Assessment of the rapid test based on an immunochromatography technique for detecting anti-Treponema pallidum antibodies

A rapid test based on an immunochromatography assay - Determine™ Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine™ test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine™ is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.

Detection of EBV-DNA in serum samples of an immunosuppressed child during a three years follow-up: association of clinical and PCR data with active infection

Twenty-four whole blood and serum samples were drawn from an eight year-old heart transplant child during a 36 months follow-up. EBV serology was positive for VCA-IgM and IgG, and negative for EBNA-IgG at the age of five years old when the child presented with signs and symptoms suggestive of acute infectious mononucleosis. After 14 months, serological parameters were: positive VCA-IgG, EBNA-IgG and negative VCA-IgM. This serological pattern has been maintained since then even during episodes suggestive of EBV reactivation. PCR amplified a specific DNA fragment from the EBV gp220 (detection limit of 100 viral copies). All twenty-four whole blood samples yielded positive results by PCR, while 12 out of 24 serum samples were positive. We aimed at analyzing whether detection of EBV-DNA in serum samples by PCR was associated with overt disease as stated by the need of antiviral treatment and hospitalization. Statistical analysis showed agreement between the two parameters evidenced by the Kappa test (value 0.750; p < 0.001). We concluded that detection of EBV-DNA in serum samples of immunosuppressed patients might be used as a laboratory marker of active EBV disease when a Real-Time PCR or another quantitative method is not available.

Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

An immunofluorescence test for diagnosis of ophthalmic herpes in a mouse corneal model

Herpes simplex virus type 1 (HSV-1) ophthalmic disease is the most common cause of corneal blindness in humans world-wide. Current culture techniques for HSV take several days and commercially available HSV laboratory based diagnostic techniques vary in sensitivity. Our study was conducted to evaluate the use of a quicker and simpler method to herpes ophthalmic diagnosis. Corneal smears were made by firm imprints of infected mouse eyes to glass slides, after smears were fixated with cold acetone, and an indirect immunofluorescence (IIF) method was performed using monoclonal antibodies in a murine model of ophthalmic herpes. Eye swabs from infected mice were inoculated in Vero cells for virus isolation. Cytology and histology of the eye were also performed, using hematoxylin-eosin routine. Mouse eyes were examined by slit-lamp biomicroscopy for evidence of herpetic disease at various times postinoculation. We made a comparative evaluation of sensitivity, specificity and speed of methods for laboratory detection of HSV. Our results indicate that this IIF method is quick, sensitive, specific and can be useful in the diagnosis of ophthalmic herpes as demonstrated in an animal model.

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.

Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates

Thirty Candida albicans isolated from oral candidosis patients and 30 C. albicans isolated from control individuals were studied. In vitro susceptibility tests were performed for amphotericin B, fluconazole, 5-flucytosine and itraconazole through the Clinical and Laboratorial Standards Institute (CLSI) reference method and E test system. The results obtained were analyzed and compared. MIC values were similar for the strains isolated from oral candidosis patients and control individuals. The agreement rate for the two methods was 66.67% for amphotericin B, 53.33% for fluconazole, 65% for flucytosine and 45% for itraconazole. According to our data, E test method could be an alternative to trial routine susceptibility testing due to its simplicity. However, it can not be considered a substitute for the CLSI reference method.

American tegumentary leishmaniasis: langerhans cells in montenegro skin test

This work analyzed the histopathology and epidermal Langerhans cells (LC) of Montenegro skin test (MST) in patients with American tegumentary leishmaniasis (ATL) in order to in situ characterize and compare the immunological reaction of the two major clinical forms of ATL, localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). MST histopathology of both LCL and MCL showed superficial and deep perivascular inflammatory infiltrate composed mainly of lymphocytes and histiocytes. Epidermal LC population was higher in MST biopsies taken from LCL patients when compared to MCL group, at 48 and 72 hours after antigen inoculation. Increased number of epidermal LC displayed in MST biopsies of LCL patients represents specific cellular immunity against parasites. The decrease of LC in MST biopsies of MCL patients does not necessarily indicate a worse specific cellular immunity in this clinical form of leishmaniasis.

Cytokine profile in Montenegro skin test of patients with localized cutaneous and mucocutaneous leishmaniasis

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL.

