Direct Staining Method Research Articles

Cryptosporidium species diagnosis in handlers of domestic pigeons in Baghdad City: Molecular and microscopic approaches.

The aim of this study is to detect the prevalence and diagnosis of molecular characteristics of Cryptosporidium infection in handlers of domestic pigeons in Baghdad City. Traditional and molecular diagnostic methods were employed to detect and identify Cryptosporidium species. Sixty stool samples were collected from the handlers of domestic pigeons, and various techniques, including direct smear, flotation concentration, staining methods, and DNA extraction coupled with nested polymerase chain reaction (PCR), were utilized. The results obtained from traditional methods indicated an overall infection rate of 55% in handlers of domestic pigeons, while significant variations were noted between male and female handlers. Age group 21-40 years were found to have higher infection rates were found to have higher infection rates. The prevalence of Cryptosporidium also displayed temporal variability throughout the study period. Molecular analysis using nested PCR revealed higher infection rates of 86% in handlers of domestic pigeon samples. Genotyping and phylogenetic analysis identified the presence of Cryptosporidium parvam species in handlers of domestic pigeons, indicating zoonotic transmission potential. These findings underscore the high prevalence of Cryptosporidium infection among handlers of domestic pigeons in Baghdad City.

Giardiasis in the Southwest Region of the United States: Causes and Preventive Measures

Giardia is a protozoan parasite of the small intestine, and a leading cause of diarrheal diseases worldwide in a variety of animals, as well as humans. Despite the decrease, Giardiasis remains the most commonly reported intestinal parasitic infection principally in the Southwestern region of the United States. The parasite is typically transmitted by fecal-oral route and may be transmitted by direct intake or indirectly from contaminated food or water. Symptomatic Giardiasis is often treated to reduce the duration of symptoms, to prevent future complications, and to minimize transmission of the parasite to other hosts. The clinical image of Giardiasis is mixed, with high variability in severity of clinical disease which can become chronic or be followed by post-infectious sequelae. At present, treatment options include nitroimidazoles derivatives; especially metronidazole, which has been the mainstay of treatment for decades and is still widely used. The Navajo Nation located in the Southwestern region of the United States has suffered from the scarcity of water expediting Navajo populations to use water resources such as underground water, river streams, lakes and surface water as their main source of accessibility. The lack of adequate domestic and municipal water has led several Navajo families to become ill with waterborne infectious diseases like Giardiasis. More recently, molecular methods have become amateur in diagnosis for testing patient samples as well as water samples for Giardiasis. Through direct immunofluorescent staining and other methods that detect intact organisms have improved the detection of Giardia trophozoites or cysts. An understanding of clinical and molecular variability of organisms is important for effective treatment and the response of remedies that are commonly used. The molecular classification of bacteria and viruses isolated from Indigenous patients might have their own specification that can be correlated with isolates from water samples collected from the same localities. This correlation could help us in source tracking and finding solutions for the communities who are already at risk due to the lack of infrastructure and resources. In this review of literature, the causes and preventive measures of Giardiasis are covered to potentially help understand the importance behind this parasitic disease, more specifically on the Navajo Nation of the Southwest of the US.

Gastrointestinal parasites in immunocompromised patients; A comparative cross-sectional study

