A novel strategy to avoid sensitivity loss in pooled testing for SARS-CoV-2 surveillance: validation using nasopharyngeal swab and saliva samples.

At the peak of the COVID-19 pandemic, pooled surveillance strategies were employed to alleviate the overwhelming demand for clinical testing facilities. A major drawback of most pooled-testing methods is the dilution of positive samples, which leads to a loss of detection sensitivity and the potential for false negatives. We developed a novel pooling strategy that compensates for the initial dilution with an appropriate concentration during nucleic acid extraction and real-time PCR. We demonstrated the proof of principle using laboratory-created 10-sample pools with one positive and corresponding individual positive samples by spiking a known amount of heat-inactivated SARS-CoV-2 into viral transport medium (VTM) or pooled negative saliva. No Ct difference was observed between a 10-sample pool with one positive vs. the corresponding individually analyzed positive sample by this method, suggesting that there is no detectable loss of sensitivity. We further validated this approach by using nasopharyngeal swab (NPS) specimens and showed that there is no loss of sensitivity. Serial dilutions of the virus were spiked into VTM and pooled with negative saliva in simulated 10-sample pools containing one positive to determine the LOD and process efficiency of this pooling methodology. The LOD of this approach was 10 copies/PCR, and the process efficiencies are ~95%-103% for N1 and ~87%-98% for N2 with samples in different matrices and with two different master mixes tested. Relative to TaqPath 1-step master mix, the TaqMan Fast Virus 1-Step master mix showed better sensitivity for the N2 assay, while the N1 assay showed no Ct difference. Our pooled testing strategy can facilitate large-scale, cost-effective SARS-CoV-2 surveillance screening and maintain the same level of sensitivity when analyzed individually or in a pool. This approach is highly relevant for public health surveillance efforts aimed at mitigating SARS-CoV-2 spread.

Epidemiological Review of Francisella Tularensis: A Case Study in the Complications of Dual Diagnoses.

INTRODUCTION: Tularemia is a rare but potentially fatal disease that develops in numerous wild and domestic animals, including lagomorphs, rodents, cats, and humans.  Francisella tularensis bacterium, the causative agent of tularemia, was identified by veterinary personnel at Fort Riley, Kansas during a routine post-mortum evaluation of a domestic feline. However, before formal diagnosis was confirmed, the sample was sent and prepared for rabies testing at the Department of Defense (DoD) U.S. Army Public Health Command Central (PHC-C), Food Analysis and Diagnostic Laboratory (FADL).  This case report provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations.  The epidemiologic characteristics of tularemia, F. tularensis as an organism of military interest, potential laboratory management of F. tularensis, and clinical findings on a case of feline tularemia are discussed. It further raises questions as to whether or not dead animals should be treated as sentinels and be pre-screened for select agents, especially in instances of dual diagnoses. METHODS: A necropsy was performed on the cat by the Fort Riley veterinarian, DNA extraction and PCR analyses were conducted by FADL microbiologists, histology and immunohistology analyses were conducted by the Kansas State Veterinary Diagnostic Laboratory, and feline tissue and blood were sent to the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) for confirmatory testing and strain identification of tularemia. RESULTS: Tularemia was identified in the spleen of the cat by the Fort Riley veterinarian and during the histological sampling of the spleen by the Kansas State Veterinary Diagnostic Laboratory.  A specific subsequent real-time polymerase chain reaction (RT-PCR) in vitro diagnostic detection of target DNA sequences of F. tularensis was conducted by the FADL microbiologists using a Joint Biological Agent Identification and Diagnostic System (JBAIDS) Tularemia Detection Kit to detect a presumptive qualitative result to detect tularemia in feline and blood samples.   USAMRIID also performed RT-PCR and identified genomic DNA from F. tularensis Type A, (SPL15.013.02), thus confirming the FADL's initial presumptive result of F. tularensis.  USAMRIID attempted to culture F. tularensis from three samples (swab, feline tissue, and transfer pipette tip), but no growth consistent with F. tularensis was observed on the cysteine heart agar with sheep blood and antibiotics (CHAB) and chocolate (CHOC) plates. DISCUSSIONS: Our case study of a dual diagnosis of presumptive F. tularensis and possible rabies exposure transmission from a pet cat to its owner provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations.  Our case study also demonstrates the obligation for cooperation between animal health, human health, and public health professionals in the management of zoonotic diseases.

A Computational Study of Charge Delocalization and Ring Fluoro Substituent Effects in 4-Fluoromethylphenoxides.

The degree of charge delocalization in 4-fluoromethylphenoxide anions has been investigated with ab initio molecular orbital calculations at the Hartree-Fock and MP2 levels and with the B3LYP density functional using the 6-31+G(d) and 6-311+G(2d,p) basis sets. The effect of aqueous solvation on the rotational profiles has been investigated using the AM1-SM2 solvation model. These anions serve as model systems for fluorinated tyrosine derivatives, which have been used as substrates for the enzyme tyrosine phenol-lyase. The importance of the double-bond/no-bond resonance structure has been assessed through an analysis of conformational profiles, geometries and charge distribution. The effect of ring fluorination on the degree of charge delocalization was investigated to aid in the interpretation of previous experimental mechanistic studies of the tyrosine phenol-lyase enzyme. Calculations were also carried out on benzyl fluoride, and the inclusion of diffuse functions was found to result in the perpendicular conformation as the predicted lowest-energy conformation by a small margin. Previous studies with smaller basis sets found the planar structure to be the lowest-energy conformation. The results indicate that the placement of an electron-donating group (O(-)) in the para ring position acts to increase the fluoride character of the fluoromethyl group and that this effect is not substantially modulated by the placement of fluorines on the ring.