The detection of trichomonas vaginalis by cytological stained tests / Монголын Анагаах Ухаан

Background: One of the confronted problems of doctors and medical personnels is sexually transmitted disease has not been decreased in our country up to now. By last 5 years propagation of trichomoniasis was 16.7-9.5% per 10000 population and infected T.vaginalis. In the practice of parasitology of our country metilen is revealed by gram method hasn’t been introduced which is used in up to date histological analyse. This became the background of our research work. Aim of our research is to diagnose trichomoniasis which infects sexually by cytological analyse and to define its specific and sensibility.Materials and Methods: A total 99 smears of 33 females aged 19-39, used cross sectional descriptive method. Finding T.vaginalis on specimens (1) Vaginal wet mount, (2) Gram staining and (3) Pap stain. Result: In our research T.vaginalis leaked out in wet mount smear was 33%, in Gram stain was (21.2%). The sensibility quality of T.vaginalis on Gram stain is 63%, specific quality is 90%, value of kappa coefficient (К=0.58 Р<0.002). In Pap stain T.vaginalis diagnosed 27,2% and sensibility quality of T.vaginalis is 81%, specific quality is 100%, value of kappa coefficient (К=0.87 Р<0.005). In the case when 3 analysis were positive case was 5 or 15.2%, in the case where 2 analysis were positive case was 6 or 18.2%. Conclusions: The Pap stain sensibility quality of T.vaginalis is 81%, specific quality is 100%, value of kappa coefficient (К=0.87 Р<0.005), that shows should be to give effect on diagnosing of sexually transmitted infectious disease.

Identification of differentially expressed cDNAs in Acanthamoeba culbertsoni after mouse brain passage

Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from 5% to 70%. Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.

Pathogenic free-living amoebae in Korea

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.

Decreasing effect of an anti-Nfa1 polyclonal antibody on the in vitro cytotoxicity of pathogenic Naegleria fowleri

The nfa1 gene was cloned from a cDNA library of pathogenic Naegleria fowleri by immunoscreening; it consisted of 360 bp and produced a 13.1 kDa recombinant protein (rNfa1) that showed the pseudopodia-specific localization by immunocytochemistry in the previous study. Based on the idea that the pseudopodia-specific Nfa1 protein mentioned above seems to be involved in the pathogenicity of N. fowleri, we observed the effect of an anti-Nfa1 antibody on the proliferation of N. fowleri trophozoites and the cytotoxicity of N. fowleri trophozoites on the target cells. The proliferation of N. fowleri trophozoites was inhibited after being treated with an anti-Nfa1 polyclonal antibody in a dose-dependent manner for 48 hrs. By a light microscope, CHO cells co-cultured with N. fowleri trophozoites (group I) for 48 hrs showed severe morphological destruction. On the contrary, CHO cells co-cultured with N. fowleri trophozoites and anti-Nfa1 polyclonal antibody (1: 100 dilution) (group II) showed less destruction. In the LDH release assay results, group I showed 50.6% cytotoxicity, and group II showed 39.3%. Consequently, addition of an anti-Nfa1 polyclonal antibody produced a decreasing effect of in vitro cytotoxicity of N. fowleri in a dosedependent manner.

Ultrastructural observation of human neutrophils during apoptotic cell death triggered by Entamoeba histolytica

Neutrophils are important effector cells against protozoan extracellular parasite Entamoeba histolytica, which causes amoebic colitis and liver abscess in human beings. Apoptotic cell death of neutrophils is an important event in the resolution of inflammation and parasite's survival in vivo. This study was undertaken to investigate the ultrastructural aspects of apoptotic cells during neutrophil death triggered by Entamoeba histolytica. Isolated human neutrophils from the peripheral blood were incubated with or without live trophozoites of E. histolytica and examined by transmission electron microscopy (TEM). Neutrophils incubated with E. histolytica were observed to show apoptotic characteristics, such as compaction of the nuclear chromatin and swelling of the nuclear envelop. In contrast, neutrophils incubated in the absence of the amoeba had many protrusions of irregular cell surfaces and heterogenous nuclear chromatin. Therefore, it is suggested that Entamoeba-induced neutrophil apoptosis contribute to prevent unwanted tissue inflammation and damage in the amoeba-invaded lesions in vivo.

