Bruno Schmidt

Serodiagnosis of Syphilis: Antibodies to Recombinant Tp0453, Tp92, and Gpd Proteins Are Sensitive and Specific Indicators of Infection by Treponema pallidum

ABSTRACT Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies). We studied a group of six recombinant T. pallidum antigens for their sensitivities and specificities with sera from individuals with syphilis (n = 43), relapsing fever (n = 8), Lyme disease (n = 8), and leptospirosis (n = 9) and from uninfected individuals (n = 15). Three recombinant proteins, Tp0155, Tp0483, and Tp0751, demonstrated sensitivity values that ranged from 28 to 42%. In contrast, three other recombinant proteins exhibited the following sensitivity and specificity values: Tp0453, 100% sensitivity and 100% specificity; Tp92 (Tp0326), 98% sensitivity and 97% specificity; and Gpd (Tp0257), 91% sensitivity and 93% specificity. Tp0453, Tp92, and Gpd also were recognized by sera from individuals with early primary syphilis that were nonreactive with the antilipoidal Venereal Disease Research Laboratory test. The reactivities of syphilis patient sera with Tp0453, Tp92, and Gpd were proportional to the titers of these sera with the treponemal test MHA-TP (microhemagglutination assay for T. pallidum). Thus, the recombinant T. pallidum antigens Tp0453, Tp92, and Gpd show promise as diagnostic antigens in the enzyme-linked immunosorbent assay-based assay.

Success and failure in the treatment of acrodermatitis chronica atrophicans

SummaryTo determine the most effective treatment for acrodermatitis chronica atrophicans, several clinical trials were undertaken in recent years to evaluate whether a 2-week course of ceftriaxone would be superior to oral antibiotics. Of the 46 patients suffering from acrodermatitis chronica atrophicans, 14 were treated with ceftriaxone 2g for 15 days. The remaining patients received either oral penicillin V 1.5 million IU t.i.d. or doxycycline 100 mg b.i.d. for 20 to 30 days. Patients were followed up for at least 1 year. Of the 14 ceftriax-one-treated patients four showed incomplete regression of the inflammatory skin changes after 6 to 12 months. Two out of five patients who were monitored forBorrelia burgdorferi DNA excretion were still positive after 12 months as compared to none of six patients who were treated orally for 20–30 days. Six out of 11 patients treated orally for only 20 days needed retreatment after 6 months because of continuing skin manifestations, neuropathy or arthralgia. A 30-day duration of treatment with oral antibiotics and not the chosen antibiotic is crucial for curing acrodermatitis chronica atrophicans. The duration of treatment with ceftriaxone needed for eradication ofBorrelia in acrodermatitis chronica atrophicans has yet to be determined in future studies.ZusammenfassungIn den letzten Jahren wurden verschiedene Therapiekonzepte zur optimalen Behandlung der Acrodermatitis chronica atrophicans durchgeführt. Aus einer Gruppe von 46 Patienten erhielten 14 Ceftriaxon 2g für 15 Tage, die übrigen Patienten Penicillin V 1,5 Mill. IE 3×1 oder Doxycyclin 100 mg 2×1 durch 20–30 Tage. Vier der 14 Patienten, die mit Ceftriaxon über nur 15 Tage behandelt worden waren, zeigten bei einer Kontrolle nach 6–12 Monaten eine inkomplette Rückbildung der Hautveränderungen. Zwei von fünf mit Ceftriaxon behandelten Patienten wiesen nach 12 Monaten eine Borrelien-DNA Ausscheidung im Harn auf. Im Vergleich dazu konnten aus dem Harn von sechs Patienten, die orale Antibiotika über 20–30 Tage erhalten hatten, nach dem gleichen Zeitraum keine Borrelien-DNA Fragmente amplifiziert werden. Sechs von 11 Patienten, die oral über 20 Tage behandelt worden waren, mußten jedoch aufgrund anhaltender entzündlicher Hautveränderungen, Neuropathie oder Arthralgien ein zweites Mal therapiert werden. Wir schließen aus den Ergebnissen, daß der Therapieerfolg bei der Acrodermatitis chronica atrophicans nicht so sehr von der Wahl des Antibiotikums sondern vielmehr von einer Therapiedauer von 30 Tagen abhängt. Der Behandlungserfolg einer möglicherweise kürzeren Behandlungsdauer mit Ceftriaxon muß noch in weiteren Studien erprobt werden.

