Morphological characteristics of microfilariae in blood smears of the common treeshrew Tupaia glis (Mammalia: Scandentia) in Gemas, Negeri Sembilan, Malaysia

@#Some filarial nematodes, such as Wuchereria bancrofti, Brugia malayi, and Brugia timori, cause lymphatic diseases in humans in the tropics, whereas other filarial parasites from wild animals cause zoonotic diseases in humans worldwide. To elucidate the prevalence and diversity of filarial parasites in Malaysia, we investigated the filarial parasites from wild animals in Gemas, Negeri Sembilan. To find adult filarial parasites, we dissected 26 animals, which included five frogs, one skink, one snake, two birds, six common treeshrews, and 11 rats. Then, we examined microfilariae in the blood smears and skin snips obtained from each animal. We found two types of microfilariae in the blood smears of common treeshrews: one was very similar to Malayfilaria sofiani and the other closely resembled Brugia tupaiae. These findings indicate an additional distribution of these filarial parasites in Gemas.

Re-evaluation of the formalin-ether sedimentation method for the improvement of parasite egg recovery efficiency.

The formalin-ether sedimentation (FES) method is considered as reliable method of fecal examination for the detection of parasites. In this study, we re-evaluated several aspects of FES such as (i) pretreatment of feces; (ii) filtration of fecal suspensions; (iii) test-tube material and (iv) substitution of ether by other organic solvents as to see an improvement in parasite egg recovery. The egg count was represented by the number of ova detected per 100 µg of sediment. Pre-treatment of feces with formalin (pH 7) increased egg detection rate remarkably compared with original FES method. Use of three layers of gauze dramatically reduced the sediment in the final product, and led to an increase in the number of ova detected. Use of polypropylene test tubes instead of glass test tubes also increased the number of egg detection. None of the organic solvents used to replace the ether produced better results. Based on these findings, we proposed a modified FES procedure. Further, we also compared the parasite positive rate and the number of ova recovered by using original FES and the modified FES procedures by examining 112 fecal samples collected from school children of parasite endemic area in Nepal. Feces collected from Nepal had many parasite ova, and these fecal samples barely displayed false-negative results even by method with low sensitivity. When the mean number of Hemenolepis nana, hookworm, T. trichiura, and A. lumbricoides ova recovered by original FES and the modified FES methods was compared, the values obtained by modified FES were superior (higher). This result suggested that the modified FES is effective and better for the recovery of parasite ova in areas of low-intensity parasitic infection.

Evaluation and modification of the formalin-ether sedimentation technique.

Formalin-ether sedimentation (MGL) is a well-known technique for the examination of faeces for parasites, but some recent reports have indicated that its efficiency is not as high as originally thought. We reevaluated the recovery efficiency of the original MGL (O-MGL) technique to modify it. We subsequently adopted the following modified MGL technique (M-MGL): filtration by three layers of gauze and washing, adjustment to pH 3, retreatment of plug, and use of 1.5 g of faeces. We also compared five faecal examination techniques (including the O-MGL and the M-MGL) for three parameters: recovery efficiency, sensitivity, and mean number of eggs detected. The highest sensitivity was obtained by the M-MGL (95%), followed by the commercially available kit (Kit; 90%), O-MGL (76%), Kato-Katz (KK; 57%), and direct smear (DS; 50%). The mean numbers of Ascaris lumbricoides eggs recovered by the techniques were in order M-MGL (148 eggs), Kit (97), O-MGL (41), KK (11), and DS (6). This M-MGL technique has the advantage not only of the above-mentioned three parameters, but also the ease of microscopic observation and the concentration index. The parameters of the O-MGL technique were not necessarily sufficient compared with the other techniques. It seems that the improved M-MGL technique in the present study is applicable for field surveys, particularly when the survey is done in areas of low parasite endemicity.

Two new variants of G6PD deficiencies in Singapore.