Gordius Worm Found in a Three Year Old Girl's Vomitus

Since the Gordius worm is a parasite of crickets and several arthropods, cases of humans infected with this worm have been rare and accidental. A Gordius worm was obtained from a three-year-old girl who consulted a local clinic in Gwangju, Kyunggi-do, Korea. She lived in a rural area, and had eaten an insect that looked like a cricket. She expelled the worm in vomitus 15 minutes later; in fact, she expelled two worms, but one was discarded. The worm had a grayish white color and an intact outer surface. It was 16 cm in length and 0.6 cm wide. The posterior end of the worm was spirally enrolled and furcated into two caudal lobes, which were nearly cylindrical but showed a somewhat concave medio- ventral surface. The cloacal aperture was round and situated anterior to the point of bifurcation of the lobes. The cloacal aperture was encircled by a dark ring, which was a little removed from the aperture. The crescent fold was reddish brown, and no hairs were noticed over the entire body surface. The worm had the morphological features of a male Gordius. Accidental human cases involving the Gordius worm are rare and this is the first such case in Korea.

Acanthamoeba sohi, n. sp., a pathogenic Korean isolate YM-4 from a freshwater fish

A new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Acanthamoeba sp. YM-4 (Korean isolate YM-4). The trophozoites were 11.0-23.0 micrometer in length and had hyaline filamentous projections. Cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Acanthamoeba YM-4 can survive at 40 degrees C, and its generation time was 19.6 hr, which was longer than that of A. culbertsoni. In terms of the in vitro cytotoxicity of lysates, Acanthamoeba YM-4 was weaker than A. culbertsoni, but stronger than A. polyphaga. On the basis of the mortality of experimentally infected mice, Acanthamoeba YM-4 was found to be highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. An anti-Acanthamoeba YM-4 monoclonal antibody, McAY7, was found to react only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster on the basis of phylogenetic distances. Thus the Acanthamoeba Korean isolate YM-4 was identified as a new species, and assigned as Acanthamoeba sohi.

Phylogenetic relationship of ribosomal ITS2 and mitochondrial COI among diploid and triploid Paragonimus westermani isolates

We compared patterns of intraspecific polymorphism of two markers with contrasting modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the lung fluke, diploid and triploid Paragonimus westermani from three geographical regions of Korea. The genetic distances between three populations of Korean diploid and triploid P. westermani showed no significant difference in the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (mtCOI) and ribosomaal second internal transcribed spacer (ITS2) genes. A highly resolved strict-consensus tree was obtained that illustrated phylogenetically useful information of the ITS2 and mtCOI sequences from diploid and triploid P. westermani.

A survey on head lice infestation in Korea (2001) and the therapeutic efficacy of oral trimethoprim/sulfamethoxazole adding to lindane shampoo

Total of 7, 495 children including 3, 908 boys and 3, 587 girls from a kindergarten and 15 primary schools were examined for head lice infestation (HLI). The overall prevalence of HLI in this study was found to be 5.8%. Head lice were much more commonly detected in girls than in boys with prevalence of 11.2% and 0.9%, respectively. Sixty-nine children with HLI were treated with 1% lindane shampoo alone (group 1), and 45 children with HLI were treated with 1% lindane shampoo and oral trimethoprim/sulfamethoxazole (group 2), and follow-up visits were conducted 2 and 4 weeks later. The children who still had HLI 2 weeks after the primary treatment were treated again. At the 2-week follow-up visit, the treatment success rates of groups 1 and 2 were 76.8% and 86.7%, respectively, and at the 4-week follow-up visit, the rates were 91.3% and 97.8%, respectively. No statistically significant synergistic effect was observed for the combination of a 1% lindane shampoo and oral trimethoprim/sulfamethoxazole.

