Drug targets and mechanisms of resistance in the anaerobic protozoa

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated ruing laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Total Daily Energy Expenditure and Incidence of Upper Respiratory Tract Infection Symptoms in Young Females

A group of 31 young females, tennis players and non-athletes, aged 16 2 years (range: 14 - 21 years), with a wide range of physical activity levels was used to investigate the relationship between total daily energy expenditure and the incidence of upper respiratory tract infection symptoms. Methods: During a 12 week winter period, habitual daily activity (excluding training) was evaluated using a 3-day physical activity record. Tennis training was quantified using a validated method of estimating energy expenditure during play. Total daily energy expenditure was calculated from the sum of daily training plus mean habitual daily activity energy expenditures. The total group of subjects was divided in quartiles for total daily energy expenditure. A validated symptom checklist was used to assess the incidence and severity of upper respiratory tract infections, on a daily basis. Results: The girls in the highest quartile of total daily energy expenditure (greater than or equal to 17322 kJ/day) and in the lowest quartile (less than or equal to 10 047 kJ/day) had the greatest incidence of URTI symptomatology, although the moderately active girls in quartile three (12290-16410 kJ/day) presented the lowest incidence. Significant differences in number of upper respiratory tract infection episodes, sickness days and symptomatology index were found between quartiles three and one (p < 0.05) and quartiles three and four (p < 0.01). Peak severity of symptoms was significantly lower in quartile three compared with all other quartiles (p < 0.05).

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng, 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids. glucose and yeastolate ultrafiltrate) into Sf9 cell cultures grown in SF900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 mug/ml of NS1 at a peak cell density of 4.2 x 10(6) cells/ml. In contrast. optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 mug ml of NS1 produced at a peak cell density of 11.3 x 10(6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10(6) cells/ml, unless the cultures were stimulated by the addition of 4 mug/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 mug/ml. which was nearly 5-fold higher than optimised batch cultures. (C) 2002 Elsevier Science B.V. All rights reserved.

Detection of Influenza type A virus by LightCycler RT-PCR

Using the Roche LightCycler we developed a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the Influenza A LightCycler RT-PCR (FA-LC-RTPCR) for the rapid detection of Influenza A. The assay was used to examine 178 nasopharyngeal aspirate (NPA) samples, from patients with clinically recognised respiratory tract infection, for the presence of Influenza A RNA. The results were then compared to a testing algorithm combining direct immunofluorescent assy (DFA) and a culture augmented DFA (CA-DFA) assay. In total, 76 (43%) specimens were positive and 98 (55%) specimens were negative by both the FA-LC-RTPCR and the DFA and CA-DFA algorithm. In addition, the FA-LC-RTPCR detected a further 4 (2%) positive specimens, which were confirmed by a conventional RT-PCR method. The high level of sensitivity and specificity, combined with the rapid turnaround time for results, makes the LC-RT-PCR assay suitable for the detection of Influenza A in clinical specimens.

Ex vivo profiling of CD8(+)-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Telehealth responses to bio-terrorism and emerging infections

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS), are of huge economic importance. They are difficult to predict. The World Health Organization has a Global Outbreak Alert and Response Network, which was involved at an early stage in the SARS outbreak in 2003. Three major lessons were learned as a result of the SARS epidemic in 2003, involving communication, evidence-based action and global partnerships. It is proposed that a series of broadband global response networks should be developed. At a technical level the networks are essentially in place, such as the Internet2 global network. Suitable peripheral devices also exist. What has not yet been created is the appropriate software to allow the use of these networks, although a number of commercial products are in the process of development.

The medicine and epidemiology of bovine respiratory disease in feedlots

Bovine Respiratory Disease (BRD) results from a complex, multifactorial interaction of stressors, animal susceptibility, and respiratory pathogens. The infectious agents associated with BRD are ubiquitous among cattle populations. Typically, one or a combination of stressors are necessary to initiate BRD. Prevention of BRD should, therefore, address management procedures to minimise stressors. Administration of vaccines against BRD agents may help reduce the incidence of BRD but is unlikely to eliminate the condition. The effectiveness of antimicrobials in the treatment of BIRD depends primarily on early recognition and treatment. The use of antioxidant vitamins, minerals or other agents in the prevention and treatment of BRD warrants further research.

Virus-Resistance Genes: The Mouse Model

Os Homens e os animais diferem grandemente nas suas respostas às infecções virais. Os vírus podem induzir, em alguns, sintomas ligeiros, enquanto que em outros podem provocar patologias graves mesmo mortais. Acumulam-se evidências que o património genético é um dos factores primordiais a condicionar e contribuir para a complexidade das interações vírus-hospedeiro. A identificação de genes com papel na resposta à infecção viral tornouse pois o tema de investigação de muitos laboratórios, com o objectivo de elucidar os processos fisiopatológicos que regem e determinam esse tipo de resposta. Neste artigo de revisão pretende-se ilustrar como o modelo murino têm sido utilizado para a identificação de genes de resistência viral, e como estes podem funcionar como base para a descoberta de genes homólogos em outras espécies. na elucidação dos mecanismos de resistência, e em novos componentes da reunir todos os genes de resistência viral descobertos em murganhos.

