Harada-Mori Culture Research Articles

A study implementing real-time PCR to identify Strongyloides species of third-stage larvae in human stool samples from Southern Vietnam

BackgroundStrongyloidiasis, a neglected disease caused by intestinal nematodes of the genus, is endemic to tropical and subtropical areas such as Vietnam. Morphological methods only identify the genus, while DNA-molecular techniques are susceptible in Strongyloides spp. detection. The study aims to determine the prevalence of dominant Strongyloides species among the population in Duc Hoa district, Long An, Vietnam. MethodsA cross-sectional study used 1190 stool specimens collected from July 2017 to November 2018. All samples were transported within 2 h, stored at 2–8°C, and processed within 48 h for microscopy smear and culture at the Laboratory of Medical Parasitology, Pham Ngoc Thach University of Medicine (PNT). Then all positive samples with the above 2 methods were verified by real-time PCR technique. Real-time PCR amplification was conducted at the Laboratory of Molecular Biology, PNT. ResultsDirect microscopy and modified Harada-Mori culture detected Strongyloides spp. larvae in 79/1190 samples (6.6%). About 94.2% of the DNA samples were Strongyloides stercoralis, 2.9% were co-infections with Strongyloides ratti and S. stercoralis, and 2.9% were patients with S. ratti. The identity of 12/14 sequences was confirmed as S. stercoralis with a high level of similarity (91.3%–100%) and over 98% for S. ratti. ConclusionDNA-molecular techniques and sequence analysis are highly suitable for identifying Strongyloides species isolated from stool samples. It is remarkable evidence of the presence of zoonosis S. ratti disease in human, not just the known S. stercoralis. It is likely to result in a certain proportion of people being infected by this animal-borne infectious pathogen.

Strongyloidiasis infection in a borderline lepromatous leprosy patient with adrenocorticoid insufficiency undergoing corticosteroid treatment: a case report

Strongyloidiasis is a soil-transmitted helminthiasis mainly caused by Strongyloides stercoralis. It is endemic to the tropics and subtropics. Sri Lanka has a 0-1.6% prevalence rate. S. stercoralis infection was identified in a 33-year-old Sri Lankan male patient treated with corticosteroids for borderline lepromatous leprosy with adrenocortical dysfunction. In March 2020, a 33-year-old Sri Lankan (Sinhalese) male patient presented with watery diarrhea, lower abdominal pain, and post-prandial abdominal fullness. Previously, he was diagnosed with borderline lepromatous leprosy and was treated with rifampicin, clofazimine, and prednisolone 60mg daily since July 2019. After developing gastrointestinal symptoms, he had defaulted leprosy treatment including the prednisolone for 3months. Duodenal biopsy revealed numerous intraepithelial nematodes within the lumina of glands in the duodenum whose appearance favored Strongyloides. Fecal wet smear revealed numerous Strongyloidisstercoralis L1 rhabditiform larvae. Larval tracks were seen in the agar plate culture. L3 filariform larvae of Strongyloidis stercoralis were seen in the Harada-Mori culture. In addition, the short synacthen test revealed adrenocortical insufficiency, and oral hydrocortisone and fludrocortisone were started with albendazole treatment against strongyloidiasis. Fecal wet smear and culture repeated after treatment with albendazole were negative for Strongyloidisstercoralis. The patient was discharged in July 2020 on oral hydrocortisone. Onemonth later his condition was reviewed and the repeated fecal wet smear and agar plate culture was normal. He is being followed up every 3months. This is the first case of strongyloidiasis diagnosed in a patient with borderline lepromatous leprosy from Sri Lanka. The patient manifested symptoms of strongyloidiasis while on high-dose steroid therapy for his lepromatous reaction. Subsequently, the patient not only discontinued his steroid therapy, but also developed adrenocortical insufficiency as a complication of leprosy. Therefore, although diagnosis of strongyloidiasis was delayed, his subsequent low steroid levels probably protected him from disseminated disease. This is an interesting case where symptomatic strongyloidiasis was diagnosed in a patient who was initially treated with high-dose steroids but subsequently developed adrenocortical insufficiency. We emphasize the need to screen all patients prior to the commencement of immunosuppressive therapy.

Clinical Performance of Real-Time Polymerase Chain Reaction for Strongyloides stercoralis Compared with Serology in a Nonendemic Setting.

