PCR enables rapid detection of dermatophytes in practice.

Dermatophytes cause superficial infections of skin, hair, and nails. Even though they rarely cause severe infections, they are relatively common, particularly in primary health care. Diagnosis of dermatophyte infections has relied on relatively slow culture-based methods. Nucleic acid-based detection (PCR) methods might provide results more rapidly. Here, we describe the transition from culture-based methods into PCR-based methods in Northern Finland with a catchment area of approximately 720,000 mostly Caucasian people. This study included 14,330 samples collected between 2019 and 2022. Our results showed that the PCR-based method has become the diagnostic test of choice for these infections in this area. Commercial real-time PCR assay DermaGenius 2.0 complete multiplex test detected more positive results than culture and covered the most important dermatophytes, Candida albicans, and few less common species. With PCR, the mean turn-around-time from sample request to results decreased from 19 days to 16 hours.IMPORTANCESuperficial fungal infections, dermatophytosis, are remarkably common worldwide, affecting an estimated 20%-25% of the global population. In the diagnosis of these infections, fast and accurate results by PCR shorten the time to diagnosis and help clinicians to avoid unnecessary antifungal treatments.

VLP-based model for the study of airborne viral pathogens.

The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus-like particles (VLPs). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by reverse transcription-loop-mediated isothermal amplification in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling the study of biosafety level 3 and biosafety level 4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins, including those of newly discovered pathogens and mutant variants; and (v) the ability to introduce viral sequences to develop nucleic acid amplification assays. IMPORTANCE: The study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

Comparative evaluation of two molecular multiplex syndromic panels with acute gastroenteritis.

We compared the Allplex Gastrointestinal V/B1/B2 Assays and Seeplex Diarrhea V/B1/B2 ACE Detection Assays in patients with acute gastroenteritis (AGE). Of the total 432 specimens, 48.8% and 54.9% samples were positive for any bacterial or viral target using Seeplex and Allplex, respectively (P = 0.002). The overall percent agreement (OPA) between the two panels was >95% and the lowest OPA was 95.4% for CdB. Allplex identified 40 samples positive for Salmonella spp., while Seeplex and OBC identified only 27 (67.5%) and 8 (20%), respectively. Shigella spp. were detected by assays in six samples, but none were identified using culture. Clostridium perfringens with Seeplex was detected in 70 (16.2%). It remained an informative species in identifying AGE although cpe gene showed only 9.8% positivity. Pathogenic Escherichia coli with Allplex could be detected in 40 (9.3%) samples, which could provide valuable information for the diagnosis of AGE.

Evaluating the cost-effectiveness of low-level HBV DNA screening in occult hepatitis B infection donors: A study from Shandong Blood Center, China.

Objective: This study aimed to assess the efficacy of individual-donation nucleic acid testing (ID-NAT) in detecting occult hepatitis B virus infection (OBI) among anti-HBc positive blood donors, compared to minipool nucleic acid testing (MP-NAT). Methods: The present study analyzed data from the Shandong Blood Center in China during the period from January 2018 to June 2022, where HBsAg-negative blood donors were screened using the 6-sample minipool nucleic acid testing (6-sample MP NAT) method. NAT-positive samples underwent subsequent anti-HBc and anti-HBs testing. Approximately 5000 samples that passed the nucleic acid mixing test were randomly selected for anti-HBc testing, and over 100 anti-HBc positive samples underwent individual donor nucleic acid testing (ID-NAT). Any HBV DNA positive samples detected by ID-NAT were subsequently confirmed using alternative nucleic acid testing methods. Results: Among 220,445 HBsAg-negative blood donors, the positivity rate of HBV DNA detection using the 6-sample minipool nucleic acid testing (MP NAT) method was found to be 0.031% (69/220,445). Of the 67 HBV DNA positive samples, 55 (82.09%) and 25 (37.31%) were found to be positive for anti-HBc and anti-HBs, respectively, using the supplementary chemiluminescent microparticle immunoassay (CMIA). Among the 4797 HBsAg-negative/MP NAT-negative samples, 909 (18.95%) tested positive for anti-HBc. Further NAT testing was performed on 164 arbitrarily selected anti-HBc-positive/MP HBV DNA-negative samples, revealing a HBV DNA positivity rate of 1.22% (2/164). Conclusion: Using individual donation nucleic acid testing can significantly increase the detection rate of occult hepatitis B virus infection in anti-HBc-positive blood donors, resulting in a detection rate of 0.22% (1.22 × 0.1895). This rate is 8.10 times higher than the detection rate achieved by mixed testing methods (0.031%) [calculated as (0.22 + 0.031)/0.031]. Therefore, it is recommended to perform single HBV DNA testing on anti-HBc-positive blood donors, discard plasma with weakly positive or negative anti-HBs but positive anti-HBc, or avoid transfusing anti-HBc-positive plasma to recipients with weakly positive or negative anti-HBs to prevent HBV infection.

