Development of an Aotus nancymaae model for Shigella Vaccine immunogenicity and efficacy studies.

Several animal models exist to evaluate the immunogenicity and protective efficacy of candidate Shigella vaccines. The two most widely used nonprimate models for vaccine development include a murine pulmonary challenge model and a guinea pig keratoconjunctivitis model. Nonhuman primate models exhibit clinical features and gross and microscopic colonic lesions that mimic those induced in human shigellosis. Challenge models for enterotoxigenic Escherichia coli (ETEC) and Campylobacter spp. have been successfully developed with Aotus nancymaae, and the addition of a Shigella-Aotus challenge model would facilitate the testing of combination vaccines. A series of experiments were designed to identify the dose of Shigella flexneri 2a strain 2457T that induces an attack rate of 75% in the Aotus monkey. After primary challenge, the dose required to induce an attack rate of 75% was calculated to be 1 × 10(11) CFU. Shigella-specific immune responses were low after primary challenge and subsequently boosted upon rechallenge. However, preexisting immunity derived from the primary challenge was insufficient to protect against the homologous Shigella serotype. A successive study in A. nancymaae evaluated the ability of multiple oral immunizations with live-attenuated Shigella vaccine strain SC602 to protect against challenge. After three oral immunizations, animals were challenged with S. flexneri 2a 2457T. A 70% attack rate was demonstrated in control animals, whereas animals immunized with vaccine strain SC602 were protected from challenge (efficacy of 80%; P = 0.05). The overall study results indicate that the Shigella-Aotus nancymaae challenge model may be a valuable tool for evaluating vaccine efficacy and investigating immune correlates of protection.

Isoniazid induces its own resistance in nonreplicating Mycobacterium tuberculosis.

Isoniazid (INH) resistance is most frequent among drug-resistant Mycobacterium tuberculosis clinical isolates. This study was conducted to investigate whether INH could induce its own resistance. During INH susceptibility testing in BACTEC 12B and MGIT 960 media, weekly subcultures were made from the drug-containing media into fresh medium without drug and susceptibility testing was performed. Rifampin (RIF) was used as a control drug. M. tuberculosis H37Rv and three clinical isolates were tested in this study. INH-resistant subcultures were analyzed for catalase activity, INH susceptibility, and mutations associated with INH resistance. With inoculum size (10(4) bacilli) smaller than a size that contains spontaneously INH-resistant mutants, INH was found to induce resistance to itself in INH-tolerant persisters but not to other drugs. The minimum time required for induction of INH resistance was 5 to 6 days. In contrast, RIF did not induce RIF resistance. Eight subcultures with INH-induced resistance were analyzed, and two had a MIC of 0.4 microg/ml INH and six had MICs of over 2 microg/ml INH. Four of the eight subcultures with INH-induced resistance had lost catalase activity, with three having katG mutations. Despite being a powerful frontline tuberculosis drug, INH has the potential drawback of inducing its own stable genetic resistance in INH-tolerant persisters. This finding helps to explain the higher frequency and prevalence of INH-resistant isolates than isolates with resistance to other drugs in patients.

Detección sensible y específica de mycobacterium tuberculosis a partir de muestras clínicas, mediante la amplificación de un elemento repetitivo de la familia rep13e12 / Sensitive and specific detection of Mycobacterium tuberculosis from clinical samples by amplification of a repeated sequence of the REP13E12 family

Objetivos: Diseñar e implementar un método de PCR para la detección sensible y específica de M. tuberculosis, orientado al oportuno diagnóstico de la tuberculosis mediante su adecuada aplicación en muestras clínicas. Materiales y métodos: Se diseñaron oligonucleótidos específicos para la amplificación de un fragmento de 318pb luego de una alineación múltiple de secuencias REP13E12 de M. tuberculosis. Se realizó la estandarización del método de PCR y se evaluó la sensibilidad y especificidad diagnóstica utilizando muestras clínicas con diagnóstico baciloscópico y cultivo. Resultados: El REP13E12-PCR detectó hasta 100 fg de ADN genómico, fue específico para la detección del complejo M. tuberculosis y capaz de distinguirlos de micobacterias atípicas. Esta evaluación preliminar proporcionó 100% de sensibilidad y especificidad en muestras clínicas de pacientes con diagnóstico de TB. Conclusiones: La amplificación de REP13E12 mediante PCR es una alternativa para el diagnóstico rápido de pacientes con TB, especialmente en casos cuyo diagnóstico pueda ser dificultoso o poco claro mediante el uso de los métodos convencionales..

Objectives: To design and implement of PCR method for sensitivity and specific detection of M. tuberculosis oriented to opportune and correctly diagnosis of tuberculosis by the application on clinical specimens. Material an methods: Specific oligonucleotides for the amplification of a fragment of 318pb after a multiple alignment of sequences REP13E12 were designed. The PCR method was standarize and also the analytical and diagnostic sensibility and specifity was evaluate; using clinical samples with smear or culture diagnosis positive and negative. Results: The REP13E12-PCR detected up to 100 fg of genomic DNA. It was specific for the detection of complex M. tuberculosis and able to distinguish them of non-tuberculous mycobacteria. This preliminary evaluation provided 100% of sensitivity and specificity in sputum samples of patients with TB diagnosis. Conclusions: The REP13E12 amplification by PCR is an alternative method for the rapid diagnosis of patients with TB, especially for cases which difficult or unclear diagnosis by the gold standard technique.