Gastrointestinal parasites (GIPs), including helminths and protozoa species, are a major health problem in many parts of the world. About 3.5 billion people are affected by the parasites worldwide. GIPs are one of the leading causes of death among immunocompromised individuals and can cause serious clinical complications, especially in people with Human Immunodeficiency Virus (HIV)/AIDS, hemodialysis patients, and transplant recipients. This study aimed to compare the prevalence of GIPs among immunocompromised patients and immunocompetent individuals in Lorestan province, West Iran. In the current study, with a sampling of 232 participants (114 hemodialysis, AIDS, and organ transplantation immunocompromised patients and 118 immunocompetent individuals as the control group), demographic characteristics and risk factors for GIPs were collected through a pre-designed questionnaire. Stool samples of patients and the control group were examined for GIPs using different diagnostic methods including direct smear (saline and Lugol's iodine), Ziehl-Neelsen staining, agar-plate culture, and concentration method (formalin ether sedimentation). To evaluate the relative status of the immune system, TCD4+ cells were counted in the blood samples of the subjects by flow cytometry. The results were analyzed using SPSS 21 software, Fisher exact, and chi-square statistical tests. Multivariate modeling of the data was performed using logistic regression. The prevalence of GIPs in immunocompromised patients was more than twice that of immunocompetent individuals in the control group (42.06% vs. 17.79%). The most prevalent parasites identified among immunocompromised patients were Cryptosporidium sp. (27.1%), Blastocystis sp. (16.7%), and Entamoeba coli (14.6%) respectively. Cryptosporidium sp. had the highest frequency among hemodialysis patients (6.49%), AIDS patients (26.92%), and transplant recipients (18.18%) respectively. Patients with AIDS had the highest positive results for Cryptosporidium sp. followed by Microsporidia sp. (23.7%). In immunocompetent individuals, the highest prevalence of GIPs was related to Blastocystis sp and Trichomonas hominis (28.57%). Statistical analysis of the data showed that there was a statistically significant difference between various age groups regarding infection with GIPs so the highest rate of GIPs infection was observed in the age group lower than 50 years (P = 0.035). The statistical difference between the variable of location and infection with GIPs was insignificant but remarkable (P = 0.070). According to the results, it can be concluded that GIP is more common in immunocompromised patients than in immunocompetent individuals with cryptosporidium sp. predominance. Due to the favorable conditions of immunocompromised patients for GIPs and considering them as one of the important sources of parasitic infections and parasite transmission in society, control, prevention, and monitoring of their social behaviors along with health issues are inevitable.

Antibacterial Property of Cotton Fabric Dyed with Vaccinium bracteatum Thunb. Leaves

Vaccinium bracteatum Thumb. leaves have a variety of nutritional values and physiological functions, and are rich in useful resources. In this study, these leaves were used as a raw material to make vegetable dyes. Cotton fabric was then dyed with them by the direct staining method. Good dyeing effects and antibacterial properties of the resulting fabrics could be obtained using an optimized dyeing process. The antibacterial properties of the fabric were directly proportional to the dye uptake ( K/S value) of the fabric. To improve dyeing, mordant and chitosan were used separately to pretreat the cotton fabrics. The K/S value and antibacterial properties of the pretreated dyed cotton fabrics were improved after pretreatment.

Presence of hyaluronan in lung alveoli in severe Covid-19: An opening for new treatment options?

Severe coronavirus disease 2019 (Covid-19) is characterized by inflammation of the lungs with increasing respiratory impairment. In fatal Covid-19, lungs at autopsy have been filled with a clear liquid jelly. However, the nature of this finding has not yet been determined. The aim of the study was to demonstrate whether the lungs of fatal Covid-19 contain hyaluronan, as it is associated with inflammation and acute respiratory distress syndrome (ARDS) and may have the appearance of liquid jelly. Lung tissue obtained at autopsy from three deceased Covid-19 patients was processed for hyaluronan histochemistry using a direct staining method and compared with staining in normal lung tissue. Stainings confirmed that hyaluronan is obstructing alveoli with presence in exudate and plugs, as well as in thickened perialveolar interstitium. In contrast, normal lungs only showed hyaluronan in intact alveolar walls and perivascular tissue. This is the first study to confirm prominent hyaluronan exudates in the alveolar spaces of Covid-19 lungs, supporting the notion that the macromolecule is involved in ARDS caused by SARS-CoV-2. The present finding may open up new treatment options in severe Covid-19, aiming at reducing the presence and production of hyaluronan in the lungs.

