Follow-up Study of Patients Previously Diagnosed with Lymphatic Filariasis in Korea.

OBJECTIVES: Korea was an endemic area for lymphatic filariasis (LF), caused by the nematode parasite Brugia malayi, until the 1970s. The World Health Organization recognized Korea as LF-free in June 2008. However, it is necessary to confirm that patients that have had LF in the past still test negative, to prevent the re-emergence of LF in Korea. METHODS: We followed up a total of 83 patients who had been diagnosed with LF between 2002 and 2010 in endemic LF areas. RESULTS: Fifty-two of the 83 subjects were negative for LF, whereas 31 subjects had re-located to a different city or province, were dead, or were unaccounted for. Most subjects with negative test results still exhibited edema in the legs or the arms, and some complained of redness and swelling in the legs or ankle joints. However, we found that these symptoms were due to diseases other than LF. CONCLUSION: In this follow-up study, we did not find any evidence indicating the potential re-emergence of LF in Korea.

Prevalence of Clonorchis sinensis Infection among Residents along 5 Major Rivers in the Republic of Korea.

Clonorchis sinensis is currently the most important parasite affecting public health problems in the Republic of Korea. We investigated the prevalence of C. sinensis infection among residents living along 5 major rivers in Korea. A total of 42,562 individual stool samples were collected from 37 localities and examined using the formalin-ether sedimentation technique. Helminth eggs were detected in 4,052 (9.5%) residents and 3,586 (8.4%) were infected with C. sinensis. The egg positive rate of C. sinensis in Nakdong, Seomjin, Geum, Yeongsan, and Han River was 11.7%, 9.9%, 6.5%, 3.1%, and 1.0%, respectively. The overall prevalence of clonorchiasis by sex was 11.2% in males and 6.2% in females. The age-prevalence was the highest in the 50-59 years band. It has been reconfirmed that the endemicity of clonorchiasis is higher in southern areas of Korea, especially along Nakdong and Seomjin Rivers. A combination of continuous control programs with health education initiatives is urgently required in these highly endemic areas of clonorchiasis in Korea.

Outbreak of Ciprofloxacin-Resistant Shigella sonnei Associated with Travel to Vietnam, Republic of Korea.

We investigated an October 2014 outbreak of illness caused by Shigella sonnei in a daycare center in the Republic of Korea (South Korea). The outbreak strain was resistant to extended-spectrum cephalosporins and fluoroquinolones and was traced to a child who had traveled to Vietnam. Improved hygiene and infection control practices are needed for prevention of shigellosis.

Detection of Encephalitozoon spp. from human diarrheal stool and farm soil samples in Korea.

Microsporidia are eukaryotic organisms that cause zoonosis and are major opportunistic pathogens in HIV-positive patients. However, there is increasing evidence that these organisms can also cause gastrointestinal and ocular infections in immunocompetent individuals. In Korea, there have been no reports on human infections with microsporidia to date. In the present study, we used real-time PCR and nucleotide sequencing to detect Encephalitozoon intestinalis infection in seven of 139 human diarrheal stool specimens (5%) and Encephalitozoon hellem in three of 34 farm soil samples (8.8%). Genotype analysis of the E. hellem isolates based on the internal transcribed spacer 1 and polar tube protein genes showed that all isolates were genotype 1B. To our knowledge, this is the first report on human E. intestinalis infection in Korea and the first report revealing farm soil samples as a source of E. hellem infection. Because microsporidia are an important public health issue, further large-scale epidemiological studies are warranted.

The first outbreak of giardiasis with drinking water in Korea.

OBJECTIVES: To identify the pathogen of the diarrhea outbreak in a village in Jeollabuk province in Korea in April 2010. METHODS: DNA extraction was performed from the 120 L of collected water, which was centrifuged at 10,000 x g for 30 min. PCR reactions were conducted in a total of 25 ul, which included PCR premix (GenDEPOT, Barker, TX, USA), 2 ul (∼100 ng) of extracted DNA, and 10 pmol of each primer. RESULTS: Nine people out of 25 had a symptom of abdominal pain accompanied by diarrhea after they used stored valley water in a water tank as a provisional water supply source without chlorine sterilization. Among them Giardia lamblia was detected in fecal samples of 7 people using the polymerase chain reaction method. Although G. lamblia was also detected from water provided by the provisional water supply system stored in the water tank and used as drinking water, it was not detected in the water tank itself. This water-borne outbreak is considered to have occurred when the provisional water supply tube was destroyed under a building construction and contaminated by G. lamblia, but its precise cause has not been clarified. CONCLUSION: This outbreak resulting from G. lamblia is very meaningful as the first outbreak of an infection by a water-borne parasite in Korea.

