Legionella in clinical specimens and hospital water supply facilities: molecular detection and genotyping of the isolates

To evaluate genus- and species-specific polymerase chain reactions [PCRs] for the detection of the genus Legionella and the species Legionella pneumophila in clinical specimens and hospital water supplies, and to establish a simple and reproducible random amplification of polymorphic DNA [RAPD]-PCR technique for genotyping of Legionella. A total of 70 respiratory tract specimens [bronchoalveolar lavage: n = 46; endotracheal secretions: n = 9; sputum: n = 15] from patients with atypical pneumonia, and 283 environmental samples [water: 20; swabs: 263] collected from water storage and supply facilities of the Mubarak Al-Kabeer Hospital, Kuwait, were tested by culture and genus-specific PCR for the detection of Legionella. The L pneumophila isolates were subsequently typed by serology and RAPD-PCR using serotype-specific sera and arbitrary primers, respectively. Of the 70 clinical samples, culture yielded 2 [2.9%] whereas genus-specific PCR detected Legionella in 20 [28.6%] samples. The 2 culture-positive specimens were also positive for L.-pneumophila-specific PCR. Testing of swab and water samples by culture and genus-specific PCR yielded 61 [21.6%] and 67 [23.7%] positive samples, respectively. All of the 61 culture-positive samples were also positive by genus-specific PCR and 45 of them were positive for L.-pneumophila-specific PCR. Serological typing of 43 L pneumophila isolates showed that 8 of these belonged to serotype 1 and 35 to serotype 3; however, RAPD-PCR analyses demonstrated polymorphisms among the isolates of both serotypes. A higher association between PCR and culture was observed for the environmental samples than for the clinical samples. The application of genus- and species-specific PCRs and RAPD is useful in the detection and typing of Legionella in clinical and environmental samples

Polymerase-chain-reaction-based detection of fetal rhesus d and Y-chromosome-specific DNA in the whole blood of pregnant women during different trimesters of pregnancy

The aim of this study was to determine whether or not a noninvasive procedure utilizing maternal peripheral blood as the source of DNA and polymerase chain reaction [PCR] could be used to detect fetal rhesus D [RhD] status as well as fetal gender during different gestational stages of pregnancy. Maternal blood samples were obtained from 54 RhD-negative pregnant women during the first trimester [6-13 weeks, n = 14], second trimester [14-26 weeks, n = 26] and third trimester [27-40 weeks, n = 14]. Genomic DNA was extracted from the whole blood and analyzed by seminested and nested PCR for detection of DNA sequences corresponding to RhD [n = 54] and Y chromosome [n = 48] using RhD and Y-chromosome-specific oligonucleotide primers, respectively. The seminested/nested PCR results were compared with the RhD status and gender of the babies after delivery. The sensitivity and specificity of seminested PCR for detection of fetal RhD positivity in whole blood of pregnant women were 81 and 100%, respectively, while the sensitivity and specificity of nested PCR for detection of male fetuses, using Y-chromosome-specific DNA as a marker, were 96 and 91%, respectively. There were no significant differences in the PCR results with samples obtained from women at different gestational stages of pregnancy. Seminested and nested PCRs for detection of fetal RhD and gender status, respectively, by using the blood of pregnant women during different gestational stages of pregnancy, are reliable noninvasive procedures with high sensitivity and specificity

Mycobacterial gene cloning and expression, comparative genomics, bioinformatics and proteomics in relation to the development of new vaccines and diagnostic reagents

Recent advances in molecular and genomic techniques have facilitated research on several aspects of mycobacteriology, such as diagnosis and the identification of new vaccines and therapeutic targets for various diseases, including tuberculosis. The aim of this review was to analyze the implications of advances in molecular and genomic techniques on the development of new vaccines for tuberculosis as well as immunological reagents to diagnose the disease. Gene cloning and expression, DNA and protein sequencing, polymerase chain reaction, comparative genomics, bioinformatics, proteomics and DNA and peptide synthesis coupled with the application of cellular immunology techniques have led to the identification of several antigens of Mycobacterium tuberculosis, which have potential for diagnosis and vaccine applications. For example, cross-reactive mycobacterial antigens like heat shock proteins, MTB32 and MTB39, have been identified as new vaccine candidates, and antigens encoded by M. tuberculosis-specific genomic regions as new reagents for diagnosis

Seroprevalence of three emerging arboviral infections in Kuwaiti nationals

Diseases caused by dengue, s and fly fever and hanta viruses pose a major health risk in many countries. We determined the threat of these arboviral infections through a serologic using enzyme linked immunosorbent assay [ELISA] based tests. Hantavirus-specific antibodies were also detected using immunofluorescence. Of 499 samples tested for dengue virus IgG antibodies l4% were as positive for dengue positive by all the ELISA tests. Among the 42 showing strong IgG reactivity, only 1 was positive for dengue virus IgM antibodies. All samples tested for IgG antibodies to s and fly fever virus were negative. Hantavirus antibodies were detected in 11% of the 46 samples from high-risk individuals. The low prevalences suggest that at present these infections are not a serious problem in Kuwait

Detection of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria by multiplex polymerase chain reactions

The ability of two-band and three-band multiplex polymerase chain reactions to detect and differentiate Mycobacterium tuberculosis complex from non-tuberculous mycobacteria was evaluated. The polymerase chain reactions differentiated between M. tuberculosis and non-tuberculous mycobacteria when standard strains and clinical isolates of mycobacteria were tested. The sensitivity of the two-band and three-band techniques to detect M. tuberculosis in clinical specimens, compared with smear and/or culture, was 88% and 75% respectively. Although both techniques showed 100% specificity, the superior sensitivity of the two-band technique suggests that it could be more useful in the diagnosis of tuberculosis and in differentiating M. tuberculosis complex from non-tuberculous mycobacteria

Promises and problems of polymerase chain reaction [PCR] in the diagnosis of tuberculosis

Tuberculosis [TB] is a major public health problem of world wide concern. Early and specific diagnosis of TB is the key to successful management of TB patients. Nucleic acid based molecular techniques are becoming popular in the specific diagnosis of infectious diseases including tuberculosis. Among these techniques, polymerase chain reaction [PCR] is the highly promising and widely attempted method The molecular basis of PCR, its promises and problems in the diagnosis of TB is presented in this article