Interferon production by Viral, Bacterial & Yeast system: A comparative overview in 2023.

Interferons play a critical role in the innate immune response against several infections and play a key role in the control of a variety of viral and bacterial infectious diseases such as hepatitis, covid-19, cancer, and multiple sclerosis. Therefore, natural or synthetic IFN production is important and had three common methods, including bacterial fermentation, animal cell culture, and recombinant nucleic acid technology. However, the safety, purity, and accuracy of the most preferred INF production systems have not been extensively studied. This study provides a comprehensive comparative overview of interferon production in various systems that include viral, bacterial, yeast, and mammalian. We aim to determine the most efficient, safe, and accurate interferon production system available in the year 2023. The mechanisms of artificial interferon production were reviewed in various organisms, and the types and subtypes of interferons produced by each system were compared. Our analysis provides a comprehensive overview of the similarities and differences in interferon production and highlights the potential for developing new therapeutic strategies to combat infectious diseases. This review article offers the diverse strategies used by different organisms in producing and utilizing interferons, providing a framework for future research into the evolution and function of this critical immune response pathway.

Continued Vaccine Breakthrough Cases of Serotype 3 Complicated Pneumonia in Vaccinated Children, Portugal (2016-2019).

We previously reported that despite the use of pneumococcal conjugate vaccines (PCVs), vaccine serotypes remained important causes of pneumonia with pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]). We cultured and performed PCR on 174 pleural fluid samples recovered from pediatric patients in Portugal from 2016 to 2019 to identify and serotype Streptococcus pneumoniae. Most PCPP cases (n = 87/98) were identified by PCR only. Serotypes 3 (67%), 14, and 8 (5% each) were the most frequent. Vaccine breakthrough cases were seen among age-appropriately, 13-valent, PCV vaccinated children (median: 3 years, range: 17 months to 7 years), mostly with serotype 3 (n = 27) but also with serotypes 14 and 19A (n = 2 each). One breakthrough was seen with serotype 14 in an age-appropriately, 10-valent, PCV-vaccinated child and another with serotype 3 in a child to whom the 23-valent polysaccharide vaccine was administered. While the relative risk of serotype 1 PCPP decreased almost 10-fold from the period of 2010 to 2015 to the period of 2016 to 2019 (relative risk [RR] = 0.106), that of serotype 3 PCPP almost doubled (RR = 1.835). Our data highlight the importance of molecular diagnostics in identifying PCPP and document the continued importance of serotype 3 PCPP, even when PCV13 use with almost universal coverage could be expected to reduce exposure to this serotype. IMPORTANCE The use of conjugate vaccines against Streptococcus pneumoniae in children has led to substantial reductions in pneumococcal invasive disease. However, the reductions seen in each of the 13 serotypes currently included in the highest-valency vaccine approved for use in children (PCV13), were not the same. It is becoming clear that most vaccine breakthroughs worldwide involve serotype 3 and are frequently associated with complicated pneumonia cases, often with empyema or pleural effusion. Here, we show that despite almost universal PCV13 use, which would be expected to reduce vaccine serotype circulation and further reinforce vaccine direct protection, pneumococci and serotype 3 remain the major causes of pediatric complicated pneumonia. Molecular methods are essential to identify and serotype pneumococci in these cases, which frequently reflect vaccine breakthroughs. A broader use of molecular diagnostics will be essential to determine the role of this important serotype in the context of PCV13 use in different geographic regions.