Identification of mutations in G6PD gene is performed as an epidemiologic investigation of G6PD deficiency in many countries. In order to understand the hereditary background of G6PD deficiency in a population, screening of mutations is required not only in exonic regions but also for intron and promoter regions. One hundred male neonatal samples diagnosed as with G6PD deficiency by newborn screening in Singapore were used in this study. The multiplex PCR using the multiple tandem forward primers and common reverse primer (MPTP) method was carried out to detect the common 11 mutations in south-east Asia such as Gaohe 95A>G, Orissa 131C>G, Vanua-Lava 383T>C, Mahidol 487G>A, Mediterranean 563C>T, Coimbra 592C>T, Viangchan 871G>A, Chatham 1003G>A, Union 1360C>T, Canton 1376G>T and Kaiping 1388G>A. Samples whose mutations were unidentified by MPTP method were scanned at cording region, intron and promoter region by direct sequencing.Out of 100 samples, 90 samples (90.0%) were identified with one of the above mentioned common mutations. Eight out of 10 samples whose mutations were unidentified by MPTP method carried exonic mutations which had been previously reported such as Murcia 209A>G, Quing Yuan 392G>T, Nankang 517T>C, Chinese5 1024C>T. Two novel mutations were identified in these samples: one had a novel mutation (25C>T); the remaining sample carried a 49 bp deletion in intron 12.

Parasite egg contamination of vegetables from a suburban market in Hanoi, Vietnam.

Helminth egg contamination of vegetables purchased at suburban market in Hanoi, Vietnam was examined. A total of 317 vegetables were examined and 82 (26%) were revealed to be positive for parasite eggs. Of the 15 varieties, 13 were positive except for horseradish and cucumber. Contamination was highest in leafy vegetables (31%), followed by root vegetables (17%) and fruit vegetables (3%). Throughout the survey, five species of parasite eggs were found: Ascaris sp., Trichuris sp., Toxocara sp., Taenia sp. and Ascaridia galli. In the interview with the villagers, 121 (81%) of 149 adult villagers stated that they usually use not only animal feces but also human feces as a fertilizer. Throughout the survey, a total of 453 eggs were recovered. Number of eggs recovered from vegetables was higher in the dry season (355 eggs) than in the rainy season (98 eggs). The study revealed that vegetables purchased at a market in suburban Hanoi (Vietnam) were highly contaminated with parasite eggs excreted by humans and animals. Considering the eating habits of the Vietnamese and the 17% embryonation rate of detected parasites, vegetables seem to play an important role in soil-transmitted helminth infection in this country.

Method for detecting circulating Toxocara canis antigen and its application in human serum samples.

Diagnosis of larval migrans (LM) is usually done by immunodiagnostic methods. These methods, however, simply show the presence or absence of antibody but not the active infection of the patients. Therefore, we aimed to establish a diagnostic method for detecting circulating Toxocara canis antigen using a sandwich, ELISA. Monoclonal antibodies (MAb) were produced against the excretory-secretory (ES) antigen of second-stage T canis larvae. Among the MAbs obtained, we selected one MAb (TCMAb12; molecular weight, 30-80 kDa, IgG) for use in the sandwich ELISA. The cross-reactivity of the sandwich-ELISA against thirteen different kinds of parasite antigens were examined. The results revealed that the antibody reacted with T canis ES antigen, T. canis female antigen, and T. canis second-stage larvae antigen, but did not react with any other antigens. From results obtained using an ES antigen concentration standard curve, we confirmed that the detection limit of the sandwich-ELISA was 5 ng/ml, which provides sufficient sensitivity for the diagnosis of toxocariasis (LM). We applied the method to suspected toxocariasis patients and examined the circulating antigen in their sera. We used nine serum samples collected from patients with suspected toxocariasis based on both their clinical symptoms and high antibody titers. Overall, five sera showed antigen-positive reactions, while the remaining four were negative. These results indicated that about 44.0% of the antibody-positive patients were antigen-negative, not ongoing active infection. The results obtained using this technique would provide us for understanding toxocariasis.

Infection dynamics of Centrocestus armatus cercariae (Digenea: Heterophyidae) to second intermediate fish hosts.

The infection dynamics of Centrocestus armatus cercariae with respect to second intermediate fish hosts were investigated using cercariae collected from naturally infected Semisulcospira libertina. Cercariae survival and infectivity were recorded at 2-hr intervals. Survival remained constant but decreased abruptly at 30 hr of age. An age-dependent model presented the best-fit curve for the survival data (r2 0.936), implying that cercariae tended to allocate resources equally among themselves and then died once those resources were depleted. Cercariae infectivity remained constant over the first 10 hr of life and then declined; an age-dependent model also provided a better fit (r2 = 0.956). The transmission rate of C. armatus cercariae to the fish host was relatively low near the time of emergence from the snail host but peaked at 85% after 1 hr and then maintained a plateau period until 5 hr of exposure to fish. The pattern of transmission was also investigated initially at varying densities of cercariae and then by maintaining a constant cercariae density but varying the total number. Results revealed that the pattern of transmission was frequency-dependent.