Strongyloides stercoralis is a nematode endemic to subtropical and tropical regions that may cause asymptomatic carriage, peripheral eosinophilia, cutaneous, gastrointestinal, and pulmonary disease, or hyperinfection syndrome. Conventional diagnostic methods for strongyloidiasis include feces microscopy and culture, with low sensitivity in chronic infection due to the low helminth burden, and serology, which may be prone to false-negative results with immunocompromise and false-positive results with other infections and immunological disorders. We evaluated a laboratory-developed real-time polymerase chain reaction (RT-PCR), detecting the 18S SSU ribosomal RNA gene, compared with conventional diagnostic methods, using serology via ELISA as the gold-standard. The population studied included tertiary hospital inpatients and outpatients residing in a nonendemic area. Seven hundred fifty unfixed stool specimens submitted sequentially between 2014 and 2018 were tested for S. stercoralis via microscopy and RT-PCR. Agar plate culture (APC), Harada-Mori culture (HMC), and ELISA were performed in conjunction with 141, 135, and 177 of the specimens, respectively. RT-PCR yielded 13 positive and 730 negative results, with inhibition in seven specimens. ELISA yielded 53 positive, 18 equivocal, and 106 negative results. Results for direct diagnostic methods obtained after treatment with ivermectin were excluded from the performance analysis. Compared with ELISA, RT-PCR, microscopy, APC, and HMC exhibited sensitivities of 38%, 6%, 3%, and 0%, respectively, and specificities of 100%. Given the low sensitivities commensurate with testing a population with remote infection and thus low parasite burden, we recommend a combination of serological and molecular diagnostic testing to achieve the best balance of sensitivity and specificity.

Comparison of Molecular and Parasitological Methods for Diagnosis of Human Trichostrongylosis.

Human trichostrongyliasis is a zoonotic disease that is prevalent among rural populations in some countries. This study was performed to evaluate various parasitological methods and polymerase chain reaction (PCR) for the diagnosis of human trichostrongyliasis. A total of 206 fresh stool samples were collected from residents of endemic villages of Northern Iran. All samples were examined using conventional parasitological methods, including wet mount, formalin ethyl acetate concentration (FEAC), agar plate culture (APC), Harada–Mori culture (HMC), and Willis, along with the PCR technique. Among the total of 206 individuals examined, 72 people (35%) were found infected with Trichostrongylus species using combined parasitological methods. By considering the combined results of parasitological methods as the diagnostic gold standard, the Willis technique had a sensitivity of 91.7% compared with 52.8% for the APC, 40.3% for the HMC, 37.5% for FEAC, and 5.6% for the wet mount technique. The diagnostic specificity of all the parasitological methods was 100%. Furthermore, the PCR method detected Trichostrongylus spp. DNA in 79 fecal samples (38.3%) with a sensitivity of 97.2% and a specificity of 93.3%. According to the current findings, the Willis method was more sensitive than are the other parasitological methods in the diagnosis of human trichostrongyliasis. However, the PCR assay was more sensitive and more reliable in the detection of human trichostrongyliasis in comparison with the parasitological methods.

Occurrence of Strongyloides stercoralis in rural areas adjacent to Dhaka city

The present study was designed to investigate Strongyloides stercoralis infection in rural communities of Savar and Gazipur. A total of 160 stool samples were collected from the inhabitants of the study areas during December 2016 to November 2017. The samples were processed by direct smear, formol-ether concentration technique and Harada-Mori culture. The overall prevalence of S. stercoralis was 10.63%; 11% in Savar and 10% in Gazipur. The elderly people were more prone to S. stercoralis infection. No respondent belonging to age group of 11-20 years was found positive for infection. Poor educational status, inferior financial condition and inadequate hygiene practice were found as potential risk factors (P<0.05). Noticeable prevalence was observed among the respondents living in mud floored house (13.46%), irregular nail clippers (22.73%), bare footers (14.89%) and laborers (27.27%) in Savar. Prevalence was high among the respondents deprived of institutional education (17.86%) in Gazipur.
 Bangladesh J. Zool. 48(2): 313-320, 2020

Evaluation of various culture techniques for identification of hookworm species from stool samples of children.