A Nucleic Acid-Based Orthopoxvirus Vaccine Targeting the Vaccinia Virus L1, A27, B5, and A33 Proteins Protects Rabbits against Lethal Rabbitpox Virus Aerosol Challenge.

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

Diagnostic Tests for Detecting Chlamydia trachomatis and Neisseria gonorrhoeae in Rectal and Pharyngeal Specimens.

Chlamydia trachomatis and Neisseria gonorrhoeae are two of the most often reported bacterial infections in the United States. The rectum and oropharynx are important anatomic sites of infection and can contribute to ongoing transmission. Nucleic acid amplification tests (NAATs) are the mainstays for the detection of C. trachomatis and N. gonorrhoeae infections owing to their high sensitivity and specificity. Several NAATs have been evaluated for testing in rectal and pharyngeal infections. A few assays recently received clearance by the Food and Drug Administration, including one point-of-care test. Those assays can be used for testing in symptomatic individuals, as well as for asymptomatic screening in certain patient populations. Routine screening for C. trachomatis in pharyngeal specimens is not recommended by the Centers for Disease Control and Prevention, though it is often performed due to the use of multiplex assays. While expanding the types of settings for screening and using self-collected rectal and pharyngeal specimens can help to increase access and uptake of testing, additional research is needed to determine the potential benefits and costs associated with increased screening for rectal and pharyngeal C. trachomatis and N. gonorrhoeae infections on a population level.

Clinical Assessment and Validation of a Rapid and Sensitive SARS-CoV-2 Test Using Reverse Transcription Loop-Mediated Isothermal Amplification Without the Need for RNA Extraction.

BACKGROUND: Amid the enduring pandemic, there is an urgent need for expanded access to rapid, sensitive, and inexpensive coronavirus disease 2019 (COVID-19) testing worldwide without specialized equipment. We developed a simple test that uses colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect severe acute resrpiratory syndrome coronavirus 2 (SARS-CoV-2) in 40 minutes from sample collection to result. METHODS: We tested 135 nasopharyngeal specimens from patients evaluated for COVID-19 infection at Massachusetts General Hospital. Specimens were either added directly to RT-LAMP reactions, inactivated by a combined chemical and heat treatment step, or inactivated then purified with a silica particle-based concentration method. Amplification was performed with 2 SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. RESULTS: Direct RT-LAMP testing of unprocessed specimens could only reliably detect samples with abundant SARS-CoV-2 (>3 000 000 copies/mL), with sensitivities of 50% (95% CI, 28%-72%) and 59% (95% CI, 43%-73%) in samples collected in universal transport medium and saline, respectively, compared with quantitative polymerase chain reaction (qPCR). Adding an upfront RNase inactivation step markedly improved the limit of detection to at least 25 000 copies/mL, with 87.5% (95% CI, 72%-95%) sensitivity and 100% specificity (95% CI, 87%-100%). Using both inactivation and purification increased the assay sensitivity by 10-fold, achieving a limit of detection comparable to commercial real-time PCR-based diagnostics. CONCLUSIONS: By incorporating a fast and inexpensive sample preparation step, RT-LAMP accurately detects SARS-CoV-2 with limited equipment for about US$6 per sample, making this a potentially ideal assay to increase testing capacity, especially in resource-limited settings.

Comparative Evaluation of Enteric Bacterial Culture and a Molecular Multiplex Syndromic Panel in Children with Acute Gastroenteritis.

