with magnesium sulfate. A total of 242,119 adult flukes (average 22,010 per person, 367-119,650 in range) was collected from diarrheic stools, all of which were identified as M. yokogawai. The results show that M. yokogawai is still highly endemic in this area.