Background & objectives:Different coproculture techniques have been developed for culturing the hookworm (HW) larvae for morphological identification in the resource-limited settings. The objective of this study was to compare the performances of Harada-Mori culture (HMC), agar plate culture (APC) and modified APC (MAPC) of HW positive stool specimens for identification of HW species in East Sikkim.Methods:This prospective study was done in East Sikkim from May 2015 to May 2016. Stool and blood samples were collected from paediatric patients with gastrointestinal symptoms. The HW positive stool specimens by microscopy were subjected to HMC, APC and MAPC techniques to harvest HW larvae. Stoll's dilution egg count for determining egg intensity and blood parameters were performed in all the 12 HW-positive patients.Results:Twelve of the 180 samples were found positive for HW by microscopy and predominance of Necator americanus (75%) over Ancylostoma duodenale (25%) was observed. Blood parameters results showed high pack cell volume (PCV) values in 78.6 per cent, anaemia in 75 per cent and high eosinophil count in most patients. Stoll's dilution egg count showed moderate infection in 66.6 per cent, light and heavy infections in 16.7 per cent each.Interpretation & conclusions:Our results showed that APC yielded 100 per cent results and was easier to perform in the laboratory compared to MAPC and HMC techniques.

Comparative evaluation of harada–Mori and agar plate culture for the identification of hookworm species under limited resources

Background: Human hookworm infection has widespread socioeconomic and public health implications. Several coproculture techniques have been developed for morphological identification of hookworm larvae under limited resource availability. The objective of this study was to compare the performances of Harada–Mori culture (HMC), agar plate culture (APC), and modified APC (MAPC) of hookworm positive stool specimen for identification of hookworm species occurring in East Sikkim, India. Materials and Methods: This prospective cohort study was performed using 180 stool specimen collected from children who attended Central Referral Hospital and Sir Thodup Namgyal Memorial Hospital, with the complained of gastrointestinal symptoms. Blood samples were also collected to correlate with the complete blood count (CBC). The hookworm positive stool specimen evaluated by microscopy was subjected to HMC, APC, and MAPC techniques to harvest hookworm larvae. Stoll's dilution egg count for determining egg intensity and CBC were also performed for the children who were positive for hookworm's eggs in their stool sample. Results: This study observed a predominance of Necator americanus (75%) over Ancylostoma duodenale (25%). CBC results showed high packed cell volume values in 9/12, low hemoglobin 9/12, and high eosinophil count in all the positive children. Stoll's dilution egg count showed moderate infection in 66.6%, light and heavy infections in 16.7% of children's. APC method was superior to HMC and MAPC in culturing and identifying hookworm species. Conclusions: APC was observed to yield better results and was easier to perform in limited resource laboratory setting compare to MAPC or Harada–Mori culture techniques.

Prevalence and Risk Factors of Strongyloides Stercoralis Infection in Selected Tea Garden of Sylhet, Bangladesh

Background: Strongyloidiasis infection is partially an asymptomatic infection and the diagnosis of patent infection is difficult using conventional parasitological methods. The residents of tea garden community of Sylhet, Bangladesh were robustly tested. Method: The collected stool samples were tested by Harada mori culture for the presence of larval stage of Strongyloides stercoralis and to reaffirm the same samples were subjected for conventional PCR, using primer sets deigned to amplify partial ribosomal DNA of S. stercoralis genome. Finally data analysis was performed by Logistic Regression procedure using STATA 13 (College Station, Texas 77845 USA) and Pearson λ2 test, with consideration of P <0.05 as an indication of significant. Result: A total of 300 stool samples freshly collected and examined among those 18 (06.00%) samples were found positive for S. stercoralis in Harada mori culture. In amplification of DNA extracted from raw samples and culture fluid of positive sample, the conventional PCR detected S. stercoralis 38 (12.67%) positive. There were 6 samples positive in Harada mori culture but did not show any response in sophisticated PCR techniques it might be due to low burden of infection. Periodic anthelmintic does not taking OR= 3.946(95% CI 1.369-11.375; P=0.011) and does not wash feet coming from out OR= 5.158(95% CI 1.656-16.068; P=0.005) significantly associated with Strongyloidiases infection. Conclusion: This study confirmed that S. stercoralis is prevalent in the tea garden community of Sylhet identified by both parasitological and molecular methods. The preventive measures by deworming are warranted. Public health education regarding properly periodic anthelmintic taking, wash feet coming from out and personal hygiene are also additional required elements.

Cultivation of parasites.

