Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples.

Due to the clinical importance of differentiating the two species of the Entamoeba histolytica/Entamoeba dispar complex, we developed a multiplex polymerase chain reaction (PCR) method that overcomes time-consuming and laborious procedures. We report here a DNA extraction protocol using non-fixed stool samples that avoid long lysis-incubation periods through the combined use of zirconium beads and a lysis-supporting buffer. We characterized 49 of 52 stool specimens from Cuban patients with amoebiosis. Among them, 36 (75.5%) were infected only with E. dispar (the nonpathogenic species), while 13 (24.5%) displayed a mixed infection with both E. dispar and E. histolytica. The multiplex PCR protocol showed a specificity of 1.00 and a sensitivity of 0.94. Furthermore, the entire procedure can be performed in one day. This approach is therefore reliable and applicable in the field for epidemiologic studies.

[Toxigenic Vibrio cholerae non-01]. / Vibrio cholerae No-01 toxigénico.

The antimicrobial susceptibility and the presence of a heat-stable toxin were researched into 100 non-01 Vibrio cholerae strains sent by 7 different health centers to the National Reference Laboratory of Acute Diarrheal Diseases in "Pedro Kourí" Tropical Medicine Institute. The presence of 20% toxigenic non-01 Vibrio cholerae was detected, a figure substantially higher than that reported in other geographic areas, except for endemic areas. This result will make it possible to set epidemiological alert in Cuba because these strains can be infected by CTX phages (element transporting genes that encode for choleric toxin) which will give such strains an epidemic potential similar to that of the etiologic agent of cholera. The identified strains could be studied as possible cholera vaccine candidates.

[Toxigenic Vibrio cholerae: identification of the ctxB gene]. / Vibrio cholerae toxigénico: identificación del gen ctxB.

BACKGROUND: A specific probe was designed to identify part of the genetic sequence of the ctxB gene which encodes for the B subunit of the cholera toxin by polymerase chain reaction (PCR) which amplifies a 318 bp segment of the ctxB gene. Marked with P32, we used this probe for colony hybridization which is a technique for identifying the production capacity of subunit B of strains of Vibrio cholerae O1 from different outbreaks in South America (Perú 1992 and Ecuador 1993-1995) and from, collection strains. This probe was tested for the identification of the ctxB gene in Vibrio cholerae O139. METHOD: Thirty-eight phylogenetically related strains were studied: 24 V. cholerae O1, 4 V. cholerae non O1, 5 Aeromonas, 4 Plesiomonas and 1 Escherichia coli. RESULTS: The probe demonstrated to be useful for the identification of the ctxB gene (which codifies for the subunit B of the cholera toxin) in 24 strains of Vibrio cholerae O1 and in the Vibrio cholerae O139 strain. The ctxB gene was not detected in the remaining strains pertaining to the Vibrio cholerae non O1 species (non O139), Plesiomonas, Aeromonas spp. and E. coli. The specificity of this product was not demonstrated since no signal of unspecific hybridization appeared with phylogenetically related strains such as Escherichia coli K88 (LT+) and Aeromonas hydrophila ATCC (LT+), producers of the thermolabile LT toxin. It is important to indicate that the ctxB gene in V. cholerae O139 has been identified, for the first time, with our probe and thus it may be said that all the strains which have genetic codification for CT up to now may be identified. CONCLUSIONS: We conclude that the system herein described provides advantages over the immunologic and biologic methods for evaluating a large number of samples in a short time and with excellent specificity and sensitivity which are important in the diagnosis and the epidemiologic surveillance of the disease.

Detección de Escherichia coli toxigénica (LT) mediante la reacción en cadena de la polimerasa [The detection of toxigenic Escherichia coli (LT) by the polymerase chain reaction].

In this paper it is described the detection enteroxigenic Escherichia coli LT (+). This method is based on the amplification of a DNA fragment of 400 pairs of bases by polymerase chain reaction (PRC). The oligonucleotides were designed by the authors and the characteristic patterns were observed when the samples were submitted to an electrophoresis in an Agarose gel at 2%. The PCR had positive results with the strains of Escherichia coli 0:149 K; 88 (LT+) collection and with 20 strains isolated from patients with acute diarrhea. Negative results were found in Escherichia coli 0:101 K:99 NM (ST+), Vibrio cholerae 01 and Aeromonas hydrophila.