[A high aged case of herpes simplex viral encephalitis associated with progressive cerebral white matter lesion].

An 80-year-old male without abnormal past medical history presented with coma, general seizures, and fever subsequent to abnormal behavior. The pressure of the cerebrospinal fluid(CSF) elevated(13.5-20.5 cm H2O), and CSF examination revealed pleocytosis with predominant mononuclear cells(80-879/mm3) and elevated protein level(32-130 mg/dl). DNAs of herpes simplex virus(HSV) type 1 and 2 in CSF were not confirmed by polymerase chain reaction method in the acute phase. The HSV(type 1) antibody(HSV-1 Ab) ratio of serum to CSF(= [serum HSV-1 Ab]/[CSF HSV-1 Ab]) was 0.98 and HSV-1 Ab index(= [CSF HSV-1 Ab]/[serum HSV-1 Ab] divided by [CSF albumin]/[serum albumin]) was 62.4. Initial fluid attenuated inversion recovery(FLAIR) (TR/TE/TI = 6,882/110/1,700 msec) axial magnetic resonance(MR) imaging showed hyperintensity in the subfrontal area, inferomedial portions of the temporal lobes, cingulate gyri, and insular cortices bilaterally. Meningoencephalitis caused by HSV-1 was diagnosed based on the values of HSV-1 Ab ratio of serum to CSF(less than 20), of HSV-1 Ab index(larger than 1.91), and the findings of MR imaging. Diffuse white matter lesions manifesting hyperintensity on FLAIR imaging in the bilateral frontal and temporal lobes close to the affected cortices developed approximately six weeks after the onset despite administration of antiviral agent and steroid. The lesion extensively involved the white matter of the bilateral frontal and temporal lobes finally. The initial value of myelin basic protein(MBP) in CSF was 0.9 ng/ml (normal value: less than 4 ng/ml). Subsequent measurement of MBP in CSF about two, six weeks, two, three, and six months after the onset showed a marked increase of 233.9 ng/ml followed by a gradual decrease of 25.4 ng/ml, 18.4 ng/ml, 7.4 ng/ml and 4.3 ng/ml, respectively. Therefore, demyelination of the lesion in the cerebral white matter was suggested by the chronological change in FLAIR imaging and MBP in CSF.

Contamination of river water by Cryptosporidium parvum oocysts in western Japan.

In Japan, only a few rivers have been inspected for Cryptosporidium parvum contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for Cryptosporidium oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for Cryptosporidium oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the C. parvum found as the bovine or human type. C. parvum was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained C. parvum oocysts. The number of C. parvum oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the C. parvum detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with C. parvum oocysts, as reported in other countries.

Chronic subdural hematoma with vasogenic edema in the cerebral hemisphere--case report.

An 80-year-old male with a history of hypertension presented with chronic subdural hematoma manifesting as progressive consciousness disturbance and left hemiparesis. T1-weighted and fluid attenuation inversion recovery (FLAIR) magnetic resonance imaging showed a fresh hematoma in the right subdural space with a midline shift of 15 mm. FLAIR and diffusion-weighted imaging showed a hyperintense area in the right paraventricular white matter compressed by the hematoma. Apparent diffusion coefficients (ADCs) corresponding to the hyperintense area in the central area of the affected cerebral hemisphere on FLAIR images were measured before and one month after the operation. The motion probing gradient was applied in the right-left direction to the body axis. Since the central area in the cerebrum includes nerve fibers perpendicular to the direction of the gradient, the measured ADC appeared to be anisotropic. Preoperative ADC in the right paraventricular white matter was anisotropic and greater than in age-matched normal subjects, so the edema was identified as the vasogenic type. The edema in the right paraventricular white matter resolved promptly with improvement of the midline shift and normalization of the ADC.

[A case of cerebral venous thrombosis with reversible brain parenchymal lesions: usefulness of diffusion weighted imaging and measurement of apparent diffusion coefficient].

