Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease.

Diagnostic tests were heralded as crucial during the Coronavirus disease (COVID-19) pandemic with most of the key methods using bioanalytical approaches that detected larger molecules (RNA, protein antigens or antibodies) rather than conventional clinical biochemical techniques. Nucleic Acid Amplification Tests (NAATs), like the Polymerase Chain Reaction (PCR), and other molecular methods, like sequencing (that often work in combination with NAATs), were essential to the diagnosis and management during COVID-19. This was exemplified both early in the pandemic but also later on, following the emergence of new genetic SARS-CoV-2 variants. The 100 day mission to respond to future pandemic threats highlights the need for effective diagnostics, therapeutics and vaccines. Of the three, diagnostics represents the first opportunity to manage infectious diseases while also being the most poorly supported in terms of the infrastructure needed to demonstrate effectiveness. Where performance targets exist, they are not well served by consensus on how to demonstrate they are being met; this includes analytical factors such as limit of detection (LOD) false positive results as well as how to approach clinical evaluation. The selection of gold standards or use of epidemiological factors such as predictive value, reference ranges or clinical thresholds are seldom correctly considered. The attention placed on molecular diagnostic tests during COVID-19 illustrates important considerations and assumptions on the use of these methods for infectious disease diagnosis and beyond. In this manuscript, we discuss state-of-the-art approaches to diagnostic evaluation and explore how they may be better tailored to diagnostic techniques like NAATs to maximise the impact of these highly versatile bioanalytical tools, both generally and during future outbreaks.

Trends in SARS-CoV-2 cycle threshold (Ct) values from nucleic acid testing predict the trajectory of COVID-19 waves.

Forecasting COVID-19 waves helps with public health planning and resource allocation. Cycle threshold (Ct) values obtained from positive SARS-CoV-2 nucleic acid amplification test (NAAT) results offer limited value for individual patient management, but real-time analysis of temporal trends of aggregated Ct values may provide helpful information to predict the trajectories of COVID-19 waves in the community. Ct value trends on 59,609 SARS-CoV-2 NAAT-positive results from 574,403 tests using a single testing assay system, between September 2021 and January 2023, were examined to monitor the trend of the proportion of positive NAAT with lower Ct values (≤28) in relation to changing COVID-19 case numbers over time. We applied regression with autoregressive integrated moving average errors modelling approach to study the relation between Ct values and case counts. We also developed an insight product to monitor the temporal trends with Ct values obtained from SARS-CoV-2 NAAT-positive results. In this study, the proportion of lower Ct values preceded by a range of 7-32 days the rising population COVID-19 testing rate reflecting onset of a COVID-19 wave. Monitoring population Ct values may assist in predicting increased disease activity.

Performances of two rapid LAMP-based techniques for the intrapartum detection of Group B Streptococcus vaginal colonization.

PURPOSE: Group B Streptococcus (GBS) is the leading cause of invasive infections in newborns. The prevention of GBS neonatal disease relies on the administration of an intrapartum antibiotic prophylaxis to GBS-colonized women. In recent years, rapid intrapartum detection of GBS vaginal colonization using real-time nucleic acid amplification tests (NAATs) emerged as an alternative to antenatal culture screening methods. METHODS: We compared the performances of two loop-mediated isothermal amplification (LAMP) tests, the Ampliflash® GBS and the PlusLife® GBS tests, to standard culture for GBS detection in vaginal specimens from pregnant women. The study was conducted from April to July 2023 in a French hospital of the Paris area. RESULTS: A total of 303 samples were analyzed, including 85 culture-positive samples (28.1%). The Ampliflash® GBS test and the PlusLife® GBS tests gave a result for 100% and 96.3% tests, respectively. The performances of the tests were as follows: sensitivity 87.1% (95% confidence interval (CI) 78.3-92.6) and 98.7% (95% CI 93.0-99.8), specificity 99.1% (95% CI 96.7-99.8), and 91.9% (95% CI 87.3-95.0), respectively. False negative results of the Ampliflash® GBS test correlated with low-density GBS cultures. Time-to-results correlated with GBS culture density only for the PlusLife® GBS test (p < 0.001). CONCLUSION: Both techniques provide excellent analytical performances with high sensitivity and specificity together with a short turnaround time and results available in 10 to 35 min. Their potential to further reduce the burden of GBS neonatal disease compared with antenatal culture screening needs to be assessed in future clinical studies.

Production of [211At]NaAt solution under GMP compliance for investigator-initiated clinical trial.