Parasite cultivation techniques constitute a substantial segment of present-day study of parasites, especially of protozoa. Success in establishing in vitro and in vivo culture of parasites not only allows their physiology, behavior and metabolism to be studied dynamically, but also allows the nature of the antigenic molecules in the excretory and secretory products to be vigorously pursued and analyzed. The complex life-cycles of various parasites having different stages and host species requirements, particularly in the case of parasitic helminths, often make parasite cultivation an uphill assignment. Culturing of parasites depends on the combined expertise of all types of microbiological cultures. Different parasites require different cultivation conditions such as nutrients, temperature and even incubation conditions. Cultivation is an important method for diagnosis of many clinically important parasites, for example, Entamoeba histolytica, Trichomonas vaginalis, Leishmania spp., Strongyloides stercoralis and free-living amoebae. Many commercial systems like InPouch TV for T. vaginalis, microaerophilous stationary phase culture for Babesia bovis and Harada-Mori culture technique for larval-stage nematodes have been developed for the rapid diagnosis of the parasitic infections. Cultivation also has immense utility in the production of vaccines, testing vaccine efficacy, and antigen - production for obtaining serological reagents, detection of drug-resistance, screening of potential therapeutic agents and conducting epidemiological studies. Though in vitro cultivation techniques are used more often compared with in vivo techniques, the in vivo techniques are sometimes used for diagnosing some parasitic infections such as trypanosomiasis and toxoplasmosis. Parasite cultivation continues to be a challenging diagnostic option. This review provides an overview of intricacies of parasitic culture and update on popular methods used for cultivating parasites.

Strongyloidiasis in a high risk community of Dhaka, Bangladesh

Residents of a slum community of Dhaka city, Bangladesh were tested by serological and faecal examination for evidence of Strongyloides stercoralis infection. In stool specimens from a total of 147 participants Strongyloides larvae were found in 34 (23.1%) by Harada-Mori culture, 15 (10.2%) by agar plate culture (APC) and one (0.7%) by microscopy. Strongyloides IgG, IgG1 and IgG4 antibodies were found in 90 (61.2%), 46 (31.3%) and 53 (36.1%) of participants, respectively. A positive correlation was observed between total IgG levels and the presence of isotypes IgG1 and IgG4 (p<0.001). Six sera (4.0%) reacted to the recombinant filaria antigen Bm 14, three of which were Strongyloides IgG positive. This indicates either there is cross reactivity or some participants are co-infected with lymphatic filariasis. No correlations were found between positive serology and Strongyloides infection in stool, socio- demographic factors or domestic hygienic practices. However, positive stool cultures showed significant associations with irregular nail trimming, walking bare-foot and irregular hand washing after defecation (p<0.05). Other enteric parasites were detected in stools of some participants but their presence showed no correlation with S. stercoralis infection or socio demographic factors. This study confirms that squatters in this slum community in Dhaka have a high prevalence of S. stercoralis infection identified both by serological and coprological methods.

Diagnosis of soil-transmitted helminthiasis in an Amazonic community of Peru using multiple diagnostic techniques

An observational descriptive study was conducted in a Shipibo-Conibo/Ese’Eja community of the rainforest in Peru to compare the Kato–Katz method and the spontaneous sedimentation in tube technique (SSTT) for the diagnosis of intestinal parasites as well as to report the prevalence of soil-transmitted helminth (STH) infections in this area. A total of 73 stool samples were collected and analysed by several parasitological techniques, including Kato–Katz, SSTT, modified Baermann technique (MBT), agar plate culture, Harada–Mori culture and the direct smear examination. Kato–Katz and SSTT had the same rate of detection for Ascaris lumbricoides (5%), Trichuris trichiura (5%), hookworm (14%) and Hymenolepis nana (26%). The detection rate for Strongyloides stercoralis larvae was 16% by SSTT and 0% by Kato–Katz, but 18% by agar plate culture and 16% by MBT. The SSTT also had the advantage of detecting multiple intestinal protozoa such as Blastocystis hominis (40%), Giardia intestinalis (29%) and Entamoeba histolytica/E. dispar (16%). The most common intestinal parasites found in this community were B. hominis, G. intestinalis, H. nana, S. stercoralis and hookworm. In conclusion, the SSTT is not inferior to Kato–Katz for the diagnosis of common STH infections but is largely superior for detecting intestinal protozoa and S. stercoralis larvae.

