Rapid diagnosis of parasitic diseases: current scenario and future needs

Background

Parasitic diseases are one of the world's most devastating and prevalent infections, causing millions of morbidities and mortalities annually. In the past, many of these infections have been linked predominantly to tropical or subtropical areas. Nowadays, however, climatic and vector ecology changes, a significant increase in international travel, armed conflicts, and migration of humans and animals have influenced the transmission of some parasitic diseases from ‘book pages’ to reality in developed countries. It has also been noted that many patients who have never travelled to endemic areas suffer from blood-borne infections caused by protozoa. In the light of existing knowledge, this new trend can be explained by the fact that in the process of migration a large number of asymptomatic carriers become a part of the blood bank donor and transplant donor populations. Accurate and rapid diagnosis represents the crucial weapon in the fight against parasitic infections.

ICPC-2--international classification of primary health care and its application in Croatian health care

A consensus reached by the medical profession, Croatian Institute of Health Insurance, Croatian Institute of Public Health, and Ministry of Health constitutes a solution to the problem of a data standard required in building an information system for primary health care. This consists of accepting ICPC-2 as a data standard for our Family Medicine, Pediatrics and Women's Health. The classification structure of the International Classification of Primary Care allows, recommends and urges that special codes be established by individual states or local authorities when registering patients' reasons for seeking medical aid or medical procedures. Namely, it urges the authorities to set the codes for such reasons about which a state or local agreement or determination has been made. This is the first public presentation of the proposal Croatia's Additions to the International Classification of Primary Care. They are essential to its implementation in our health insurance, health statistics and medical informatics.     

National public health information system

Information production and its communication being a key public health activity, developing modern information systems is a precondition for its fulfilling these assignments. A national public health information system (NPHIS) is a set of human resources combined with computing and communication technologies. It enables data linkage and data coverage as well as undertaking information production and dissemination in an effective, standardized and safe way. The Croatian Institute of Public Health LAN/WAN modules are under development. Health Safety System, Health Workers Registry, and Digital Library are among the Institute's developmental priorities. Communication between NPHIS participants would unfold over the Internet by using every relevant data protection method. Web technology-based applications would be run on special servers. Between individual applications, use would be made of the transaction module of communication through an exchange of the HL7 standard-based xml messages. In the conditions of transition, the health system must make an optimal use of the resources, which is not feasible without applying modern information and communication technologies.     

Systemic fungal infections in immunocompromised patients

Opportunistic fungal infections are becoming more frequent complications during cancer therapy, after organ transplantation and in AIDS infections, especially after better control of bacterial infections in immunocompromised patients. Periods of prolonged neutropenia with neutrophil count less than 0.5 x 10(9)/L longer than 7 days, are the most important risk factors for the development of systemic fungal infections. Especially susceptible are the patients during treatment of acute leukemia, or after bone marrow transplantation. The most frequent causing agents of systemic fungal infections are Candida and Aspergillus species, than Cryptococcus neoformans and Mucor. Some other unusual species such Fusarium, Trichosporon, non-albicans Candida species of Candida are becoming more frequent, and is frequently resistant to conventional therapy. The difficulties in early and precise diagnosis of fungal infections, and the lack of adequate and efficient drugs are responsible for the high mortality of immunocompromised patients, even in potentially curable diseases. The recognition of risk factors, introduction of prophylactic measures, application of empirical antifungal therapy, are the procedures for the reduction of morbidity and mortality of invasive fungal infections. Fluconazole administration in prevention of systemic fungal infections, has become the standard approach, especially after bone marrow transplantation, while the oral itraconazole solution, has even more extended activity. Fluconazole appears successful also in the treatment of systemic Candidiasis. Conventional amphotericin-B is still the "gold standard" in the treatment of fungal infections. The new lipid formulations of amphotericin-B, intravenous itraconazole, has an identical efficacy, but are less toxic than conventional amphotericin-B. Several new promising agents are in the stage of clinical investigation like voriconazole, caspofungin, mycafungin and some other.     

Modified Dynabeads method for enumerating CD4+ T-lymphocyte count for widespread use in resource-limited situations

The Dynabeads method showed the potential for enumerating CD4 T lymphocytes (CD4 count) in HIV-1-infected individuals. The large volume of Dynabeads required for 1 sample and complex procedure made the method expensive and not easy for use, however. To decrease the cost and simplify the procedure, we reduced the volume of the Dynabeads, added wash times, and skipped over the staining step so as to count the CD4 cells directly under an optical microscope. The CD4 count of 246 blood samples using our modified Dynabeads method (DynabeadsCD4) showed a significant correlation with that obtained by flow cytometry (FlowcytoCD4) (r = 0.91 [P < 0.0001]; slope = 1.03, intercept = -16). The sensitivity and specificity for a CD4 count less than 200 cells/microL were 79% and 94%, and for a CD4 count less than 350 cells/microL, the sensitivity and specificity were 95% and 88%, respectively. The positive and negative predictive values for a CD4 count less than 350 cells/microL were 97% and 83%, respectively. The systematic error was 8 cells/microL (95% confidence interval [CI]: 0.4-16). The cost of Dynabeads for 1 sample was less than $1.00; thus, the estimated cost per DynabeadsCD4 test is less than $3.00, including the cost of other disposable materials. Our modified method is simple, economic, and accurate enough to monitor antiretroviral therapy in resource-limited situations.     

Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan syndrome and other fibrillinopathies

Marfan Syndrome (MFS) is an autosomal dominant disorder of the connective tissue due to mutations of Fibrillin-1 gene (FBN1) in more than 90% of cases and Transforming Growth Factor-Beta-Receptor2 gene (TGFB2R) in a minority of cases. Genotyping is relevant for diagnosis and genotype-phenotype correlations. We describe the FBN1 genotypes and related phenotypes of 81 patients who were referred to our attention for MFS or Marfan-like phenotypes. Patients underwent multidisciplinary pertinent evaluation in the adult or paediatric setting, according to their age. The diagnosis relied on Ghent criteria. To optimise DHPLC analysis of the FBN1 gene, all coding regions of the gene were directly sequenced in 19 cases and 10 controls: heterozygous amplicons were used as true positives. DHPLC sensitivity was 100%. Then, DHPLC was used to screen 62 other cases. We identified 74 FBN1 mutations in 81 patients: 64 were novel and 17 known. Of the 81 mutations, 41 were missense (50.6%), 27, either nonsense or frameshift mutations and predicted a premature termination codon (PTC) (33%), 11 affected splice sites (13.6%), and two predicted in-frame deletions (2.5%). Most mutations (67.9%) occurred in cbEGF-like modules. Genotype was clinically relevant for early diagnosis and conclusion of the diagnostic work-up in patients with incomplete or atypical phenotypes.