Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis.

Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations.

Enzyme-linked immunosorbent assay to diagnose human leptospirosis: a meta-analysis of the published literature.

We report an evaluation of the accuracy of ELISA for the detection of Leptospira-specific antibodies in humans. Eighty-eight studies published in 35 articles met all inclusion criteria and were submitted to meta-analysis. Pooled sensitivity and specificity were 0·779 (95% CI 0·770-0·789) and 0·913 (95% CI 0·908-0·917), respectively, and the area under the curve was 0·964. Heterogeneity across studies was statistically significant, but none of the sources of heterogeneity (disease stage, antigen used, antibody detected) could fully explain this finding. Although the convalescent stage of disease was significantly associated with higher diagnostic accuracy, IgM ELISA was the best choice, regardless of the stage of disease. Negative ELISAs (IgG or IgM) applied in the acute phase do not rule out leptospirosis due to the possibility of false-negative results. In this case it is advisable to request a second blood sample or to apply a direct method for leptospiral DNA.

Clinical characteristics and risk factors of human leptospirosis in Argentina (1999-2005).

There is scarce data on the burden of leptospirosis and its epidemiological characteristics in Argentina. This study aimed to evaluate distribution of leptospirosis cases and identify risk factors for the disease during national laboratory-based surveillance. From January 1999 to December 2005, 812 suspected cases were referred to the national reference laboratory, of which 182 and 463 had respectively, laboratory confirmed and unconfirmed diagnosis of leptospirosis. The diagnosis of leptospirosis was discarded in 167 cases. The most prevalent presumptive infecting serogroup was Icterohaemorrhagie followed by Pomona, Ballum and Canicola. The majority of cases occurred during the worm and rainy months. Confirmed cases were predominantly adults and males, who presented with fever, headache and myalgias. Severe clinical manifestations included jaundice and acute renal insufficiency. Conjunctival suffusion, a hallmark clinical sign of leptospirosis, was found in 55% of confirmed cases, and 43% of the cases with discarded diagnosis (p=0.036). After multivariate analyses, age >30 years (OR=2.16; 1.05-4.41), occupation in a rural setting (OR=3.41; 1.45-8.06), contact with contaminated surface water (OR=2.17; 1.01-4.68), and contact with floods (OR=4.49; 1.17-17.25) were significantly associated with leptospirosis. In conclusion, although activities associated with rural occupations remain important risk factors in Argentina, exposures occurring during flooding events have emerged to be the major risk factor for leptospirosis.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins / Reconocimiento por anticuerpos de péptidos sintéticos que imitan regiones inmunodominantes de las proteínas p24 y p17 de VIH-1

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.

El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

[Evaluation of synthetic peptides for the detection of antibodies against human immunodeficiency virus]. / Evaluación de péptidos sintéticos para la detección de anticuerpos contra el virus de inmunodeficiencia humana.

The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18% and 100%, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.

Evaluación de péptidos sintéticos para la detección de anticuerpos contra el virus de inmunodeficiencia humana. / [Evaluation of synthetic peptides for the detection of antibodies against human immunodeficiency virus]

The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18

and 100

, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.

Evaluación de péptidos sintéticos para la detección de anticuerpos contra el virus de inmunodeficiencia humana / [Evaluation of synthetic peptides for the detection of antibodies against human immunodeficiency virus]

The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18

, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.