Antifungal activity of titanium dioxide nanoparticles against Candida albicans

The unregulated release of titanium dioxide nanoparticles into the environment has raised concern, in particular due to the impact of the nanoparticles on indigenous micro-biome in our ecosystem. This paper reports a study on antifungal activity of titanium dioxide nanoparticles on a healthy growing fungi species, Candida albicans, a known opportunistic pathogen. A quantification of the total cell death was performed using a direct staining method, Trypan blue exclusion assay. Exposure to nanoparticles not only altered the growth rate, but also affected the onset and length of Candida albicans growth phases. The log and the onset of the death phase were shortened and accelerated, respectively. Up to 65% of the Candida albicans were killed after exposure to 100 μg/mL of the anatase titanium dioxide nanoparticles, while only 33% were killed with rutile. A higher dosage and incubation time of the nanoparticles increased their toxicity. Cells suffered from morphological changes upon the nanoparticle exposure, which correlates well with the results showing an altered growth phase culture.

The relationship between hygiene-social variables and Trichomonas vaginalis

Purpose: Trichomonas vaginalis (T. vaginalis) is the most common parasitic infection among nonviral, sexually transmitted diseases in the urogenital system. The incidence of the disease can vary according to the socio-economic status of the societies or different social groups. This research was performed on women who had vaginal complaints and who applied to Eskisehir Cancer Research Early Diagnosis and Screening Center (KETEM) and Eskisehir State Hospital Obstetrics and Gynecology Clinics. Methods & Materials: T. vaginalis was investigated by stained and unstained direct microscopic examination, culture and Real Time PCR methods, in a total of 406 female vaginal swabs taken from women applied to these two hospitals for control examination. During the collection of samples, an information form was applied to patients, in order to investigate the relationship between the prevalence of T. vaginalis and some social parameters. Results: T. vaginalis was detected by at least one of the methods in 35 (8.6%) of 406 swab samples. The distribution of 35 cases of T. vaginalis was; Twenty-eight (6.9%) were identified by direct microscopy and 27 (6.7%) by Giemsa staining and 31 (7.6%) by culture method and 35 (8.6%) by Real-Time PCR. In addition, direct microscopic examination, gram staining and routine culture methods were performed in all samples for other microorganisms that are likely to cause vaginal discharge. In 85 (20%) of all samples Candida spp., in 9 (2.2%) T. vaginalis and Candida spp. coexistence, in 25 (6.2%) Gardnerella vaginalis, in 11 (2.7%) G. vaginalis and Candida spp. coexistence was detected. There was no statistically significant relationship between the prevalence of T. vaginalis and social variables. Conclusion: When the sensitivity and specificity of the diagnostic methods are compared with their determined performance, it was suggested that the use of PCR method in addition to direct and stained microscopic examination and culture method in trichomoniasis would increase the sensitivity and would be helpful in accurate diagnosis of the agent.

Sublethal Injury and Viable but Non-culturable (VBNC) State in Microorganisms During Preservation of Food and Biological Materials by Non-thermal Processes.

The viable but non-culturable (VBNC) state, as well as sublethal injury of microorganisms pose a distinct threat to food safety, as the use of traditional, culture-based microbiological analyses might lead to an underestimation or a misinterpretation of the product’s microbial status and recovery phenomena of microorganisms may occur. For thermal treatments, a large amount of data and experience is available and processes are designed accordingly. In case of innovative inactivation treatments, however, there are still several open points with relevance for the investigation of inactivation mechanisms as well as for the application and validation of the preservation processes. Thus, this paper presents a comprehensive compilation of non-thermal preservation technologies, i.e., high hydrostatic pressure (HHP), pulsed electric fields (PEFs), pulsed light (PL), and ultraviolet (UV) radiation, as well as cold plasma (CP) treatments. The basic technological principles and the cellular and molecular mechanisms of action are described. Based on this, appropriate analytical methods are outlined, i.e., direct viable count, staining, and molecular biological methods, in order to enable the differentiation between viable and dead cells, as well as the possible occurrence of an intermediate state. Finally, further research needs are outlined.