Consideraciones sobre el aislamiento en exudados vaginales de Streptococcus morbillorum / The isolation of Streptococcus morbillorum from vaginal exudates

De el estudio de 195 exudados vaginales enviados por el Servicio de Ginecologia de este hospital, durante el periodo 1988-1990, hemos seleccionado aquellos en los que el cultivo fue positivo para estreptococos, 58 (30 por ciento) de los cuales 26 (44.8 por ciento) correspondia a Streptococcus morbillorum, 9 (15.5 por ciento) a Gardnerella vaginalis, 5 (8.6 por ciento) a Enterococcus faecalis-durans, y a Streptococcus agalactiae, 3 (5.1 por ciento) a Streptococcus mitis y Styreptococcus milleri, 2 (3.4 por ciento) a Streptococcus bovis y Streptococcus cremoris y 1 (1.7 por ciento) a Streptococcus salivarius, Streptococcus equinus y Strptococcus sanguis II respectivamente. En todos los casos se observó antecedentes de actuacción medicoquirurjica en el tracto genital, y en el 52,8 por ciento de los casos fuè concomitante con el diagnostico clinico-micologico de candidiasis vaginal. La identificacción bacteriologica se realizó mediante el sistema API 20 STREP (sistema api bioMérieux GmbH, Nüttingen, Alemania) dando un patron tipico ("excelente identificacción") para el Streptococcus morbillorum.

[The isolation of Streptococcus morbillorum from vaginal exudates]. / Consideraciones sobre el aislamiento en exudados vaginales de Streptococcus morbillorum.

We have tested 195 vaginal secretions sent by Gynecology Service of this hospital between the years 1988-1990. We achieved positive culture for streptococci in 58 (30%) of these cultures, 26 (44.8%) corresponding to Streptococcus morbillorum 9 (15.5%), to Gardnerella vaginalis 5 (8.6%), to Enterococcus faecalis-durans and to Streptococcus agalactiae, 3 (5.1%) to Streptococcus mitis and milleri 2 (3.4%), to Streptococcus bovis and cremoris, and 1 (1.7%) to Streptococcus salivarius, equinus and sanguis II respectively. We previously found that 52.8% of these patients were positive for vaginal candidiasis. The bacteriological identification done by the API 20 STREP System (bioMerieux GmbH, Nútingen, Germany) provides a typical pattern ("good identification") for the Streptococcus morbillorum.

[Ampicillin resistance mediated by the R plasmid in strains of Shigella flexneri]. / Resistencia a la ampicilina mediada por plásmido R en cepas de Shigella flexneri.

Forty Shigella flexneri strains isolated from children attended to at the Children's Hospital of Camagüey during an outbreak of acute diarrheal disease were studied; the minimal inhibitory concentration of ampicillin was determined. 33 strains (82.5%) were resistant to higher concentrations: 8 to 16 micrograms/mL, and 7 were susceptible to 4 micrograms/mL concentrations. Resistance plasmid (R) extraction was carried out in all the isolated strains and a common plasmid was found this plasmid was purified and transferred to Escherichia coli HE 101. Resistance transmission was tested.

Survey for secondary dapsone and rifampicin resistance in Cuba.

A total of 1211 Cuban multibacillary leprosy patients treated for at least 5 years were clinically and bacteriologically examined. They were being treated according to a 2-phase monotherapy regimen with RMP first and DADDS afterwards. On skin-smear examination 50 patients were found positive, of which 9 showed a BI of 3+ or higher at any site. With regard to the clinical status the only cases found with clinical signs of relapse were 5 out of 7 long-standing patients with BI of 4+ and 5+. A 6th patient of this high BI group who showed a good clinical condition, except for a heavy infiltration of both earlobes, was receiving a second RMP course when examined and biopsied for this research. These 9 patients were biopsied and susceptibility tests to RMP and DDS performed. The results showed that in 1 case the Mycobacterium leprae were resistant to both drugs; the organisms from 2 other patients were susceptible to RMP but low-grade resistant to DDS. Those from another patient were susceptible to RMP and fully resistant to DDS. In 3 other cases the bacilli did not multiply in any of the mice but 1 of these strains was from the patient taking a second RMP course, therefore this strain might also be susceptible to RMP and resistant to DDS. In the last 2 cases multiplication was only observed in 2 of the controls and in 1 of the 0.0001% DDS treated mice; therefore, these experiments were not conclusive, and the AFB recovered were inoculated into fresh mice to repeat the tests but these failed to multiply.