Development and validation of an ELISA for the detection of leptospire-specific antibodies in rodents.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies in rodents was developed and validated with the microscopic agglutination test (MAT) and leptospiral cultures. Sonicated antigen from cultures of serovars tarassovi and pyrogenes was used. As conjugate, a combination of anti-rat and anti-hamster IgG labeled with peroxidase was used. The optimal cut-off point was determined by plotting the sensitivity and specificity for various cut-off point values by means of receiver operating characteristic (ROC) curve. Concordance between ELISA and each of the MAT titers was measured by kappa (kappa). Proportions of positive results were compared by means of McNemar's test. Total 214 rodents were trapped, but only 117 could be processed by the three techniques (culture, ELISA, MAT; 1:20, 1:40, 1:50) and used for statistical analysis. Although, MAT titers in rodents infected with the serogroup Ballum tended to be lower than those infected with the serogroup Icterohaemorrhagiae, all (20/20) were ELISA-positive and almost all (19/20) were MAT-positive.The percentage of positive results obtained by ELISA, 47.0% exceeded significantly the 40.2% obtained by MAT (1:50). Difference between ELISA and MAT (1:40) was not significant and no differences were observed between ELISA and MAT (1:20). Agreement, specificity, sensitivity and the consequent area under the ROC curve between ELISA and MAT were higher as MAT cut-off points were lowered, being optimal at 1:20. The fact that differences between ELISA and MAT were significant at 1:50, but not at 1:40 or 1:20, supports the suggestion that lower MAT titers should be considered positive in rodents. The ELISA developed to detect leptospire-specific antibodies had optimal sensitivity and specificity in relation to MAT and it is concluded that it may constitute a very useful indicator for epidemiological purposes of past or present leptospiral infection in rodents.

Empleo del serodiagnóstico para detectar amebiasis invasiva en niños con disentería / Application of serodiagnosis to detect invasive amoebiasis in children with dysentery

La amebiasis es una parasitosis que afecta al 10 por ciento de la población mundial. Nueve de cada 10 infecciones son causadas por E. dispar, pero es diagnosticada y tratada como E. histolytica, por presentar quistes iguales. En cambio la infección por E. histolytica es invasiva, sintomática y presenta anticuerpos séricos específicos antiameba. La OMS recomienda el diagnóstico diferencial, dado que el tratamiento es innecesario si la infección es causada por E. dispar. Se evaluó la utilidad de las técnicas de la inmunofluorescencia indirecta (IFI) y el enzimoinmunoanálisis en fase sólida (ELISA) para el diagnóstico diferencial de estas infecciones. Se estudiaron 65 niños asistidos clasificados según clínica y examen coproparasitológico en: Grupo 1-A: asintomáticos, con coproparasitológicos negativos; Grupo 1-B: asintomáticos y con al menos un enteroparásito en el coproparasitológico; Grupo 2-A: con disentería, sospecha clínica de amebiasis y sin trofozoítos y/o quistes de E. histolytica / E. dispar en el coproparasitológico; Grupo 2-B: con disentería, sospecha clínica de amebiasis y con quistes y/o trofozoítos de E. histolytica / E. dispar en el coproparasitológico. Se realizaron exámenes coproparasitológicos directos y seriados y en muestras de suero se practicaron los inmuoensayos ELISA e IFI para la detección de anticuerpos específicos antiameba. Se observó que no hubo reactividad inespecífica con otras infecciones parasitarias y que la serología resultó reactiva cuando se hallaron trofozoítos hematófagos, patognomónicos de E. histolytica. La serorreactividad en los pacientes con quistes y/o trofozoítos no patognomónicos es fuertemente indicativa de una infección reciente. La probabilidad de tratarse de una infección remota es baja, ya que la proporción de anticuerpos específicos estudiada en 50 pacientes del grupo asintomático fue nula. Ambas técnicas presentaron resultados homólogos, a excepción de uno de los pacientes estudiados en quien, por serología, se pudo detectar una infección activa, pese a no haber sido detectada por el examen coproparasitológico. Se concluye que el empleo de técnicas de ELISA e IFI es una herramienta útil para mejorar el diagnóstico de invasión amebiana, permite aplicar una terapia acorde a las recomendaciones de la OMS y evita el tratamiento en infecciones por E. dispar (AU)

Empleo del serodiagnóstico para detectar amebiasis invasiva en niños con disentería / Application of serodiagnosis to detect invasive amoebiasis in children with dysentery

