Islay Rodríguez

Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

Sistema de aglutinación con látex para el diagnóstico rápido de la leptospirosis en Cuba

OBJETIVOS: Evaluar la sensibilidad, la especificidad, la reproducibilidad y la estabilidad de cinco sistemas de aglutinacion con latex disenados para detectar anticuerpos contra leptospira en sueros de humanos y de animales, basados en los serogrupos de Leptospirade mayor circulacion en Cuba. METODOS: Se realizo un estudio analitico descriptivo con 706 sueros humanos (65 sueros positivos a anticuerpos contra leptospira mediante microaglutinacion (MAT) y hemaglutinacion (HA); 156 sueros negativos, segun MAT y HA; 485 sueros de 424 pacientes con signos clinicos o epidemiologicos de leptospirosis); y 29 sueros de animales (16 equinos, 6 bovinos, 5 porcinos, 1 canino y 1 ovino). Todas las muestras se evaluaron con cinco conjugados de latex con celulas enteras de Leptospira interrogans de los cuatro serogrupos de mayor circulacion en Cuba en el periodo entre 2002 y 2004. Con las celulas obtenidas de los cultivos celulares de cepas tipo se obtuvieron cuatro conjugados especificos (latex-Canicola, latex-Icterohaemorrhagiae, latex-Pomona y latex-Sejroe) y un conjugado de latex con la mezcla de las celulas de esos cuatro serogrupos a partes iguales (latex-Pool). Adicionalmente, las muestras se evaluaron con el sistema comercial de aglutinacion con latex Lepto Tek Dri Dot (bioMerieux, Francia). La estabilidad y la reproducibilidad de los conjugados de latex se evaluaron mediante controles mensuales durante 6 meses con sueros positivos y negativos. RESULTADOS: De los sistemas evaluados, la mejor combinacion de sensibilidad y especificidad se observo con el conjugado latex-Pool (93,8% y 90,4%, respectivamente). La mejor combinacion de valores predictivos positivos y negativos se observo con el conjugado latex-Sejroe (90,9% y 95,8%, respectivamente), seguido del conjugado latex-Pool (94,2% y 96,6%, respectivamente). Los valores predictivos positivo y negativo del sistema comercial Lepto Tek Dri Dot fueron 78,5% y 88,4%, respectivamente. De las 137 muestras de pacientes positivas a alguno de los serogrupos estudiados segun MAT, los conjugados de latex lograron identificar correctamente 107 (78,1%), mientras que el conjugado latex-Pool detecto como positivos 116 sueros (84,7%). Al evaluar los sueros de animales, el conjugado latex-Pool detecto como positivos el mayor numero de sueros y tuvo la mayor coincidencia con MAT (93,1%). Se observo una adecuada estabilidad y reproducibilidad de los conjugados estudiados. CONCLUSIONES: Los conjugados de latex con celulas enteras de leptospira de los serogrupos de mayor circulacion en Cuba demostraron un grado de coincidencia con MAT similar o superior al observado con el sistema comercial Lepto Tek Dri Dot, tanto en sueros de humanos como de animales. Se recomienda extender el uso del conjugado latex-Pool en Cuba para el tamizaje inicial de anticuerpos contra leptospira.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

