Yuichi Chigusa

Cross-reactivity in rapid diagnostic tests between human malaria and zoonotic simian malaria parasite Plasmodium knowlesi infections

Plasmodium knowlesi has a relatively broad host range extending to humans, in whom it causes zoonotic malaria. Recent studies have shown that human infection with P. knowlesi is widely distributed in forested areas of Southeast Asia. In the present study, we evaluated commercial rapid diagnostic tests (RDTs) for human malaria to assess their reactivity and sensitivity in detecting P. knowlesi parasites using blood samples obtained from infected monkeys. The blood samples were assayed using two commercial RDTs based on immunochromatographic assays: (i) the OptiMAL-IT, designed to detect parasite lactate dehydrogenase (pLDH) of both P. falciparum and other plasmodia, and (ii) the Entebe Malaria Cassette (MC), designed to detect P. falciparum-specific histidine-rich protein 2 (PfHRP2) and P. vivax-specific pLDH. Interestingly, when the P. knowlesi-infected blood samples were examined with the RDTs, OptiMAL test results were interpreted as falciparum malaria-positive, while Entebe MC test results were interpreted as vivax malaria-positive. The sensitivities of both tests in detecting P. knowlesi parasite were similar to those for P. falciparum and higher than P. vivax. Thus, commercial RDTs based on detection of pLDH should be used with great caution, and should not replace conventional microscopy in the diagnosis of suspected cases of P. knowlesi malaria.

Identification and differentiation of human schistosomes by polymerase chain reaction.

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.

Imaging diagnosis of schistosomiasis japonica-the use in Japan and application for field study in the present endemic area

For detecting lesions-related schistosomiasis japonica, X-rays, scintillation scanning, ultrasonography (US), computed tomography (CT), magnetic resonance (MR) and endoscopic examinations with biopsies have been used in Japan. Liver fibrosis and calcified changes are detected by US and CT. Most of the lesions that are detected by endoscopic examinations are due to deposited ova of Schistosoma japonicum. Portal hypertension is detected by US, CT and gastroscopic examination. Because schistosome infection decreased rapidly in Japan, most of the studies on imaging diagnosis were performed on chronic lesions or sequelae of schistosomiasis. Most of the techniques were used on admitted patients in well-equipped hospitals. US was introduced in the 1970s as a safe, rapid, non-invasive and inexpensive technique and has been used for diagnosis in hospitals and screening in the fields. As a typical US image of the liver, septal formation by high echogenic bands like mosaic was described, and this network pattern was reported in the other endemic countries; China and Philippines. As an appropriate technique, US has been broadly used in developing countries. Not only for diagnosis in a hospital, but also for monitoring changes of morbidity, US is used in the community level. Network pattern related to the severity of S. japonicum infection, has not been described in S. mansoni or S. haematobium infection. Appearance of network pattern depends on pathological changes such as periportal fibrosis, postnecrotic fibrosis and calcified ova. For advanced studies on morbidity of schistosomiasis japonica, further research on pathological basis of network pattern and standardization of US diagnosis are necessary.

Detection of active schistosome infection by cell-free circulating DNA of Schistosoma japonicum in highly endemic areas in Sorsogon Province, the Philippines.

The current status of schistosomiasis in highly endemic areas is difficult to determine by ovum detection because of the superficially low parasite load after mass drug administration, whereas the parasite transmission rates are still high. Cell-free parasite DNA is fragments of parasite-derived DNA existing in the hosts body fluids. We conducted population-based studies to test the presence of cell-free schistosome DNA in endemic areas of Sorsogon Province, the Philippines. Schistosome DNA in the serum and urine of Kato-Katz (KK)-positive subjects was detected by PCR (100% sensitivity). Schistosome DNA was also detected from KK-negative subjects (9/22 serum and 10/41 urine samples). Schistosome DNA was found to be network echogenic pattern (NW)-positive (serum 53.3%, urine 42.9%) or NW-negative (serum 25.5%, urine 20.8%) and enzyme-linked immunosorbent assay (ELISA)-positive (serum 47.1%, urine 40%) or ELISA-negative (serum 33.3%, urine 13.3%). These results indicate that cell-free schistosome DNA is a promising diagnostic marker for active schistosome infection in the case of light infection.