Development of a Diagnostic Kit to Detect Cryptosporidium parvum and Giardia lamblia.

OBJECTIVES: This study aims to develop a high-sensitivity antibody diagnostic kit that will enable a rapid and accurate detection of Cryptospofidium parvum and Giardia lamblia in patients with diarrhea. METHODS: The cultivated C. parvum oocysts and G. lamblia cysts in each calf and dog were injected to mice to obtain antibodies, which were titrated. Spleen cells of the immunized mouse were separated and blended with myelomas to produce hybrid cell lines that form monoclonal antibodies. Using ELISA method, antibodies that specifically respond to C. parvum and G.lamblia were then selected. The cells were injected into the abdominal cavity of a BALB/c mouse to isolate hydrops abdominis containing high level of antibodies. The IgG antibody was purified using protein G gel. RESULTS: The detection limit of monoclonal antibodies for Cryptosporidium parvum and Giardia lamblia was 125 oocysts/mL and 1250 cysts/mL, respectively. In addition, during testing they did not show cross-reactivity to viruses (n = 15), bacteria (n =17), and parasites (n = 9). CONCLUSION: The rapid diagnostic antibody kit developed in this study, which specifically responds to C. parvum and G. lamblia, will be useful in detecting and monitoring diarrheal infections.

Cryptosporidium hominis infection diagnosed by real-time PCR-RFLP.

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.

Surveillance and vector control of lymphatic filariasis in the republic of Korea.

OBJECTIVES: Until the early 2000s, lymphatic filariasis would commonly break out in the coastal areas in Korea. Through steady efforts combining investigation and treatment, filariasis was officially declared eradicated in 2008. This study surveyed the density of vector species of filariasis in past endemic areas, and inspected filariasis DNA from collected mosquitoes for protection against the reemergence of filariasis. METHODS: Between May and October 2009, mosquitoes were caught using the black night trap in past endemic coastal areas: Gyeongsangnam-do, Jeollanamdo, and Jeju-do. The collected mosquitoes were identified, and the extracted DNA from the collected vector mosquitoes was tested by polymerase chain reaction for Brugia malayi filariasis. RESULTS: Ochletotatus togoi, Anophel es (Hyrcanus) group and Culex pipiens were most frequently caught in Jeollanam-do (Geomun Island, Bogil Island, Heuksan Island), Jeju-do (Namone-ri, Wimi-ri). and Gyeongsangnam-do (Maemul Island). DNA of B malayi was not found in Och Togoi and An (Hyrcanus) group as main vectors of filariasis. CONCLUSION: Lymphatic filariasis was not found in the vector mosquitoes collected in past endemic areas. However, considering that the proportion of vector species is quite high, there is a potential risk that filariasis could be reemerging through overseas travel or trade. Thus, there is a need to continuously monitor vector mosquitoes of lymphatic filariasis.

PCR diagnosis of Entamoeba histolytica cysts in stool samples.

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

Seasonal prevalence of mosquitoes, including vectors of Brugian filariasis, in southern islands of the Republic of Korea.

A survey of mosquitoes, including the vector status of Brugia malayi filariasis and their relative larval density, was conducted from 2002 to 2005 at several southern remote islands of Jeollanam-do (province), Gyeongsangnam-do, and Jeju-do, Korea, where filariasis was previously endemic. Overall, a total of 9 species belonging to 7 genera were collected. Ochlerotatus togoi (formerly known as Aedes togoi), Anopheles (Hyrcanus) group, and Culex pipiens were the predominant species captured at all areas. Oc. togoi larvae were most frequently collected at salinity levels <0.5% during June and July, with densities decreasing sharply during the rainy season in August. The most likely explanation for the eradication of filariasis in these areas is suggested to be an aggressive treatment program executed during the 1970s and the 1990s. However, high prevalence of the vector mosquitoes may constitute a potential risk for reemerging of brugian filariasis in these areas.

Infection status of hospitalized diarrheal patients with gastrointestinal protozoa, bacteria, and viruses in the Republic of Korea.

