Rapid Tuberculosis Diagnostics Including Molecular First- and Second-Line Resistance Testing Based on a Novel Microfluidic DNA Extraction Cartridge.

Xpert MTB/RIF testing has improved tuberculosis (TB) diagnostics and rifampicin (Rif) resistance testing worldwide. However, it has weaknesses, such as its restriction to Rif resistance testing and the inability to use extracted DNA for further testing. Herein, a holistic diagnostic workflow, including TB detection and resistance testing toward Rif, isoniazid, and important second-line drugs (SLDs), based on a novel microfluidic DNA extraction cartridge (TB-Disk), is presented. DNA from 73 precharacterized sputum samples was extracted with TB-Disk, including 45 clinical and bacteriologically confirmed TB samples, nine TB-negative samples, and 19 sputum samples spiked with twofold dilutions of TB bacteria. The extracted DNA was subjected to further testing with FluoroType MTB (FT-MTB), GenoType MTBDRplus (GT-plus), and GenoType MTBDRsl. A total of 100% (20/20) and 72% (18/25) of smear-positive and smear-negative TB samples were identified as Mycobacterium tuberculosis complex positive. A total of 79% (33/42) of subsequently GT-plus tested samples yielded a valid result. Eight samples were identified as multidrug-resistant TB by GT-plus and further tested for resistance toward SLDs using GenoType MTBDRsl, yielding 75% (6/8) valid results. FT-MTB with cartridge-based DNA extraction (Disk-DNA) and DNA extracted with FluoroLyse yielded similar analytical sensitivities. FT-MTB with Disk-DNA was 100% specific. TB-Disk in combination with FT-MTB enables sensitive TB detection. The Disk-DNA can be further used for screening resistance toward first-line drugs and SLDs.

A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB.

Rapid diagnosis of tuberculosis (TB) and antibiotic resistances are imperative to initiate effective treatment and to stop transmission of the disease. A new generation of more sensitive, automated molecular TB diagnostic tests has been recently launched giving microbiologists more choice between several assays with the potential to detect resistance markers for rifampicin and isoniazid. In this study, we determined analytical sensitivities as 95% limits of detection (LoD95) for Xpert MTB/Rif Ultra (XP-Ultra) and BD-MAX MDR-TB (BD-MAX) as two representatives of the new test generation, in comparison to the conventional FluoroType MTB (FT-MTB). Test matrices used were physiological saline solution, human and a mucin-based artificial sputum (MUCAS) each spiked with Mycobacterium tuberculosis in declining culture- and qPCR-controlled concentrations. With BD-MAX, XP-Ultra, and FT-MTB, we measured LoD95TB values of 2.1 cfu/ml (CI95%: 0.9-23.3), 3.1 cfu/ml (CI95%: 1.2-88.9), and 52.1 cfu/ml (CI95%: 16.7-664.4) in human sputum; of 6.3 cfu/ml (CI95%: 2.9-31.8), 1.5 cfu/ml (CI95%: 0.7-5.0), and 30.4 cfu/ml (CI95%: 17.4-60.7) in MUCAS; and of 2.3 cfu/ml (CI95%: 1.1-12.0), 11.5 cfu/ml (CI95%: 5.6-47.3), and 129.1 cfu/ml (CI95%: 82.8-273.8) in saline solution, respectively. LoD95 of resistance markers were 9 to 48 times higher compared to LoD95TB. BD-MAX and XP-Ultra have an equal and significantly increased analytical sensitivity compared to conventional tests. MUCAS resembled human sputum, while both yielded significantly different results than normal saline. MUCAS proved to be suitable for quality control of PCR assays for TB diagnostics.

Automated high-throughput immunomagnetic separation-PCR for detection of Mycobacterium avium subsp. paratuberculosis in bovine milk.

Two monoclonal antibody-mediated immunomagnetic separation PCR kits (AnDiaTec ParaTub-S IMS-PCR-ELISA and ParaTub-SL IMS-real time PCR) were developed and evaluated for the automated high-throughput detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in bulk milk of naturally infected dairy herds and made commercially available. M. paratuberculosis are first isolated from milk by high-throughput immunomagnetic bead separation using a completely automated magnetic particle pipetting robot within 45 min and released subsequently for analysis directly into PCR amplification mixtures for real time PCR or for PCR-ELISA. The threshold detection level and specificity of the tests were evaluated first with different M. paratuberculosis pure cultures and artificially contaminated (spiked) bulk milk samples. Both experiments proved a good detection limit, specificity and reliability of the tests that consistently detected 20 or less M. paratuberculosis organisms from cattle, deer and mufflon in 1 ml milk. Experiments with more than 200 bulk milk samples that were tested in parallel with the PCR methods and with the cultural method in a second evaluation study demonstrated that both PCR tests are superior to culture and sufficiently sensitive to detect single shedders in pooled milk samples. The experiments proved that the newly developed tests are sensitive, specific and fast, and thus for the first time allow the standardized large-scale routine M. paratuberculosis screening of bulk milk samples at acceptable costs.

Development and evaluation of a sensitive PCR-ELISA for detection of adenoviruses in feces.

OBJECTIVE: Most of the PCR-based assays for the detection of adenoviruses require gel electrophoresis for resolution, which is time- and work-consuming and interpretation difficulties often occur. Therefore our objective was to develop and evaluate a PCR-ELISA for broad detection of adenoviruses, that is easy to perform and leads to an increase of specificity in virus detection. METHODS: A total of 64 fecal specimens from patients with acute gastroenteritis, previously tested negative for other viruses than adenovirus and for bacterial pathogens, were tested for adenovirus in parallel with an Antigen-ELISA, virus isolation in cell culture, with a previously published hybridization-based PCR and our newly developed semi-nested PCR-ELISA. RESULTS: The new PCR-ELISA turned out to be the best method with 26 samples positive for adenovirus. Of these, 22 became positive by Ag-ELISA, 19 by virus isolation in cell culture and only 14 by the previously published hybridization-based PCR. CONCLUSION: The semi-nested PCR-ELISA is a sensitive, reliable and rapid method for the detection of enteric adenoviruses from fecal samples.

AnDiaTec Salmonella sp. PCR-ELISA for analysis of food samples.

A commercially available PCR kit (AnDiaTec Salmonella sp. PCR-ELISA) was developed and evaluated for the detection of Salmonella sp. in food samples. The test is based on PCR amplification and hybridization of the amplified DNA to a microtiter plate followed by the detection of PCR product in the manner of an enzyme-linked immunosorbent assay test. The sensitivity and specificity were evaluated first with Salmonella pure cultures and artificially contaminated food samples, including food types for which an inhibition of the PCR reaction was expected. Both experiments proved a very good sensitivity, specificity, and reliability of the test with a very broad range of food types. In a second evaluation study, more than 1,100 food samples of different types were tested in parallel with the PCR method and with the International Standardization Organization 6579 bacteriological reference method. The results of this evaluation study and the results from other experiments on dilutions of artificially contaminated food samples led to the establishment of a positive-negative cutoff value (optical density at 450 nm of more than 0.9) with respect to the conventional bacteriological method. Using this positive-negative cutoff, 98% agreement to the bacteriological method was obtained.

First case of confirmed rotavirus meningoencephalitis in Germany.

We report the case of a 9-year-old girl in Germany with acute meningoencephalitis associated with rotavirus gastrointestinal infection. Sequence analysis revealed a genetic relationship of the strain to rotaviruses with subgroup II specificity. Reverse transcription-PCR was positive for rotavirus.