Although enteric multianalyte syndromic panels are increasingly employed, direct comparisons with traditional methods and the inclusion of host phenotype correlations are limited. Luminex xTAG gastrointestinal pathogen panel (GPP) and culture results are highly concordant. However, phenotypic and microbiological confirmatory testing raises concerns regarding the accuracy of the GPP, especially for Salmonella spp. A total of 3,089 children with gastroenteritis submitted stool specimens, rectal swab specimens, and clinical data. The primary outcome was bacterial pathogen detection agreement for shared targets between culture and the Luminex xTAG GPP. Secondary analyses included phenotype assessment, additional testing of GPP-negative/culture-positive isolate suspensions with the GPP, and in-house and commercial confirmatory nucleic acid testing of GPP-positive/culture-negative extracts. The overall percent agreement between technologies was >99% for each pathogen. Salmonella spp. were detected in specimens from 64 participants: 12 (19%) by culture only, 9 (14%) by GPP only, and 43 (67%) by both techniques. Positive percent agreement for Salmonella spp. was 78.2% (95% confidence interval [CI], 64.6%, 87.8%). Isolate suspensions from the 12 participants with specimens GPP negative/culture positive for Salmonella tested positive by GPP. Specimens GPP positive/culture negative for Salmonella originated in younger children with less diarrhea and more vomiting. GPP-positive/culture-negative specimen extracts tested positive using additional assays for 0/2 Campylobacter-positive specimens, 0/4 Escherichia coli O157-positive specimens, 0/9 Salmonella-positive specimens, and 2/3 Shigella-positive specimens. For both rectal swab and stool samples, the median cycle threshold (CT ) values, determined using quantitative PCR, were higher for GPP-negative/culture-positive samples than for GPP-positive/culture-positive samples (for rectal swabs, 36.9 [interquartile range {IQR}, 33.7, 37.1] versus 30.0 [IQR, 26.2, 33.2], respectively [P = 0.002]; for stool samples, 36.9 [IQR, 33.7, 37.1] versus 29.0 [IQR, 24.8, 30.8], respectively [P = 0.001]). GPP and culture have excellent overall agreement; however, for specific pathogens, GPP is less sensitive than culture and, notably, identifies samples false positive for Salmonella spp.

The new screening program to prevent cervical cancer using HPV DNA: getting the balance right in maintaining quality.

Along with the reduction in human papillomavirus (HPV) infection and cervical abnormalities as a result of the successful HPV vaccination program, Australia is adopting a new screening strategy. This involves a new paradigm moving from cervical cytological screening to molecular nucleic acid technology (NAT), using HPV DNA assays as primary screening methodology for cervical cancer prevention. These assays must strike a balance between sufficient clinical sensitivity to detect or predict high-grade cervical intraepithelial lesions, the precursor to cervical cancer, without being too sensitive and detecting transient infection not destined for disease. Ensuring the highest quality HPV NAT is thus a priority in order to reduce the possibility of falsely negative screens and manage the risk associated with false positive HPV NAT test results. How to do this needs informed discussion and on-going refinement of the screening algorithm. This is of relevance as more countries move to more sensitive HPV NAT tests for secondary prevention of cervical cancer and as more HPV assays become available.

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens.

Poor efficacy of nucleic acid testing in identifying occult HBV infection and consequences for safety of blood supply in Italy.

BACKGROUND & AIMS: In Italy, DNA screening of blood donations for hepatitis B virus (HBV) was introduced to prevent the transmission of window period and occult HBV infection. Anti-HBc screening is not recommended in order to avoid shortage of the blood supply. To contain costs, donor samples are generally pooled before testing. We evaluated the safety of this national policy using a prospective repository of donors/recipient pairs. METHODS: We used highly sensitive nucleic acid testing (NAT) assays to test repository and follow-up samples from donors who were initially classified as negative by minipool NAT assays (6-MP), but were later found to carry occult HBV DNA. When available, we also analysed recipients' pre- and post-transfusion samples, collected in the context of a repository financed by the European Commission (the BOTIA project). RESULTS: Between 2008 and 2011 6-MP NAT assays identified 18 carriers of occult HBV infection among 12,695 donors; 28 samples from previous donations were available from 13 of these carriers. Highly sensitive HBV DNA detection methods showed that 6-MP HBV DNA screening failed to identify 14/28 (50%) viraemic donations, that were released for transfusion. HBV marker testing of such blood product recipients revealed two cases of transfusion transmitted HBV infection, documented by donor-recipient sequence identity. CONCLUSIONS: Viraemic blood donations from occult HBV infection carriers remain undetected by current minipool HBV DNA screening, and transfusion transmission of HBV continues to occur in susceptible patients. More effective individual HBV DNA screening and/or tests for antibodies to HBV core antigen should be considered to improve blood safety.