Helminthic Infections in School Children in Puducherry, South India

Intestinal parasites are widely prevalent in developing countries, probably due to poor sanitation and inadequate personal hygiene. It is estimated that as much as 60% of the world's population is infected with intestinal parasites, which may play a significant role in morbidity due to intestinal infections. This study was designed to estimate the prevalence of helminthic infections among school children in Puducherry, South India. The prevalence of helminthic infections was studied in 1,172 children in public schools in Puducherry (Pondicherry), South India, between March and September 2006. The stools collected were concentrated using the formol-ether sedimentation technique, screened using conventional iodine and saline wet mounts, and examined by direct microscopy. The prevalence of helminthic infection was found to be 34.56% in school children; Ascaris lumbricoides constituted 43.21% of total infections; Ancylostoma duodenale (hookworm), 28.89%; Trichuris trichiura, 10.87%; Hymenolepis nana, 7.66%; Taenia spp., 7.41%; and Enterobius vermicularis, 1.98%. Co-infections were also common; dual infections accounted for 8.40% and multiple infections for 2.47%, respectively, of total infection. The stool samples found to be positive for hookworm ova were further processed for culture using the Harada-Mori culture technique. Sixty-five (55.56%) hookworm ova-positive stools were found to be culture positive. The infection prevalent in school children in this area is Ascariasis.

Screening for Intestinal Parasitic Infections Among Myanmar Migrant Workers in Thai Food Industry: A High-Risk Transmission

The impact of intestinal parasitic infections on public health has been neglected. Millions of Myanmar natives have migrated to work in Thailand. We performed a study of intestinal parasitic infections in Myanmar-migrants working in the Thai food industry. A total of 338 Myanmar migrant workers in a food plant at Samut Sakhon Province, Thailand, were recruited for this study. 284 (84%) returned requested stool samples. Samples were examined for intestinal parasites by means of simple smear, formalin-ether concentration, Locke-Egg-Serum medium, and Harada-Mori culture methods. We found parasites in 177 (62.3%) migrants (29 of 46 males; 148 of 238 females). The majority (89.3%) were infected with parasites transmitted by fecal-oral route, including Blastocystis hominis (41.5%), Trichuris trichiura (22.2%), Giardia lamblia (14.1%), and Ascaris lumbricoides (1.8%). Mixed infections were common (40.7%). The highest prevalence (73.3%) was found among migrants from Kohsong city, Myanmar. This high parasite infection rate in Myanmar migrant workers is an obvious public health hazard.

Responses to Albendazole Treatment for Hookworm Infection in Ethnic Thai and Immigrant in West-central Thailand

Ethnic Thai and immigrant schoolchildren and villagers of Bo-ong, a village in Pilok sub-district, Thong Pha Phum District, Kanchanaburi Province, western Thailand, were investigated for helminth infections in September 2003 and July 2004. Among the 143 schoolchildren, total cumulative hookworm prevalence in both surveys was 58.7%, with 47.6% for Thais and 63.4% for immigrants, while among the 183 villagers, it was 69.4% (Thais: 60.6%; immigrants: 75.0%). The efficacy of 400 mg single-dose albendazole among different hookworm-infected racial/ethnic groups was analyzed 21 days' post-treatment. Kato-Katz and polyethylene tube cultivation methods were used for stool examination. Among the 211 hookworm-positive cases in both surveys, only 82 cases from the last survey were followed up. By Kato-Katz technique, for the schoolchildren and villagers combined, the cure rate tended to be higher among the immigrants (65.0%) than the Thais (54.6%) (p=0.445). By Sasa's modified Harada-Mori culture technique, the cure rates also differed by racial grouping, and were higher (46.3%) among the immigrants and lower (27.8%) among the Thais (p=0.269). However, similar egg reduction rates were found for the two racial groups, at 96.0 and 92.6%, respectively. In addition, a higher intensity of hookworm infection tolerated albendazole therapy, lower cure rates were obtained in moderate-to-heavy infections (56.3%) and higher rates for light infections (63.6%) among the total population. There were no significant differences in drug efficacy among the 2 ethnic groups of Thai and immigrants (p>0.05) in Kanchanaburi Province, Thailand.

Some gastro-intestinal tract parasites in wild De Brazza's monkeys (Cercopithecus neglectus) in Kenya.