BACKGROUND: The alpha emitter astatine-211 (211At) is garnering attention as a novel targeted alpha therapy for patients with refractory thyroid cancer resistant to conventional therapy using beta emitter radioiodine (131I). Herein, we aimed to establish a robust method for the manufacturing and quality control of [211At]NaAt solution for intravenous administration under the good manufacturing practice guidelines for investigational products to conduct an investigator-initiated clinical trial. RESULTS: 211At was separated and purified via dry distillation using irradiated Bi plates containing 211At obtained by the nuclear reaction of 209Bi(4He, 2n)211At. After purification, the 211At trapped in the cold trap was collected in a reaction vessel using 15 mL recovery solution (1% ascorbic acid and 2.3% sodium hydrogen carbonate). After stirring the 211At solution for 1 h inside a closed system, the reaction solution was passed through a sterile 0.22 µm filter placed in a Grade A controlled area and collected in a product vial to prepare the [211At]NaAt solution. According to the 3-lot tests, decay collected radioactivity and radiochemical yield of [211At]NaAt were 78.8 ± 6.0 MBq and 40 ± 3%, respectively. The radiochemical purity of [211At]At- obtained via ion-pair chromatography at the end of synthesis (EOS) was 97 ± 1%, and remained > 96% 6 h after EOS; it was detected at a retention time (RT) 3.2-3.3 min + RT of I-. LC-MS analysis indicated that this principal peak corresponded with an astatide ion (m/z = 210.988046). In gamma-ray spectrometry, the 211At-related peaks were identified (X-ray: 76.9, 79.3, 89.3, 89.8, and 92.3 keV; γ-ray: 569.7 and 687.0 keV), whereas the peak at 245.31 keV derived from 210At was not detected during the 22 h continuous measurement. The target material, Bi, was below the 9 ng/mL detection limit in all lots of the finished product. The pH of the [211At]NaAt solution was 7.9-8.6; the concentration of ascorbic acid was 9-10 mg/mL. Other quality control tests, including endotoxin and sterility tests, confirmed that the [211At]NaAt solution met all quality standards. CONCLUSIONS: We successfully established a stable method of [211At]NaAt solution that can be administered to humans intravenously as an investigational product.

Development and performance evaluation of a qPCR-based assay for the fully automated detection of group B Streptococcus (GBS) on the Panther Fusion Open Access system.

Streptococcus agalactiae [group B Streptococcus (GBS)] poses a major threat as the primary cause of early-onset neonatal invasive disease, particularly when mothers are colonized rectovaginally. Although culture remains the gold standard for antepartum GBS screening, quantitative polymerase chain reaction (qPCR) offers advantages in terms of sensitivity and turnaround time. The aim of this study was to validate the clinical utility of an automated qPCR laboratory-developed test (LDT) for antepartum GBS screening using the Panther Fusion Open Access system (Hologic, California, USA). The LDT targeted a conserved region of the GBS surface immunogenic protein gene, demonstrating no cross-reactivity and high coverage (99.82%-99.99%). The limit of detection (LoD) was 118 CFU/mL. Comparison with commercial qPCR assays (Panther Fusion GBS and VIASURE Streptococcus B Real-Time) revealed an overall agreement of 99.7%, with a robust Cohen's kappa coefficient of 0.992. Testing of 285 rectovaginal swabs from pregnant women and 15 external quality assessment samples demonstrated exceptional diagnostic performance of the LDT, achieving a diagnostic sensitivity and specificity of 100%, underscoring its accuracy. Prevalence and predictive values were also determined to reinforce test reliability. Our research highlights the limitations of culture-based screening and supports the suitability of our qPCR-based LDT for GBS detection in a clinical setting.IMPORTANCERectovaginal colonization by GBS is a major risk factor for early-onset invasive neonatal disease. The most effective approach to reducing the incidence of early-onset disease (EOD) has been described as universal screening, involving assessment of GBS colonization status in late pregnancy and intrapartum antibiotic prophylaxis. Despite its turnaround time and sensitivity limitations, culture remains the gold standard method for GBS screening. However, nucleic acid amplification-based tests, such as qPCR, have been utilized due to their speed and high sensitivity and specificity. This study validated the clinical usefulness of an automated qPCR-LDT for antepartum GBS screening through the Panther Fusion Open Access system (Hologic). Our study addresses the critical need for more robust, sensitive, and rapid strategies for GBS screening in pregnant women that could favorably impact the incidence of EOD.

External quality assessment for molecular detection of Ureaplasma urealyticum in China.