Importance of Sample Preparation for Molecular Diagnosis of Lyme Borreliosis from Urine

ABSTRACT Urine PCR has been used for the diagnosis of Borrelia burgdorferi infection in recent years but has been abandoned because of its low sensitivity and the irreproducibility of the results. Our study aimed to analyze technical details related to sample preparation and detection methods. Crucial for a successful urine PCR were (i) avoidance of the first morning urine sample; (ii) centrifugation at 36,000 × g; and (iii) the extraction method, with only DNAzol of the seven different extraction methods used yielding positive results with patient urine specimens. Furthermore, storage of frozen urine samples at −80°C reduced the sensitivity of a positive urine PCR result obtained with samples from 72 untreated erythema migrans (EM) patients from 85% in the first 3 months to <30% after more than 3 months. Bands were detected at 276 bp on ethidium bromide-stained agarose gels after amplification by a nested PCR. The specificity of bands for 32 of 33 samples was proven by hybridization with a GEN-ETI-K-DEIA kit and for a 10 further positive amplicons by sequencing. By using all of these steps to optimize the urine PCR technique, B. burgdorferi infection could be diagnosed by using urine samples from EM patients with a sensitivity (85%) substantially better than that of serological methods (50%). This improved method could be of future importance as an additional laboratory technique for the diagnosis of unclear, unrecognized borrelia infections and diseases possibly related to Lyme borreliosis.

Course of Borrelia burgdorferi DNA shedding in urine after treatment.

Diagnosis of Lyme borreliosis by urine polymerase chain reaction (PCR) has been recognized as having better diagnostic sensitivity in patients with erythema migrans than serological methods. We made serial tests with 192 urine specimens from 70 patients with erythema migrans and 60 urine specimens from 21 patients with acrodermatitis chronica atrophicans to evaluate the course of positive urine PCR after antibiotic treatment. Before treatment, urine samples from patients with erythema migrans showed a positive PCR in 27/34 samples (79%), and those from patients with acrodermatitis chronica atrophicans in 7/11 (63%). The specificity of bands was proven by hybridization with GEN-ETI-KTM-DEIA kit in 40/41 samples. Borrelia DNA in urine decreased gradually within the observation period of one year in both patients with erythema migrans and acrodermatitis chronica atrophicans, and persisted without clinical symptoms in 4/45 patients with erythema migrans (8%) after 12 months. Urine PCR can serve as a diagnostic method in early Lyme borreliosis and also in seropositive patients with unclear clinical symptoms.

Evaluation of a New Particle Gel Immunoassay for Determination of Antibodies against Treponema pallidum

ABSTRACT A new particle gel immunoassay (DiaMed AG, Cressier sur Morat, Switzerland) with three recombinant Treponema pallidum antigens was evaluated with serum samples from patients with syphilis (n = 124) and patients without syphilis (n = 490). It proved to be a simple, rapid (20 min), and useful test with sensitivity, specificity, and positive and negative predictive values of 91.9, 99.8, 99.2, and 98%, respectively.

Inability of One-Step Real-Time PCR To Detect Borrelia burgdorferi DNA in Urine

We recently described successful molecular diagnosis of B. burgdorferi DNA by nested PCR in urine in patient-derived samples only after extraction with DNAzol (1). The aim of this study was to examine whether Borrelia burgdorferi DNA can also be detected in urine by quantitative one-step real-time PCR (Q-PCR). Q-PCR was performed after extraction with DNAzol (Molecular Research Center Inc., Cincinnati, Ohio), using the same protocol as recently published, and also after extraction with the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) and the High Pure PCR template preparation kit (Roche Diagnostics, Mannheim, Germany). Q-PCR was performed according to the assay method of Schwaiger et al. (2) and also by using newly designed primers and probes derived from the flagellin gene (B. burgdorferi sensu lato: TaqMan probe, 5′ FAM-ATT GCT GAT CAA GCT CAA TAT AAC CAA ATG CAC A-TAMRA 3′; forward primer, 5′-TGA AAT AGA GCA ACT TAC AGA CGA AAT T-3′; reverse primer, 5′-CAT TTT GAG AAG CAG ATT TGT TTG A-3′). For the latter PCR, 5 μl of DNAzol-extracted DNA or 10 μl of QIAamp- or Roche-extracted DNA, respectively, was mixed with 12.6 μl of 2× TaqMan buffer, 200 nM TaqMan probe, 600 nM (each) primer, and H2O to a final volume of 25 μl. Amplification was performed on a GeneAmp 5700 system and, for control purposes, on a GeneAmp 7000 system (Applied Biosystems, Foster City, Calif.) starting at 95°C for 10 min, followed by 45 cycles at 95°C, each for 15 s, and then 60°C for 1 min using the TaqMan universal PCR master mix (Applied Biosystems) with the human TaqMan beta-actin detection reagents (Applied Biosystems) for control purposes. DNA of all three B. burgdorferi genospecies could be detected down to 50 borrelia cells/PCR for Borrelia afzelii and Borrelia garinii and 100 cells/PCR for B. burgdorferi sensu stricto in B. burgdorferi-spiked urine samples after extraction with DNAzol (Fig. ​(Fig.1).1). After extraction with the QIAamp and Roche kits, positive signals could be seen only when urine samples were spiked with 500 borreliae (data not shown). Neither 10 urine samples positive by nested PCR after DNAzol extraction nor 12 freshly obtained urine samples from patients with erythema migrans gave a positive signal by Q-PCR, regardless of which extraction procedure was used, although beta-actin was positive in all samples (Fig. ​(Fig.1).1). The requirements for positive patient samples in earlier experiments were a detection rate of five borrelia cells/PCR in spiked urine samples. Although optimizing procedures using further primer-probe sequences, primer concentrations, and cycling conditions could improve the outcome of this assay, we assume that unlike nested PCR, one-step Q-PCR as performed by two different primers and probes is not sensitive enough to detect the few borreliae present in urine from patients with erythema migrans. FIG. 1. Results of Q-PCR in spiked urine samples (1 to 4; 8 to 10) and patient urine samples (5 to 7) after extraction with DNAzol, QIAamp, or Roche. Forty-two PCR cycles are shown performed with the newly designed assay. 1, beta-actin assay as control example, ...