Reliability of nasal auscultation and their relation to middle ear condition – a clinical interest to pediatrics respiratory physiotherapy

Background: Acute respiratory infections are usual in children under three years old occurring in upper respiratory tract, having an impact on child and caregiver’s quality of life predisposing to otitis media or bronchiolitis. There are few valid and reliable measures to determine the child’s respiratory condition and to guide the physiotherapy intervention. Aim: To assess the intra and inter rater reliability of nasal auscultation, to analyze the relation between sounds’ classification and middle ear’s pressure and compliance as well as with the Clinical Severity Score. Methods: A cross-sectional observational study was composed by 125 nursery children aged up to three years old. Tympanometry, pulmonary and nasal auscultation and application of Clinical Severity Score were performed to each child. Nasal auscultation sounds’ were recorded and sent to 3 blinded experts, that classified, as “obstructed” and “unobstructed”, with a 48 hours interval, in order to analyze inter and intra rater reliability. Results: Nasal auscultation revealed a substantial inter and intra rater reliability (=0,749 and evaluator A - K= 0,691; evaluator B - K= 0,605 and evaluator C - K= 0,724, respectively). Both ears’ pressure was significantly lower in children with an "unobstructed" nasal sound when compared with an “obstructed” nasal sound (t=-3,599, p<0,001 in left ear; t=-2,258, p=0,026 in right ear). Compliance in both ears was significantly lower in children with an "obstructed" nasal sound when compared with “unobstructed” nasal sound (t=-2,728, p=0,007 in left ear; t=-3,830, p<0,001 in right ear). There was a statistically significant association between sounds’ classification and tympanograms types in both ear’s (=11,437, p=0,003 in left ear; =13,535, p=0,001 in right ear). There was a trend to children with an "unobstructed" nasal sound that had a lower clinical severity score when compared with “obstructed” children. Conclusion: It was observed a good intra and substantial inter reliability for nasal auscultation. Nasal auscultation sounds’ classification was related to middle ears’ pressure and compliance.

Serologic studies on the behaviour of influenza virus type A in persons of greater São Paulo during 1976, 1978 and 1979

Sera of persons of different age groups collected in 1976, 1978 and 1979 were tested for the presence of HI antibodies against various strains of the H3N2 and H1N1 subtypes of influenza virus. The occurrence of infection by H3N2 subtype was recorded during the 1976-1978 period but in 1979, circulation of this subtype of virus was limited. The prevalence of antibody against A/São Paulo/1/78 (H1N1) was significantly higher than that of antibody to A/USSB/90/77 (HIND in 1978. However in 1979 the predominant strain was A/USSR/90/77 (HIND. Persons under 20 years of age were the most affected by H1N1 subtype. Antibodies to H1N1 subtype were detected in sera of individuals older than 20 years in 1976, before the re-emergence of this strain. Serological results indicate that infections by H3N2 subtype in 1978 occurred in 65.4% of young children (0-4 year group). About 47.0% of children from the 0-4 year group had antibodies to H1N1 subtype in 1979. Antibodies to swine influenza virus were detected in 60% of 60+ year old people.

Cytomegalovirus and other herpesviruses infections in heart and bone marrow transplant recipients

From January 1988 to January 1989 all the heart transplant and bone marrow recipients at the Instituto do Coração of the Hospital das Clínicas of the University of São Paulo Medical School were studied for the incidence and morbidity associated with herpesviruses infections after transplantation. Five bone marrow and 5 heart transplant recipients were followed for a mean of 4.2 months post-transplantation. All the patients were seropositive for cytomegalovirus (CMV) before admission and 80% experienced one or more recurrences during the observation period. Of the 12 episodes of CMV infection, that were identified in this study, 83% were accompanied by clinical or laboratory abnormalities. However, there was only one case of severe disease. The overall incidence of infection for herpes simplex (HSV) was 50%. Although most of HSV reactivations were oral or genital, one case of HSV hepatitis occurred. One of the 6 episodes of HSV infections that were treated with acyclovir showed an unsatisfactory response and was successfully managed with ganciclovir. All the individuals had anti-varicella zoster virus antibodies, but none of them developed infection. The study emphasizes the importance of active diagnostic surveillance of herpesvirus infections in transplant patients. Both CMV and HSV reactivations showed high incidence and important morbidity and thus, deserve prophylactic therapy.

The frequency of blood-born viral infections in a population of multitransfused Brazilian patients

The frequency of viral markers for hepatitis B (HBV) and C (HCV), human immunodeficiency virus-1 (HIV-1) and human T-lymphotropic virus-1 (HTLV-1) was evaluated in 32 Brazilian ß-thalassemia multitransfused patients. Additionaly the serum concentrations of ferritin and alanine aspartate transaminase (ALAT) were determined. The results show a high prevalence of markers of infection by HBV (25.0%) and HCV (46.8%) and a low prevalence of markers for HIV-1 and HTLV-1. No correlations were demonstrated between the presence of the hepatitis markers and the number of units transfused or the serum concentrations of ferritin and ALAT.

Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients

Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15% and reached 86.5% for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8% of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7% (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.