Frequency of human toxocariasis in a rural population from Cajamarca, Peru determined by DOT-ELISA test

The aim of this study was to estimate the frequency of human toxocariasis in Cauday district, Cajamarca, Peru, using a dot-ELISA test. From June to October 2005, a total of 256 adult subjects were studied. Blood samples were collected for serology by a dot-ELISA test and for hematological examination. Parasitological examination was also carried out in stool samples to check cross-reactions in the dot-ELISA. The frequency observed was 44.92%, with a significant higher proportion of positivity in male subjects. From subjects with positive serology, 45.6% had respiratory symptoms, 40.44% abdominal pain, 32.35% hepatic symptoms, 14.7% cutaneous signs, 13.23% ocular manifestations, 43.38% eosinophilia, and all of these were statistically associated to serology. Among the population evaluated, 90.23% (231/256) were parasitized. From subjects with positive serology, 92.17% had at least one intestinal parasite and the most frequent were: Blastocystis hominis (68.38%), Giardia lamblia (28.68%), Hymenolepis nana (20.0%), Ascaris lumbricoides (15.65%), Entamoeba histolytica/E. dispar (13.24%), Cyclospora cayetanensis (4.41%), Cryptosporidium sp. (1.47%), Enterobius vermicularis (0.87%), Strongyloides stercoralis (0.87%), Taenia sp. (0.87%), and Trichuris trichiura (0.87%). The rate of false positives in the dot-ELISA test was improved by serum absorption each with A. suum antigens, with a decrease of cross-reactions. In conclusion, human toxocariasis is highly frequent in this population and some risk factors like dog/cat ownership, presence of pets within house, and previous history of geophagia were observed in the present study.

Rapid HIV diagnostic test in undocumented pregnant women applied at an inner-city teaching hospital

A significant number of Brazilian gestational-age women are still not tested for HIV, representing a high risk of transmission to their newborns. The current study sought to identify the number of pregnant women with no previous testing or undocumented for HIV referred to the Gynecology and Obstetrics Department of a Regional Teaching Hospital and included diagnosis of HIV infection determined by a rapid test and perinatal transmission in pregnancy. Medical records of all pregnant women admitted to hospital from January 2001 to December 2005 were reviewed. Pregnant women without HIV results were submitted to a rapid HIV test. Those who tested positive were further tested by ELISA and confirmed by indirect immunofluorescence assay (IIA) or Western blot (WB). The viral load from babies born to HIV-infected mothers was assessed by bDNA. Of the 16,424 pregnant women analyzed (6.6%), 1,089 were undocumented for HIV. Eleven women were positive in rapid testing and 10 were confirmed by ELISA, IIA or WB, with 0.9% seropositivity. Mother/infant pairs received zidovudine monotherapy prophylaxis and infant viral load was lower than 50 copies/mL. A higher number of pregnant women previously tested for HIV during antenatal care was verified, compared to that obtained nationwide.

A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol.

Validation of a rapid stool antigen test for diagnosis of Helicobacter pylori infection

The aim of this study was to validate the rapid lateral flow Helicobacter pylori stool antigen test (One step H. pylori antigen test, ACON laboratories, San Diego, USA; Prime diagnostics, São Paulo), using 13C-Urea Breath Test as the gold standard for H. pylori infection diagnosis. A total of 98 consecutive patients, asymptomatic or dyspeptic, entered the study. Sixty-nine were women, with a mean age of 45.76 ± 14.59 years (14 to 79 years). In the H. pylori-positive group, the rapid stool antigen test detected H. pylori antigen in 44 of the 50 positive patients (sensitivity 88%; 95% CI: 75.7-95.5%), and six false-negative; and in the H. pylori-negative group 42 presented negative results (specificity 87.5%; 95% CI: 74.7-95.3%), and six false-positive, showing a substantial agreement (Kappa Index = 0.75; p < 0.0001; 95% CI: 0.6-0.9). Forty four of fifty patients that had positive stool antigen were H. pylori-positive, the PPV of the stool antigen test was 88% (95% CI: 75.7-95.5%), and 42 patients with negative stool antigen test were H. pylori-negative, the NPV of the stool antigen test was 87.5% (95% CI: 74.7-95.3%). We conclude that the lateral flow stool antigen test can be used as an alternative to breath test for H. pylori infection diagnosis especially in developing countries.

Evaluation of Helicobacter pylory colonization by serologic test (IgG) and dyspepsia in volunteers from the countryside of Monte Negro, in the Brazilian western Amazon region

The present study intended to analyze the seroprevalence of Helicobacter pylori, IgG, and its relation to dyspepsia in a population from the western Amazon region. During the "Projeto Bandeira Científica", a University of São Paulo Medical School program, in Monte Negro's rural areas, state of Rondônia, 266 blood samples were collected from volunteers. The material was tested for IgG antibodies anti-Helicobacter pylori by ELISA method and the participants were also interviewed on dyspepsia, hygiene and social aspects. Participants aged between five and 81 years old (34 years on average), 149 (56%) were female and 117 (44%) male. We found 210 (78.9%) positive, 50 (18.8%) negative and six (2.3%) undetermined samples. Dyspeptic complaints were found in 226 cases (85.2%). There was no statistical association between dyspepsia and positive serology for H. pylori. We concluded that the seroprevalence in all age categories is similar to results found in other studies conducted in developing countries, including those from Brazil. On the other hand, the seroprevalence found in Monte Negro was higher than that reported in developed countries. As expected, there was a progressive increase in the positivity for H. pylori in older age groups.