Comparison of Treponema-specific immunoglobulin G (IgG) index with Treponema pallidum particle agglutination (TPPA) index for detection of intrathecal Treponema-specific antibody synthesis for serological diagnosis of neurosyphilis.

The determination of Treponema-specific intrathecal immunoglobulin synthesis with the Treponema pallidum particle agglutination (TPPA) index is a well-established method recommended in German guidelines for the diagnosis of neurosyphilis. However, the TPPA test is no longer available. The aim of this study was to evaluate whether the determination of a Treponema-specific immunoglobulin G (IgG) index can substitute the TPPA index. Serum and cerebrospinal fluid (CSF) samples from patients with confirmed (n=6) and probable (n=3) neurosyphilis as well as patients with adequately treated syphilis without neurosyphilis (n=4) were investigated. In addition to index calculation further CSF parameters were determined. The results of the Treponema IgG and the TPPA index were consistent in all patients with confirmed neurosyphilis and non-neurosyphilis patients. In two patients with probable neurosyphilis the IgG index appeared more plausible than the TPPA index when taking into account all available laboratory and clinical data of the patients. In conclusion, the determination of Treponema-specific intrathecal immunoglobulin synthesis with the IgG index appears to be a suitable alternative to the TPPA index.

A family cluster of schistosomiasis acquired in Solenzara River, Corsica (France) - Solenzara River is clearly a transmission site for schistosomiasis in Corsica.

We report a patient with urogenital schistosomiasis and three cases of subclinical infection within one family acquired from Solenzara River, Corsica, in 2019. Our cases confirm that transmission of schistosomiasis in Corsica is ongoing and has been extended from the Cavu River to the Solenzara River. Solenzara River is clearly a transmission site for schistosomiasis in Corsica. Public health efforts are recommended to uncover and prevent further cases.

Evaluation of the SARS-CoV-2-IgG response in outpatients by five commercial immunoassays.

Commercially available immunoassays have been developed for sensitive and specific detection of antibodies against SARS-CoV-2. While high sensitivity has been reported in hospitalized COVID-19 patients, little is known about the performance of the assays in ambulatory patients. Therefore, we evaluated the SARS-CoV-2-IgG response in 51 SASR-CoV-2-PCR-confirmed outpatients with five commercial immunoassays. The sensitivity in serum samples, collected at a median of 24 days after onset of symptoms, detected by the Anti-SARS-CoV-2-ELISA IgG (Euroimmun), EDI™ Novel Coronavirus COVID-19 IgG ELISA (Epitope Diagnostics), Liaison® SARS-CoV-2 S1/S2 IgG (Diasorin), SARS-CoV-2 IgG on the Architect™ i2000 (Abbott), and Elecsys® Anti-SARS-CoV-2 (IgM/IgA/IgG) on the cobas™ e801 (Roche) was 84.3%, 78.4%, 74.5%, 86.3%, and 88.2%, respectively. The sensitivity in serum samples, collected >20 days after onset of symptoms, varied between 75.0% and 90.0%, and in samples, collected at least 28 days after onset of symptoms, did not increase, except in the Anti-SARS-CoV-2-ELISA IgG by Euroimmun (90.0%). There was not an obvious association between the type of the antigen (N versus S protein) and the overall sensitivity of the assays. Our results show significant individual differences of the IgG response against SARS-CoV-2, additionally confirmed in three patients with follow-up serum samples and seven asymptomatic but PCR-positive contact persons. In conclusion, our study shows that commercially available immunoassays detect SARS-CoV-2-IgG or total antibodies in outpatients with a satisfying sensitivity, but lower than that reported for hospitalized patients. In asymptomatic persons the SARS-CoV-2-IgG response may even be absent in a relevant percentage of persons.

SARS-CoV-2-IgG response is different in COVID-19 outpatients and asymptomatic contact persons.