A case of breast sparganosis

A 29-year-old Korean woman visited the Department of Surgery in MizMedi Hospital with a palpable itching mass on the right breast that had existed for the past 7 months. She had no history to eat either frogs or snakes, but had the history of drinking impure water. Sonography revealed a serpiginous hypoechoic tubular structure associated with partial fat necrosis in breast parenchymal layer and subcutaneous fat layer. It also revealed oval cystic lesions. At operation, an ivory white opaque ribbon-like worm that measured 16.5 cm in length and 0.5 cm in width was extracted. Anti-sparganum specific serum IgG level in the patient's serum (absorbance = 0.71), measured by ELISA, was found to be significantly higher than those of normal controls (cut off point = 0.21). Sonography and ELISA appear to be helpful to diagnose sparganosis. Breast sparganosis is rarely found throughout the world.

Status of intestinal parasites infection among primary school children in Kampongcham, Cambodia

A survey was made to find the extent of intestinal parasite infection in Kampongcham, Cambodia in February 2002. A total of 251 fecal specimens were collected from Tonlebat primary school children and examined by formalin-ether sedimentation technique. The overall infection rate of intestinal parasite was 54.2% (males, 57.3%; females, 50.8%). The infection rate of intestinal helminths by the species were as follows: Ascaris lumbricoides 26.3%, Echinostoma sp. 15.6%, hookworm 6.4%, Opisthorchis sp. 4.0%, Rhabditis sp. 2.4%, and Trichuris trichiura 0.4%. The infection rate of intestinal protozoa were as follows: E. coli 7.6%, G. lamblia 3.2%, I. butschlii 3.2%, and E. histolytica 0.8%. More than two different kinds of parasites were found in 16.7% of the stool samples. All the children infected were treated with albendazole, praziquantel and metronidazole according to parasite species. The results showed that intestinal parasites are highly endemic in this area.

The first Korean case of human pulmonary dirofilariasis

Human pulmonary dirofilariasis has been documented from many parts of the world, but not in Korea so far. We experienced a patient of pulmonary dirofilariasis who had visited a local clinic because of chest pain for 1 month. On chest radiograph, a coin lesion of 2 cm diameter and enlargement of the mediastinal lymph node were shown. An exploratory lung resection was done. Pathologically the lesion was a pulmonary dirofilariasis complicated with necrotic pneumonia, fibrosis, and infarction. At the center of the lesion, degenerated nematode sections with multilayered cuticle, thick musculature, and bilateral internal ridges on each side were found, which was identified to be Dirofilaria immitis. This is the first report of human pulmonary dirofilariasis in Korea.

Acanthamoebiasis in Korea: two new cases with clinical cases review

The first case was 7-month-old immunodeficiency girl in whom the diagnosis of Acanthamoeba pneumonia was established by culture of a bronchial washing. The patient had been ill for a month when she was admitted due to neonatal thrombocytopenia with respiratory difficulty and treated with gammaglobulin and steroid. Her chest X-ray showed diffuse alveolar consolidation on the left lung with interstitial hazziness and a partial sign of hyperinflation on the right lung. Laboratory tests showed that the Candida antigen was negative and Pneumocystis carinii was not detected. Mycoplasma antigen was negative. All the immunoglobulin levels (IgG, IgA, IgM) were below the normal range. Five days later the patient expired. The second case was an immunosuppressed 7-year-old boy in whom Acanthamoeba trophozoites were found in the skin biopsy, followed by meningitis leading to death. About five days after a laceration on the region of the left eyebrow, a painful bean-sized nodule developed at the suture site and it was treated with antibiotics and corticosteroid. The skin biopsy showed severe inflammatory cell infiltration. Trophozoites were scattered near the blood vessels throughout the inflammatory zone. From one weak prior to admission, the patient had suffered from vomiting, indigestion and mild fever. Skin nodules with tenderness appeared all over his body surface. Examination of cerebrospinal fluid showed clear, Gram stain was negative, bacterial culture negative, India ink preparation negative, and organism on wet smear negaive. On admission day 10, focal seizure of the left extremity occurred. Brain CT revealed calcific density on the left parietal lobe area and hypodensity on the left basal ganglia. He became comatous and died immediately after discharge. Until now in Korea, two cases that are described in this paper, one Acanthamoeba meningoencephalitis case and seven Acanthamoeba keratitis cases including two unreported keratitis cases that are reported in this paper have been presented.