BACKGROUND AND AIMS: A pilot external quality assessment (EQA) scheme for molecular detection of Ureaplasma urealyticum (UU) was conducted by the National Center for Clinical Laboratories (NCCL) to evaluate the testing capabilities of clinical laboratories and the actual performance of DNA-based nucleic acid amplification tests (NAAT) and RNA-based NAATs when applied in clinical settings. MATERIALS AND METHODS: The EQA panel contained twelve lyophilized samples, including positive samples containing inactivated cell culture supernatants of UU at different concentrations and sterile saline for negative samples. The positive samples were further divided into three groups of high, moderate and low concentrations. The panels were distributed to the participants and the datasets were analyzed according to the qualitative results. RESULTS: A total of 365 laboratories participated in the EQA scheme, and 360 results submitted by 338 laboratories were collected, of which 96.11 % (346/360) of the returned results and 95.86 % (324/338) of the laboratories were deemed competent. The positive percentage agreement (PPA) was ≥ 97.5 % for high and moderate concentration samples, but varied significantly for low concentration samples, decreasing from 86.94 % to 51.94 % as the sample concentration decreased. Additionally, for low concentration samples, RNA-based NAAT showed higher PPAs than DNA-based NAATs, but these results were specific to UU supernatants used in this study. CONCLUSION: Most of UU detection assays employed by the participants were generally consistent with their estimated limit of detection (LOD), and the majority of participants can reliably detect UU samples with high and moderate concentrations, while the poor analytical performance for low concentration samples requires further improvement and optimization.

Comparison of the Simplexa GBS Direct and ARIES GBS assays for the detection of S. agalactiae in broth-enriched swab specimens.

We conducted a comparative evaluation of the FDA-cleared Simplexa GBS Direct and ARIES GBS molecular assays for the detection of Streptococcus agalactiae (Group B Streptococcus, GBS) in 386 prospectively collected, broth-enriched vaginal/rectal swab specimens. The sensitivity of each test was 96.2% and specificity was ≥98.7% when compared to a combined direct and enriched culture method using chromogenic culture medium. A total of four specimens were called positive by both molecular assays but negative by culture, likely representing specimens with a low burden of GBS in these specimens. Two specimens were reported positive by culture but negative by both molecular assays. One of these specimens demonstrated atypically colored colonies on chromogenic agar; the other yielded typically colored colonies only observed after broth enrichment. Our data demonstrate equivalent performance of Simplexa and ARIES molecular assays for the detection of GBS in clinical specimens.IMPORTANCEClinical laboratories often face decisions regarding which of the multiple available molecular platforms would best fit their needs based on cost, workflow, menu, and diagnostic performance. Therefore, objective clinical comparisons of similar molecular tests are valuable resources to aid these decisions. We provide a clinical comparison of two FDA-cleared tests to routine culture and to each other that can be used by clinical laboratories when determining which of the available molecular platforms would best fit their laboratory in terms of workflow, cost, and performance.

The Past, Present, and Future in the Diagnosis of a Neglected Sexually Transmitted Infection: Trichomoniasis.

More than one million curable sexually transmitted infections occur every day. Trichomonas vaginalis is one of the main infections responsible for these epidemiological data; however, the diagnosis of this protozoan is still mainly based on microscopic and culture identification. The commercialization of immunological tests and the development of molecular techniques have improved the sensitivity of classical methods. Nevertheless, the fact that trichomoniasis is a neglected parasitic infection hinders the development of novel techniques and their implementation in routine diagnosis. This review article shows the different methods developed to identify T. vaginalis in population and the difficulties in diagnosing male and asymptomatic patients. The importance of including this parasite in routine gynecological screening, especially in pregnant women, and the importance of considering T. vaginalis as an indicator of high-risk sexual behavior are also discussed.

Rapid detection of Mucorales in human blood and urine samples by functionalized Heusler magnetic nanoparticle assisted customized loop-mediated isothermal amplification.

Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in polymerase chain reaction, offering a straightforward means for improving diagnoses as a near-point-of-care test. The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. The assay was tested with various species of Mucorales, and the limit of detections for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus, and Cunninghamella bertholletiae were 1 fg, 1 fg, 0.1 pg, 0.1 pg, and 0.01 ng, respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of > 83%. Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in < 45 min, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples.

Mucormycosis is a challenging disease to diagnose early. This study introduces a sensitive and rapid diagnostic approach using Loop-mediated isothermal amplification technology. Testing blood and urine samples from 30 patients revealed promising sensitivity and repeatability, indicating its potential for non-invasive diagnosis.

Comparison of Clostridioides difficile nucleic acid amplification test (NAAT) results using fresh and frozen stool specimens and rectal swabs.

IMPORTANCE: Nucleic acid amplification tests (NAATs) are frequently used in Clostridioides difficile research and diagnostic testing, but the effect of freezing specimens on C. difficile NAAT performance is not well characterized. This study evaluated the concordance of NAAT results between fresh and frozen specimens (fecal and rectal swabs) and found it to be very good to excellent. The results indicate that frozen fecal and rectal swab specimens may be used for C. difficile NAAT testing in research when fresh specimens are not available.