Fresh faecal droppings of wild group of De Brazza's monkeys (Cercopithecus neglectus), earmarked for translocation, were collected between January and July 1998. The samples were analysed using direct smears, ether-sedimentation and the Harada-Mori culture techniques for gastro-intestinal tract parasites (GIT). Two species of helminths and three of protozoa were detected. Entamoeba coli was found in all 40 samples screened from 11 monkeys. Entamoeba histolytica was detected in 71.8% of the total samples screened, Streptopharagus spp. in 12.8% and Strongyloides spp. and Balantidium coli each in 7.7% of the samples. E. histolytica and Streptopharagus spp. were most prevalent in faecal samples of juveniles while Strongyloides spp. and B. coli were mostly found in adult females. This, to our knowledge, is the first report of GIT parasites in a wild population of De Brazza's monkeys and our results are baseline.

INCIDENCE OF PARASITIC INFECTION AMONG THE REFUGEES OF VIETNAM BY STOOL EXAMINATION

The stools of twenty-one persones which drifted from Democratic Republic of Vietnam to Japan were examined against the eggs of intestinal parasites by the Tween 80 citric acid ether sedimentation method and Harada-Mori's culture method. The results of examinations proved that they were harbored by roundworms (38.1%), whipworms (9.5%) and hookworms (4.8%). Hookworms were carefully examined for the infective larvae by the culture method and adult worms (especially based on the bursa and inner ventral teeth) from infected dogs and all resembled Ancylostoma ceylanicum as describedby Biocca (1951) and Yoshida (1974). Although A. ceylanicum was not found in stray dogs captured in Kagoshima City which located in the main land of Kyushyu, it was found in Amami Oshima and Tokunoshima (Yoshida and Okamoto, 1972) and Okinawa (Oohama, 1941). Although A. ceylanicum are frequently encountered in cats and dogs of various areas, man is regarded as an abnormal host for A. ceylanicum (Chowdhury and Schad, 1972; Lie and Tan, 1959). However, the potential medical importance of this species lies therein, so that their pathogenicity of this s pecies is strong. Infections with these hookworms are easily overlooked, as the diagnosis is possible only by a careful examination of the ventral teeth present in the oral cavity and the bursa of male. Accordingly, this hookworm species may occur more frequently than the existing reports would suggest.

Development of Spirometra and Paragonimus Eggs in Harada-Mori Cultures

The Harada-Mori type of hookworm culture which consists of a thin film of feces on a strip of filter paper in an upright test tube containing a shallow reservoir of water was found to be a simple and effective medium for incubating Spirometra and Paragonimus eggs. When the eggs of S. mansonoides in feces of experimentally infected cats were cultured by this method, essentially all of the eggs contained coracidia in 2 weeks at room temperature (22 to 26 C). When the feces-smeared filter paper from the culture was placed in water, there usually was prompt hatching and swarming of coracidia into the water. Eggs of P. kellicotti similarly incubated in feces of an experimentally infected cat contained fully developed miracidia in 17 to 20 days and these remained viable in cultures for 3 months. When placed in water, a high proportion of the miracidia hatched, especially if subjected to a sudden change of temperature. Hatching in water possibly was not necessary for infecting snails, however, for of 67 Pomatiopsis lapidaria allowed to feed directly on the leached culture feces, six became infected and shed cercariae in 60 to 68 days. The observations suggest that development of the eggs of these helminths may occur under nonaquatic natural conditions similar to those provided by the Harada-Mori culture. In the laboratory, the eggs of trematodes and cestodes having aquatic intermediate hosts usually are incubated in water. In nature, the infection of such hosts by miracidia and coracidia generally is believed to occur only in aquatic habits. However, the final hosts of many of these parasites frequently-more often than not in some instances-defecate at the water's edge rather than in the water, and a high proportion of the egg-bearing feces therefore rests on and becomes a part of the damp soil at the margin of streams and other bodies of water. Moreover, the intermediate hosts are in some instances amphibious-as, for example, those of Paragonimus kellicotti and Schistosoma japonicum--or are adapted to temporary ponds and to bodies of water having widely fluctuating water levels-as, for example, those of Fasciola hepatica, Fascioloides magna, and Schistosomatium douthitti. Under these conditions the intermediate hosts and the developmental stages of the parasites are for relatively long periods of time in or on the mud and wet ooze of nonaquatic habitats. The type of fecal culture described by Harada and Mori (1955) provides a habitat which in a number of respects resembles those of the mud-ooze situations just referred to. As a preliminary investigation of the possibilities of nonaquatic incubation of eggs and of subsequent natural infection of amphibious and aquatic hosts in and out of water the following observations were made.