A case of loiasis

The prevalence of Loa loa infections in non-endemic areas such as Korea is very low, even though it is quite common in the endemic regions of West and Central Africa. We describe a patient who presented with temporary localized edema (classical Calabar swellings) after travelling to Cameroon and in whom the diagnosis of loiasis was made by ELISA. This is the second reported case of loiasis in Korea. As international travel is becoming more frequent, Loa loa infection should be considered in the differential diagnosis for patients with eosinophilia and Calabar swellings in Korea.

Serodiagnosis of human sparganosis by a monoclonal antibody-based competition ELISA

Competition ELISA test using sparganum specific monoclonal antibodies (Mab) was investigated to improve the diagnostic specificity of sparganosis. By cell fusion, one hybridoma clone secreting anti-sparganum specific Mab was selected (Sp-20), which reacted on bands of 32 kDa and 38 kDa. Sp-20 reacted on calcium corpuscles on IFA. By micro-ELISA, 16 of 17 sparganosis cases (95%) were found positive, but 1 of 18 clonorchiasis cases (5%), 4 of 16 cysticercosis cases (25%) and 2 of 16 normal controls (11%) showed false positive reactions. On the other hand, by competition ELISA using a sparganum specific Mab (Sp-20), 16 out of 17 (95%) of sparganosis cases were found positive, but 2 of 18 clonorchiasis cases (10%), 2 of 16 cysticercosis cases (12%), 3 of 16 paragonimiasis cases (18%) and 1 of 16 normal controls (6%) showed false positive reactions.

Detection of specific serum IgE in clonorchiasis cases and analysis of Clonorchis sinensis allergens

Specific serum IgE levels of Clonorchis sinensis in infected humans were measured by avidin-biotin ELISA, and allergens from C. sinensis were identified by immunoblot and autoradiography. Then, allergens fractionated by Sephadex G-200 gel filtration were analyzed, and cross-reactive allergenic components of C. sinensis reacted with paragonimiasis sera were revealed. Fourteen out of 15 C. sinensis egg-positives were found to be serum IgE positive (absorbance > 0.27). Of 14 IgE-reacting allergen bands visualized, major allergens of 66, 61.5, 45, 37, 28.5, 23.5 and 15.5 KD were recognized by more than 50% of the sera of infected humans. The considerable individual variations of IgE immune responses to C. sinensis allergenic components were also noticed. C. sinensis extract was separated into 5 fractions by Sephadex G-200 gel filtration. Seventy-four KD allergen was recognized in the first fraction, 50, 45, 37, 29.5 and 28.5 KD in the third, and 15.5 KD in the fourth. Cross-reactive allergens with sera of paragonimiasis cases were identified as 66, 45, 28.5, 13 and 7.5 KD.

A comparative study on hydrolase activities in Acanthamoeba culbertsoni and A. royreba

Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta- N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80 % of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba extracts than in culture media up to 120 hours after cultivation, but after 120 hours of cultivation those activities were detected higher in culture media than in the amoeba lysates. Neutral proteinase activity in A. culbertsoni increased more in EBSS medium than in the lysate specimens although the activity in the extracts was generally steady according to the cultivation time. Summarizing the above results, it is concluded that there were differences in hydrolase activities between pathogenic A. culbertsoni and non-pathogenic A. royreba, and that some hydrolase activities were detected remarkably higher in A. culbertsoni which revealed strong cytotoxicity to the target CHO cell line.

