A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Clinical application of T-spot test of Mycobacterium tuberculosis infection for diagnosis of suspected pulmonary tuberculosis patients / 解放军医学杂志

Objective To explore the application value of T-spot test of Mycobacterium tuberculosis infection (T-SPOT.TB) on diagnosis and differential diagnosis of pulmonary tuberculosis.Methods From Apr.2014 to Dec.2016,700 patients with suspected pulmonary tuberculosis were collected,venous blood (5ml) was drawn off and sputum was collected from each patient separately for T-SPOT.TB and pathogens identification (including TB).Chest CT,bronchoscopy brush or biopsy histopathological examination were followed up,cultivation of My.tuberculosis and of common bacteria with sputum or lavage fluid when needed.T-SPOT.TB test was performed according to the kit instruction operation.2.5 × 105 peripheral blood mononuclear cells (PBMCs) were added into the pre-coated anti-human γ-interferon antibody,and co-incubated separately with two specific My.tuberculosis antigens,namely early secretory targeting 6 (ESAT-6) and culture filtration protein 10 (CFP-10),and then the spot forming cells (SFCs) were counted.The gold standard for present study were set as follows:1) My.tuberculosis smear positive or culture positive;2) Clinical diagnosis (meet any one is positive).The efficacy of T-SPOT.TB on diagnosing active TB was observed,and then the optimal critical value for diagnosing active TB was determined.Patients diagnosed as active TB were divided into 4 subgroups:initial treatment group,retreatment group,smear or culture positive group,and smear or culture negative group.T-SPOT.TB was carried out to detect A and B antigen,and the difference of formed SFCs was then compared.The present study was approved by the Ethics Committee of Xinjiang Uygur Autonomous Region Chest Hospital.Results Of 700 cases suspected of pulmonary tuberculosis enrolled in present study,528 out of 624 definite cases (84.6％) were finally diagnosed as active tuberculosis (active TB group) and 96 cases (15.4％) were as without TB infection (non-TB group).Positive results of T-SPOT.TB test were found in 414 cases in active TB group,and 47 cases in non-TB group were reported with T-SPOT.TB negative.The sensitivity and specificity of T-SPOT.TB test for diagnosing active TB were 78.4％ and 49％,respectively.The positive predictive value,negative predictive value,positive likelihood ratio and negative likelihood ratio were 89.4％,29.2％,1.537 and 0.441,respectively.ROC curve showed that the specificity increased significantly (from 49％ to 62.5％) while the sensitivity decreased (from 78.4％ to 72.7％) when antigen A (cut-off:16.0 SFCs/2.5 × 105 PBMC) was combined with antigen B (cut-off:7.0 SFCs/2.5 × 105 PBMC) for analysis.In addition,the number of A and B antigen spots in active TB group was significantly higher than that in non-TB group (P＜0.01).The number of B antigen spots in positive TB group was significantly higher than that in negative TB group (P＜0.05).There was no significant difference among the other groups.Conclusions Since the high sensitivity and low specificity of T-SPOT.TB in the diagnosis of active TB,the final diagnosis should be combined with clinical manifestations.When the A antigen is 16.0 SFCs/2.5 × 105 PBMC and the B antigen is 7.0 SFCs/2.5 × 105 PBMC,the specificity of T-SPOT.TB will be improved.Higher number of spots has a certain reference diagnostic value for active TB.

Prevalence of Norovirus and epidemiology of acute gastroenteritis in children

Pediatric gastroenteritis is a major cause of childhood mortality and morbidity worldwide, especially in developing countries. Diarrhoea can be caused by a variety of different pathogens including bacteria, viruses and parasites. Among the viruses; Rotavirus has been extensively studied and is responsible for 44% of GE cases. As the Rotavirus vaccination coverage improves, the causative agent’s shift may be more towards the Calciviruses (Norovirus, Sappovirus) and other similar viruses, and consequently the investigations should focus on these viruses in future. This study was conducted in a Teaching hospital, Hyderabad, Telangana State included 118 cases of Gastroenteritis of which 6 cases were positive for Norovirus (NoV) i.e.; 5% of cases were NoV positive by RIDASCREEN EIA and RIDA QUICK, the rapid test for NoV virus. These 6 cases were children between 7 months and one and half year old. The age profile showed a fall in the number of diarrhoea cases as the child’s age increases. 63 (53.3%) were male children and 55 (46.6%) were female children. In children < 2 years (n=83), 22 (26.5%) were breast fed, 30 (36.1%) were bottle fed and 7 (8.4%) were on mixed Alimelu M, Radha Mohan M., Vindhya Tuladi, Sudhershan Reddy P, Shailaja V.V., Preeti Nagaraj G. Prevalence of Norovirus and epidemiology of acute gastroenteritis in children. IAIM, 2016; 3(6): 157-163. Page 158 feeds. Among mothers 25.42% never washed their hands with soap, 60.16% used soap occasionally and only 14.4% always used soap. 43.2 % presented with no dehydration, 27.96% presented with some dehydration and 28.8% presented with severe dehydration. 70.3% of mothers continued to feed during diarrhoea. Regarding the treatment used for diarrhoea before admission in hospital, 42.37% used ORS, 27.11% used both ORS and antibiotics, 55.08% were on antibiotics and 11% took no treatment at all.