Flow cytometry as a tool for measuring the kinetics of IgM-positive cells in the gill and spleen of sea bream juveniles after bath immunization against Photobacterium damselae subsp. piscicida (Phdp)

ABSTRACTFlow cytometry (FC) is a straightforward, highly specific and sensitive procedure, which provides objective and quantitative recording of fluorescent signals from individual cells. FC has been used widely in the area of aquaculture research due mainly to the development of monoclonal antibodies (mabs). Phagocyte activity of head–kidney macrophages of several fish species, antibody production against specific antigens or detection of viruses inside immune cells are some of the several possible analysis approached by this assay. The aim of this work is to analyse and to compare kinetics of IgM-positive cells in the gill and spleen of sea bream juveniles after bath immunization against Photobacterium damselae subsp. piscicida (Phdp) using a direct staining method. Fish were immunized with three different inactivated vaccines prepared with 94/99 antigen and a commercial vaccine was used as a positive control. The effect on IgM-positive cells post-vaccination was compared among groups. Results show that the direct staining method with mabs is an effective and repeatable method for staining sea bream IgM-positive cells.

Direct fast scarlet 4BS染色液調整法の検討

Direct fast scarlet 4BS染色液調整法の検討

Study on Epitenon Fibroblast Migration after Incising and Suturing the Tendon with Modified Kessler in New Zealand Rabbits

Introduction: In our study, we present a direct staining method of epitenon fibroblasts with the dye Dil (1,1΄- dioctadecyl -3,3,3΄,3΄-tetramethylindocarbocyanine percholate) using an animal model, and furthermore we observe the migration of these cells. Our methodology tries to imitate what actually happens in clinical practice, as far as possible. Material and methods: In 36 New Zealand rabbits, the flexor digitorum profundus tendon of right anterior leg was pulled out of its sheath, incised and sutured with modified Kessler. The tendon was stained with the dye Dil and placed back to its sheath, where it was fixed distally. Tendon specimens were taken from 6 rabbits and examined under fluorescence microscopy at 1, 3, 5, 7, 14 and 28 days postoperatively. In order to evaluate our results, the fibroblasts' migration was divided into 4 phases: first phase, all the stained cells are on the tendon surface; second phase, there is only sporadic migration of fibroblasts under the tendon surface; third phase, there is massive migration of cells below the tendon surface; fourth phase, the cells are deeper into the tendon substance. Results: The majority of epitenon's fibroblasts in the first 24 hours were found to be in the first phase (all stained cells were at the tendon's surface and only some cell clusters were found to be in the second phase). On the 3 rd day, the majority was found to be in the third phase (massive migration under tendon's surface), and on the 5 th day

Study on the Extraction of Ginkgo and Dyeing Tussah

In this study the natural dye was extracted from Ginkgo leaves with the ethanol solution and then the obtained dye was applied to dye tussah silk strips with direct staining methods. The best dye conditions of the tussah silk strips could be obtained when the bath ratio was 1:50, the concentration of ginkgo pigment was 10%, the temperature of dyeing was 70°C and the time of dyeing was 60 minutes. The different colors could be obtained under different experimental conditions. The color of sample which was dyed with pigment which reacted with NaOH solution was bright and yellowish green and the color of sample which was directly dyed with pigment was orange.

Kingella Kingae Osteoarticular Infections in Young Children: Clinical Features and Contribution of a New Specific Real-time PCR Assay to the Diagnosis