Commercially available immunoassays have been developed for sensitive and specific detection of antibodies against SARS-CoV-2. While a fast and reliable IgG response has been reported for samples from hospitalized COVID-19 patients, less is known about ambulatory patients. We evaluated the SARS-CoV-2-IgG response by the Anti-SARS-CoV-2-ELISA IgG (Euroimmun) in a defined cohort of SARS-CoV-2-PCR-confirmed outpatients and asymptomatic contact persons including 137 serum samples from PCR-confirmed outpatients (n = 111) and asymptomatic but PCR-positive contact persons (n = 26) sent to our laboratory as part of routine diagnostics for determination of SARS-CoV-2-IgG. Overall positivity rate for SARS-CoV-2-IgG was 81.1 % in outpatients (irrespective of sampling before or after day 21 after onset of symptoms) but significantly lower in asymptomatic contact persons (15.4 %, p < 0.0001). In contact persons without symptoms the ct values of the PCR assays were significantly higher (5-7 threshold cycles) than in outpatients, and ct values were significantly negative correlated to the SARS-CoV-2-IgG ratio, suggesting a lower viral load as a possible explanation for lower rate of seropositivity. In summary, our study shows that serological response to SARS-CoV-2 in outpatients including asymptomatic persons is less pronounced than in hospitalized patients. Further controlled studies are urgently needed to determine serological response in outpatients and asymptomatic persons since this is the main target population for seroepidemiological investigations.

Analysis of humoral immune responses to recombinant Chlamydia pneumoniae antigens.

OBJECTIVES: Chlamydia pneumoniae is a difficult to diagnose respiratory pathogen. This study was performed to systematically characterize humoral immune responses to selected C. pneumoniae antigens in order to provide novel serodiagnostic perspectives for clinical and epidemiological issues. METHODS: Based on a literature search, gene library screening, and serological proteome analysis, 15 immunogenic surface-associated, virulence-associated, and hypothetical C. pneumoniae antigens were selected, recombinantly expressed, and lined on a nitrocellulose strip. Specific IgM and IgG reactivity was measured in a total of 172 PCR- and micro-immunofluorescence testing (MIF)-characterized serum samples from patients with respiratory infections. A theoretical model was conceived to approximate a putative course of C. pneumoniae antigen expression and assess the potential of early and late antigens. RESULTS: While surface antigens performed poorly, the virulence-associated TARP was a reliable antigen for IgM detection, with a sensitivity of 80.0% and a diagnostic specificity of 90.2%. The hypothetical protein YwbM proved powerful for IgG detection with MIF-correlative sensitivities of up to 94.4% and a diagnostic specificity of 95.1%. CONCLUSIONS: This study provides new insights into antibody profiles to immunogenic proteins in C. pneumoniae infection. The study findings offer antigen candidates for more reliable and standardized serological investigations of C. pneumoniae infections, including studies on seroprevalence and epidemiology.

Evaluation of a new combined Western and line blot assay (EUROLINE-WB) for diagnosis and species identification of Echinococcus infection in humans.

Serological detection of echinococcosis is crucial for diagnosis and management. We evaluated the new blot assay Euroline-WB (ELB, Euroimmun) which consists of a Western blot with Echinococcus multilocularis (E.m.) vesicle antigens and a line blot part with recombinant antigens from E. granulosus (E.g., genus-specific EgAgB) and E.m. (species-specific Em18 and Em95), in comparison to a commercial Western Blot (EWB, LDBio) for detection and species differentiation of echinococcosis within routine laboratory diagnostics. Thirty-five serum samples from 35 patients classified according to a standardized classification were included in the analysis. Out of 24 cases of proven and probable infection with E.m. or E.g. 16 (66.7%) and 15 (62.5%) were correctly identified on species level by EWB and ELB, respectively. False Echinococcus species were assigned in two cases by EWB but none by ELB. Negative blot results in patients with proven infections were noticed in 8.3% (ELB) compared to 4.2% (EWB), but were limited to patients with antiparasitic therapy or post-surgery indicating a treatment-induced loss of antibody activity. Thus, identification of Echinococcus infection at least on the genus level was possible in 23/24 (95.8%) and 19/24 (79.2%) of patients by EWB and ELB (or 22/24 patients (91.7%) including borderline results of ELB), respectively. Recombinant Em18 and Em95 were highly specific for detection of E.m. infection but differed in sensitivity (Em18 56% and 80 %, and Em95 22% and 20% in proven and probable infections, respectively). Advantages of ELB are the standardized analysis of the banding pattern by EUROLineScan software and a faster turn-around-time.

