Study of dirofilariasis in a selected area in the Western Province.

INTRODUCTION: Human dirofilariasis is a zoonotic infection caused by the filarial worm, Dirofilaria (Nochtiella) repens, whose primary host is the dog. This infection is on the increase over the past decade in Sri Lanka and the prevalence of canine dirofilariasis in the country is also believed to be high. We present here a study on public awareness of dirofilariasis and the prevalence of this infection in dogs in Negombo, an urban area that has a high domestic canine population. OBJECTIVE: To assess the awareness of dirofilariasis infection among residents and study the prevalence of this infection in domestic dogs in Negombo. DESIGN: Prospective study. SETTING: Department of Parasitology, Faculty of Medicine, University of Colombo, Colombo 8, Sri Lanka. METHODS: A descriptive cross-sectional study within the city of Negombo during September and November 2003 using a pre-tested, interviewer-administered questionnaire with cluster sampling was done. Two hundred seventy adults, including 132 dog owners, were included in the study. Data analysis was done using the Epilnfo programme. The prevalence of canine dirofilariasis was studied in a group of 65 dogs over the age of 1 year. They were selected by cluster sampling with random choice of the streets within the study area. Stained thick blood films, made following an earlobe-prick at any time during the day, were microscopically examined for the presence of microfilariae. RESULTS: Forty nine of the respondents (18%) were aware of the existence of canine dirofilariasis while human dirofilariasis was known only to 6%. Awareness was related to the level of education. Knowledge of canine filariasis was better among pet owners (32/132) when compared to others (18/138; p < 0.05). Veterinary surgeons were acknowledged as the source of information by 38% of those who were aware of the disease. Forty five per cent (n = 29) of the dogs screened were positive for microfilariae. Out of these 18 and two dogs were infected with D repens. and B. ceylonensis, respectively, while nine others were co-infected with the two parasites. CONCLUSIONS: The knowledge of dirofilariasis is extremely poor in the study area. The high prevalence of filarial infections in dogs highlights the importance of improving the public awareness of this disease, especially among the dog owners. Proper management of this condition in dogs-the reservoirs of infection for human dirofilariasis, is important for the control of this zoonotic infection.

Semiautomated data analysis of flow cytometric estimation of fetomaternal hemorrhage in D- women.

BACKGROUND: Accurate and reliable measurement of the volume of fetal D+ cells in D- women is required for adequate anti-D prophylaxis. A semiautomated flow cytometry assay based on a standardized calibration curve that was created with simulated fetomatemal hemorrhage (FMH) mixtures was developed. STUDY DESIGN AND METHODS: A calibration range of 0.083- to 2-percent D+ cells in the D-RBC mixtures (2-44 mL calculated FMH) was analyzed by use of a flow cytometer (XL-MCL, Coulter Electronics Ltd). Linear regression analysis of the calibration curve data with computer software (Excel, Microsoft) allowed semiautomated determination of the FMH volume. To optimize the assay, fresh versus frozen and thawed RBCs, RBCs from adults who are heterozygous for D or cord RBCs, and indirect- or direct-labeling techniques were evaluated by use of MoAbs. RESULTS: Fresh RBCs from adults heterozygous for D were chosen for routine use, although equivalent calibration curves were obtained with all cells tested (n = 12 calibration assays; r2 = 0.999; mean SD, 14%). A monoclonal anti-D reagent (Therad 10, Diagnostics Scotland) worked well in both indirect-(anti-IgG F(ab)-FITC) and direct-(anti-D-FITC) labeling methods compared to the use of BRAD-3 FITC. In routine practice, the FMH volumes obtained were mainly lower than those obtained in the Kleihauer Betke test when there was less than 4 mL of FMH. CONCLUSION: Semiautomated data acquisition and calibration curve analysis represents a further step toward standardization of flow cytometry for accurate FMH quantification and facilitates evaluation and control of day-to-day variations between laboratories, flow cytometers, and operators.

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.

V(D)J germline gene repertoire analysis of monoclonal D antibodies and the implications for D epitope specificity.

BACKGROUND: The D antigen is a highly immunogenic human RBC antigen. Alloimmunization against the D antigen produces high-affinity antibodies that cause hemolytic transfusion reactions and HDN. STUDY DESIGN AND METHODS: Cloning and subsequent sequence analysis of 11 new samples of monoclonal anti-D was performed in an attempt to identify V(D)J germline gene usage. Sequences were compared and analyzed with 37 previously published samples of anti-D for identification of V(H) and V(L) pairings, canonical structures, and conformation of restricted germline gene usage. RESULTS: The V(H) and V(L) pairings used by the new D MoAbs resulted in seven canonical combinations, three of which had not been described previously. Preferential usage of gene segments from the VH3 and VH4 families and of D3, D6, JH6, and DPK9 germline gene segments was also determined. Three samples of anti-D from different donors were found to use similar V(H) and V(kappa) germline genes, despite the fact that two of the antibodies recognized epD6/7 and the third recognized epD1. From the cumulative analysis of the anti-D IgG, 24 V(H) and V(L) gene pairings were identified, resulting in only 10 canonical structures. CONCLUSIONS: Despite the potential for diversity, only a minority of V(H) and V(L) germline genes are used by anti-D. Consequently, V(H) and V(L) pairings and the resulting canonical structures are similarly restricted.

Comparison between hybridoma and Fab/phage anti-RhD: their V gene usage and pairings.

