BACKGROUND:The general assumption that spirochetemia does not occur at the early localized stage of Lyme disease is due to a lack of sensitive and specific methods for molecular diagnosis. METHODS:During a Lyme disease season in 2023, the platelet-rich plasma specimens of 145 people residing in Lyme disease-endemic areas in the United States were immediately separated from the blood cells following venous blood collection to prevent the spirochetes, if any, from invading the lymphocytes in the test tube. The entire DNA content was extracted from the platelet pellet and used for split sample polymerase chain reaction (PCR) amplification; Sanger sequencing was performed on the nested PCR products to detect the Borrelia burgdorferi flaB and 16S rRNA genes. RESULTS:In 98 of the people who were clinically suspected of having early localized Lyme disease irrespective of the presence or absence of a skin lesion, 33 of their blood specimens (33.7%) were positive for Borrelia burgdorferi (B. burgdorferi), including 17 positive for flaB gene only, 15 positive for both the flaB and 16S rRNA genes, and one positive for 16S rRNA gene only. Eight (17.0%) of the 47 asymptomatic resident controls were positive for flaB PCR only. CONCLUSIONS:The flaB gene is a more sensitive chromosomal target than the 16S rRNA gene for molecular detection of one to three B. burgdorferi cells due to spirochetes gaining or retaining flaB paralogs at the early localized stage of Lyme disease.

It is generally assumed that spirochetemia does not take place at the early localized stage of Lyme disease. This misassumption is due to the lack of sensitive and specific methods for detection. This paper reports that separation of the platelet-rich plasma immediately after venous blood collection to prevent the spirochetes from invading the lymphocytes in the test tube is crucial in molecular detection of a single Borrelia burgdorferi cell spun down in platelet pellets. In 2023, we tested the venous blood specimens of 145 people residing in Lyme disease-endemic areas in the United States. In 98 of them, who were suspected of having early localized Lyme disease irrespective of the absence or presence of a skin lesion, we found 33 specimens (33.7%) positive for B. burgdorferi, including 17 positive for FlaB gene only, 15 positive for both FlaB gene and 16S rRNA gene, and 1 positive for 16S rRNA gene only. Eight (17.0%) of the 47 asymptomatic resident controls were positive for FlaB PCR only. FlaB is a more sensitive chromosomal target than the 16S rRNA gene for the detection of 1-3 B. burgdorferi cells in 1 mL of platelet-rich plasma due to spirochetes gaining or retaining FlaB paralogs at the early localized stage of Lyme disease.

Chordomas are rare tumors believed to be arising from the notochord remnant in the axial skeleton. Diagnosis is often difficult since they show overlapping imaging features with other more common disease including metastases. Since individualized papers are only discussing the imaging features at different locations, the aim of this pictorial review is to have a comprehensive review on the common imaging findings of chordomas along the entire neuroaxis with a series of pathological proven cases in a local tertiary hospital in Hong Kong.

Both SARS-CoV-2 and SARS-CoV initially appeared in China and spread to other parts of the world. SARS-CoV-2 has generated a COVID-19 pandemic causing more than 6 million human deaths worldwide while the SARS outbreak quickly ended in six months with a global total of 774 reported deaths. One of the factors contributing to this stunning difference in the outcome between these two outbreaks is the inaccuracy of the RT-qPCR tests for SARS-CoV-2, which generated a large number of false-negative and false-positive test results that have misled patient management and public health policy-makers. This article presented Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV can in fact detect SARS-CoV-2 accurately due to the well-known nonspecific PCR amplification of DNAs with similar sequences. Using nested RT-PCR followed by Sanger sequencing to retest 50 patient samples collected in January, 2022 and sold as RT-qPCR positive reference confirmed 21 (42%) were false-positive. Although the other 29 positive isolates were categorized as Omicron variant by partial sequencing of the N gene, and the RBD and the NTD of the S gene, 9 (31%) showed focal to complete sequencing failure in the S gene segments due to multi-allelic SNPs. During the course of the study, an Omicron variant isolate containing a BA.1 NTD and a BA.2 RBD in its S gene was also detected. Routine partial S gene sequencing of all PCR-positive samples can timely discover multi-allelic SNPs and viral recombination in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have a potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein, along with certain mutations in other regions within the S protein as specific variants, in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.

