Non-culture based diagnostic tests for mycotic infections.

Non-culture methods being developed and evaluated for mycotic infections include polymerase chain reaction (PCR), galactomannan (GM) antigenemia, Western blot (WB) to detect antibodies, and detection of the fungal metabolites D-arabinitol and (1,3)-beta-D-glucan. Sample preparation for PCR from blood specimens depends on fractionation of peripheral blood, its pre-incubation in blood culture broth, or a total DNA method, which does not rely on fractionation, or pre-incubation. Targets for PCR of fungi in the 18S or ITS2 subunits of the ribosomal RNA genes facilitated the design of Aspergillus and Candida genus and species probes. Amplicons were identified using PCR-enzyme linked immunosorbent assay (ELISA) or reverse line-blot formats. A pilot study indicated that PCR tests on blood specimens were positive at least once in patients with confirmed invasive aspergillosis (IA). When serum-PCR and serum-GM tests were compared in IA patients, antigenemia was more often positive. PCR detected Aspergillus DNA in bronchoalveolar lavage specimens from patients at risk even when cultures were negative. D-Arabinitol can be detected as a marker of candidiasis with gas chromatography-mass spectrometry or enzyme dependent-fluorometry. Each method can differentiate the microbial D- and host L-enantiomers. (1,3)-beta-D-Glucan is produced by most genera of pathogenic fungi and can be detected in plasma by the 'G-test'. In patients with febrile neutropenia the efficacy of azole therapy correlated with plasma (1,3)-beta-D-glucan concentrations of > or = 10 pg ml(-1). The diagnosis of early acute pulmonary histoplasmosis can be improved by a WB test utilizing deglycosylated M antigen, a 94-kDa glycoprotein. The identity of M antigen as a catalase was deduced from the sequence of the cloned gene. PCR identification of Histoplasma capsulatum cultures was accomplished with primer pairs selected from H and M antigen gene sequences.

A randomized, double-blind, placebo-controlled trial of single-dose ciprofloxacin versus erythromycin for the treatment of chancroid in Nairobi, Kenya.

A randomized, double-blind, placebo-controlled clinical trial was conducted in Nairobi, Kenya, to compare single-dose ciprofloxacin with a 7-day course of erythromycin for the treatment of chancroid. In all, 208 men and 37 women presenting with genital ulcers clinically compatible with chancroid were enrolled. Ulcer etiology was determined using culture techniques for chancroid, serology for syphilis, and a multiplex polymerase chain reaction for chancroid, syphilis, and herpes simplex virus (HSV). Ulcer etiology was 31% unmixed chancroid, 23% unmixed syphilis, 16% unmixed HSV, 15% mixed etiology, and 15% unknown. For 111 participants with chancroid, cure rates were 92% with ciprofloxacin and 91% with erythromycin. For all study participants, the treatment failure rate was 15%, mostly related to ulcer etiologies of HSV infection or syphilis, and treatment failure was 3 times more frequent in human immunodeficiency virus-infected subjects than in others, mostly owing to HSV infection. Ciprofloxacin is an effective single-dose treatment for chancroid, but current recommendations for empiric therapy of genital ulcers may result in high treatment failure due to HSV infection.

Detection of Treponema pallidum, Haemophilus ducreyi, and Herpes Simplex Virus by Multiplex PCR.

The three major causes of genital ulcer disease (GUD) are herpes simplex virus (HSV), Treponema pallidum, and Haemophilus ducreyi. Although techniques exist for the laboratory diagnosis of all three organisms, constraints of cost, availability of equipment and expertise, and the lack of sensitivity and specificity of available tests, result in clinical presentation being primarily used for the diagnosis of GUD both in the United States and in developing countries. Due to the overlapping clinical presentation of the three diseases caused by these etiologic agents, and due to coinfection, these diseases are often misdiagnosed (1). It is now recognized that not only is GUD a cofactor in HIV transmission, but also that treatment of sexually transmitted diseases can reduce the incidence of HIV (2-4), thus efficient and early diagnosis and treatment of GUD is of utmost importance.

An investigation of genital ulcers in Jackson, Mississippi, with use of a multiplex polymerase chain reaction assay: high prevalence of chancroid and human immunodeficiency virus infection.

