Evolution of genotypes of Group A streptococci from colonization and infection

Dissertação para obtenção do Grau de Doutor em Biologia

Single step polymerase chain reaction (PCR) for the diagnosis of the Leishmania (Viannia) subgenus

In Brazil, the main etiologic agent of Leishmaniasis that frequently presents with mucosal involvement belongs to the Viannia subgenus. The therapeutic conduct in this disease depends on the parasitological diagnosis, and classical methods are restricted in identifying the agent. In this paper we describe a polymerase chain reaction (PCR), which uses primers designed from mini-exons repetitive sequences. The PCR amplifies a 177bp fragment that can distinguish (Viannia) from (Leishmania) subgenus. This test could be a useful diagnostic tool.

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore, our results confirm the potential usefulness of PCR serial screening and the clinical applicability in everyday routine. PCR screening offers a noninvasive repeatable aid to the diagnosis of IFI.

Clinical characteristics and evolution of syphilis in 24 HIV+ individuals in Rio de Janeiro, Brazil

A total of 24 patients with syphilis and HIV infection were treated from January 1997 to March 2003 at the Infectious Dermatology Outpatient Clinic of the Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. The caseload consisted of 20 males (83.3%) and four females (16.7%), with a mean age of 38.04 years and mean T CD4+ count of 389.5 cells/mL. Syphilis was diagnosed as secondary in 16 (62.5%) patients, late latent in eight (33.3%), and tertiary in one (4.2%). Manifestations of secondary syphilis were palmar and plantar erythematopapulous cutaneous lesions in nine (37.5%), papulous exanthema in four (16.7%), patchy alopecia in 3 (12.5%) and osteochondritis in one patient (4.2%). Tertiary syphilis was characterized by verrucous lesions. Neurosyphilis was diagnosed in four patients (16.7%), with headache as the only manifestation in two patients. Drugs used in treatment included benzathine penicillin, ceftriaxone, erythromycin, and crystalline penicillin. Cure was achieved in 18 patients (75%). Five patients (20.8%) were retreated, three of whom presented a history of re-exposure. This study confirms the importance of establishing the diagnosis of neurosyphilis in patients with HIV infection, in addition to performing follow-up on treatment for syphilis.

Abdominal angiostrongyliasis: a case with severe evolution

A case of acute abdomen disease caused by abdominal angiostrongyliasis is reported. A 42-year-old otherwise healthy patient presented with a complaint of nine days of abdominal pain, constipation, disury, fever and right iliac fossa palpable mass. Exploratory laparotomy was performed. After surgical treatment the patient presented serious complications.

Comparison of serology, antigenemia assay and the polymerase chain reaction for monitoring active cytomegalovirus infections in hematopoietic stem cell transplantation patients

Forty-six allogeneic hematopoietic stem cell transplantation (HSCT) patients were monitored for the presence of CMV antibodies, CMV-DNA and CMV antigens after transplantation. Immunoenzymatic serological tests were used to detect IgM and the increase in CMV IgG antibodies (increase IgG), a nested polymerase chain reaction (N-PCR) was used to detect CMV-DNA, and an antigenemia assay (AGM) was used to detect CMV antigens. The presence of CMV-IgM and/or CMV-increase IgG antibodies was detected in 12/46 (26.1%) patients, with a median time between HSCT and the detection of positive serology of 81.5 days. A positive AGM was detected in 24/46 (52.2%) patients, with a median time between HSCT and antigen detection of 62 days. Two or more consecutive positive N-PCR results were detected in 32/46 (69.5%) patients, with a median time between HSCT and the first positive PCR of 50.5 days. These results confirmed that AGM and mainly PCR are superior to serology for the early diagnosis of CMV infection. Six patients had CMV-IgM and/or CMV-increase IgG with a negative AGM (five cases) or N-PCR assay (one case). In five of these cases the serological markers were detected during the first 100 days after HSCT, the period of highest risk. These findings support the idea that serology may be useful for monitoring CMV infections in HSCT patients, especially when PCR is unavailable.

Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants

A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4%) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4%) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.

The influence of gait cadence on the ground reaction forces and plantar pressures during load carriage of young adults

Biomechanical gait parameters—ground reaction forces (GRFs) and plantar pressures—during load carriage of young adults were compared at a low gait cadence and a high gait cadence. Differences between load carriage and normal walking during both gait cadences were also assessed. A force plate and an in-shoe plantar pressure system were used to assess 60 adults while they were walking either normally (unloaded condition) or wearing a backpack (loaded condition) at low (70 steps per minute) and high gait cadences (120 steps per minute). GRF and plantar pressure peaks were scaled to body weight (or body weight plus backpack weight). With medium to high effect sizes we found greater anterior-posterior and vertical GRFs and greater plantar pressure peaks in the rearfoot, forefoot and hallux when the participants walked carrying a backpack at high gait cadences compared to walking at low gait cadences. Differences between loaded and unloaded conditions in both gait cadences were also observed.