Currently, molecular tests for SARS-CoV-2 infection are primarily based on reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) on cell-free fluid samples of respiratory tract specimens. These tests measure the rate of fluorescent signal accumulation as a surrogate for direct DNA sequence determination and are known to generate false-negative and false-positive results. The author has developed a routine protocol to test the cellular components of respiratory tract specimens instead of cell-free fluids only and to use conventional nested RT-PCR to amplify the target nucleic acid for high detection sensitivity. A 398-bp heminested PCR amplicon is used as the template for direct DNA sequencing to ensure no false-positive test results. Using this protocol to re-test 20 reference samples prepared by the Connecticut State Department of Public Health, the author found 2 positives among 10 samples classified as negative by RT-qPCR assays. One of these two positive samples contained a mutant with a novel single nucleotide insertion in the N gene and a wild-type parental SARS-CoV-2. Of the 10 samples classified as positive by RT-qPCR assays, only 7 (7/10) were confirmed to contain SARS-CoV-2 by heminested PCR and DNA sequencing of a 398-bp amplicon of the N gene. One of the latter 7 positive SARS-CoV-2 isolates belongs to a newly discovered mutant first isolated from a specimen collected in the State of New York on March 17, 2020, according to information retrieved from the GenBank database. Routine sequencing of a 398-bp PCR amplicon can categorize any isolate into one of 6 clades of SARS-CoV-2 strains known to circulate in the United States. The author proposes that extremely accurate routine laboratory tests for SARS-CoV-2 be implemented as businesses attempt to return to normal operation in order to avoid raising false alarms of a re-emerging outbreak. False-positive laboratory test reports can easily create unnecessary panic resulting in negative impacts on local economies.

The spirochetal bacterium Borrelia miyamotoi is a human pathogen and has been identified in many countries throughout the world. This study reports for the first time the presence of Borrelia miyamotoi in Ireland, and confirms prior work with the detection of B. garinii and B. valaisiana infected ticks. Questing Ixodes ricinus nymph samples were taken at six localities within Ireland. DNA extraction followed by Sanger sequencing was used to identify the species and strains present in each tick. The overall rate of borrelial infection in the Irish tick population was 5%, with a range from 2% to 12% depending on the locations of tick collection. The most prevalent species detected was B. garinii (70%) followed by B. valaisiana (20%) and B. miyamotoi (10%). Knowledge of Borrelia species prevalence is important and will guide appropriate selection of antigens for serology test kit manufacture, help define the risk of infection, and allow medical authorities to formulate appropriate strategies and guidelines for diagnosis and treatment of Borrelia diseases.

Lyme disease, initially described as Lyme arthritis, was reported before nucleic-acid based detection technologies were available. The most widely used diagnostic tests for Lyme disease are based on the serologic detection of antibodies produced against antigens derived from a single strain of Borrelia burgdorferi. The poor diagnostic accuracy of serological tests early in the infection process has been noted most recently in the 2018 Report to Congress issued by the U.S. Department of Health and Human Services Tick-Borne Disease Working Group. Clinical Lyme disease may be caused by a diversity of borreliae, including those classified as relapsing fever species, in the United States and in Europe. It is widely accepted that antibiotic treatment of Lyme disease is most successful during this critical early stage of infection. While genomic sequencing is recognized as an irrefutable direct detection method for laboratory diagnosis of Lyme borreliosis, development of a molecular diagnostic tool for all clinical forms of borreliosis is challenging because a "core genome" shared by all pathogenic borreliae has not yet been identified. After a diligent search of the GenBank database, we identified two highly conserved segments of DNA sequence among the borrelial 16S rRNA genes. We further developed a pair of Borrelia genus-specific PCR primers for amplification of a segment of borrelial 16S rRNA gene as a "core genome" to be used as the template for routine Sanger sequencing-based metagenomic direct detection test. This study presented examples of base-calling DNA sequencing electropherograms routinely generated in a clinical diagnostic laboratory on DNA extracts of human blood specimens and ticks collected from human skin bites and from the environment. Since some of the tick samples tested were collected in Ireland, borrelial species or strains not known to exist in the United States were also detected by analysis of this 16S rRNA "core genome". We recommend that hospital laboratories located in Lyme disease endemic areas begin to use a "core genome" sequencing test to routinely diagnose spirochetemia caused by various species of borreliae for timely management of patients at the early stage of infection.

The most extensively used approach to identify potentially hormone-responsive breast cancers is to measure soluble estrogen receptor (ER) proteins in the supernatant, the so-called cytosol fraction of the tumor tissue homogenates, using a dextran-coated charcoal or a sucrose density gradient technique. The urgent need for a practical histochemical technique which can be used to distinguish between ER-negative and ER-positive cancers was quickly recognized by the pathologists as soon as the technology of the ER protein assays extended beyond the boundaries of the few large, federally funded biochemical laboratories. One important application of the histochemical technique is to use it as a means to identify potentially hormone-responsive breast cancers. A histochemical technique can provide a means to evaluate a function of a specific cell type, for example, the infiltrating cancer cells, using the number of cancer cells under observation as the common denominator.

