Evaluation of the stability of Yamakagashi (Rhabdophis tigrinus) Equine Antivenom after 20 years storage

@#In 2000, an equine Yamakagashi (Rhabdophis tigrinus) antivenom (Lot 0001) was testmanufactured as an unapproved drug for treatment of Yamakagashi bites. It was stocked on the premise of super-legal use from the viewpoint of emergency health crisis management. The antivenom showed a strong neutralizing ability against the hemorrhagic and coagulation activity of the Yamakagashi venom in its potency test. One vial of the antivenom can effectively neutralize at least about 4 mg of Yamakagashi venom. Its efficacy has also been confirmed in patients with severe cases of R. tigrinus bite that has been used in emergency. In 2020, this antivenom (Lot 0001) has reached 20 years after its production. To evaluate the integrity and potency of the antivenom, quality control, safety and potency tests had been conducted almost every year since 2012. Physical and chemical tests (property test, moisture content test, insoluble foreign matter test, osmotic pressure ratio test, pH test, protein content test, endotoxin test, sterility test) of the antivenom, showed no significant changes throughout the years, when compared to the results immediately after its production in 2000. All the parameters measured were also within the standard values. In animal safety tests (test for absence of toxicity and pyrogen), there was no change in the test results during the storage period and no abnormalities were observed. The potency test (anti-coagulant activity) after 20 years of the product, showed the same potency as those recorded immediately after production. Therefore, in all of the stability monitoring tests conducted so far, the product did not show any significant change compared to the results immediately after production. This confirms the stability of the product during the stockpiling period to the present, that is, 20 years after production.

Seasonal distribution of enteropathogens detected from diarrheal stool and water samples collected in Kathmandu, Nepal.

A total of 334 diarrheal fecal samples (from 210 males and 124 females) collected in Kathmandu, Nepal, were studied for various kinds of enteropathogens. Overall, 33% (111/334) fecal samples were positive for one or more enteropathogens. There was no difference in detection rates between males and females. Enteropathogen detection rates in summer, winter, spring, and autumn were 61% (40/66), 52% (45/87), 31% (25/81), and 25% (25/100), respectively. Altogether eight species of bacteria, three genera of viruses, and five species of protozoan parasites were detected with considerable seasonal variations. Among the bacterial isolates, enteropathogenic Escherichia coli topped the list followed by Vibrio sp. Only one sample had Shigella (S. sonnei). Rotavirus type A was the most frequently detected among the enteric viruses, followed by human enterovirus and human adenovirus, respectively. Among the enteric protozoan parasites, Giardia intestinalis was the most frequently detected followed by Cryptosporidium parvum. Detection of bacterial and protozoan pathogens showed a slightly high tendency in the summer season compared with that in the other seasons (p>0.05), whereas the detection of viruses was significantly high in the winter season (p<0.05). Of the total 57 water samples, 43 (75%) showed one or more bacterial species out of which 51% (22/43) were E. coli. Among the E. coli isolates, 68% were EPEC. Enterohemorrhagic E. coli (O157) was not detected.

Contamination of soil with helminth parasite eggs in Nepal.

Contamination of soil with helminth eggs in Kathmandu Valley (n=122) and outside of Valley (n=34) in Nepal was investigated with the use of centrifugal floatation technique using sucrose solution (sp gr 1.200). The overall soil contamination rate was 36.5% (57/156). The prevalence was uniform in Kathmandu Valley (36.9%) and outside of the valley (35.3%). A mean of six helminth eggs per sample were detected out of which more than half were embryonated (potentially infective). In Kathmandu Valley, soil contamination rate was higher (48.3%) during wet season compared with that observed in dry season (33.3%) but without significant difference (p>0.05). Multiple species of helminth eggs were detected in 22.8% of total positive samples (n=57). Altogether five species of nematoda (Ascaris lumbricoides, Toxocarta sp, Trichuris trichiura, Capillaria sp and Trichostrongylus sp) and two species of cestoda (Hymenolepis nana and H. diminuta) were recovered. A. lumbricoides was predominant in Kathmandu Valley while Trichostrongylus was the commonest one in outside of valley.

Toxoplasma antibody prevalence in Nepalese pregnant women and women with bad obstetric history.