Kingella kingae is an emerging pathogen that may be recognized as the most common bacteria responsible for osteoarticular infections (OAI) in young children. However, its diagnosis remains a challenge and thus little evoked in infants, because K. kingae is a difficult germ to isolate on solid medium, and clinical signs are often mild. The main objective of this prospective study is to describe the clinical, biologic, and radiologic features of children with OAI caused by K. kingae. In addition, we describe the usage of a new specific real-time PCR assay in children under 4 years admitted for OAI with a probe that detects 2 independent gene targets from the K. kingae RTX toxin. All children less than 4 years admitted in our institution between January 2007 and November 2009 for suspected OAI were enrolled in this prospective study (43 cases). Age, gender, clinical signs, duration of symptoms, bone or joint involved, imaging studies, and laboratory data, including bacterial investigations, full blood count, erythrocyte sedimentation rate, and serum C-reactive protein were collected for analysis. Identification of the microorganism was possible for 28 cases (65.1%) yielding K. kingae in 23 cases (82.1%). Mean age of children with K. kingae OAI was 19.6 months. Less than 15% of these patients were febrile during the admission, but 46% of them presented a history of fever-peak superior to 38.5 degrees C before admission. Thirty-nine percent of the children with K. kingae OAI had normal C-reactive protein; WBC was elevated in only 2 cases, whereas 21 patients had abnormal erythrocyte sedimentation rate, and 13 abnormal platelet counts. Direct Gram staining and classical isolation methods were negative for all cases subsequently detected as K. kingae OAI by specific real-time PCR. This study confirms that K. kingae is the major bacterial cause of OAI in children less than 4 years. The real-time PCR assay, specific to the K. kingae RTX toxin, provides interesting diagnostic performance when implemented in the routine microbiologic laboratory. Needless to say, a bigger cohort is required to adequately study this new qPCR assay, but the results so far seem promising. The most important additional finding is the mild-to-moderate clinical, radiologic, and biologic inflammatory response to K. kingae infection with the result that these children present few criteria evocative of OAI. II.

Detection of fetal nucleated red blood cells in the maternal circulation by Kleihauer test

Maternal blood was obtained from 18 pregnant women at 7 to 25 weeks of gestation. After Percoll discontinuous density gradient centifugation, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test. Positive fetal cells appeared with an intense red cytoplasmic staining while maternal cells with adult haemoglobin were colourless. Individual positive NRBC was collected by micromanipulator and whole genome amplification was then performed to determine sex and STR status. This allowed the simultaneous verification of the fetal origin of NRBC and prenatal diagnosis of genetic diseases. The non-invasive prenatal genetic diagnosis of 9 fetuses at high risk of Duchenne muscular dystrophy (DMD) was completed successfully. The Kleihauer test is a rapid, simple and direct chemical staining method to select fetal cells and can be applied in prenatal diagnosis.

Cytotoxicity and antimicrobial test of the bioactive compound isolated from Stylissa flabelliformis sponge

A research of cytotoxicity and antimicrobial test of bioactive compound of Stylissa flabelliformis sponge have been done. Cytotoxicity test were conducted on myeloma cells, while antimicrobial test were conducted against Staphylococus aureus,Eschericia coli and Candida albican. The research was initiated with the isolation of bioactive compound from the active fraction of sponge S. Stylissa monitored by Brine shrimp Lethality tes (BST). The active fraction was chromatograped with silica gel as its immobile phase and the mixture of hexane and ethyl acetate (1:1 ) as the mobile phase to obtain compound 1,2,3 and 4. Compound 3 has the most toxic character because it could kill A. salina equal to 100% at dose 25 mg/ml, and has LC50 of 0,9 mg/ml. Cytotoxicity test with direct staining method using tripan blue on myeloma cell at density of 4,5x10 cell / 100.ul showed that the compound 3 had a high activity against myeloma cell, having LC50 equal to 0,08 mg/ml. The result of antimicrobial effect showed that compound 3 was a fungicide against Candida albicans but having no activity on Staphylococcus aureus and Eschericia coli Key words: sponge, Stylissa flabelliformis , cytotoxicity, fungicide

High Peripheral Blood CD34 Count in Patients with Idiopathic Myelofibrosis (IF) Is Not Due to Fibrosis and Altered Marrow Microenvironment.

