Sensitivity and specificity of anti-double-stranded RNA immunofluorescence for universal detection of viral infection in respiratory specimens

The turnaround times for conventional methods used to detectMycobacterium tuberculosisin sputum samples and to obtain drug susceptibility information are long in many developing countries, including Panama, leading to delays in appropriate treatment initiation and continued transmission in the community. We evaluated the performance of a molecular line probe assay, the Genotype MTBDRplusversion 2.0 assay, in detectingM. tuberculosiscomplex directly in respiratory specimens from smear-positive tuberculosis cases from four different regions in Panama, as well as the most frequent mutations in genes conferring resistance to isoniazid (katGandinhA) and rifampin (rpoB). Our results were confirmed with the nitrate reductase assay and genomic sequencing.M. tuberculosiscomplex was detected by the Genotype MTBDRplus2.0 assay with 100% sensitivity and specificity. The sensitivity and specificity for rifampin resistance were 100% and 100%, respectively, and those for isoniazid resistance were 90.7% and 100%. Isoniazid monoresistance was detected in 5.2% of new cases. Genotype MTBDRplus2.0 is highly accurate in detectingM. tuberculosiscomplex in respiratory specimens and is able to discriminate isoniazid-monoresistant cases from multidrug-resistant cases within 2 days.

ABSTRACT Currently, diagnosis of Pneumocystis jirovecii pneumonia (PJP) relies on analysis of lower respiratory specimens, either by microscopy or quantitative real-time PCR (qPCR). Thus, bronchoscopy is required, which is associated with increased risk of respiratory failure. We assessed the value of noninvasive serologic β-d-glucan (BDG) testing for laboratory diagnosis of PJP using a newly available turbidimetric assay. We identified 73 cases of PJP with positive qPCR results from lower respiratory specimens for Pneumocystis and serology samples dating from 1 week before to 4 weeks after qPCR. In addition, 25 sera from controls with suspected PJP but specimens negative for Pneumocystis by qPCR were identified. Sera were tested with a turbidimetric BDG assay (Fujifilm Wako Chemicals Europe GmbH, Neuss, Germany), using an 11-pg/ml cutoff. Sensitivity and specificity were calculated based on qPCR test results as a reference. The turbidimetric BDG assay identified 63/73 patients with positive or slightly positive qPCR tests for an overall sensitivity of 86%; after exclusion of cases with only slightly positive qPCR results, sensitivity was 91%. No correlation between serum BDG levels and respiratory specimen DNA levels was found. Serologic BDG testing was negative in 25/25 controls with negative qPCR for a specificity of 100% using the predefined cutoff. In 22/25 samples (88%), no BDG was detected. Serologic BDG testing using the turbidimetric assay showed high sensitivity and specificity compared to qPCR of lower respiratory specimens for the diagnosis of PJP. Both turnover time and test performance will allow clinicians to delay or in some cases forego bronchoscopy.

AbstractNucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.

Abstract Dendritic cells (DCs), contribute to the initiation of immune responses to viral infection. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and initiate antimicrobial immune responses. TLR3 in DCs recognizes viral double-stranded RNA and triggers downstream signals to activate the NF?B and the interferon ß promoter. Double-stranded RNA may also be produced by double-stranded DNA viruses, such as HCMV, through bidirectional transcription from the genome during infection. Here we investigated whether TLR3 mediates the interaction between monocyte-derived immature DCs (iDCs) and HCMV after either active viral replication or viral penetration. We observed that HCMV strains differ in their interactions with iDCs. Strains that show no tropism for DCs, such as AD169, only penetrate iDCs, whereas the DC-tropic strains, e.g. TB40-E, actively replicate in iDCs. This difference provides an opportunity to study different forms of virus-DC interaction. Genome-wide expression array analysis showed that although 23 genes encoding cytokines, chemokines, and transcription factors are upregulated in iDCs after incubation with either strain, subsets of genes are induced specifically by DC-tropic or DC-nontropic strains. Only interaction with the DC-tropic HCMV strain TB40E, which replicates and produces mature virions, led to up-regulation of the TLR3 gene as well as genes downstream of TLR3 in the TLR3-signaling pathway, including class I interferon genes, NF?B, TRAF family member-associated NFKB activator (TANK), TANK-binding kinase 1 (TBK1), CXCL10, and CXCL11. The DC-nontropic HCMV strain AD169, which penetrates iDCs without replicating, did not upregulate genes of the TLR3 pathways. For selected genes, array data were confirmed by quantitative real-time PCR assay and ELISA to detect the gene products. To further confirm that the DC-tropic HCMV strain TB40E interacts with iDCs via TLR3, we transfected DCs with TLR3-specific siRNA prior to infection. TLR3 gene expression was potently silenced, while levels of the hALAS housekeeping gene mRNA remained normal. After these transfected DCs were infected with TB40E, HCMV-induced TLR3 gene expression was still markedly downregulated (−219 x), as were the downstream genes of the TLR3-signaling pathway (IFNa, −2.8 x; IFNß, −12.8 x; NF?B, −7.7 x; CCL5, −14.4 x; CXCL10, −16.5 x; CXCL11, −10.9 x). In contrast, TLR3 siRNA alone did not significantly modulate the expression of NF?B, CCL5, CXCL10, and class I interferons. Our results are consistent with those of McWirther et al., who reported that mice with a deficiency of TBK1 which is downstream of TLR3 show marked defects in IFNa and IFNß gene expression after viral infection or after engagement of TLR3 by double-stranded RNA. Thus, a key mediator of HCMV-DC interaction, which activates both a MyD88-dependent pathway that leads to early NF?B activation and a MyD88-independent pathway that leads to a class I interferon response (IFNa and IFNß) via interferon regulatory factor 3 (IRF3). This activation of the TLR3 signalling pathway was not observed when the DC-nontropic HCMV strain AD169 penetrated DCs without replicating. The identification of pathways that enhance innate antiviral immune responses may provide new avenues of therapeutic intervention for viral infections.