Diagnosis of pulmonary pneumocystosis by microscopy on wet mount preparations

We have compared the searching of the presence of "honeycomb" structures by direct microscopy on wet mount preparations with the direct immunofluorescence (DIF) for the diagnosis of Pneumocystis carinii pneumonia (PCP) in 115 bronchoalveolar (BAL) fluids. The samples belonged to 115 AIDS patients; 87 with presumptive diagnosis of PCP and 28 with presumptive diagnosis other than PCP. The obtained results were coincident in 114 out of 115 studied samples (27 were positive and 87 negative) with both techniques. A higher percentage of positive results (32.18%) among patients with presumptive diagnosis of PCP with respect to those with presumptive diagnosis other than PCP (3.57%) was observed. One BAL fluid was positive only with DIF, showed scarce and isolated P. carinii elements and absence of typical "honeycomb" structures. The searching for "honeycomb" structures by direct microscopy on wet mount preparations could be considered as a cheap and rapid alternative for diagnosis of PCP when other techniques are not available or as screening test for DIF. This method showed a sensitivity close to DIF when it was applied to BAL fluids of AIDS patients with poor clinical condition and it was performed by an experienced microscopist.

Fatal disseminated paracoccidioidomycosis in a two-year-old child

A two year-old female child was admitted at the Pediatric Intensive Care Unit in a septic shock associated with a lymphoproliferative syndrome, with history of fever, adynamia and weight loss during the last two months. On admission, the main clinical and laboratory manifestations were: pallor, jaundice, disseminated enlarged lymph nodes, hepatosplenomegaly, crusted warts on face, anemia, eosinophilia, thrombocytopenia, increased direct and indirect bilirubin, alkaline phosphatase, and gammaglutamyl transpeptidase. A parenteral administration of fluids, dobutamine and mechanical ventilation was started, without improvement of the clinical conditions. A direct examination of exsudate collected from cervical lymph node revealed numerous oval-to-around cells with multiple budding, like a "pilot wheel" cell, suggesting Paracoccidioides brasiliensis. Even though treatment with intravenous sulfamethoxazole-trimethoprine was soon started, the child died 36 hours after hospital admission. Disseminated paracoccidioidomycosis was confirmed in the autopsy. This is the youngest case of paracoccidioidomycosis in children reported in the literature.

Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification

Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.

Dapsone hypersensitivity syndrome in an adolescent during treatment during of leprosy

A 12 y old girl was admitted 24 days after start a WHO multidrug therapy scheme for multibacillary leprosy (dapsone, clofazimine and rifampicin) with intense jaundice, generalized lymphadenopathy, hepatoesplenomegaly, oral erosions, conjunctivitis, morbiliform rash and edema of face, ankles and hands. The main laboratory data on admission included: hemoglobin, 8.4 g/dL; WBC, 15,710 cells/mm³; platelet count, 100,000 cells/mm³; INR = 1.49; increased serum levels of aspartate and alanine aminotransferases, gamma-glutamyl transpeptidase, alkaline phosphatase, direct and indirect bilirubin. Following, the clinical conditions had deteriorated, developing exfoliative dermatitis, shock, generalized edema, acute renal and hepatic failure, pancytopenia, intestinal bleeding, pneumonia, urinary tract infection and bacteremia, needing adrenergic drugs, replacement of fluids and blood product components, and antibiotics. Ten days after admission she started to improve, and was discharged to home at day 39th, after start new supervised treatment for leprosy with clofazimine and rifampicin, without adverse effects. This presentation fulfils the criteria for the diagnosis of dapsone hypersensitivity syndrome (fever, generalized lymphadenopathy, exfoliative rash, anemia and liver involvement with mixed hepatocellular and cholestatic features). Physicians, mainly in geographical areas with high prevalence rates of leprosy, should be aware to this severe, and probably not so rare, hypersensitivity reaction to dapsone.

FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL

Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients. The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures, abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%) were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%) C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates. In vitroresistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B, flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex species, mainly with the OPA09 marker.

