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.

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.

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection

In Amazonian Brazil, the Cebus apella monkey (Primates: Cebidae) has been associated with the enzootic cycle of Leishmania (V.) shawi, a dermotropic parasite causing American cutaneous leishmaniasis (ACL). It has also been successfully used as animal model for studying cutaneous leishmaniasis. In this work, there has been investigated its susceptibility to experimental Leishmania (L.) infantum chagasi-infection, the etiologic agent of American visceral leishmaniasis (AVL). There were used ten C. apella specimens, eight adult and two young, four males and six females, all born and raised in captivity. Two experimental infection protocols were performed: i) six monkeys were inoculated, intra-dermal via (ID), into the base of the tail with 2 x 10(6) promastigotes forms from the stationary phase culture medium; ii) other four monkeys were inoculated with 3 x 10(7) amastigotes forms from the visceral infection of infected hamsters by two different via: a) two by intravenous via (IV) and, b) other two by intra-peritoneal via (IP). The parameters of infection evaluation included: a) clinical: physical exam of abdomen, weigh and body temperature; b) parasitological: needle aspiration of the bone-marrow for searching of amastigotes (Giemsa-stained smears) and promastigotes forms (culture medium); c) immunological: Indirect fluorescence antibody test (IFAT) and, Delayed-type hypersensitivity (DTH). In the six monkeys ID inoculated (promastigotes forms) all parameters of infection evaluation were negative during the 12 months period of follow-up. Among the four monkeys inoculated with amastigotes forms, two IV inoculated showed the parasite in the bone-marrow from the first toward to the sixth month p.i. and following that they cleared the infection, whereas the other two IP inoculated were totally negative. These four monkeys showed specific IgG-antibody response since the third month p.i. (IP: 1/80 and IV: 1/320 IgG) toward to the 12th month (IP: 1/160 and IV: 1/5120). The DTH-conversion occurred in only one IV inoculated monkey with a strong (30 mm) skin reaction. Considering these results, we do not encourage the use of C. apella monkey as animal model for studying the AVL.

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.

PREVALENCE OF PARACOCCIDIOIDOMYCOSIS INFECTION BY INTRADERMAL REACTION IN RURAL AREAS IN ALFENAS, MINAS GERAIS, BRAZIL

This study aimed to estimate the prevalence of paracoccidioidal infection by intradermal reaction (Delayed-Type Hypersensitivity, DTH) to Paracoccidioides brasiliensis in rural areas in Alfenas, Southern Minas Gerais (MG) State, Brazil, and to assess risk factors (gender, occupation, age, alcohol intake and smoking) associated with infection. We conducted a population-based cross-sectional study using intradermal tests with gp 43 paracoccidioidin in 542 participants, who were previously contacted by local health agents and so spontaneously attended the test. Participants underwent an interview by filling out a registration form with epidemiological data and were tested with an intradermal administration of 0.1 mL of paracoccidioidin in the left forearm. The test was read 48 hours after injection and was considered positive if induration was greater than or equal to 5 mm. Out of 542 participants, 46.67% were positive to the skin test. Prevalence increased in accordance with an increase of age. There was statistical significance only for males. Occupation, alcohol intake and smoking habits were not significantly associated with the risk of paracoccidioidomycosis infection. There is relevance of paracoccidioidomycosis infection in such rural areas, which suggests that further epidemiological and clinical studies on this mycosis should be done in the southern part of Minas Gerais State.

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

Mortality of Dipetalogaster maximus (uhler) in response to temperature and humidity

Observations were made on the mortality of Dipetalogaster maximus in relation to humidity and temperature in controlled conditions. The bugs survived longer at higher relative humidities and at lower temperatures, but when these results were plotted against vapour pressure déficit, no independent temperature effect was seen. The results may be explained by thefaster depletion of water reserves at higher vapour pressure deficits. D. maximus did not increase its resistance to water vapour transferat higher vapour pressure deficits. In orderto increase survival rates when D. maximus is used for xenodiagnosis in field conditions it should be protected against high temperatures and low humidities.

Aspects of the granulomatous reaction in the liver of mice infected and reinfected with two different geographical strains of Schistosoma mansoni

In this study, which was undertaken in relation to the histopathologic behavior of two different strains (LE-Belo Horizonte, MG and SJ - São José dos Campos, SP) in infections and reinfections (homologous or heterologous) with Schistosoma mansoni, the authors confirmed a more accentuated pathogenicity of the SJ strain. All the reinfections showed the presence of typical granulomas of the acute phase, when performed either with the same strain (homologous) or with a different strain (heterologous) of the parasite of the primo infection. The possible mechanisms responsible for reactivation of the immunopathologic response in reinfections are discussed.

Low sensitivity of polymerase chain reaction for diagnosis of tuberculous meningitis in southeastern Brazil

Two polymerase chain reaction (PCR) protocols showed low sensitivity (36% and 53% for TB AMPLICOR and MPB64 nested PCR, respectively), when compared with classic microbiological methods (73% and 54% for Ziehl-Neelsen staining and culture, respectively), in the diagnosis of tuberculous meningitis in 91 patients in southeastern Brazil. Only three PCR-positive, microbiologically negative patients were found. Analysis of sequential cerebrospinal fluid samples by nested PCR detected Mycobacterium tuberculosis DNA up to 29 days after the introduction of antituberculosis chemotherapy.

Paradoxical reaction to the treatment of tuberculosis uncovering previously silent meningeal disease

The development of paradoxical clinical worsening following initiation of tuberculosis treatment may complicate the clinical course of both HIV-infected and uninfected patients. We report a severe manifestation of the so called paradoxical reaction to the treatment of tuberculosis that unmasked previously silent meningeal disease in a 34-year-old HIV-infected male patient.

Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

Recommendations for the detection of Leptospira in urine by PCR

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi

Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.