Phylogeny of haemosporidian blood parasites revealed by a multi-gene approach.

The apicomplexan order Haemosporida is a clade of unicellular blood parasites that infect a variety of reptilian, avian and mammalian hosts. Among them are the agents of human malaria, parasites of the genus Plasmodium, which pose a major threat to human health. Illuminating the evolutionary history of Haemosporida may help us in understanding their enormous biological diversity, as well as tracing the multiple host switches and associated acquisitions of novel life-history traits. However, the deep-level phylogenetic relationships among major haemosporidian clades have remained enigmatic because the datasets employed in phylogenetic analyses were severely limited in either gene coverage or taxon sampling. Using a PCR-based approach that employs a novel set of primers, we sequenced fragments of 21 nuclear genes from seven haemosporidian parasites of the genera Leucocytozoon, Haemoproteus, Parahaemoproteus, Polychromophilus and Plasmodium. After addition of genomic data from 25 apicomplexan species, the unreduced alignment comprised 20,580 bp from 32 species. Phylogenetic analyses were performed based on nucleotide, codon and amino acid data employing Bayesian inference, maximum likelihood and maximum parsimony. All analyses resulted in highly congruent topologies. We found consistent support for a basal position of Leucocytozoon within Haemosporida. In contrast to all previous studies, we recovered a sister group relationship between the genera Polychromophilus and Plasmodium. Within Plasmodium, the sauropsid and mammal-infecting lineages were recovered as sister clades. Support for these relationships was high in nearly all trees, revealing a novel phylogeny of Haemosporida, which is robust to the choice of the outgroup and the method of tree inference.

High local diversity of Trypanosoma in a common bat species, and implications for the biogeography and taxonomy of the T. cruzi clade.

The Trypanosoma cruzi clade is a group of parasites that comprises T. cruzi sensu lato and its closest relatives. Although several species have been confirmed phylogenetically to belong to this clade, it is uncertain how many more species can be expected to belong into this group. Here, we present the results of a survey of trypanosome parasites of the bat Artibeus jamaicensis from the Panamá Canal Zone, an important seed disperser. Using a genealogical species delimitation approach, the Poisson tree processes (PTP), we tentatively identified five species of trypanosomes - all belonging to the T. cruzi clade. A small monophyletic group of three putative Trypanosoma species places at the base of the clade phylogeny, providing evidence for at least five independent colonization events of these parasites into the New World. Artibeus jamaicensis presents a high diversity of these blood parasites and is the vertebrate with the highest number of putative trypanosome species reported from a single locality. Our results emphasize the need for continued efforts to survey mammalian trypanosomes.

Occurrence of immunoglobulin M antibodies against several bacterial and viral pathogens in acute hantavirus infection.

Elevated levels of immunoglobulin M antibodies against various pathogens, most frequently Epstein-Barr-virus and Coxiella burnetii, were detected by immunoassay in 15 of 48 patients (31.3%) with acute Puumala virus infections. Although the mechanisms leading to this IgM response are not clear yet, polyspecific immunoglobulin M antibodies have to be taken into account to avoid misinterpretation of serological results in acute hantavirus infection.

TcBat a bat-exclusive lineage of Trypanosoma cruzi in the Panama Canal Zone, with comments on its classification and the use of the 18S rRNA gene for lineage identification.