Lyme disease (LD), the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA) gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain.

Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.

The diagnoses of Lyme disease based on clinical manifestations, serological findings and detection of infectious agents often contradict each other. We tested 52 blind-coded serum samples, including 20 pre-treatment and 12 post-treatment sera from clinically suspect Lyme disease patients, for the presence of residual Lyme disease infectious agents, using nested PCR amplification of a signature segment of the borrelial 16S ribosomal RNA gene for detection and direct DNA sequencing of the PCR amplicon for molecular validation. These archived sera were split from the samples drawn for the 2-tier serology tests performed by a CDC-approved laboratory, and are used as reference materials for evaluating new diagnostic reagents. Of the 12 post-treatment serum samples, we found DNA evidence of a novel borrelia of uncertain significance in one, which was also positive for the 2-tier serology test. The rest of the post-treatment sera and all 20 control sera were PCR-negative. Of the 20 pre-treatment sera from clinically suspect early Lyme disease patients, we found Borrelia miyamotoi in one which was 2-tier serology-negative, and a Borrelia burgdorferi in two-one negative and one positive for 2-tier serology. We conclude that a sensitive and reliable DNA-based test is needed to support the diagnosis of Lyme disease and Lyme disease-like borreliosis.

The newly gained knowledge of the viral etiology in cervical carcinogenesis has prompted industrial interests in developing virology-based tools for cervical cancer prevention. Due to the long incubation period from viral infection to developing an invasive cancer, a process whose outcome is influenced by numerous life-style and genetic factors, the true efficacy of the genotype-specific human papillomavirus (HPV) vaccines in cervical cancer prevention cannot be determined for another 30 years. Most HPV DNA test kits designed to replace the traditional Papanicolaou (Pap) smears for precancer detection lack the analytical sensitivity and specificity to comprehensively detect all potentially carcinogenic HPVs and to perform reliable genotyping. The authors implemented the classic nested PCR and Sanger DNA-sequencing technology for routine HPV testing. The results showed a true negative HPV PCR invariably indicates the absence of precancerous cells in the cytology samples. However, 80.5% of single positive HPV-16 tests and 97.3% of single positive HPV-18 tests were associated with a negative or a largely self-reversible Pap cytology. Routine sensitive and reliable HPV type-specific or perhaps even variant-specific methods are needed to address the issues of persistence of HPV infection if a virology-based primary cervical screen is used to replace the Pap cytology screening paradigm.

Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results.

A highly conserved 357-bp segment of the 16S ribosomal RNA gene (16S rDNA) of Borrelia burgdorferi sensu lato and the correspondent 358-bp segment of the Borrelia miyamotoi gene were amplified by a single pair of nested polymerase chain reaction (PCR) primers for detection, and the amplicons were used as the templates for direct Sanger DNA sequencing. Reliable molecular diagnosis of these borreliae was confirmed by sequence alignment analysis of the hypervariable regions of the PCR amplicon, using the Basic Local Alignment Search Tool (BLAST) provided by the GenBank. This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.

A low temperature (LoTemp?) polymerase chain reaction (PCR), conducted at cycling temperatures not to exceed 85℃and catalyzed by a novel highly processive HiFi? DNA polymerase with proofreading function, was used to study the topological conformational changes of the human papillomavirus (HPV) L1 gene DNA fragments bound to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant in the quadrivalent HPV vaccine, Gardasil?. L1 gene DNA fragments of HPV-11, HPV-18 and HPV-16 were detected in the AAHS particles by nested PCR, but all were lacking a region that was amplifiable by an MY09 degenerate primer. In addition, a pair of degenerate consensus GP6/MY11 primers was able to amplify a target segment of the HPV-11 L1 gene DNA and the HPV-18 L1 gene DNA bound to the AAHS particles as expected for any HPV DNA in the B-conformation. However, there was no co-amplification of the HPV-16 L1 gene DNA known to coexist in the same samples. The lack of co-amplification was verified by direct DNA sequencing of the PCR amplicons. The companion HPV-16 L1 gene DNA in the same sample required repeated PCRs with a pair of modified non-degenerate GP6/ MY11 primers for detection. This melting profile of the HPV-16 L1 gene DNA was similar to that of the HPV-16 L1 gene DNA recently discovered in the postmortem blood of a young woman who suffered a sudden unexpected death 6 months after Gardasil? vaccination. The findings suggest that the topological conformational changes in the HPV L1 gene DNA residues bound to the AAHS adjuvant may be genotype-related. The special non-B-conformation may prevent the HPV-16 L1 gene DNA from being degraded in the body of the vaccine recipients after in- tramuscular injection.

As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'–5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.