La amebiasis es una parasitosis que afecta al 10 por ciento de la población mundial. Nueve de cada 10 infecciones son causadas por E. dispar, pero es diagnosticada y tratada como E. histolytica, por presentar quistes iguales. En cambio la infección por E. histolytica es invasiva, sintomática y presenta anticuerpos séricos específicos antiameba. La OMS recomienda el diagnóstico diferencial, dado que el tratamiento es innecesario si la infección es causada por E. dispar. Se evaluó la utilidad de las técnicas de la inmunofluorescencia indirecta (IFI) y el enzimoinmunoanálisis en fase sólida (ELISA) para el diagnóstico diferencial de estas infecciones. Se estudiaron 65 niños asistidos clasificados según clínica y examen coproparasitológico en: Grupo 1-A: asintomáticos, con coproparasitológicos negativos; Grupo 1-B: asintomáticos y con al menos un enteroparásito en el coproparasitológico; Grupo 2-A: con disentería, sospecha clínica de amebiasis y sin trofozoítos y/o quistes de E. histolytica / E. dispar en el coproparasitológico; Grupo 2-B: con disentería, sospecha clínica de amebiasis y con quistes y/o trofozoítos de E. histolytica / E. dispar en el coproparasitológico. Se realizaron exámenes coproparasitológicos directos y seriados y en muestras de suero se practicaron los inmuoensayos ELISA e IFI para la detección de anticuerpos específicos antiameba. Se observó que no hubo reactividad inespecífica con otras infecciones parasitarias y que la serología resultó reactiva cuando se hallaron trofozoítos hematófagos, patognomónicos de E. histolytica. La serorreactividad en los pacientes con quistes y/o trofozoítos no patognomónicos es fuertemente indicativa de una infección reciente. La probabilidad de tratarse de una infección remota es baja, ya que la proporción de anticuerpos específicos estudiada en 50 pacientes del grupo asintomático fue nula. Ambas técnicas presentaron resultados homólogos, a excepción de uno de los pacientes estudiados en quien, por serología, se pudo detectar una infección activa, pese a no haber sido detectada por el examen coproparasitológico. Se concluye que el empleo de técnicas de ELISA e IFI es una herramienta útil para mejorar el diagnóstico de invasión amebiana, permite aplicar una terapia acorde a las recomendaciones de la OMS y evita el tratamiento en infecciones por E. dispar

Epitope contiguous chains and antibody recognition in HIV-1 synthetic peptide antigens.

Five peptides derived from human immuno deficiency virus (HIV-1) gp41 transmembrane protein have been synthesized: M9 (610-618), M12 (598-609), M15 (600-614), M21 (584-604) and M23 (587-609). These sequences partially overlap in the region vicinal to the immunodominant epitope CSGKLIC, between two cysteine residues 603-609 and three of them (M12, M15 and M23) include this complete heptapeptide. M23, the longer peptide, includes an hydrophilic chain in addition to the heptapeptide loop. The purpose of this work was to determine the influence of contiguous chains to the heptapeptide loop on antibody recognition in fluid and solid phases, and dissociation constants (KD) of each sequence with human anti-HIV-1 antibodies. Two peptides, M13 and M23, overlapped on this loop, were found to be more reactive. Antigen-antibody dissociation constants were determined for both peptides by competition enzyme-linked immunosorbent assay, using each peptide alternatively as the solid phase-immobilized antigen. In addition to the influence of solid-phase antigen on calculated dissociation constants (a phenomenon described by Seligman, 1994), the inhibitory effect of M15 in liquid phase on antibody binding to solid phase M23 was higher than exerted by M23 in solution over antibody binding to M15 on solid phase. On the basis of peptide sequence and predicted antigenicity, this behavior appeared to be contradictory. It is assured that the possible origin of this phenomenon is due to unfavorable conformation of the longer peptide. Even though synthetic peptides mimic mainly sequential epitopes, conformational preferences in fluid or solid phase play an important role in epitope functionality. In particular, addition of residues to known immunodominant sequences may not always amplify antibody recognition if conformation provokes steric hindrance in the native epitope.