The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the ...outbreak is more widespread than initially thought, and international spread through travellers does already occur.
We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.
Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.
The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project.
The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
Mutations of the NPM1 gene (NPM1mut) are among the most common genetic alterations in acute myeloid leukemia and are suitable for minimal residual disease detection. We retrospectively investigated ...the prognostic impact of NPM1mut-based minimal residual disease detection from bone marrow for development of relapse by using a newly developed real-time polymerase chain reaction based on locked nucleic acid–containing primers in 174 patients, 155 of whom were treated within prospective protocols. The prognostic value of 5 cutoff values after completion of treatment or after allogeneic transplantation was studied by using cause-specific hazard models. Subsequent validation using cross-validated partial likelihood analysis revealed that an increase of more than 1% NPM1mut/ABL1 was most prognostic for relapse after chemotherapy, whereas an increase of more than 10% NPM1mut/ABL1 was most prognostic for relapse after allogeneic transplantation. Univariate and multivariate analysis of disease-free survival and overall survival revealed a significantly worse outcome in patients with >1% NPM1mut/ABL1 and >10% NPM1mut/ABL1, respectively, which remained significant after adjustment for FLT3–internal tandem duplication status. Our results in a large data set define and optimize cutoff values for early diagnosis of molecular relapse. These results may be especially important for defining triggers for early therapeutic intervention.
• NPM1 RT-PCR levels >1% are associated with poor overall and disease-free survival in AML patients treated with chemotherapy.• NPM1 MRD levels >10% are associated with poor overall and disease-free survival in AML patients after allogeneic transplantation.
Establishing nucleic acid-based assays for genetic newborn screening (NBS) provides the possibility to screen for genetically encoded diseases like spinal muscular atrophy (SMA), best before the ...onset of symptoms. Such assays should be easily scalable to 384-well reactions that make the screening of up to 2000 samples per day possible. We developed a test procedure based on a cleanup protocol for dried blood spots and a quantitative (q)PCR to screen for a homozygous deletion of exon 7 of the survival of motor neuron 1 gene (SMN1) that is responsible for >95% of SMA patients. Performance of this setup is evaluated in detail and tested on routine samples. Our cleanup method for nucleic acids from dried blood spots yields enough DNA for diverse subsequent qPCR applications. To date, we have applied this approach to test 213,279 samples within 18 months. Thirty patients were identified and confirmed, implying an incidence of 1:7109 for the homozygous deletion. Using our cleanup method, a rapid workflow could be established to prepare nucleic acids from dried blood spot cards. Targeting the exon 7 deletion, no invalid, false-positive, or false-negative results were reported to date. This allows timely identification of the disease and grants access to the recently introduced treatment options, in most cases before the onset of symptoms. Carriers are not identified, thus, there are no concerns of whether to report them.
We examined fecal specimens of patients with diarrhea from 3 continents for Tropheryma whipplei and enteropathogens. T. whipplei was most common in South Africa, followed by Singapore and Germany. ...Its presence was associated with the presence of other pathogens. An independent causative role in diarrhea appears unlikely.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background The standard diagnosis of Ascaris lumbricoides and other soil-transmitted helminth (STH) infections relies on the detection of worm eggs by copromicroscopy. However, this method is ...dependent on worm patency and shows only limited accuracy in low-intensity infection settings. We aimed to decipher the diagnostic accuracy of different antibodies using various Ascaris antigens in reference to copromicroscopy and quantitative PCR (qPCR), four months after national STH preventative chemotherapy among school children in western Kenya. Methodology STH infection status of 390 school children was evaluated via copromicroscopy (Kato-Katz and mini-FLOTAC) and qPCR. In parallel, Ascaris-specific antibody profiles against larval and adult worm lysates, and adult worm excretory-secretory (ES) products were determined by enzyme-linked immunosorbent assay. Antibody cross-reactivity was evaluated using the closely related zoonotic roundworm species Toxocara cati and Toxocara canis. The diagnostic accuracy of each antibody was evaluated using receiver operating curve analysis and the correspondent area under the curve (AUC). Principal findings Ascaris was the predominant helminth infection with an overall prevalence of 14.9% (58/390). The sensitivity of mini-FLOTAC and Kato-Katz for Ascaris diagnosis reached only 53.5% and 63.8%, respectively compared to qPCR. Although being more sensitive, qPCR values correlated with microscopic egg counts (R = -0.71, P<0.001), in contrast to antibody levels. Strikingly, IgG antibodies recognizing the ES products of adult Ascaris worms reliably diagnosed active Ascaris infection as determined by qPCR and microscopy, with IgG1 displaying the highest accuracy (AUC = 0.83, 95% CI: 0.75-0.91). Conclusion IgG1 antibody responses against adult Ascaris-ES products hold a promising potential for complementing the standard fecal and molecular techniques employed for monitoring Ascaris infections. This is of particular importance in the context of deworming programs as the antibody diagnostic accuracy was independent of egg counts.