A 47-year-old man with a history of thrombophlebitis of his left leg for several years presented with a mild left hemiparesis and ipsilateral hypesthesia. Magnetic resonance imaging showed subacute thrombosis of the superior sagittal sinus and a cortical vein of the right cerebral hemisphere. A linear hyperintense area was found in the white matter of the right postcentral gyrus on T 2- and diffusion weighted axial imagings on the 7 days after the onset. The patient was treated conservatively, and his clinical course was uneventful. His neurological dysfunctions recovered within approximately three weeks after the onset. The white matter lesion in the right postcentral gyrus also disappeared one month later. The apparent diffusion coefficients (ADCs) in the white matter of the pre- and postcentral gyrus were measured bilaterally on the ADC mapping imaging. In the subacute stage, the ADC values in the white matter of the right pre- and postcentral gyrus were 0.50 x 10(-3) mm2/sec and 0.91 x 10(-3) mm2/sec, respectively. The %ADC indicating the ratio of ADC value of the lesion to that of the contralateral brain tissue was calculated. The %ADCs in the white matter of the pre- and postcentral gyrus were 64.9% and 124.5% respectively. In the chronic stage, the ADC values in the white matter of the right pre- and postcentral gyrus were 0.96 x 10(-3) mm2/sec and 0.99 x 10(-3) mm2/sec, and the %ADCs improved to 106.7% and 106.5% respectively. The lesions in the white matter of the right pre- and postcentral gyrus were reversible. The former was thought to be mainly ascribed to cellular edema and the latter was vasogenic edema. The present case showed when %ADC of the ischemic lesion in cerebral venous thrombosis was higher than 60%-70%, conservative therapy alone is sufficient effective for the improvement of neurological deficits.

Successful treatment of a ruptured enlarged infundibular widening of the posterior communicating artery--case report.

A 67-year-old female with a history of hypertension and cerebral infarction presented with subarachnoid hemorrhage (SAH) (Hunt and Kosnik grade 4). Brain computed tomography (CT) revealed a clot dominantly on the right (Fisher's classification Group 4). Cerebral angiography showed funnel-shaped widenings at the origins of the bilateral posterior communicating arteries. The maximum diameter of the widening was greater than 3 mm, so this widening was called an enlarged infundibular widening. Angiography showed a small bulge protruding posterolaterally from the wall of the right enlarged infundibular widening, and the right posterior communicating artery arose from the apex of the enlarged infundibular widening. Based on the findings of the brain CT and cerebral angiography, the diagnosis was SAH due to rupture of the right enlarged infundibular widening. Approximately 12 hours after the onset, the clot was evacuated through the right pterional approach. Bleeding from the small bulge of the right enlarged infundibular widening occurred intraoperatively, so a right-angled ring clip was applied parallel to the right internal carotid artery to obliterate the rupture point. Postoperatively, she was discharged without neurological deficit, and follow-up CT showed no other new infarction. We recommend clipping rather than wrapping or coating for similar cases of ruptured enlarged infundibular widening.

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.

Cryptococcal meningoencephalitis presenting with an unusual magnetic resonance imaging appearance--case report.

A 61-year-old female with a past history of gastric cancer presented with altered mental status, a few seizures, and low-grade fever. Lumbar puncture revealed elevated cerebrospinal fluid (CSF) pressure, lymphocytic pleocytosis, elevated protein level, remarkably decreased glucose level, and presence of cryptococcal antigen. Cryptococcus neoformans was identified by India ink staining and culture of CSF. The patient was given antifungal agents intravenously and intrathecally. CSF findings improved and C. neoformans could not be detected in CSF one month after the onset. Cerebral sulcal hyperintensity was identified in the bilateral frontal and parietal lobes on fluid-attenuated inversion recovery (FLAIR) magnetic resonance (MR) imaging one month after the onset, but no leptomeningeal enhancement was detected in the affected sulci on T1-weighted MR imaging. The sulcal hyperintensity on FLAIR imaging developed in the bilateral temporal and occipital lobes 2 months after the onset. CSF findings obtained by lumbar puncture were within the normal range except for pressure. However, neurological deterioration and reconfirmation of C. neoformans in CSF indicated recurrent cryptococcal inflammation. The sulcal hyperintensity on FLAIR imaging may indicate a high CSF protein concentration in the subarachnoid space. Such cerebral sulcal hyperintensity is an unusual MR imaging finding of cryptococcal meningoencephalitis, and may be an early sign of procrastinating process or recurrent inflammation even if the findings of CSF obtained by lumbar puncture are normal.

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.

Prevalence of Cryptosporidium parvum infection and pattern of oocyst shedding in calves in Japan.