Do antiborrelial antibodies suggest Lyme disease in Cuba

To the Editor: Lyme disease is the most common vector-borne disease in the United States and parts of Eurasia (1). It represents a considerable emerging infectious disease threat because of its consequences to human health and the difficulties in preventing and controlling it (2,3). In Cuba, Lyme borreliosis has never been reported. However, in the last 20 years ixodid ticks, mainly Amblyomma cajennenses, have been found in the human population in the Cuban village of Las Terrazas, Pinar del Rio. These ixodid bites were frequent and widespread, especially in children, many of whom were hospitalized without a confirmatory laboratory diagnosis. Affected persons had symptoms associated with Lyme disease such as erythematous macules or papules, fever, fatigue, malaise, headache, arthralgias, myalgias, meningitis, peripheral radiculoneuropathies, and myocarditis (4). A Cuban researcher, a specialist in ixodid ticks, was bitten several times by the ticks; dermatologic and neurologic symptoms compatible with Lyme disease (skin lesions, hyperesthesia with loss of reflexes, loss of muscular coordination, and fecal incontinence) developed. Borreliosis was not diagnosed at this stage; the diagnosis was either myeloradiculitis or Guillain-Barre syndrome. Three years later, a serologic diagnosis of Lyme disease was made by indirect immunofluorescence in a laboratory in the Czech Republic (5). During 1998, serum samples from 14 persons who lived in the village Las Terrazas and had epidemiologic and clinical evidence of Lyme disease, were studied in our laboratory. We used an immunoglobulin (Ig) G and IgM–enzyme-linked immunoabsorbent assay (ELISA) kit (Enzygnost Borreliosis, Behring, Marburg, Germany), in which each strip contained wells coated with inactivated borrelial antigen (detergent extract from strain isolate PKo [Borrelia afzelii]), to detect specific antibodies to B. burgdorferi complex. The assays were performed according to the manufacturers instructions. In our study, five serum samples had positive IgM titers and one near the cutoff value by IgM and IgG. ELISA has been widely used to detect antibodies to B. burgdorferi; however, this assay is not standardized, which results in different levels of sensitivity and specificity. False-positive results may occur, especially when serum samples are obtained from persons with other illnesses (6). To study possible cross-reactions with other infectious illnesses, different serologic tests were applied to the positive serum samples by using ELISA. One sample was weakly reactive to human leptospirosis (indirect hemagglutination assay with erythrocyte-sensitive substance antigen [Labiofam, Havana, Cuba]), but no samples were reactive to syphilis (rapid plasma reagin [Imefa, Havana, Cuba] and hemagglutination of Treponema pallidum [Oxoid, Diagnostic Reagents, Basingstoke, UK]). No indication of other infectious diseases was found. All serum samples positive by ELISA were also analyzed by IgG and IgM Western blotting in the spirochete laboratory at the University of Trieste, Italy. The Western blotting was performed with a protein profile from whole–cell strain PKo and by applying the criteria of positivity described by Hauser et al. (7). Two serum samples showed clear IgM antibody bands to 41- and 23-kDa proteins. No IgG bands were observed. This test reportedly is more sensitive than ELISA for IgM detection (6). We investigated the clinical manifestations of the patients with positive Western blotting. We found that one of the patients had been bitten several times by ticks and had an erythematous rash around the different bite sites; the rashes reddened and expanded over the course of a few days, with partial central clearing. The patient also had fever, hepatosplenomegaly, adenopathies, joint pain, and some nonspecific symptoms. He was given erythromycin before the laboratory results were confirmed and had a satisfactory recovery. In similar situations, repeat testing would be highly advisable. This was the same patient with low levels of antibodies to Leptospira. Investigating the symptoms of the other patient was not possible. The presence of IgM antibodies is frequently confirmed in the early stage of Lyme disease (6). The patients history of being bitten by an A. cajennenses tick, clinical manifestations of Lyme borreliosis, and specific antibodies to B. burgdorferi complex suggest the diagnosis of Lyme disease. A. cajennenses has not been reported as a vector for Lyme disease. However, it is very abundant and aggressive in Cuba, and bites from this species are common. The genus Ixodes, the main vector of B. burgdorferi sensu lato, has not been reported in the area of the study. Several articles describe a new species in the United States, B. lonestari. B. lonestari in A. americanum has been confirmed in humans with erythema migrans (8,9). No serologic test is available for antibodies to B. lonestari. That we found antiborrelial-complex antibodies may suggest the presence of a new species in this antigenic complex containing cross-reactive antigens, but many other studies are necessary to confirm it. This study represents the first serologic report of antiborrelial antibodies in Cuba. It suggests that Lyme borreliosis is present and that new cases can be expected in our country. Further laboratory studies are necessary for a more accurate diagnosis of this emerging infectious disease in Cuba.