We report TcBat, a recently described genetic lineage of Trypanosoma cruzi, in fruit-eating bats Artibeus from Panama. Infections were common (11.6% prevalence), but no other T. cruzi cruzi genotypes were detected. Phylogenetic analyses show an unambiguous association with Brazilian TcBat, but raise questions about the phylogenetic placement of this genotype using the 18S rRNA gene alone. However, analyses with three concatenated genes (18S rRNA, cytb, and H2B) moderately support TcBat as sister to the discrete typing unit (DTU) TcI. We demonstrate that short fragments (>500 bp) of the 18S rRNA gene are useful for identification of DTUs of T. cruzi, and provide reliable phylogenetic signal as long as they are analyzed within a matrix with reference taxa containing additional informative genes. TcBat forms a very distinctive monophyletic group that may be recognized as an additional DTU within T. cruzi cruzi.

Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view.

Polymerase chain reaction (PCR)-based techniques allow more rapid and sensitive detection of pathogens compared with conventional blood culture. Nevertheless, the climate of opinion of relevant studies is that currently PCR can supplement but not replace blood culture. In numerous studies, combined detection rate of both methods was significantly higher compared with PCR or blood culture alone. Also, complete determination of antibiotic resistance can currently be performed only by blood culture. Further increase of the panel of multiplex PCR is complicated, because the vast majority of sepsis pathogens are already included, primer interactions leading to primer heteromers limit the amount of targets detectable within one PCR tube, and an array of too many individual PCR reactions for investigation of a single specimen leads to high cost and workload. Except for diagnostics of patients in whom unusual, not culturable, or fastidious pathogens are detected more often, such as immunosuppressed patients with suspected parasitic infection, etc., it might even not be necessary to further increase the spectrum of detectable species. If the primary aim of PCR diagnostics is to decrease inappropriate empirical treatment and improve patient outcome, detection should focus on those pathogens or resistance determinants that are not covered by guideline-recommended treatment regimens and that have been identified as the major cause of inappropriate treatment according to current studies. In our opinion, such a narrower assay is more cost effective, may achieve higher accuracy due to reduced intratest interference, and would better address current and emerging clinical needs.

Evaluation of two automated chemiluminescence immunoassays, the LIAISON Treponema Screen and the ARCHITECT Syphilis TP, and the Treponema pallidum particle agglutination test for laboratory diagnosis of syphilis.

BACKGROUND: Automated Treponema pallidum-specific chemi-luminescence immunoassays (CLIA) run on random-access analyzers allow for rapid diagnosis of syphilis infection. METHODS: We evaluated the LIAISON Treponema Screen (LIA) and the ARCHITECT Syphilis TP (ARCH) in comparison to the Treponema pallidum particle agglutination (TPPA) test, as a screening test for syphilis. We performed a prospective study using 577 sera submitted for diagnosis of syphilis, including 318 samples from pregnant women. In addition, 42 stored sera from 32 patients with clinically and serologically characterized syphilis infection were investigated. RESULTS: In the prospective study, the sensitivity and specificity of LIA, ARCH, and TPPA were 100% (18/18), 100% (17/17), and 100% (18/18), and 100% (558/558), 99.8% (552/553), and 99.6% (556/558), respectively. In pregnant women, the specificity of LIA and ARCH was 100% (317/317) and of TPPA 99.7% (316/317). One sample from a child with assumed exposure to maternal antitreponemal antibodies was omitted from analysis. LIA, ARCH, and TPPA were also positive in all investigated sera from patients with known syphilis. CONCLUSIONS: Both automated CLIA demonstrated excellent diagnostic sensitivity and specificity when evaluated as a screening test for syphilis under routine conditions of a diagnostic laboratory. Thus, these may be used independently as an alternative to the manual TPPA screen.

Two novel parvoviruses in frugivorous New and Old World bats.