Cryptosporidium parvum infection and the pattern of oocyst shedding were observed in calves. A total of 480 fecal samples were collected from 30 calves (age, < or =30 days) over a period of 10 months from June 1998 to March 1999. A sucrose centrifugal flotation technique revealed 28/30 (93%) calves were passing Cryptosporidium oocysts. Oocyst shedding was first detected on the sixth day after birth, with 8% of the calves testing positive. This rate increased day by day and reached approximately 80% by day 15. Oocyst shedding varied from 1 to 13 days, with a mean of 7 days. Calves infected with C. parvum had a significantly higher rate of diarrhea (33%) than non-infected calves (8%) (P<0.05), suggesting C. parvum infection as the likely cause. The mean number of oocysts excreted by calves < or =30 days old was approximately 6x10(7) per gram of feces. These results indicated that one calf would excrete some 6x10(11) oocysts in the first month after birth, taking both the quantity of feces in a day and the period of excretion into consideration. Accordingly, it is clear that calves are important in the spread of cryptosporidiosis to calves and humans.

Differentiation of Toxocara canis and T. cati eggs by light and scanning electron microscopy.

In this study, we investigated the morphological identification of Toxocara canis and T. cati eggs on the basis of light and scanning electron microscopic (SEM) observations. T. canis and T. cati eggs used in this study were recovered from the uteri of respective gravid female worms. Measurement of egg size was not helpful in the differentiation of these species, because approximately 90% of eggs measured were of similar size. Using SEM, we were able to differentiate T. canis eggs from T. cati eggs based on their respective characteristic surface structures. Both species have subspherical eggs with markedly pitted surfaces like those of a golf ball, but the surface pitting in T. canis is more coarse than that in T. cati. In this study, however, these differences were not absolute, as 16% of T. canis and 29% of T. cati eggs showed surface pitting that was uncharacteristic of their species. Of the 16% of T. canis eggs that could not be differentiated by surface structure, 3% had pitting resembling T. cati, and the remaining 13% showed intermediate type surface pitting. Similarly, 5% of T. cati eggs resembled T. canis, and 25% of these were of intermediate type. Light microscopic observation yielded results similar to those of SEM, indicating that light microscopy is also a useful tool for the identification of Toxocara eggs.

[Intracranial vertebral artery stenosis successfully revascularised by cerebral angioplasty and stenting in chronic stage: a case report of prompt improvement of the neurological impairment].

A 72-year-old man with a history of hypertension had a left cerebellar infarction and followed by a right cerebellar infarction within about one and a half months after the initial stroke. Brain magnetic resonance images(MRI) showed infarctions in both middle cerebellar peduncles and in the mid-portion of lower pons. Right veretebral artery(VA) terminated in posterior inferior cerebellar artery(PICA). Left intracranial VA has a high-grade eccentric atherosclerotic stenosis(91%) proximal to the left PICA. No collateral circulation was developed from bilateral carotid arteries. Three months after the final ischemic episode, the patient had remained bed ridden and needed a whole assistance for regular daily life because of severe ataxia of four limbs and truncs and of left hemiparesis. The patient and his family gave us informed written consent, then cerebral angioplasty and stenting(CAS) was performed for the left VA stenosis, which was sufficiently dilated. Iodine-123 iodoamphetamine(123I-IMP) single photo emission computed tomography (SPECT) showed hypoperfusion in both cerebellar hemispheres before CAS. Post CAS 123I-IMP SPECT scans demonstrated improvement of the hypoperfusion in the left cerebellar hemisphere. Ataxia of four limbs, left hemiparesis and his will for physical therapy improved in a short period after the treatment. Ten months later, the left VA had a mild stenosis and patient presented mild truncal ataxia and needed less assistance for regular daily life. The present case indicated that improvement of neurological impairment was expected by the endovascular revascularization even in a chronic stage.

Short report: possible Cryptosporidium muris infection in humans.

Oocysts of cryptosporidia whose morphology resembled that of Cryptosporidium muris were found in the stool of 2 healthy girls in Surabaya, Indonesia. The oocysts were predominantly oval and measured 7.75+/-0.17 x 5.55+/-0.13 microm (mean+/-SD). The number of oocysts excreted were more than 10(5) per gram of stool. The oocysts were well stained with fluorescein-conjugated monoclonal antibody to Cryptosporidium. The specimens from both girls containing the oocysts showed a positive result by the polymerase chain reaction (PCR) using primers specific for the genus Cryptosporidium, but a negative result by the PCR using primers specific for C. parvum. The 2 girls passed oocysts for 5 and 6 days, respectively. They did not complain of any symptoms during the passage of oocysts.