The hamster (mesocricetus auratus) as experimental model in chagas' disease: parasitological and histopathological studies in acute and chronic phases of Trypanosoma cruzi infection

This research characterizes the acute and chronic phases of Chagas ' disease in hamster through parasitological and histopathological studies. The acute phase was achieved with 44 young hamsters injected intraperitoneally with 100.000 blood trypomastigotes of Benedito and Y strains of T. cruzi. The chronic phase was induced in 46 hamsters injected intraperitoneally with 35.000 trypomastigotes ofVicentina, Benedito and Y strains. Animals were sacrificed at regular intervals of 24 hours of acute phase and from the 3rd to the 10th month of infection ofchronic phase. In the acute phase, parasites were easily recoveredfrom all animals and there was an inflammatory reaction characterized by mononuclear and polymorphous leukocyte infiltration of variable degree in the majority of tissues and organs, specially in the connective loose and fatty tissues, smooth muscle myocardium and skeletal muscle. In the chronic phase the lesions occurred in the same tissues and organs, but the inflammatory response was less severe and characterized by mononuclear infiltration mainly with focal or zonalfibrosis in the myocardiun. In 50% of infected animals parasites were found inmyocardiun and recoveredfrom pericardic, peritoneal and ascitic fluids in some animals. Signs of heart failure, sudden death and enlargement of bowel were observed regularly. We concluded that the hamster is an useful model for Chagas' disease studies.

Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

Fluids in which Mycobacterium tuberculosis are seldom found, such as pleural and cerebrospinal liquids, are good candidates to be studied using PCR techniques. We detail our experience with a PCR assay applied to pleural and cerebrospinal fluids using the primer MPB64. Seventy three specimens were analyzed: 30 pleural fluids (PF), 26 pleural biopsies (PB) and 17 cerebrospinal fluids (CSF). The gold standard for the diagnosis of tuberculous meningitis was the positive culture for M. tuberculosis in CSF. Tuberculous pleural effusion was diagnosed when cultures of PF and/or PB were positive for M. tuberculosis, or the PB histology showed granulomas. Our results, compared to the gold standards employed, showed a sensitivity of 70%, specificity of 88%, positive predictive value of 82% and negative predictive value of 80%. The high specificity of the MPB64 fragment while still retaining a good sensitivity makes it very well suited for pleural and cerebrospinal tuberculosis diagnosis.

Seroprevalence of hepatitis B virus infection and seroconvertion to anti-HBsAg in laboratory staff in Goiânia, Goiás

Were analyzed 648 serum samples from laboratory staff in Goiânia, Goiás aiming detection of three serological markers of HBV: HBsAg, anti-HBsAg and anti-HBcAg. The HBsAg and anti-HBcAg positive samples were also analyzed for HBeAg, anti-HBeAg and anti-HBcAgIgM markers. HBV infection rate of 24.1% was observed and, from them, 0.7% were positive for HBsAg. Viral DNA was detected by PCR in two HBsAg positive samples. A vaccination index of 74.5% and a global index of 89.9% of serological response to vaccination were observed. The direct work with biological fluids as well as cleaning workers represented significant risks for acquisition of HBV infection. The data from the present study showed an increase of the vaccination index among laboratory staff but the rates of HBV infection did not change through the years in the region.

Quantification of HIV-1 viral RNA in the blood in needles used for venous puncture in HIV-infected individuals

