The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.

IPTp with SP increases the prevalence of resistance markers in the placenta and in HIV-infected women at delivery, which suggests that host immunity is key for the clearance of drug-resistant infections. However, this effect of IPTp is limited to the period when blood levels of SP are likely to be significant and does not translate into more-severe infections or adverse clinical outcomes.

Real-time PCR can rapidly identify Mycobacterium tuberculosis in paraffin-embedded tissue in the absence of microbiological culture. In a comparison of single-copy and multicopy PCR targets in 70 tissue samples, the sensitivities were 26% and 54%, respectively, with 100% specificity. Sensitivity was 75% for newer samples and was not decreased for acid-fast bacillus (AFB) stain-negative specimens.With a third of the world's population infected by Mycobacterium tuberculosis complex, accurate and timely diagnosis of tuberculosis is critical for management of this global epidemic (12). Although tuberculosis is often diagnosed by sputum smear and microbiological culture, the disease can also present as extrapulmonary mass lesions, which are frequently biopsied without clinical suspicion for infection (3). Under histopathologic examination, biopsy sections typically demonstrate necrotizing granulomas with or without acid-fast bacilli, neither of which is specific for tuberculosis (1, 5). Without an alternative method for diagnosis, a repeat biopsy is often necessary to procure tissue for mycobacterial culture.More recently, real-time PCR has been utilized for detection of M. tuberculosis in culture and sputum (4, 6, 9, 10). Real-time PCR is faster than conventional PCR and does not require postamplification specimen handling (10). Several studies have also evaluated the usefulness of real-time PCR for M. tuberculosis in formalin-fixed, paraffin-embedded tissue, with sensitivities ranging from 67% to 100% (1,3,5,8,13). However, most of these studies did not have complete microbiological culture results to use as their reference standard and instead relied on histologic findings and/or clinical data to identify presumptive tuberculosis cases. Furthermore, none evaluated multiple tissue sites or other factors that may have impacted the sensitivity of real-time PCR in fixed tissue. An older study using conventional PCR suggested that IS6110, a multicopy PCR target, is more sensitive than a single-copy PCR target; however, this study also did not use culture as the reference standard, and the assays were compared across different laboratories using different instruments and reagents (11). Thus, the purpose of this study was to evaluate the sensitivity and specificity of real-time PCR for detection of culture-positive M. tuberculosis in formalin-fixed paraffin-embedded tissue and to identify factors that enhance or impede the sensitivity of this assay.A retrospective study was performed on 70 formalin-fixed paraffin-embedded tissue biopsy specimens collected at a major academic hospital over a 10-year period. Biopsies of 35 consecutive specimens with M. tuberculosis complex, 23 specimens with nontuberculous mycobacteria, and 12 noninfectious specimens were included. Biopsy sites included lung (n ϭ 30), lymph nodes (n ϭ 17), bone (n ϭ 5), skin (n ϭ 5), and other sites (n ϭ 13), such as soft tissue, with sample types evenly distributed over the study time period. Microbiological culture using reference methods (10) from tissu...

The combination of congenital diaphragmatic hernia (CDH) and omphalocele is quite rare but can be seen in several syndromes. We report on a female newborn with this combination that had not been diagnosed prenatally. The patient suffered respiratory failure that persisted despite intensive care support, suggesting severe secondary pulmonary hypoplasia. Autopsy revealed the combination of an anterolateral CDH and omphalocele in the absence of other anomalies. We believe this to be the first such case to be reported in the literature.

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