SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

AN ELISA SUITABLE FOR THE DETECTION OF RABIES VIRUS ANTIBODIES IN SERUM SAMPLES FROM HUMAN VACCINATED WITH EITHER CELL-CULTURE VACCINE OR SUCKLING-MOUSE-BRAIN VACCINE

An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.

FATAL GROUP A STREPTOCOCCAL TOXIC SHOCK-LIKE SYNDROME IN A CHILD WITH VARICELLA: REPORT OF THE FIRST WELL DOCUMENTED CASE WITH DETECTION OF THE GENETIC SEQUENCES THAT CODE FOR EXOTOXINS SPE A AND B, IN SÃO PAULO, BRAZIL

A previously healthy seven-year-old boy was admitted to the intensive care unit because of toxaemia associated with varicella. He rapidly developed shock and multisystem organ failure associated with the appearance of a deep-seated soft tissue infection and, despite aggressive treatment, died on hospital day 4. An M-non-typable, spe A and spe B positive Group A Streptococcus was cultured from a deep soft tissue aspirate. The criteria for defining Streptococcal toxic shock-like syndrome were fulfilled. The authors discuss the clinical and pathophysiological aspects of this disease as well as some unusual clinical findings related to this case.

Detection of a Giardia lamblia coproantigen by using a commercially available immunoenzymatic assay, in Belo Horizonte, Brazil

It is known that fecal examination to detect Giardia lamblia cysts or trophozoites produces a high percentage of false-negative results. A commercially available immunoenzymatic assay (ProSpecT Giardia Microplate Assay, Alexon, Inc., BIOBRÁS) to detect G. lamblia specific coproantigen was evaluated for the first time in Brazil. A total of 90 specimens were tested. Each specimen was first tested as unpreserved stool, and then it was preserved in 10% Formalin to be tested 2 months later. The assay was able to identify all the 30 positive patients (sensitivity = 100.0%) by visual or spectrophotometric examination in the unpreserved specimens and was negative in 57 of the 60 patients without G. lamblia (specificity = 95.0%). The assay identified 27 of the 30 positive patients (sensitivity = 90.0%) and was negative in 59 of the 60 negatives (specificity = 98.3%) in the preserved stools according to both readings. A marked difference was observed in the optical densities in both groups, preserved and unpreserved stools, when the G. lamblia-positive specimens were compared to the negative or positive for other intestinal parasites than G. lamblia. The assay seems a good alternative for giardiasis diagnosis, especially when the fecal examination was repeatedly negative and the patient presents giardiasislike symptoms.

Serological diagnosis of toxoplasmosis: usefulness of IgA detection and IgG avidity determination in a patient with a persistent IgM antibody response to Toxoplasma gondii

We report the detection of specific IgA antibodies and the determination of IgG avidity in sequential serum samples from a patient exhibiting significant levels of Toxoplasma-specific IgM antibodies for seven years after the onset of the clinical symptoms of toxoplasmosis. IgM antibodies were detected by an indirect immunofluorescence test and by three commercial enzyme-linked immunosorbent assays (ELISA). Anti-T. gondii IgA was quantified by the a-capture ELISA technique using a commercial kit. As defined by the manufacturer of the IgA ELISA test used, most patients with acute toxoplasmosis have antibody levels > 40 arbitrary units per ml (AU/mL). At this cut-off level, the patient still had a positive ELISA result (45 AU/mL) in a serum sample taken one year after the beginning of clinical manifestations. The IgG avidity-ELISA test was performed with the Falcon assay screening test (F.A.S.T.®) - ELISA system. Avidity indices compatible with a recent Toxoplasma infection were found only in serum samples taken during the first 5 months after the onset of the clinical symptoms of toxoplasmosis. These results show that the interpretation of positive IgM results as indicative of recently acquired toxoplasmosis requires additional laboratory confirmation either by other tests or by the demonstration of a significant rise in the antibody titers in sequential serum samples.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Total IgE detection in paired cerebrospinal fluid and serum samples from patients with neurocysticercosis

Neurocysticercosis (NC), the presence of Taenia solium metacestodes in tissues, is the most frequent and severe parasitic infection of the central nervous system. We investigated the presence of total IgE by an automated chemiluminescence assay in 53 paired cerebrospinal fluid (CSF) and serum samples from patients with NC (P) and in 40 CSF samples from individuals with other neurological disorders as the control group (C). Total IgE concentration ranged from 1.2 to 6.6 IU/ml (mean = 1.4 IU/ml, standard deviation-sd = 1.1 IU/ml) in 28.3% of CSF samples from the P group, a value significantly higher than for the C group (£1.0 IU/ml). The serum samples from the P group showed concentrations ranging from 1.0 to 2330.0 IU/ml (mean = 224.1 IU/ml, sd = 452.1 IU/ml), which were higher than the normal value cited by the manufacturer (<100.0 IU/ml) in 32.1% of the samples. A significant difference was observed in CSF samples from the P and C groups (p = 0.005) and in serum samples from the P group compared to the normal value (p = 0.005), with sera showing more frequent abnormal results.