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Intense transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa might promote emergence of variants. We describe 10 SARS-CoV-2 lineages in Benin during early 2021 that ...harbored mutations associated with variants of concern. Benin-derived SARS-CoV-2 strains were more efficiently neutralized by antibodies derived from vaccinees than patients, warranting accelerated vaccination in Africa.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder, which causes loss of renal proximal tubular function and progressive loss of glomerular function, finally leading to ...end stage renal failure at school age. In the course of the disease most patients will need kidney transplantation if treatment has not been started before clinical manifestation. With an effective treatment available, a newborn screening assay is highly demanded. Since newborns with cystinosis usually do not show symptoms within the first months of life and no biochemical markers are easily detectable, a DNA-based method seems to be an obvious tool for early diagnosis. Screening was performed using high-throughput nucleic acid extraction followed by 384-well qPCR and melting analysis for the three most frequent variants (57 kb deletion NC_000017.11:g.3600934_3658165del (GRCh38); c.18_21del GACT; c.926dupG) responsible for the defective lysosomal membrane protein cystinosin (CTNS). To increase sensitivity, all heterozygous samples identified in qPCR assay were verified and screened for additional variants by applying next generation sequencing. From January 2018 to July 2019 nearly 292,000 newborns were successfully screened. We identified two newborns with a homozygous 57 kb deletion and a second one with heterozygous 57 kb deletion and a G>C substitution at position c.-512 on the second allele. Cystinosis is an example for diseases caused by a limited number of high prevalence and a high number of low prevalence variants. We have shown that qPCR combined with NGS can be used as a high throughput, cost effective tool in newborn screening for such diseases.
Novel SARS-CoV-2 variants are emerging at an alarming rate. The delta variant and other variants of concern (VoC) carry spike (S)-protein mutations, which have the potential to evade protective ...immunity, to trigger break-through infections after COVID-19 vaccination, and to propagate future waves of COVID-19 pandemic. To identify SARS CoV-2 variants in Bangladesh, patients who are RT-PCR-positive for COVID-19 infections in Dhaka were screened by a RT-PCR melting curve analysis for spike protein mutations. To assess the anti-SARS CoV-2 antibody responses, the levels of the anti-S -proteins IgA and IgG and the anti-N-protein IgG were measured by ELISA. Of a total of 36 RT-PCR positive samples (75%), 27 were identified as delta variants, with one carrying an additional Q677H mutation and two with single nucleotide substitutions at position 23029 (compared to Wuhan-Hu-1 reference NC 045512) in the genome sequence. Three (8.3%) were identified as beta variants, two (5.5%) were identified as alpha variants, three (8.3%) were identified as having a B.1.1.318 lineage, and one sample was identified as an eta variant (B.1.525) carrying an additional V687L mutation. The trend of higher viral load (lower Cp values) among delta variants than in the alpha and beta variants was of borderline statistical significance (p = 0.045). Prospective studies with larger Bangladeshi cohorts are warranted to confirm the emergence of S-protein mutations and their association with antibody response in natural infection and potential breakthrough in vaccinated subjects.
Genomic surveillance and seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) in Bangladesh is paramount for COVID-19 pandemic preparedness yet lagging the high-income ...countries due to limited resources.
SARS-CoV-2 variants, COVID-19 symptoms, and serology were prospectively evaluated in a cross-sectional study of Bangladeshi adults testing RT-PCR positive in 2021 and 2022.
SARS CoV-2 Omicron variants of asymptomatic or mild COVID-19 in 2022 replaced Delta variant infections requiring hospitalization and oxygen support. The omicron XBB became predominant in July 2022 and associated with cough, headache or body ache and loss of smell; 47 of 68 (69 %), 30 of 68 (44 %) and 27 of 68 (40 %) respectively at higher frequency than BA.1/BA.2; 16 of 88 (18 %), 13 of 88 (15 %) and 0 of 88 (0 %) p < 0.01, p < 0.01 and p < 0.0001. Linear regression analysis reveals no associations between the number of previous infections and the number of symptoms, r = −0.084, p = 0.68. The anti-nucleoprotein (N)-protein IgG post COVID-19 and anti-Spike (S) protein IgG post-COVID-19 vaccination were similar between BA.2, BA.4/BA.5 and XBB and significantly lower than the levels in delta variant infections (p < 0.001).
Omicron XBB subvariants emerged in Bangladesh two months prior to previous reports and include unique patterns of S-protein mutations not assigned in PANGO lineage. The SARS CoV-2 omicron break-through infections persist in the presence of sustained antibody responses and vaccinations, underscoring the importance of molecular surveillance in low-income countries.
This study was performed to comparably assess two commercial real-time PCR assays for the identification of
DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for ...infections with either
or apathogenic
were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for
and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both
and
without further discrimination. To discriminate between
- and
-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94)
-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite
. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with
in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of
was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of
from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic
according to the RealStar assay may be a disadvantage in areas of co-circulation with
, while the test performance of the two compared assays will be quite similar in geographic settings where
infections are unlikely.
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