7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Dr. med.

Professor

Accuracy of a fluorescence flow cytometer (Sysmex XN-31) for diagnosing malaria: two years of experience in a tertiary care hospital

Diagnostic and Clinical Microbiology

Question: Early and accurate diagnosis is essential for effective management of malaria. In non-endemic areas, laboratory staff may lack experience with malaria and fail to detect parasites when examining blood smears microscopically. Non-microscopy based methods like immunochromatographic assays, PCR and more recently fluorescence flow cytometry using Sysmex XN-31 (Sysmex Deutschland GmbH, Norderstedt) has been registered to support malaria diagnosis. XN-31 is fully automated with a limit of detection of 20 parasites/µl. We assessed the diagnostic accuracy of the XN-31 within a prospective study for malaria diagnosis compared to microscopy and PCR. Methods: 203 blood samples were analysed by XN-31, thin blood film after automatic Giemsa staining with Aerospray Pro (Kreienbaum GmbH, Langenfeld) and RealStar Malaria Screen &amp; Type PCR Kit 1.0 (altona Diagnostics GmbH, Hamburg). An immunchromatographic assay was also performed but results were not taken into account in ths study. In order to assess interlaboratory performance of XN-31, a small subset of samples (n=14) was evaluated in a second laboratory. Results: Over two years, 137 negative and 70 malaria positive blood samples were analysed initially and 145 follow-up samples. Compared to microscopy, XN-31 showed high sensitivity (98.4%), specificity (100%) as well as excellent negative predictive value (NPV, 99,3%) and positive predictive value (PPV, 100%) for the initial diagnosis of malaria. Agreement was high with Cohen`s kappa of 0.988. In comparison to PCR, values were similar: sensitivity 92.6%, specificity 100%, NPV 96%, PPV 100% and high agreement (Cohen`s kappa 0.941). In five cases, the XN-31 result was not available. XN-31 separated P. falciparum from non-falciparum successfully and parasitaemia determination was performed within one minute. Interlaboratory comparison showed high reproducibility of XN-31. Conclusions: Malaria diagnosis can be improved in non-endemic areas by use of XN-31 combined with microscopy and PCR. Parasite load is provided in significant shorter time by the device making diagnosis of malaria faster. Especially in follow-up samples microscopy can likely be replaced by XN-31.

Oral Presentation

Development and diagnostic accuracy field testing in Sudan of a novel serodiagnostic POC for Visceral Veishmaniasis

IntroductionVisceral leishmaniasis (VL) is the most severe form of leishmaniasis leading to death if not properly diagnosed and treated. Reliable diagnosis of infected patients and reservoir animals (dogs) is vital for controlling the spread of leishmaniasis. East-African countries suffer from a very high incidence of VL and although several diagnostic tests are available, point of care diagnosis (POC) continues to be a big challenge in these countries due to low sensitivity and specificity in these countries. GoalThe aim was to develop a reliable, stable and easy to handle POC test that significantly improves the VL-diagnostic efficiency in East-Africa and other VL-endemic countries in both, humans and dogs. Material &amp; MethodsBioinformatic and biochemical analysis of B-cell epitopes of kinesins from different East African VL-strains lead to the identification of a new diagnostic kinesin antigen (rKLi8.3) with high B-cell affinity. A novel cloning and expression strategy was developed to express rKLi8.3 for the production of a new and improved lateral flow test (LFT) POC. ResultsProspective comparative field-testing of the rKLi8.3 LFT with the standard rK39 LFT in Sudan (Gedaref) on 107 VL suspects revealed 98.7% sensitivity and 100% specificity. No cross-reactivity was observed in patients with malaria, typhoid, brucellosis and tuberculosis and trypanosomiasis. Retrospective and comparative testing of the rK39 and rKLi8.3 ELISAs on 300 sera from Sudanese patients and controls revealed a sensitivity of 93.2% to 97.2% and a specificity of 93.6% to 99.2%, respectively. Similar results were observed in VL-patients from Brazil. In contrast to rK39, the rKLi8.3 based ELlSA and LFT showed no cross-reactivity with African and American Trypanosomiasis. Similar results were also obtained in VL-infected dogs (CanL) in Brazil and Croatia. SummaryrKLi8.3 based LFTs offer substantially improved diagnostic efficiency for VL in East Africa and other endemic areas, compared to currently available sero-diagnostic tests. Further, rKLi8.3 LFT are easy to handle and very heat stable, thus excellent for the use in tropical and remote endemic areas.