Serological investigation of vector-borne disease in dogs from rural areas of China

<p><b>OBJECTIVE</b>To evaluate the Anaplasma phagocytophilum (A. phagocytophilum), Ehrlichia canis (E. canis), Dirofilaria immitis (D. immitis) (canine heartworm), Borrelia burgdorferi (B. burgdorferi) infections in countryside dogs from Yunnan, Hainan and Anhui provinces.</p><p><b>METHODS</b>Serum samples were collected from 26 dogs in Yunnan, Hainan and Anhui provinces. The samples were tested using a commercial ELISA rapid diagnostic assay kit (SNAP(®) 4Dx(®); IDEXX Laboratories, Inc. U.S.A.). Meanwhile, indirect immunofluorescence assay (IFA) recommended by WHO was conducted to detect IgG to A. phagocytophilum. Two methods were analyzed and compared.</p><p><b>RESULTS</b>The number of serologically positive dogs for IgG to A. phagocytophilum was only 2 which was from Hainan province and none of the 26 dogs responded positive for E. canis, D. immitis (canine heartworm), and B. burgdorferi by ELISA rapid diagnostic method. The number of serologically positive dogs for IgG to A. phagocytophilum was 13 (50%) by IFA method. Data of the two methods were analyzed by statistical software and the difference was statistically significant (P=0.002).</p><p><b>CONCLUSIONS</b>It can be concluded that IFA method was more sensitive than ELISA rapid diagnostic method. However, we need conduct further and intensive epidemiology survey on tick-born diseases pathogens including A. phagocytophilum, E. canis, D. immitis (canine heartworm), and B. burgdorferi which have public health significance.</p>

Serological investigation of vector-borne disease in dogs from rural areas of China

Objective:To evaluate the Anaplasma phagocytophilum (A. phagocytophilum), Ehrlichia canis (E. canis), Dirofilaria immitis (D. immitis) (canine heartworm), Borrelia burgdorferi (B. burgdorferi) infections in countryside dogs from Yunnan, Hainan and Anhui provinces. Methods: Serum samples were collected from 26 dogs in Yunnan, Hainan and Anhui provinces. The samples were tested using a commercial ELISA rapid diagnostic assay kit (SNAP? 4Dx?; IDEXX Laboratories, Inc. U.S.A.). Meanwhile, indirect immunofluorescence assay (IFA) recommended by WHO was conducted to detect IgG to A. phagocytophilum. Two methods were analyzed and compared. Results: The number of serologically positive dogs for IgG to A. phagocytophilum was only 2 which was from Hainan province and none of the 26 dogs responded positive for E. canis, D. immitis (canine heartworm), and B. burgdorferi by ELISA rapid diagnostic method. The number of serologically positive dogs for IgG to A. phagocytophilum was 13 (50%) by IFA method. Data of the two methods were analyzed by statistical software and the difference was statistically significant (P=0.002). Conclusions: It can be concluded that IFA method was more sensitive than ELISA rapid diagnostic method. However, we need conduct further and intensive epidemiology survey on tick-born diseases pathogens including A. phagocytophilum, E. canis, D. immitis (canine heartworm), and B. burgdorferi which have public health significance.

A Reliable "Direct from Field" PCR Method for Identification of Mycorrhizal Fungi from Associated Roots

A very reliable and specific method for the identification of fungi in ectotrophic mycorrhizal symbiosis was developed using a specific PCR assay based on the amplification of the ITS1 region. To obtain specific data, an ITS-diagnostic assay was carried out that reveals genera and species specific sequences. Here, an application of one method is presented, which covers the identification of pure mycelia, basidiocarps as well as mixed samples such as ectomycorrhizal roots that were mingled with remains of the host plant. For this purpose a protocol was established that allowed the extraction of DNA from single mycorrhizal roots. In order to perform a specific ITS analysis we generated a new ITS-primer (ITS8) by a multiple alignment of five different genera and species of mycorrhizal fungi. The utilization of ITS1 and ITS8 resulted in specific PCR amplicons, which were characterized by sequencing without purification steps, even when the template DNA was associated with roots.