Idiopathic myelofibrosis (IF) is due to an acquired somatic mutation(s) of a single hematopoietic multipotent progenitor and thus circulating myeloid, erythroid, megakaryocyte, B and T cells (Reeder et al Blood . 2003; 101: 1981–1983) are clonal. It has been suggested that fibrosis may occur secondary to cytokines secreted by the malignant clone. Although marked elevation of circulating CD34 cells occurs in patients with IF, it is not known whether it is a consequence of the primary defect of the hematopoietic stem cell, or secondary to fibrosis and impaired marrow microenvironment. To answer this question, we studied CD34 count in peripheral blood of patients with IF and secondary myelofibrosis due to pulmonary hypertension. Nineteen patients with either primary or secondary pulmonary hypertension due to connective tissue disease and 11 patients with IF were studied. Bone marrow biopsy revealed fibrosis in all 19 patients with PH: 11 severe, 3 moderate, and 5 mild. All eleven patients with IF had severe fibrosis. Clonality studies in 13/14 informative patients with PH showed polyclonal platelets and granulocytes in contrast to monoclonal platelets and granulocytes in patients with IF. Peripheral blood was assayed for CD34+ cells using a three-color direct immunofluorescent staining method. Mean absolute CD34 count was significantly (P=0.02) lower - 6/ul (range: 1–18/ul) in patients with PH compared to 142 /ul (range 4–833/ul) in patients with IF. We therefore conclude that high CD34 count is not seen in patients with PH with secondary myelofibrosis; furthermore, elevated CD34 in patients with IF is not due to marrow fibrosis and altered microenvironment.

한국에서 분리한 Tobacco rattle virus(TRV-K)의 특성

Tobacco rattle virus(TRV) was detected from Gladiolus hybridus, Crocus spp. and Narcissus spp. leaves show-ing notched or stripe on the leaf and malformation symptoms collected from Daegu and Kyungbuk province by electron microscopy (EM), immunosorbent electron microscopy (ISEM) and host range study. Direct negative staining method by EM showed rigid rod long particles 170~20022 nm and rigid rod short particles 40~11422 m. TRV-K isolated from G. hybridus propagated with Nicotiana tabacum. TRV coat protein(CP) gene was amplified using specific oligonucleotide primer by RT-PCR. Sequence analysis of amplified CP gene showed 99.5％ nucleotide similarity to TRV-ORY.