Detection of Francisella tularensis holarctica specific SNP by a qPCR using T-Blocker technology

Francisella tularensis is a Gram-negative bacterium causing Tularemia. While the high pathogenic subspecies Francisella tularensis tularensis has been isolated nearly exclusively in Northern-America the moderate pathogenic subspecies Francisella tularensis holarctica is distributed over the complete northern hemisphere. Especially, Francisella tularensis tularensis has a high potential to be misused as biowarfare agent. Therefore, the assessment of the subspecies of Francisella tularensis is of great importance for patient´s prognosis or as an indication of intentional exposition if Francisella tularensis tularensis is found outside of Northern-America.To discriminate Francisella tularensis holarctica from other Francisella species and subspecies a SNP polymorphism in the 23s rRNA gene can be used. Besides probe based qPCR systems SNP can be detected by T-Blocker PCR. This technology uses forward primers that end at the SNP position. This is followed by a blocking-primer that binds, if the SNP is not complemetary to the forward primer and prevents amplification. So the amplification is less effective and by comparing ct values of T-Blocker qPCR for both alleles the allele can be distinguished. The set up and validation of this T-Blocker qPCR assay to confirm the occurence of Francisella tularensis holarctica in a sample is presented.

Prof. Dr. med.

PD. Dr.

A head-to-head comparison of three MALDI-TOF mass spectrometry systems with 16S rRNA gene sequencing

Background: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized diagnostics in bacteriology. To date, virtually all MALDI-TOF MS based diagnostics is carried out using systems of only two manufacturers, i.e., Bruker Daltonics (MALDI biotyper system) and bioMérieux (VITEK MS system). Recently, a new competitor (Zybio) launched the novel EXS2600 MALDI-TOF MS device. This study aimed to evaluate the performance of the three test systems by comparing the results to 16S rRNA gene sequencing.Methods: A set of 356 previously collected difficult-to-identify bacterial isolates was tested in parallel with the three systems relying only on the direct smear method and simple formic acid extraction.Results: MALDI Biotyper, EXS2600, and Vitek MS yielded valid results for 98.6 %, 94.4 %, and 93.3 % of all isolates, respectively. Of all valid results, agreement with sequencing data was achieved in 98.9 % (MALDI Biotyper), 98.5 % (EXS2600), and 99.7 % (Vitek MS). Misidentification rates were 0 %, 2.6 %, and 1.1 % for MALDI Biotyper, EXS2600, and Vitek MS, respectively, when only the isolates with valid measurements at single species level were considered. Minor differences concerning the handling were observed between the respective devices and software systems.Conclusion: The three systems provided comparable results. Our data demonstrate that all systems are suitable for the use in medical diagnostic laboratories.

Performance of mycobacterium growth indicator tube incubated at 30°C for detection of mycobacteria in CF and tissue samples compared to conventional growth conditions of liquid and solid media