Sera from randomly selected 345 pregnant Nepalese women aged 16-36 years and 13 women with bad obstetric history (BOH) were tested for the presence of Toxoplasma antibodies using microlatex agglutination (MLA) and ELISA methods. The overall prevalence was 55.4% (191/345). Prevalence was slightly higher (59.0%) in older age-group (27-36 years) compared with younger age-group (16-26 years) (52.2%). No significant difference in antibody prevalence in women belonging to two different ethnic-groups (Tibeto-Burmans 57.8%, Indo-Aryans 52.7%) was observed (p>0.05). MLA antibody titer ranged from 1:16 to 1:2,048. Over three-fourth of the women showed either high (1:510 or over) or low (1:16 or 1:32) antibody titer. Three percent (6/191) of MLA antibody positive subjects had Toxoplasma IgM antibodies by IgM-ELISA. All six IgM antibody positive pregnant women had MLA antibody titer of over 1:510. Of the total 13 women with BOH, 5 (38.5%) had Toxoplasma antibodies of which 2 (40.0%) were positive for Toxoplasma-IgM antibodies.

Use of polymerase chain reaction in the diagnosis of toxocariasis: an experimental study.

In this paper we report the usefulness of polymerase chain reaction technique in the diagnosis of visceral larva migrans in a mouse model. Liver samples obtained from two set of experimentally infected mice (10, 100, 1,000 and 10,000 embryonated Toxocara canis eggs per mouse) along with the eggs of T. canis, T. cati and Ascaris suum were included in this study. Polymerase chain reaction (PCR) was performed using Toxocara primers (SB12). The first PCR product electrophoresis revealed very thin positive bands or no bands in liver samples. However, on second PCR a clear-cut bands were observed. No positive band was shown by A. suum eggs. Our findings thus indicate the usefulness of PCR technic in the diagnosis of visceral larva migrans (VLM) in liver biopsy materials specifically by means of double PCR using the primer SB12.

Seroepidemiological study of Toxoplasma infection in central and western regions in Nepal.

The present study was carried out to ascertain the seroprevalence rate in different geographical areas in Central and Western Regions in Nepal. A total of 1,237 serum samples collected from Nuwakot (217), Kathmandu valley (402) and Chitawan (159) districts in Central Region, and Mustang (143), Surkhet (64) and Banke (252) districts in Western Region in Nepal were included in this study. Toxoplasma antibodies were detected by micro-latex agglutination (MLA) and enzyme linked immunosorbent assay (IgM-ELISA) methods. The seropositive rate in Central and Western Regions were found to be 48% and 49%, respectively; with an overall positive rate of 48 percent. Districtwise, the seropositive rate in Nuwakot, Kathmandu valley, Chitawan, Mustang, Surkhet and Banke districts were 38, 46, 64, 51, 67 and 44%, respectively. Interestingly, the relatively newly inhabited Surkhet district in Western Region and Chitawan district in Central Region showed significantly higher seropositive rate compared with those of two other districts in the respective Regions (p < 0.05). Ethnically, Tibeto-Burmans showed higher seropositive rates in Central Region (p > 0.05). In contrast, Indo-Aryans showed higher seropositive rate in Western Region (p > 0.05). Age related increase in seropositivity was observed only in Central Region. One percent of Toxoplasma antibody positive samples also showed Toxoplasma IgM antibody positivity.

Seroepidemiological study of Toxocara infection in Nepal.

Seroepidemiological study of Toxocara infection in Nepalese people aged more than fourteen years was carried out by enzyme linked immunosorbent assay (ELISA) using T. canis soluble embryonated egg antigen. Of the total 200 subjects included in this study, 162 (81%) were found to possess antibodies to Toxocara spp. Males showed higher (85%) antibody positive rates than females (77%). Inhabitants of Kathmandu valley showed higher antibody positive rates (84%) compared with those living outside of the valley (78%). These differences, however, were not significant statistically (p > 0.05). Ethnically, Newar, Rai/Limbu, Tamang/Sherpa, Brahmin/Chhetri, Jha/Yadavs, and others (Kami, Damai) showed Toxocara antibody positive rates of 85, 85, 83, 70, 77 and 100%, respectively. Half of the Toxocara antibody positive subjects (49%) showed an elevated level (two plus) of antibody.