Bats, a globally distributed group of mammals with high ecological importance, are increasingly recognized as natural reservoir hosts for viral agents of significance to human and animal health. In the present study, we evaluated pools of blood samples obtained from two phylogenetically distant bat families, in particular from flying foxes (Pteropodidae), Eidolon helvum in West Africa, and from two species of New World leaf-nosed fruit bats (Phyllostomidae), Artibeus jamaicensis and Artibeus lituratus in Central America. A sequence-independent virus discovery technique (VIDISCA) was used in combination with high throughput sequencing to detect two novel parvoviruses: a PARV4-like virus named Eh-BtPV-1 in Eidolon helvum from Ghana and the first member of a putative new genus in Artibeus jamaicensis from Panama (Aj-BtPV-1). Those viruses were circulating in the corresponding bat colony at rates of 7-8%. Aj-BtPV-1 was also found in Artibeus lituratus (5.5%). Both viruses were detected in the blood of infected animals at high concentrations: up to 10E8 and to 10E10 copies/ml for Aj-BtPV-1 and Eh-BtPV-1 respectively. Eh-BtPV-1 was additionally detected in all organs collected from bats (brain, lungs, liver, spleen, kidneys and intestine) and spleen and kidneys were identified as the most likely sites where viral replication takes place. Our study shows that bat parvoviruses share common ancestors with known parvoviruses of humans and livestock. We also provide evidence that a variety of Parvovirinae are able to cause active infection in bats and that they are widely distributed in these animals with different geographic origin, ecologies and climatic ranges.

Low endotoxin release from Escherichia coli and Bacteroides fragilis during exposure to moxifloxacin.

BACKGROUND: Bacterial endotoxin is known to act as a potent trigger of disseminated coagulation and septic shock. During clinical antibiotic treatment, endotoxin may be released from Gram-negative bacteria. It is known that antibiotic classes differ in their ability to induce endotoxin release. AIM: It was the aim of this study to test the endotoxin-liberating potential of different antibiotics with activity against Escherichia coli and Bacteroides fragilis. METHODS: In vitro test models were used to evaluate the endotoxin-liberating potential of moxifloxacin, a 4th-generation quinolone with antianaerobic activity. Bacteria were exposed to moxifloxacin at 2×, 10× and 50× the minimal inhibitory concentration. Endotoxin release was measured by enzyme-linked immunosorbent and Limulus amoebocyte lysate assays. Comparator drugs were ceftazidime and imipenem, i.e. antibiotics with known high and low endotoxin-liberating potential, respectively. As a parameter for biological responses to endotoxin, the release of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1ß) from monocytes/macrophages was quantified with bioassays. RESULTS: In all test systems, release of endotoxin during exposure of bacteria to moxifloxacin was minimal or low and comparable with that of imipenem. CONCLUSIONS: Moxifloxacin has a low potential to cause endotoxin-mediated detrimental clinical effects. Concerning its endotoxin-releasing properties, moxifloxacin appears to be a choice equivalent to the carbapenems.

Accelerated identification of Staphylococcus aureus from blood cultures by a modified fluorescence in situ hybridization procedure.

This study evaluated fluorescence in situ hybridization (FISH) for rapid identification of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) directly from blood cultures. Initially, 360 blood cultures containing Gram-positive cocci were investigated by a previously described microwave-FISH procedure: 44/49 (89.8 %) S. aureus and 298/299 (99.7 %) CoNS were correctly identified. Because FISH proved useful and reliable but handling was found to be inconvenient, the method was modified by employing a recently developed slide chamber. This reduced the time required from 60 to 30 min. The simplified execution allowed integration of the method into the workflow of a routine laboratory without difficulty. The modified method proved to be highly reliable, identifying 37/37 (100 %) S. aureus and 169/172 (98.2 %) CoNS directly from blood cultures.

Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis.