BackgroundMycobacterial culture using mycobacterium growth indicator tube (MGIT) liquid culture incubated at 36°C has a superior performance for mycobacteria detection compared to solid media. For cystic fibrosis (CF) patients and tissue samples incubation at 30°C may additionally improve performance. We evaluate MGIT incubated at 30°C compared to MGIT 36°C and solid media incubated at 36°C and 30°C in samples from CF patients and tissue samples.MethodsAll samples (n=1446) of CF patients and tissue samples submitted for mycobacterial diagnostics between February 2021 and September 2023 to the University Medicine Essen were inoculated on MGIT liquid medium (Becton Dickinson), Lowenstein Jensen (LJ) and Stonebrink solid media with PACT (Oxoid, Wesel, Germany) and were incubated at both temperatures, 30°C and 36°C. MGIT cultured at 36°C were incubated in a Bactec MGIT 960TM device (Becton Dickinson) for 6 weeks, all other cultures were incubated for 8 weeks. MGIT 30°C cultures were incubated in a 30°C incubator and analysed for growth using MicroMGIT reader (BD). Ziehl-Neelsen staining was primarily performed for all positive cultures to confirm mycobacterial growth. Identification of mycobacteria was primarily performed using GenoType CM, AS or NTM-DR (Hain Lifescience).Results Of 1446 samples, a total of 187 mycobacterial isolates were cultured. The highest detection rate was from MGIT 30°C with 86.1% (161 of 187 mycobacterial isolates). MGIT 36°C recovered 59.8% (112 of 187), LJ 36°C 48,1% (90), Stonebrink 36°C 50.8% (95), LJ 30°C 50.3% (94) and Stonebrink 30°C 55.1% (103) of grown isolates. MGIT 30°C detected 34 mycobacterial isolates that were not recovered by any other culture condition. MGIT 30°C detected the highest number of isolates for M. abscessus complex (39), M. chelonae (6), M. chimaera (20), M. fortuitum (1), M. gordonae (21), M. intermedium (1), M. mucogenicum (3), M. simiae complex (1), M. spec. (6), M. nebraskense (1) and M. tuberculosis (23).ConclusionsFor CF patients and tissue samples, MGIT cultivated at 30°C for eight weeks improves performance of mycobacterial culture.

Evaluation of FT-IR spectroscopy and machine learning for the discrimination of Escherichia coli and Shigella spp.

Background. The differentiation of Escherichia coli (Ec) from Shigella spp., the identification of Shigella at species level, as well as the discrimination of Ec at serotype level to delineate pathogenic lineages, are challenging with common routine methods. They require serological/genomic typing approaches, which present some disadvantages in terms of costs, ease-of-use and applicability in routine settings. In this study, we evaluated the discriminative power of Fourier-Transform Infrared (FT-IR) spectroscopy to distinguish E. coli isolates at serotype level and to delineate E. coli from Shigella species.Material/Methods. 225 genomically or serologically typed strains (n=132 Ec belonging to 71 serotypes, n=93 Shigella spp.) were investigated. FT-IR analysis was performed applying the IR Biotyper® system (IRBT - Bruker Daltonics, Bremen, Germany) following the manufacturer&quot;s instructions. Exploratory data analysis was performed by principal component analysis (PCA) and linear discriminant analysis (LDA). Machine learning was applied to create an automated classifier for the delineation of E. coli from Shigella spp., using the algorithms included in the IRBT software. One hundred and twenty-three isolates were used to build the training set, representing all 75 groups (Ec serotypes + the 4 Shigella species). The remaining 102 isolates were used as a testing set.Results. Exploratory data analysis showed that IRBT clustering is correlated with the E. coli O-H serotypes and the Shigella species. PCA/LDA showed that the E. coli serotypes and the 4 Shigella species are separable. The classifier showed an accuracy of 99% (101/102), with one O157:H7 isolate misclassified as S. sonnei.Conclusion. IR Biotyping showed the potential of delineating E. coli at serotype level, and of discriminating E. coli from Shigella spp., demonstrating its potential suitability for infection control, public health and epidemiological assessments. Further investigation is underway to confirm and strengthen these promising preliminary results.

Oral Session
Innovative Diagnostic Methods

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Oral SessionInnovative Diagnostic Methods

Appointment

Chair

Topic

Description

Programme

Oral Session
Innovative Diagnostic Methods