Indications of carbapenem resistance evolution through heteroresistance as an intermediate stage in Acinetobacter baumannii after carbapenem administration

We describe an in vivo evolution of an antimicrobial profile from susceptibility to full-resistance to carbapenems, with heteroresistance as an intermediate stage, in an Acinetobacter baumannii strain. Heteroresistance was characterized by the growth of sub-populations within the susceptibility halo in both disk-diffusion and Etest. PCRs for the main A. baumannii carbapenemases were negative. The exact resistance mechanism, diagnostic methods and clinical relevance of heteroresistance in A. baumannii warrant further investigations. This is the first description of such phenomenon in vivo and the second report of heteroresistance to carbapenems in A. baumannii.

Accidental infection by Trypanosoma cruzi follow-up by the polymerase chain reaction: case report

We report a case of accidental infection by Trypanosoma cruzi in a 42-year-old female patient who presented an inoculation chagoma. Laboratory confirmation was based on examination of fresh blood, Giemsa-stained blood smear, immunoenzyme test (EIA-IgG), indirect immunofluorescence (IIF-IgM, IgG) and polymerase chain reaction (PCR). Only the PCR gave a positive result, and the EIA test was inconclusive. Two treatments with benznidazole were necessary. PCR was the only technique that continued to give positive results for approximately two months (65 days, or 2.2 months) following the second treatment and negative results from 96 days (3.2 months) to 850 days (28.3 months). We concluded that the presence of an inoculation chagoma and use of PCR were important and decisive for diagnosis and follow-up of the case.

Effects of vitamin C supplementation on acute phase Chagas disease in experimentally infected mice with Trypanosoma cruzi QM1 strain

The tissue changes that occur in Chagas disease are related to the degree of oxidative stress and antioxidant capacity of affected tissue. Studies with vitamin C supplementation did not develop oxidative damage caused by Chagas disease in the host, but other studies cite the use of peroxiredoxins ascorbate - dependent on T. cruzi to offer protection against immune reaction. Based on these propositions, thirty "Swiss" mice were infected with T. cruzi QM1 strain and treated with two different vitamin C doses in order to study the parasitemia evolution, histopathological changes and lipid peroxidation biomarkers during the acute phase of Chagas disease. The results showed that the parasite clearance was greater in animals fed with vitamin C overdose. There were no significant differences regarding the biomarkers of lipid peroxidation and inflammatory process or the increase of myocardium in animals treated with the recommended dosage. The largest amount of parasite growth towards the end of the acute phase suggests the benefit of high doses of vitamin C for trypomastigotes. The supplementation doesn't influence the production of free radicals or the number of amastigote nests in the acute phase of Chagas disease.

USE OF THE POLYMERASE CHAIN REACTION FOR THE DIAGNOSIS OF ASYMPTOMATIC Leishmania INFECTION IN A VISCERAL LEISHMANIASIS-ENDEMIC AREA

The diagnosis of asymptomatic infection with Leishmania (Leishmania) chagasi has become more important over recent years. Expansion of visceral leishmaniasis might be associated with other routes of transmission such as transfusion, congenital or even vector transmission, and subjects with asymptomatic infection are potential reservoirs. Moreover, the identification of infection may contribute to the management of patients with immunosuppressive conditions (HIV, transplants, use of immunomodulators) and to the assessment of the effectiveness of control measures. In this study, 149 subjects living in a visceral leishmaniasis endemic area were evaluated clinically and submitted to genus-specific polymerase chain reaction (PCR), serological testing, and the Montenegro skin test. Forty-nine (32.9%) of the subjects had a positive PCR result and none of them developed the disease within a follow-up period of three years. No association was observed between the results of PCR, serological and skin tests. A positive PCR result in subjects from the endemic area did not indicate a risk of progression to visceral leishmaniasis and was not associated with a positive result in the serological tests.

EVOLUTION OF PATIENTS WITH AIDS AFTER cART: CLINICAL AND LABORATORY EVOLUTION OF PATIENTS WITH AIDS AFTER 48 WEEKS OF ANTIRETROVIRAL TREATMENT

SUMMARY Combination Antiretroviral Therapy (cART) aims to inhibit viral replication, delay immunodeficiency progression and improve survival in AIDS patients. The objective of this study was to compare two different schemes of cART, based on plasma viral load (VL) and CD4+ T lymphocyte count, during 48 weeks of treatment. For this purpose, 472 medical charts of a Specialized Outpatient Service were reviewed from 1998 to 2005. Out of these, 58 AIDS patients who had received a triple drug scheme as the initial treatment were included in the study and two groups were formed: Group 1 (G1): 47 individuals treated with two nucleoside reverse-transcriptase inhibitors (NRTI) and one non-nucleoside reverse-transcriptase inhibitor; Group 2 (G2): 11 patients treated with two NRTI and one protease inhibitor. In G1 and G2, 53.2% and 81.8% respectively were patients with an AIDS-defining disease. The T CD4+ lymphocyte count increased progressively up until the 24th week of treatment in all patients, while VL became undetectable in 68.1% of G1 and in 63.6% of G2. The study concluded that the evolutions of laboratory tests were similar in the two treatment groups and that both presented a favorable clinical evolution.