Use of Cell-Free Circulating Schistosome DNA in Serum, Urine, Semen, and Saliva To Monitor a Case of Refractory Imported Schistosomiasis Hematobia

ABSTRACT This case of imported refractory schistosomiasis has highlighted the usefulness of cell-free parasite DNA as a diagnostic marker to assess active schistosome infection. In contrast to the rapid disappearance of ova in urine, parasite DNA remained persistent in several other specimen types even after the fourth treatment with praziquantel. This result was consistent with the presence of morphologically intact ova in bladder biopsy samples and with the corresponding symptoms.

Utilization of ELISA using thioredoxin peroxidase-1 and tandem repeat proteins for diagnosis of Schistosoma japonicum infection among water buffaloes.

Background The presence of animal reservoirs in Schistosoma japonicum infection has been a major obstacle in the control of schistosomiasis. Previous studies have proven that the inclusion of control measures on animal reservoir hosts for schistosomiasis contributed to the decrease of human cases. Animal surveillance should therefore be included to strengthen and improve the capabilities of current serological tests. Methodology/Principal Findings Thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) were initially evaluated against human sera. The previous test showed high sensitivity and specificity for antibody detection against SjTPx-1 and Sj7TR. In this study, the immunodiagnostic potential of these recombinant proteins was evaluated using enzyme-linked immunoassay on 50 water buffalo serum samples collected in Cagayan, the Philippines as compared with the soluble egg antigen (SEA). For specificity, 3 goat serum samples positive with Fasciola hepatica were used and among the antigens used, only SEA showed cross-reaction. Stool PCR targeting the S. japonicum 82 bp mitochondrial NAD 1 gene was done to confirm the true positives and served as the standard test. Twenty three samples were positive for stool PCR. SjTPx-1 and Sj1TR gave the highest sensitivity among the recombinant proteins tested for water buffalo samples with 82.61% and 78.26% respectively which were higher than that of SEA (69.57%). Conclusions/Significance These results prove that SjTPx-1 works both for humans and water buffaloes making it a good candidate antigen for zoonotic diagnosis. Sj1TR showed good results for water buffaloes and therefore can also be used as a possible candidate for detecting animal schistosome infection.

Human Antibody Response to Thioredoxin Peroxidase-1 and Tandem Repeat Proteins as Immunodiagnostic Antigen Candidates for Schistosoma japonicum Infection

Schistosomiasis continues to be a public health problem in many tropical and subtropical countries. Improving the diagnostic tools for surveillance and monitoring in areas that have reached elimination level will help hasten the possible elimination of this disease. This study therefore aims to develop enzyme-linked immunosorbent assay through the use of recombinant proteins such as thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, and Sj7TR). Cutoff values were calculated using 38 serum samples from healthy Japanese volunteers. Sera from 35 schistosomiasis-confirmed patients, four cured from the disease by chemotherapy, and 15 endemic negative controls were used to assess these antigens. SjTPx-1 and Sj7TR both had 85.71% sensitivity. Furthermore, these antigens were also tested against human sera positive for other parasitic infections and showed no or very minimal cross-reaction. These results suggest the potential defined antigens for development of an accurate diagnostic test for schistosomiasis.

Field survey focused on Opisthorchis viverrini infection in five provinces of Cambodia.