Specific IgM tests in the diagnosis of syphilis

Zusammenfassung. Der Aussagewert von 3 Treponema-pallidum-spezifischen IgM-Testen, dem 19S-IgM-FTA-ABS-, dem 19S-IgM-Solid-Phase-Haemadsorption-Assay (IgM-SPHA) sowie dem IgM-Captia-Test wurde an 359 Sera von unbehandelten Patienten mit bekannter Syphilisinfektion untersucht und die Ergebnisse miteinander sowie mit den Befunden der Routineserologie (FTA-ABS-, TPHA-, VDRL-Test) verglichen. Im Primärstadium (n=38) waren die Proben sämtlicher untersuchter Patienten (37/37) im 19S-IgM-FTA-ABS- und im IgM-Captia-Test, jedoch nur 14/35 (40%) im IgM-SPHA-Test reaktiv. Der FTA-ABS zeigte bei 36/38 (94,7%), der TPHA bei 32/38 (84,2%), der VDRL bei 27/38 (71,1%) die Infektion an. Im Stadium der Frühlatenz (n=53) war der 19S-IgM-FTA-ABS-Test bei 48/50 (96%) reaktiv, der IgM SPHA bei 38/52 (73,1%), der IgM-Captia bei 44/49 (89,8%), der FTA-ABS bei 53/53 (100%), der TPHA bei 51/53 (96,2%) und der VDRL bei 48/53 (90,6%). Im Sekundärstadium (n=28) war der 19S-IgM-FTA-ABS-Test bei 28/28 (100%) reaktiv, der IgM-Captia bei 26/27 (96,3%), der 19S-IgM-SPHA bei 36/52 (69,2%), der FTA-ABS, der TPHA- und der VDRL-Test bei 28/28 (100%). Bei quantitativer Auswertung (ELISA-Einheiten – Absorptions-Grenzwert) des IgM-Captia-Testes lagen die Mittelwerte im Primärstadium deutlich höher (2,25) als im Sekundärstadium (1,7). Die Sera von Patienten mit Neurosyphilis (n=45) waren im 19S-IgM-FTA-ABS-Test in 19/38 (50%) der Proben, im IgM-SPHA-Test in 45/45 (100%), im IgM-Captia in 6/26 (23,1%), im FTA-ABS in 45/45 (100%), im TPHA in 45/45 (100%) und im VDRL in 43/45 (95%) reaktiv. Schließlich wurden die Sera von 195 Personen mit latenter Syphilis von unbekannter Dauer untersucht.Abstract. A total of 359 sera of untreated patients with syphilis were examined by three methods for the detection of Treponema pallidum specific IgM antibodies, the 19S-IgM-FTA-ABS test, the IgM solid phase haemadsorption assay (IgM-SPHA), and the IgM Captia assay. The results were compared and evaluated. In primary syphilis, the 19S-IgM-FTA-ABS and IgM-Captia yielded reactive results in all patients, whereas only 40% were positive in the IgM-SPHA; the corresponding values for early latent syphilis were 96.0%, 91.5% and 73.1%, respectively. In secondary syphilis, the reactivity of █ serum out of 27 was missed by IgM-Captia and that of another, by the IgM-SPHA. Mean values (ELISA units=extinction/cut-off) of IgM-Captia were higher in primary (2.25) than in secondary syphilis (1.70). In neurosyphilis, only the IgM-SPHA test detected reactivity in all sera, sensitivity for 19S-IgM FTA-ABS and IgM-Captia was 50.0% and 23.1%, respectively. Specificity of the IgM-Captia test results, determined in 386 sera, was 91.2%. The results of specific IgM tests are essential in the diagnosis of congenital syphilis as well as in the recognition of reinfection; they indicate the need for treatment and are useful in the assessment of the effectiveness of therapy.