In 1994, an apparent outbreak of atypical genital ulcers was noted by clinicians at the sexually transmitted disease clinic in Jackson, Mississippi. Of 143 patients with ulcers tested with a multiplex polymerase chain reaction (PCR) assay, 56 (39%) were positive for Haemophilus ducreyi, 44 (31%) for herpes simplex virus, and 27 (19%) for Treponema pallidum; 12 (8%) were positive for > 1 organism. Of 136 patients tested for human immunodeficiency virus (HIV) by serology, 14 (10%) were HIV-seropositive, compared with none of 200 patients without ulcers (P < .001). HIV-1 DNA was detected by PCR in ulcers of 6 (50%) of 12 HIV-positive patients. Multivariate analysis indicated that men with chancroid were significantly more likely than male patients without ulcers to report sex with a crack cocaine user, exchange of money or drugs for sex, and multiple sex partners. The strong association between genital ulcers and HIV infection in this population highlights the urgency of preventing genital ulcers in the southern United States.

Comparison of clinical diagnosis and standard laboratory and molecular methods for the diagnosis of genital ulcer disease in Lesotho: association with human immunodeficiency virus infection.

A multiplex polymerase chain reaction (M-PCR) assay for Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) was compared with clinical and standard laboratory methods for the diagnosis of genital ulcer disease (GUD) in 105 patients; 36% were human immunodeficiency virus (HIV)-seropositive. Chancroid (80%), syphilis (8%), and genital herpes (8%) were the most frequent diagnoses. H. ducreyi and HSV were isolated from ulcers of 43% and 18% of patients, respectively; in 35%, all cultures were negative and the laboratory diagnosis indeterminate. M-PCR detected H. ducreyi, T. pallidum, and HSV in 56%, 23%, and 26% of patients, respectively; (no definitive diagnosis, 6%). The proportion of patients with more than one agent was 4% by culture and 17% by M-PCR (P = .002). Resolved sensitivities of M-PCR for H. ducreyi and HSV cultures were 95% and 93%, respectively. The sensitivities of H. ducreyi and HSV cultures were 75% and 60%, respectively. HSV, detected in 47% of specimens from HIV-infected versus 16% from HIV-uninfected patients (P < .001), may be emerging as a more frequent cause of GUD.

Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers.

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media. HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respectively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the detection of HSV, H. ducreyi, and T. pallidum from genital ulcers.

Identification of transposition proteins encoded by the bacterial transposon Tn7.

The bacterial transposon, Tn7, encodes an elaborate array of transposition genes, tnsABCDE. We report here the direct identification of the TnsA, TnsB, TnsC and TnsD polypeptides by immunoblotting. Our results demonstrate that the complexity of the protein information devoted to Tn7 transposition is considerable: the aggregate molecular size of the five Tns polypeptides is about 300 kDa. We also report the sequence of the tnsA gene and of the 5' ends of tnsB and tnsD. This analysis reveals that all five tns genes are oriented in the same direction within Tn7.

Recognition of Escherichia coli attTn7 by transposon Tn7: lack of specific sequence requirements at the point of Tn7 insertion.

Transposon Tn7 inserts at high frequency into a specific site in the Escherichia coli chromosome called attTn7. We show that the point of Tn7 insertion in attTn7 lies within the transcriptional terminator of the bacterial glmS gene. We have exploited the glmS transcription terminator to isolate mutants with altered sequences at the point of Tn7 insertion and have used these mutants to show that the nucleotide sequence at the point of Tn7 insertion is irrelevant to attTn7 target activity. Thus, the nucleotides which provide attTn7 target activity are distinct from the point of Tn7 insertion. We have also examined the effect of transcription on the capacity of attTn7 to act as a target for Tn7 transposition. Our results suggest that transcription of attTn7 does not modulate its Tn7 target activity.

Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion.

Transposon Tn7 transposes at high frequency to a specific site, attTn7, in the Escherichia coli chromosome. We devised a quantitative assay for Tn7 transposition in which Tn7-end derivatives containing the cis-acting transposition sequences of Tn7 transpose from a bacteriophage lambda vector upon infection into cells containing the Tn7-encoded transposition proteins. We used this assay to identify a 68-base-pair DNA segment containing the sequences essential for attTn7 target activity. This segment is positioned asymmetrically with respect to the specific point of Tn7 insertion in attTn7 and lacks obvious homology to the sequences at the ends of Tn7 which participate directly in transposition. We also show that some sequences essential for attTn7 target activity are contained within the protein-coding sequence of a bacterial gene.