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged < or = 5 years was significantly higher, while C. perfringens among bacterial infection was higher for > or = 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.

Cryptosporidium parvum in Korea: prevalence in individuals residing in three major river valleys and genetic characteristics of the isolates.

Cryptosporidiosis is a diarrheal illness caused by apicomplexa parasite Cryptosporidium spp. In this study, to examine the overall infection status of Cryptosporidium spp. in individuals residing in southern parts of Korea, eight counties around Yeongsan, Seomjin and Nakdong River valleys was surveyed. The investigation was carried out from April to October 2005. A total of 9,498 stool samples were collected from individuals. Stool samples were analyzed for modified acid-fast stains, and DNA fragment extracted from positive samples was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for 18S rRNA polymorphic region. Oocysts of Cryptosporidium spp. were detected in 239 specimens (2.5%) by a modified acid-fast stain. Infection rate was not significantly different between male (2.2%) and female (2.8%) individuals examined (P>0.05). In the infection rate by age, totally 1-9 (4.8%) and 80< (3.7%) age group were shown to the highest, and there was shown to significant differences (P<0.05). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 18S rRNA gene from 51 isolates showed that all the isolates were identified as C. parvum. Our data collectively suggested that C. parvum infection is prevalent in the studied areas of Korea and more comprehensive nation-wide epidemiological studies are needed to elucidate the infection status of Cryptosporidium infection in Korea.

Prevalence of Clonorchis sinensis Infections Along the Five Major Rivers in Republic of Korea, 2007.

OBJECTIVES: The prevalence of Clonorchis sinensis infection was investigated among residents of the five major river basins, that is, Hangang, Nakdonggang, Seomjingang, Yeongsangang, and Geumgang River basins in Korea. METHODS: From January to December 2007, a total of 31,268 stool samples were collected from 29 localities and examined by the formalin-ether sedimentation technique. RESULTS: Intestinal parasite eggs and/or protozoan cysts were detected from 2957 (9.5%) inhabitants. Number of residents harbouring helminth eggs in the faeces was 2542 (8.1%) for C. sinensis, 255 (0.8%) for Heterophyes spp., 36 (0.1%) for Echinostoma spp., 30 (0.1%) for Trichuris trichiura, 8 (0.03%) for Ascaris lumbricoides, 7 (0.02%) for Gymnophalloide seoi, and 50 (0.02%) for Trichostrongylus orientalis. Number of residents harbouring protozoan cysts in the faeces was 133 (1.3%) for Entamoeba spp. and 50 (0.2%) for Giardia lamblia. The positive rates of C. sinensis in Nakdonggang, Seomjingang, Yeongsangang, Geumgang, and Hangang River basins were 12.2%, 9.5%, 3.3%, 3.0%, and 1.0%, respectively. The egg positive rate of C. sinensis was higher in male (10.6%) than in female (6.1%), and the age group of 50s had the highest positive rate (10.4%). CONCLUSION: The result of this study revealed little decrease in positive rate of C. sinensis compared with the result of southern endemic areas of Korea in 2006.

Successful control of lymphatic filariasis in the Republic of Korea.

A successful experience of lymphatic filariasis control in the Republic of Korea is briefly reviewed. Filariasis in the Republic of Korea was exclusively caused by infection with Brugia malayi. Over the past several decades from the 1950s to 2006, many investigators exerted their efforts to detection, treatment, and follow-up of filariasis patients in endemic areas, and to control filariasis. Mass, combined with selective, treatments with diethylcarbamazine to microfilaria positive persons had been made them free from microfilaremia and contributed to significant decrease of the microfilarial density in previously endemic areas. Significant decrease of microfilaria positive cases in an area influenced eventually to the endemicity of filariasis in the relevant locality. Together with remarkable economic growth followed by improvement of environmental and personal hygiene and living standards, the factors stated above have contributed to blocking the transmission cycle of B. malayi and led to disappearance of this mosquito-borne ancient disease in the Republic of Korea.

Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory-secretory products of Clonorchis sinensis adult worms.