A PCR detection method for testing Mycoplasma contamination of veterinary vaccines and biological products.

A rapid test method was developed for detecting mycoplasma contamination in veterinary biological products. The method reduces testing time by 2 weeks and shows comparable sensitivity to the current agar-based detection model. The primary goals for the development of the test were to reduce the testing time, incorporate a method that was easily adaptable across the veterinary biologics industry and reduce the subjective interpretation of results. We found that biological enrichment is necessary to maintain sensitivity of the detection method when compared to the standard culture-based test and that periodic sampling of enrichment cultures is essential to detect a wide variety of mycoplasma species that may be present as contaminants. The PCR detection method is comparable to the agar-based model and can reduce the overall testing time by up to 14 days.

Transfusion transmitted infections in thalassaemics: need for reappraisal of blood screening strategy in India.

The aim of the study was to assess the blood safety in India through prevalence in thalassaemic population. Safety of the blood supply is a subject of great concern for all recipients. This review attempts to assess the relevance and format of tests for viruses in the context of transfusion transmitted infection (TTI) prevalence in India. Serological marker testing for human immunodeficiency virus-1/2 (HIV-1/2), hepatitis C virus (HCV) and hepatitis B virus (HBV) is mandatory in India. Numerous TTI incidents in the repeat recipients supported by results from nucleic acid technology (NAT) testing indicate the deficiencies in blood safety. The ß-thalassaemic population (3-17%) in India has been used to reflect on blood safety. The prevalence of HIV-1/2, HCV and HBV in the Indian donor population, the limitations in accessing safe donors, quality of serological tests and the impact on repeat recipients is evaluated. The reports point to prevalence of ˜2% of viral diseases in the blood donor population, and the insufficiency of serology testing resulting in up to 45% TTIs in thalassaemics. The revelation by individual donation (ID) NAT testing, of 1 per 310 units being serology negative-NAT reactive is alarming. Extrapolating the serology negative NAT reactive yields, for an annual blood supply of 7.9 million units, 23,700 units or nearly 100,000 blood components are likely to be infectious. Though the cost for ID-NAT testing is considered unaffordable for a medium development country such as India, the enormity of TTIs will place an unmanageable cost burden on the society.

Factors in enhancing blood safety by nucleic acid technology testing for human immunodeficiency virus, hepatitis C virus and hepatitis B virus.

In the last few decades through an awareness of transfusion transmitted infections (TTI), a majority of countries have mandated serology based blood screening assays for Human immunodeficiency virus (HIV), Hepatitis C virus (HCV), and Hepatitis B virus (HBV). However, despite improved serology assays, the transfusion transmission of HIV, HCV, and HBV continues, primarily due to release of serology negative units that are infectious because of the window period (WP) and occult HBV infections (OBI). Effective mode of nucleic acid technology (NAT) testing of the viruses can be used to minimize the risk of TTIs. This review compiles the examples of NAT testing failures for all three viruses; analyzes the causes for failure, and the suggestions from retrospective studies to minimize such failures. The results suggest the safest path to be individual donation testing (ID) format for highest sensitivity, and detection of multiple regions for rapidly mutating and recombining viruses. The role of blood screening in the context of the donation and transfusion practices in India, the donor population, and the epidemiology is also discussed. World wide, as the public awareness of TTIs increases, as the recipient rights for safe blood are legally upheld, as the possibility to manage diseases such as hepatitis through expensive and prolonged treatment becomes accessible, and the societal responsibility to shoulder the health costs as in the case for HIV becomes routine, there is much to gain by preventing infections than treating diseases.