Malaria vectors in the Brazilian Amazon: Anopheles of the subgenus Nyssorhynchus

Various species of Anopheles (Nyssorhynchus) were studied in the Amazon with the objective of determining their importance as malaria vectors. Of the 33 known Anopheles species occurring in the Amazon, only 9 were found to be infected with Plasmodium. The different species of this subgenus varied both in diversity and density in the collection areas. The populations showed a tendency towards lower density and diversity in virgin forest than in areas modified by human intervention. The principal vector, An. darlingi, is anthropophilic with a continuous activity cycle lasting the entire night but peaking at sunset and sunrise. These species (Nyssorhynchus) are peridomiciliary, entering houses to feed on blood and immediately leaving to settle on nearby vegetation. Anopheles nuneztovari proved to be zoophilic, crepuscular and peridomiciliary. These habits may change depending on a series of external factors, especially those related to human activity. There is a possibility that sibling species exist in the study area and they are being studied with reference to An. darlingi, An. albitarsis and An. nuneztovari. The present results do not suggest the existence of subpopulations of An. darlingi in the Brazilian Amazon.

Detection of hepatitis A antibodies by ELISA using saliva as clinical samples

The possibility of detecting acute infection and immunity using body fluids that are easier to collect than blood, mainly in children, would facilitate the investigation and follow-up of outbreaks of hepatitis A (HAV). Our study was carried out to evaluate the detection of anti-HAV IgM, IgA and total antibodies in saliva using serum samples as reference. Forty three paired serum and saliva samples were analyzed. From this total, 24 samples were obtained from children and 1 from one adult during the course of acute hepatitis A; an additional 18 samples were obtained from health professionals from Adolfo Lutz Institute. The sensitivity to detect anti-HAV IgM was 100% (95%CI: 79.1 to 100.0%), employing saliva as clinical samples. In detecting anti-HAV IgA, the sensitivity was 80.8% (95%CI: 60.0 to 92.7%) and for the total antibodies was 82.1% (95%CI: 62.4 to 93.2%). The specificity was 100% for each. The rate of agreement was high comparing the results of serum and saliva samples for detecting HAV antibodies. We conclude that saliva is an acceptable alternative specimen for diagnosing acute hepatitis A infection, and for screening individuals to receive hepatitis A vaccine or immunoglobulin.

An initial examination of the epidemiology of malaria in the State of Roraima, in the Brazilian Amazon Basin

This study firstly describes the epidemiology of malaria in Roraima, Amazon Basin in Brazil, in the years from 1991 to 1993: the predominance of plasmodium species, distribution of the blood slides examined, the malaria risk and seasonality; and secondly investigates whether population growth from 1962 to 1993 was associated with increasing risk of malaria. Frequency of malaria varied significantly by municipality. Marginally more malaria cases were reported during the dry season (from October to April), even after controlling for by year and municipality. Vivax was the predominant type in all municipalities but the ratio of plasmodium types varied between municipalities. No direct association between population growth and increasing risk of malaria from 1962 to 1993 was detected. Malaria in Roraima is of the "frontier" epidemiological type with high epidemic potential.

Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat

The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat. Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10-7, 10-8 or 10-9 CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass). The assay was able to detect until 10-9 CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol. As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

Detection of enteroviruses in cases of neurological disorders in the State of Pará, Brazil

Eighty-one cerebrospinal fluid (CSF) samples mainly from cases of aseptic meningitis and motor deficiency syndrome were sent to the Virology Section of Evandro Chagas Institute, Belém Pará, in the period of January 1995 to January 1996 in order to isolate viruses. All samples were inoculated onto HEp-2 cell culture and newborn mice, with negative results. The probability of isolating viruses by these methods is reduced because of the low concentration of viral particles in these specimens. In order to obtain more information about the etiology of these cases, a group of 23 samples were selected to be tested by a more sensitive technique than the virus isolation - the reverse transcription polymerase chain reaction (RT-PCR). Specific primers directed to conserved regions in the enterovirus genome were used, considering that this group of viruses is frequently associated with these neurological disorder. The age of the patients ranged from 1 to 55 years and nearly all of them lived in Belém, State of Pará, North of Brazil. Of 15 samples analyzed by RT PCR nine (60%) were positive; of these, 6 (66.6%) had motor deficiency and 3 (33.3%) developed aseptic meningitis. These results show that it is important to investigate enterovirus as cause of these syndromes.

A rapid latex agglutination test for the detection of anti-cysticercus antibodies in cerebrospinal fluid (CSF)

Simple and rapid latex-based diagnostic tests have been used for detecting specific antigens or antibodies in several diseases. In this article, we present the preliminary results obtained with a latex agglutination test (LAT) for diagnosing neurocysticercosis by detection of antibodies in CSF. A total of 43 CSF samples were assayed by the LAT: 19 CSF samples from patients with neurocysticercosis and 24 CSF samples from patients with other neurologic disorders (neurosyphilis, n = 8; neurotoxoplasmosis, n = 3; viral meningitis, n = 4, chronic headache, n = 9). The LAT exhibited 89.5% sensitivity and 75% specificity. The use of LAT seems to be an additional approach for the screening of neurocysticercosis with advantage of simplicity and rapidity. Further studies could be performed using purified antigens and serum samples.