INTRODUCTION: Occupational HIV infection among healthcare workers is an important issue in exposures involving blood and body fluids. There are few data in the literature regarding the potential and the duration of infectivity of HIV type 1 (HIV-1) in contaminated material under adverse conditions. METHODS: We quantified HIV-1 viral RNA in 25×8mm calibre hollow-bore needles, after punctures, in 25 HIV-1-infected patients selected during the sample collection. All of the patients selected were between the ages of 18 and 55. Five samples were collected from 16 patients: one sample for the immediate quantification of HIV-1 RNA in the plasma and blood samples from the interior of 4 needles to be analyzed at 0h, 6h, 24h, and 72h after collection. In nine patients, another test was carried out in the blood from one additional needle, in which HIV-1 RNA was assessed 168h after blood collection. The method used to assess HIV-1 RNA was nucleic acid sequence-based amplification. RESULTS: Up to 7 days after collection, HIV-1 RNA was detected in all of the needles. The viral RNA remained stable up to 168h, and there were no statistically significant differences among the needle samples. CONCLUSIONS: Although the infectivity of the viral material in the needles is unknown, the data indicate the need to re-evaluate the practices in cases of occupational accidents in which the source is not identified.

Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

Field evaluation of an exoantigen-containing Babesia vaccine in Venezuela

Bovine babesiosis is endemic in Venezuela, causing significant losses in highly susceptible imported cattle. Current immunoprphylatic methods include the less desirable use of live parasites. Inactivated vaccines derived from exoantigen-containing supernatant fluids of in vitro Babesia bovis and B. bigemina cultures have been developed and constitute a major improvement in vaccine safety, stability and ease of handling. Vaccination trials conducted under field conditions provide the final evaluation of a culture-derived B. bovis-B. bigemina vaccine. During a 5-year period, approximately 8,000 cattle were vaccinated and 16 clinical trials carried out in. 7 states of Venezuela Clinical, serologic and parasitologic data were collected monthly from 10% of the animals over a 2-year period. Data were also collected from a similar number of nonvaccinated control cattle. Analysis of results from these trials demonstrated a reduction in the incidence of clinical disease among vaccinated animals and complete protection against mortality among vaccinated and nonvaccinated cattle. Use of this inactivated vaccine offers the best combination od safety, potency and efficacy for thew immunoprophylatic control of bovine babesiosis.

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

One-tube nested Polymerase Chain Reaction for detection of Chlamydia trachomatis

Here we present a one-tube nested PCR test, which allows the detection of minimal quantities of Chlamydia trachomatis in human fluids. This assay includes the use of an internal control to avoid false negative results due to the presence of inhibitors. The results obtained show that this assay is robust enough to be used for clinical diagnosis.

Preparation of sand fly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies

Phlebotominae sand fly specimens were prepared for histological and physiological studies. Different fixatives were tested on sectioned and whole bodied adult females in order to obtain good fixation and provide satisfactory penetration of the embedding media. All fixed specimens were infiltrated (up to seven days under 5ºC) and embedded in hydroxyethyl metacrylate. Two-three µm sections were stained, mounted in Canada balsam and observed by light microscopy. Best results were achieved when whole bodied insects were double fixed in Bouin's and Carnoy's fluids (4 h/2 h) and stained in Hematoxilin/Eosin or fixed in calcium formaldehyde and stained in mercury bromophenol blue.

Immunodetection of Helicobacter sp. and the associated expression of ABO blood group antigens in the gastric mucosa of captive and free-living New World primates in the Amazon region

The histo-blood group ABH antigens were first described in humans. These antigens are only present on erythrocytes from great apes and humans, while in more primitive animals they are found in tissues and body fluids. The ABH antigens are mainly distributed in tissues exposed to the external environment and potentially serve as ligands for pathogens or inhibitors of tissue connections. The objective of this paper was two-fold: (i) to determine the presence of Helicobacter sp. in the gastric mucosa of 16 captive and 24 free-living New World monkeys and (ii) to evaluate the presence of histopathological alterations related to bacterial infection and the associated expression of ABH antigens in the tissue. Stomach tissues from 13 species of monkey were assessed using haematoxylin-eosin and modified Gram staining (Hucker) methods. An immunohistochemical analysis of the tissue revealed the presence of infectious bacteria that were characteristic of the genus Helicobacter sp. The results demonstrate that various species of monkey might be naturally infected with the Helicobacter sp. and that there is an increased susceptibility to infection. This study serves as a comparative analysis of infection between human and non-human primates and indicates the presence of a new species of Helicobacter.