Our 11 anti-RhD's in conjunction with 37 previously published RhD antibodies, produced by hybridoma technology were analysed for germline gene usage and restriction in VH and VL pairings. The 17 VH germline genes used by the hybridoma anti-RhD IgG were derived from 4 VH families (VH1, VH2, VH3 and VH4). Eighteen kappa chains were restricted to only 5 germline genes from only 2 V kappa families (V kappa 1 and V kappa 3). However, the 13 lambda chains were not as restricted, using 10 V lambda germline genes from 4 families (V lambda 1, V lambda 2, V lambda 3 and V lambda 8). Fifty six unique Fab/phage anti-RhD were also analysed. In all cases the Fab/phage VH germline genes were derived from the VH3 family (41/41). The 29 kappa chains were restricted to 4 germline genes primarily from V kappa 1 (97%) and the 24 lambda chains used 10 V lambda germline genes from 5 families (V lambda 1, V lambda 2, V lambda 3, V lambda 4 and V lambda 7). The VH germline genes of the Fab/phage were restricted to 4 of the 17 used by the hybridoma anti-RhD IgG (DP46, DP49, DP50 and DP77). Ninety percent of the Fab/phage were restricted to 1 of the 5 V kappa germline genes used by the IgG (DPK9). However, the repertoire of the V lambda germline genes used in these two systems is different, with analysis showing greater diversity in V lambda gene usage with 8 unique germline genes used by 76% Fab/phage compared to 4 unique genes used by 46% of the IgG hybridoma anti-RhD.

Proteolytic fragmentation of the murine prion protein: role of Tyr-128 and His-177.

The prion protein (PrP) has been proposed to display sequence and structural similarities to membrane-anchored signal peptidases [Glockshuber et al. (1998) FEBS Lett. 426, 291-296]. We have investigated the role of Tyr-128 and His-177 in the proteolytic fragmentation of murine PrP by mutating these residues to Phe and Leu, respectively, and expressing the resultant mutants in the human neuroblastoma SH-SY5Y. Both PrP-Y128F and PrP-H177L were expressed at the cell surface as glycosyl-phosphatidylinositol-anchored forms and were localised in detergent-insoluble membrane domains similar to wild type PrP. Following deglycosylation, the 19 kDa proteolytic fragment PrP-II was present in cells expressing either mutant, indicating that Tyr-128 and His-177 are not involved in the proteolytic fragmentation of PrP.

Changing trends in maternity care in Sri Lanka.

PIP: Sri Lanka's health policy calls for health services to be accessible to all people, which requires appropriate training of physicians, nurses, and paramedicals. Increasing the training and cadre of public health midwives allows Sri Lanka to make free domiciliary and field health services available to all parts of the country. Sri Lanka must adopt realistic measures to improve guidance and supervision of midwives to derive maximum benefits. Sri Lanka first instituted primary health care (PHC) in 1926 (Health Unit at Kalutara). PHC provides 3 stages of maternal and child health care through home and/or clinic visits: prenatal care, trained assistance during delivery, and postpartum care. In most parts of Sri Lanka, 1 family health worker is available for every 3000 people. Yet, maternal mortality is rather high. A UNICEF study shows that family health workers register only about 50% of pregnant women. A lack of personnel to provide maternal health services is a main reason for high maternal mortality. Many medical officers (1979, 52%) have not done an internship in gynecology and obstetrics. Many (1979, 38%) are assigned to peripheral facilities where they practice obstetrics without any help from experienced physicians. They are reluctant to do simple obstetric measures, e.g., removal of a retained placenta. This reluctance keeps some physicians from admitting mothers in labor, so they transfer them to upper-level hospitals. The Postgraduate Institute of Medicine needs to implement courses in obstetrics and gynecology so the district hospitals can have competent obstetricians on staff. The leading causes of maternal death are, in order of frequency, hemorrhage, abortion complications, eclampsia, sepsis, and obstructed labor. Anemia and retained placenta are the reasons for hemorrhage. Insufficient access to life-saving treatment in emergencies is the single most critical weakness in the maternal health care system, indicating a need to establish intensive care units.^ieng

Sri Lanka [Population education in countries of the region].

PIP: Increases in the educational level and allied factors such as late marriage have led to a decrease in the birth rate to 27.6/1000 in Sri Lanka. The World Fertility Survey in 1975 revealed that the average number of children desired was only 2.4 although the average number per family was 3.9, indicating the need for population education especially at the school level. Population education components were introduced to the junior secondary level, grades 6-9, in stages beginning in 1974 following institution of the Population Education In-School Project at the Curriculum Development Centre of the Ministry of Education. Population components were introduced into language, mathematics, science, health science, and social studies curricula already being taught. The longrange objectives of the program are to prepare future citizens who will be knowledgeable about the impact of population growth on the quality of life, and to promote responsible attitudes and decisions regarding family size. Immediate objectives are to promote understanding of population dynamics and encourage responsible attitudes. The program strives to avoid conflict with sociocultural and socioreligious norms of the various population sectors. Teacher training was provided in residential seminars, and through course guides and reference books. Reorganization of the educational structure led to a brief period of inactivity for the Population Education Unit, but work began again in 1980. The Non-Formal Education Branch of the Ministry of Education is ragarded as a possible area for introduction of population education components in adult education. Most population education programs directed toward adults are conducted by other ministries. The main problems in implementation of the junior secondary level population education program have been initial training of teachers and provision of resource and reference materials; use of the mobile library donated by the UNESCO Population Education Clearinghouse is expected to help alleviate the latter problem. An innovative method of inservice teacher training utilized by the program is the use of a network of master teachers as inservice advisors.^ieng