Clonorchis sinensis, the Chinese liver fluke, is the causative agent of clonorchiasis as well as liver and biliary diseases. The excretory-secretory products (ESPs) of the parasites play important roles in host-parasite interactions. In this study, we have investigated the proteome of ESPs obtained from C. sinensis adult worms. Although the full genome database of C. sinensis is not yet available, we have successfully identified 62 protein spots using 2-DE-based mass analysis and EST database of C. sinensis. The proteins identified include detoxification enzymes, such as glutathione S-transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly. In order to identify potential targets for the diagnosis and therapy of clonorchiasis, we conducted immunoblot analysis of the ESPs proteome using the sera obtained from clonorchiasis patients and identified legumains and cysteine proteases as antigens present in the ESPs. Although the cysteine proteases were previously reported to elicit antigenicity, the legumains are found herein for the first time as a serological antigen of C. sinensis. To confirm these findings, we expressed recombinant legumain in Escherichia coli and verified that recombinant legumain also functions as a potent antigen against the sera of clonorchiasis patients. Our results illustrate the validity of immuno-proteomic approaches in the identification of serodiagnostic antigens in the parasites.

Identification and characterization of a mitochondrial iron-superoxide dismutase of Cryptosporidium parvum.

Cryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals. In this study, we identified a gene encoding mitochondrial iron-superoxide dismutase of C. parvum (Cp-mtSOD) and characterized biochemical properties of the recombinant protein. Multiple sequence alignment of the deduced amino acid sequence of Cp-mtSOD with those of previously reported iron-containing SODs (Fe-SODs) from other protozoan parasites showed that Cp-mtSOD shares common metal-binding residues and motifs that were conserved in Fe-SODs. However, the N-terminal 26-amino acid residues of Cp-mtSOD did not show sequence identities to any other Fe-SOD sequences. Further analysis of the N-terminal presequence of Cp-mtSOD suggested that it shares common physiochemical characteristics found in mitochondria targeting sequences and predicted localization of Cp-mtSOD in the mitochondria. The recombinant Cp-mtSOD showed typical biochemical properties with other characterized Fe-SODs, including molecular structure, broad pH optimum, and sensitivity to hydrogen peroxide.

Genotypic characterization of cryptosporidium oocysts isolated from healthy people in three different counties of Korea.

To investigate Cryptosporidium infection among healthy people, we collected stool samples from 150 healthy individuals in Gokseong, Muan, and Imshil Counties, southwest Korea, where neighbors on both an animal farm and a river respectively. In 12 of 150 samples, Cryptosporidium oocysts were detected by means of modified acid-fast staining. The bovine genotype, Cryptosporidium parvum, was identified by PCR/RFLP and 18S rRNA sequencing. C. parvum existed endemically in these areas, and the residents showed a relatively higher infection rate for C. parvum than that for C. hominis. Our results indicate that countermeasures against Cryptosporidium infection must be taken in these areas to ensure human health.

First case of human babesiosis in Korea: detection and characterization of a novel type of Babesia sp. (KO1) similar to ovine babesia.

We report on the first case of human babesiosis in Korea. The intraerythrocytic parasite (KO1) in the patient's blood mainly appeared as paired pyriforms and ring forms; but Maltese cross forms were not seen, and the parasite showed morphological features consistent with those of the genus Babesia sensu stricto. The sequence of the 18S rRNA gene of KO1 was closely related to that of Babesia spp. isolated from sheep in China (similarity, 98%). The present study provides the first evidence of the presence of a hitherto unidentified, new type of Babesia parasite capable of infecting humans.

Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores.

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.

Effect of the lower molecular capsule released from the cell surface of Bacillus anthracis on the pathogenesis of anthrax.

Bacillus anthracis enters the body as an endospore, and encapsulation and toxin production occur after germination. Capsule is proposed to be an antiphagocytic factor, and toxin induces cytokine production for systemic shock. The dep gene, adjacent to the cap region for the encapsulation, degrades the high-molecular weight capsule (H-capsule) to the lower-molecular weight capsule (L-capsule), which releases into the culture supernatant. This study analyzed the biological function of the cap-dep region. The dep null mutant Sm-1, which formed H-capsule but not L-capsule, was avirulent. However, Sm-1 with an intact dep gene or with purified L-capsule recovered its pathogenicity. Sm-1 was subjected to phagocytosis by macrophages more easily than its parent strain, Sm, in vitro; in vivo, it cleared without L-capsule and grew well with L-capsule, which suggests that L-capsule is essential for in vivo multiplication. Moreover, a new name, capD, might be appropriate, because of the part of the cap operon involved in both polymerization and depolymerization of the capsule.