땅콩(Arachis hypogaea)에서 분리한 Bean common mosaic virus와 Peanut mottle virus

한국산 땅콩에 감염되어 있는 바이러스를 동정하기 위하여 땅콩 재배지 에서 모자이크, 괴저를 동반한 얼룩무늬, 황화, 줄무늬, 엽맥녹대, 위축 등의 바이러스 감염 증상을 나타내는 땅콩잎 및 땅콩을 채집하였다. 이들 시료로부터 기주범위, 면역전자현미경(ISEM), 감염 세포내의 바이러스의 존재양식, direct immune staining assay(DISA), RT-PCR 등에 의하여 Bean common mosaic virus(BCMV-PSt)와 Peanut mottle virus(PeMoV)를 분리하였다. 이들 시료를 DN법에 의거하여 전자현미경으로 관찰한 바 길이가 약 780 nm의 사상형 입자와 세포질 봉입체를 관찰하였다. 초박절편의 시료 관찰에서도 길이 약 700 nm의 사상 입자가 엽육세포 등의 세포질과 액포에 산재 혹은 병행 배열로 존재해 있는 영상 및 세포질 봉입체가 반드시 확인되었다. 또한, 이들 시료를 BCMV-PSt와 PeMoV의 항혈청으로 ISEM을 실시한 결과, 두 항체에 모두 decoration 되었음을 확인하였다. 두 바이러스가 종자전염이 되는 지를 확인하기 위하여 땅콩을 직접 BCMV-PSt와 PeMoV의 항혈청으로 DISA를 실시하였다. 그 결과, BCMV-PSt 및 PeMoV 항혈청에 발색이 되었으며, 이들을 발아시켜 유식물체를 획득하였다. 이들 유식물체에서도 바이러스 감염 증상인 얼룩무늬 증상이 관찰되었고, 이들을 BCMV-PSt와 PeMoV의 항혈청으로 ISEM방법으로 검경한 결과, 두 항체에 모두 decoration 되었다. 또한, 자연감염 식물체 및 DISA에 의해 바이러스 감염이 확인된 종자를 발아시킨 2년생 식물체로부터 생물검정 법 및 ISEM에 의해 두 종의 바이러스가 검출되었다. Rl-PCR을 실시한 결과, 약 1.2Kb크기의 BCMV-PSt coat protein유전자가 증폭 되었다. 이 연구결과, 한국산 땅콩에 BCMV-PSt가 가장 많이 감염되어 있음을 확인하였다. For the survey of viruses infected in peanut cultivated in Korea, peanut seeds and leaves showing viral symptoms were collected from their growing areas. Typical symptoms on virus infected peanut leaves including mosaic, mottle with necrosis, yellowing, stripe or vein banding and stunts were observed. Two viruses isolated from the naturally infected peanuts were identified as Bean common mosaic virus(BCMV-PSt) and Peanut mottle virus(PeMoV) by their host range, immunosorbent elcetron microscopy(ISEM), direct immuno staining assay(DISA), RT-PCR, and intracellural symptoms. Direct negative staining method by electron microscope showed filamentous particles of about 780 m in length as well as inclusion bodies. In ultrathin sections of BCMV-PSt and PeMoV infected tissues, cytoplasmic cylindrical inclusions as well as filamentous virus particles were observed in the cytoplasm of parenchyma cells. ISEM revealed filamentous particles strongly decorated with antiserums of BCMV-PSt and PeMoV Peanut seeds were stained with BCMV-PSt and PeMoV antisera indicating the possibility of seed transmission far these viruses. Seedlings germinated from peanut seeds which reacted with antiserums of BCMV-PSt by DISA showed mild mottle or stripe symptoms while mosaic and necrotic mottle symptoms were observed for PeMoV-positive seedlings. Filamentous particles were strongly decorated with each antiserum under ISEM observation. BCMV-PSt coat protein gene of about 1.2 Kbp was amplified by RT-PCR. Altogether these results indicate that BCMV-PSt is the most prevalent virus infecting peanut in Korea.

A direct-staining method to evaluate the mucoadhesion of polymers from aqueous dispersion

A novel technique to evaluate polymer adhesion to human buccal cells following exposure to aqueous polymer dispersion, both in vitro and in vivo, is described. Adhering polymer has been visualised by staining with 0.1% (w/v) of either Alcian blue (60 min) or Eosin (10 min) solution, uncomplexed dye being removed by 0.25 M sucrose washings. The extent of polymer adhesion was quantified by measuring the relative staining intensity of control and polymer-treated cells by image analysis. In vitro, Carbopol 974P, polycarbophil (Noveon AA-1) and chitosan (CL 113) were found to adhere to human buccal cells from 0.10% (w/w) aqueous dispersions of these polymers. Following in vivo administration as a mouthwash, these polymers persisted upon the human buccal mucosa for at least 1 h.

Use of the Kleihauer test to detect fetal erythroblasts in the maternal circulation

Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythrocytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during pregnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythroblast enrichment step. In the present study, after an erythroblast enrichment step on a ficoll gradient followed by a positive immuno-magnetic selection with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless. Preservation of the cytoplasmic integrity allows one to distinguish morphological characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was tested by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogenetic or sonographic sex determination. For 12 pregnancies fetal sex was determined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer test. The selection method was applied to a pregnancy at risk for cystic fibrosis (CF). After a Kleihauer test, fetal erythroblasts were collected by microdissection, whole genomic DNA was amplified by primer extension pre-amplification (PEP) followed by a nested CF PCR. The fetal genotype was successfully characterized and confirmed by conventional prenatal diagnosis.