BACKGROUND Opisthorchiasis is endemic in Thailand and Lao Peoples Democratic Republic and constitutes a major public health problem throughout the Mekong Basin. Although Cambodia is located in the Mekong Basin, the status of O. viverrini infection in that country was not previously clarified. This research was conducted to document the extent and distribution of O. viverrini infection in Cambodia. METHODS Surveillance was conducted in 55 villages in five Cambodian provinces. Research tools included stool examination using the Kato-Katz thick-smear technique, identification of intermediate hosts, and interviews covering factors related to O. viverrini infection. Some larvae and egg-positive stool samples were examined using PCR to detect O. viverrini DNA. RESULTS A total of 16,082 stool samples from the 55 villages were examined, of which 1232 were egg positive. In 15 villages with egg-positive rates of greater than 10%, eggs were found in 998 of 3585 stool samples, for an egg-positive rate of 27.8%. PCR analysis showed that 30 of 33 samples were positive for O. viverrini DNA from five villages in Kampong Cham and Kampong Thom provinces. The first intermediate host Bithynia siamensis siamensis was identified in the target areas of Takaev, Kandal, and Kampong Cham provinces. Cercariae were identified morphologically as O. viverrini and some were confirmed using PCR. Metacercariae of O. viverrini were identified by morphologic observations, animal experiments, or PCR in six species of fish in the target areas. DISCUSSION AND CONCLUSIONS Four Cambodian provinces were identified as endemic areas of O. viverrini infection. Careful planning is necessary for effective field surveys, because complex environmental factors might be involved in the distribution of O. viverrini infection-endemic areas in Cambodia. Many problems remain to be resolved regarding the status of O. viverrini infection in Cambodia, and a nationwide baseline survey is necessary.

New endemic foci of schistosomiasis infections in the Philippines

Schistosomiasis affects 28 provinces in the Philippines found along the southeastern part where there is continuous rainfall throughout the year. In 2002 and 2005 respectively, two new endemic foci were reported in the northernmost (Gonzaga, Cagayan) and central (Calatrava, Negros Occidental) parts of the country. This study conducted in March 2008-March 2009 confirmed the presence of the disease by determining its prevalence using four diagnostic tests - Kato-Katz, circumoval precipitin test (COPT), ELISA and ultrasonography. Oncomelania hupensis quadrasi was identified through snail surveys conducted in possible snail habitats in the seven new endemic villages. Animal surveys through stool examination confirmed the presence of schistosomiasis infection in animals in Gonzaga but not in Calatrava. Compared to Calatrava, Gonzaga demonstrated markedly higher prevalence of schistosomiasis using all four diagnostic methods. Proximity of snail habitats to human habitation including higher snail density and snail infection rate could be responsible for the high prevalence. Snail sites were more widespread in Gonzaga whereas those in Calatrava were confined only in areas not frequented by the general population except by farmers. GIS maps showing spatial distribution of snails in Gonzaga and Calatrava indicated differences in elevation among the snail sites. It is hypothesized that the snail intermediate host has been in these sites for sometime but discovered only lately. Migration of people from endemic provinces into Gonzaga and Calatrava brought in cases and in the presence of snail intermediate hosts, emergence of disease was just a matter of time.

Prevalence of asthma and allergic rhinitis among adult population in Ulaanbaatar, Mongolia

Background Mongolia is changing lifestyle, unhealthy habits, increase of air pollution, increasing life expectancy have led to an up rise of chronic respiratory diseases. Over 10 years ago, the prevalence of asthma and allergic rhinoconjunctivitis in Mongolia were in the lower range reported from previous studies. Objective The main aim of the survey is to know the prevalence of asthma and allergic rhinitis among adult population of Ulaanbaatar city, Mongolia and their risk factors. Methods Total of approximately 1,200 adults aged 20 years and over were planned to be randomly selected. The questionnaire was developed on the basis of WHO Protocol for Assessment of Prevalence of Major Respiratory Diseases and modified by local risk factors assessment and by other international survey approach including Global Initiative for Asthma and European Community Respiratory Health Survey. Results Prevalence of current wheezer in all age group was 15.7% (95% CI: 14.7-16.8). Age and sex segregated distribution of current wheezer were defined among male and female and prevalence was 14.5% (95% CI: 13.3-16.2) in male and female 16.6% (95% CI: 15.2-18.3) respectively. Prevalence of diagnosed asthma among adults was 4.7% (95% CI: 4.3-5.6) in all age group, 3% (95% CI: 2.4-3.7) in male and 6.8% (95% CI: 5.8-7.9) in female. Prevalence of rhinoconjunctivitis was 14.6% in all age group. 28.4% out of subjects with allergic rhinitis has current asthma, while 11.6% of subjects without allergic rhinitis has asthma (p < 0.01). Conclusion The prevalence of asthma increased for one decade in Ulaanbaatar. Prevalence of diagnosed asthma is approximately 5% and current wheezer is approximately 15% in adults of population, which is close to other Asia and European countries. Allergic rhinitis is a risk factor for asthma.