In a prospective, multicenter study of 342 blood samples from 187 patients with systemic inflammatory response syndrome, sepsis, or neutropenic fever, a new commercial PCR test (SepsiTest; Molzym) was evaluated for rapid diagnosis of bacteremia. The test comprises a universal PCR from the 16S rRNA gene, with subsequent identification of bacteria from positive samples by sequence analysis of amplicons. Compared to blood culture (BC), the diagnostic sensitivity and specificity of the PCR were 87.0 and 85.8%, respectively. Considering the 34 BC-positive patients, 28 were also PCR positive in at least one of the samples, resulting in a patient-related sensitivity of 82.4%. The concordance of PCR and BC for both positive and negative samples was (47 + 247)/342, i.e., 86.0%. In total, 31 patients were PCR/sequencing positive and BC negative, in whom the PCR result was judged as possible or probable to true bacteremia in 25. In conclusion, the PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTest appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients.

Characterization of clinical Enterococcus faecalis small-colony variants.

In this report, we present a clinical case of chronic aortic valve endocarditis caused by Enterococcus faecalis small-colony variants (SCVs), with ensuing characterization of the SCV phenotype in comparison to the clonally related normal phenotype with respect to alterations in microscopic and ultrastructural morphology, growth behavior, and metabolic pathways. In contrast to the normal phenotype, light and electron microscopy of the Enterococcus SCVs demonstrated the presence of heterogeneous cells of different sizes with aberrant shapes. Furthermore, SCVs showed excessive production of an intercellular substance and alterations in cell division displayed by a thick, coarse cell wall and incomplete, branched, and multiple cross walls without obvious cell separation. In addition, empty "ghost" cells were visible. In growth experiments, SCVs displayed an extended lag phase with delayed entrance into the stationary phase. Interestingly, SCV cells growing under aerobic conditions did not attain the growth and viability of the normal phenotype or those of SCVs growing under microaerobic conditions, suggesting impaired growth behavior and enhanced vulnerability in the presence of oxygen. By metabolite analysis, SCVs failed to produce significant amounts of acetate or lactate under aerobic growth conditions but were able to produce lactate under microaerobic growth conditions, implicating the induction of a fermentative metabolism. In conclusion, the observed structural alterations and changes in the cellular growth and metabolic pathways facilitated the survival of Enterococcus SCVs under microaerobic conditions in vitro and thus presumably in vivo during endocarditis.

Evaluation of new selective culture media and a rapid fluorescence in situ hybridization assay for identification of Clostridium difficile from stool samples.

Two new Clostridium difficile-selective agars, from Oxoid (according to Brazier) and from BD, were compared with cycloserine-cefoxitin-fructose agar (Oxoid) for their sensitivity of recovery of toxigenic C. difficile from stool samples. For the culture-positive samples, the sensitivities were 84.0, 42.6 and 90.4 %, respectively. In addition, a C. difficile-specific fluorescence in situ hybridization assay was developed, facilitating rapid and reliable identification of cultured isolates.

Rapid diagnosis of candidaemia by real-time PCR detection of Candida DNA in blood samples.

This study prospectively evaluated an 18S rRNA gene-targeted real-time PCR approach in comparison with standard blood culture (BC) diagnostics for rapid diagnosis of candidaemia in a large study population of 384 patients, including 902 whole blood samples from 468 infectious episodes (IEs) of 329 adults and 55 children with haematological malignancies and various forms of immunodeficiency, and intensive care unit patients. Seven out of eight BC-proven cases (87.5 %) of candidaemia and seven out of twelve BC-positive samples (58.3 %) were positive by the Candida-specific PCR. A positive PCR result was also obtained for 28/460 BC-negative samples from IEs, including 8 patients with culture-confirmed Candida infection at primary sterile body sites. Of the PCR-positive, culture-negative patients, more than 50 % received systemic antifungal therapy. In 432/460 BC-negative IEs, the Candida specific-PCR was negative, resulting in a negative predictive value of 99.8 %. In conclusion, the Candida specific-PCR approach facilitates rapid detection of Candida DNA in blood samples of patients at risk of candidaemia within a few hours. Although standard BC diagnostics appear to remain indispensable for the detection of all cases of candidaemia, this PCR assay allowed the detection of candidaemia at a mean of 3 days earlier than BC diagnostics. Thus, it enables earlier antifungal therapy for patients with suspected candidaemia and may prevent further complications.