Passive immunity by splenocyte transfer against amebic meningoencephalitis in mice

The role of passive cell-mediated transfer of immunity against primary amoebic meningoencephalitis(PAME) in mice was studied. Naegleria fowleri, ITMAP 359, were cultured in CGVS medium. The ICR mice used were six week-old males of average weight of 15 g. Immunization was done by three intraperitoneal injections of l x 10(6) N. fowleri trophozoites at the interval of one week. Splenocytes were obtained from normal and immune mice spleens, and 1 x 10(7) cells were administered intraperitoneally into mice 3 days before challenge infection. Mice were infected intranasally with 7 x 10(4) N. fowleri trophozoites in a 3 microliter suspension under secobarbiturate anesthesia. Transplants of normal or immune splenocytes seem to alter the pattern of the PAME development. The splenocytes transferred from immune mice reduced the mortality rate in the N. fowleri infected mice, as compared with the mice transferred with the same number of normal splenocytes or without splenocyte. The blastogenic response of the splenocytes to both lipopolysaccharide and concanavalin A was elevated on day 7 after infection the mice transinoculated with immune splenocytes. The serum antibody titers in the mice transferred with immune splenocytes were increased gradually from day 7 up to day 20 after infections by mean of ELISA. It is suggested that the transfer of splenocytes from immunized mice conferred immunity against N. fowleri infection.

Blastogenic responses of splenic lymphocytes to Naegleria fowleri lysates and T-cell mitogen in mice with primary amoebic meningoencephalitis

This study was to observe the changes of blastogenic responses of splenic lymphocytes to T-cell mitogens, N. fowleri lysate and concanavalin A, and serum antibody titer during the course of experimental PAM in mice. Naegleria fowleri, strain 0359, was cultured in the CGVS medium axenically and inoculated intranasally with 7 x 10(4) trophozoites for the development of experimental PAM in mice. The amoebae were subjected to ultrasonication and centrifuged at 20,000 g for 60 minutes, and filtered through 0.2 micro-m filter membrane. The supernatant, N. fowleri lysate, was used as T-cell mitogen, and antigen for ELISA. The serum antibody was examined by ELISA using peroxidase conjugate. Two hundred micro-l of 10(6) splenocytes in RPMI 1640 containing 10% fetal calf serum were added to each well of a microtiter plate. To each well was added T-cell mitogens, 100 micro-g/ml of N. fowleri lysate or 4 micro-g/ml of con. A, and the plates were incubated for 42 hours at 37 C in 5% CO(2) incubator. Cultures were pulsed with 1 micro-Ci of methyl-(3H)-thymidine 6 hour before harvesting. The mean blastogenic response of the splenocytes to N. fowleri lysate was reduced, whereas that to con. A was also reduced up to on day 11 after infection. Both of these results were statistically significant compared with those of uninfected control group. The serum antibody titers were increased gradually up to day 15. The results indicated that there was an impairment of the blastogenic response of splenocytes to N. fowleri lysate during the acute course of experimental PAM in mice.

A case of human thelaziasis in Korea

The 15th case of human thelaziasis is reported from Korea. The patient was a 15-year-old girl from Seoul. Both of the worms which were removed from her eye were female and were identified by the number of transverse cuticular striations (281, 172 and 162/mm in the oesophageal, middle and posterior portions, respectively), as Thelazia callipaeda. The patient owned a pet dog ADDITIONAL ABSTRACT: The 15-year-old patient had herself removed a worm from one eye. Two worms were identified morphologically as Thelazia callipaeda. The source of infection was uncertain, but the patient was known to have close contact with a dog. The authors refer to 14 previously reported cases of thelaziasis in Korea.