Prevalence of antibodies to Borrelia burgdorferi sensu stricto in humans from a Cuban village

UNLABELLED Lyme disease has not been officially reported in Cuba. However, clinical cases have been serologically reported. Seroprevalence survey of Borrelia burgdorferi sensu stricto antibodies in humans in the country has not been conducted. OBJECTIVE To estimate the prevalence of borrelial antibodies in inhabitants of a village with historically high level of tick infestation. METHODS Serum specimens from 247 persons randomly selected from the population of the village were examined by IgG Western blot using B31 strain for estimating the prevalence of antibodies profile. RESULTS A seroprevalence value interval (95% CI) of 0.6%-7.2% was estimated for the studied population. The prevalent borrelial protein bands on immunoblots were 41, 72, 90/93, 34, 47, 60, 58, 56, 65/66 and 31 kDa in a decreasing order of significance. CONCLUSION These results support the previous serological findings, suggesting the presence of this borreliosis in Cuba.

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

First molecular evidence of Coxiella burnetii infecting ticks in Cuba.

Coxiella burnetii is the causative agent of Q fever. In order to explore the occurrence of C. burnetii in ticks, samples were collected from horses, dogs and humans living in a Cuban occidental community. The species most commonly recovered were Amblyomma mixtum (67%), Rhipicephalus sanguineus s.l. (27%) and Dermacentor nitens (6%). Specific IS1111 PCR and amplicon sequencing allowed the identification of C. burnetii DNA in A. mixtum collected from a domestic horse. These findings, for first time in Cuba, indicate the need for an in-depth assessment of the C. burnetii occurrence in hosts and humans at risk of infection.

First Report of the 23S rRNA Gene A2058G Point Mutation Associated With Macrolide Resistance in Treponema pallidum From Syphilis Patients in Cuba.

This study aimed to assess the presence of macrolide-resistant Treponema pallidum subtypes in Havana, Cuba. Samples from 41 syphilis patients were tested for T. pallidum 23S rRNA gene mutations. Twenty-five patients (61%) harbored T. pallidum with the A2058G mutation, which was present in all 8 subtypes that were identified. The A2059G mutation was not detected.

An Alternative and Rapid Method for the Extraction of Nucleic Acids from Ixodid Ticks by Potassium Acetate Procedure

Four variants of the potassium acetate procedure for DNA extraction from ixodid ticks at different stage of their life cycles were evaluated and compared with phenol-chloroform and ammonium hydroxide methods. The most rapid and most efficient variant was validated in the DNA extraction procedure from the engorged ticks collected from bovine, canine as well as from house ticks for the screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. The ammonium hydroxide procedure was used for non-engorged ticks. All the variants were efficient and allowed obtaining PCR-quality material according to the specific amplification of 16S rRNA gene fragment of the original tick. DNA extracted from the ticks under the study was tested by multiplex PCR for the screening of tick-borne pathogens. Anaplasma spp. and Babesia spp. amplification products were obtained from 29/48 extracts. Ammonium hydroxide protocol was not efficient for two extracts. Detection of amplification products from the PCR indicated that DNA had been successfully extracted. The potassium acetate procedure could be an alternative, rapid, and reliable method for DNA extraction from the ixodid ticks, mainly for poorly-resourced laboratories.

The Cuban Experience in the Elimination of Mother-to-Child Transmission of Congenital Syphilis

The article focuses on efforts by Cuba to eliminate mother-to-child transmission of congenital syphilis (CS). It states Cubas public health program for preventing and controlling syphilis has existed since 1972 and reduced the incidence of CS to under 0.5 cases for every 1,000 live births. It mentions the Cuban Ministry of Public Health seeks to expand and strengthen free access to medical and reproductive services to prevent sexually transmitted infections and HIV and promote health behaviors.