In-vivo expressed mycobacterial transcripts as diagnostic targets for pulmonary tuberculosis.

The nucleic acid amplification tests (NAATs) such as Xpert MTB/RIF have transformed the TB diagnostic field by significantly increasing the case detection. However, newer improved diagnostic assays are still needed to meet the WHO targets to end TB. Present study is based on a novel approach of utilizing the in-vivo expressed specific mycobacterial transcriptomic biomarkers for the diagnosis of pulmonary tuberculosis (PTB). Total 61 subjects were recruited including smear positive (smear+; n = 15), smear negative (smear-; n = 30) PTB patients and disease controls (n = 16). Transcripts of three mycobacterial genes Rv0986, Rv0971c and Rv3121 were analyzed using real time PCR (qRT-PCR) in sputum samples. qRT-PCR with Rv0986, Rv0971c and Rv3121 identified smear + PTB patients with 100 %, 78.6 % and 86.7 % sensitivity respectively. In smear- PTB patients, both Rv0986 and Rv0971c based qRT-PCR resulted in 63 %, sensitivity whereas Rv3121 identified these patients with ∼40 % sensitivity only. The sensitivity of the assay for smear-patients increased to 85 % when combinatorial analysis of qRT-PCR data for all the three genes was used. Thus, in-vivo expressed mycobacterial transcripts have promising potential as biomarkers for PTB diagnosis.

Utility of nucleic acid amplification test in the detection of tuberculosis in biological fluids from suspected TB patients in a cardiovascular center in the Philippines.

BACKGROUND: TB NAAT is highly sensitive and can therefore be a helpful tool used in confirming M. tuberculosis. In a prospective study, we evaluated the utility of TB NAAT in the detection of tuberculosis in biological fluids from suspected TB patients. METHODS: We compared tuberculosis nucleic acid amplification test and acid-fast bacilli smears with Lowenstein-Jensen culture, from patients with a clinical suspicion of tuberculosis disease. We calculated the sensitivity, specificity, PPV and NPV. RESULTS: Using the Lowenstein-Jensen culture as the gold standard for detection of Mycobacterium tuberculosis, the TB-NAAT showed sensitivity of 66.67 %, specificity of 93.67 %, and gave a positive predictive value of 44.44 %. CONCLUSION: We conclude that the TB-NAAT is a quick and consistent diagnostic test for TB detection. However, due to a comparably lower sensitivity than other previous studies, the utility of TB-NAAT alone may not be sufficient in the screening of TB patients. Likewise, the TB-NAAT cannot detect non-tuberculous mycobacteria, for which additional analysis may be needed.

A population-based study of the trend in SARS-CoV-2 diagnostic modalities from the beginning of the pandemic to the Omicron surge in Kyoto City, Kyoto, Japan.

BACKGROUND: The coronavirus disease 2019 (COVID-19) presents critical diagnostic challenges for managing the pandemic. We investigated the 30-month changes in COVID-19 testing modalities and functional testing sites from the early period of the pandemic to the most recent Omicron surge in 2022 in Kyoto City, Japan. METHODS: This is a retrospective-observational study using a local anonymized population database that included patients' demographic and clinical information, testing methods and facilities from January 2020 to June 2022, a total of 30 months. We computed the distribution of symptomatic presentation, testing methods, and testing facilities among cases. Differences over time were tested using chi-square tests of independence. RESULTS: During the study period, 133,115 confirmed COVID-19 cases were reported, of which 90.9% were symptomatic. Although nucleic acid amplification testing occupied 68.9% of all testing, the ratio of lateral flow devices (LFDs) rapidly increased in 2022. As the pandemic continued, the testing capability was shifted from COVID-19 designated facilities to general practitioners, who became the leading testing providers (57.3% of 99,945 tests in 2022). CONCLUSIONS: There was a dynamic shift in testing modality during the first 30 months of the pandemic in Kyoto City. General practitioners increased their role substantially as the use of LFDs spread dramatically in 2022. By comprehending and documenting the evolution of testing methods and testing locations, it is anticipated that this will contribute to the establishment of an even more efficient testing infrastructure for the next pandemic.

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay.

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.

Disseminated Gonococcal Infection With Dermatitis-Arthritis Syndrome.

Gonorrhea is a sexually transmitted infection caused by gram-negative diplococci, Neisseria gonorrhoeae. Disseminated gonorrhea is diagnosed infrequently, partly due to low suspicion at the time of presentation, and at times, due to overlapping symptoms associated with non-infectious conditions like systemic lupus erythematosus (SLE). In this article, we present a 42-year-old sexually active female with knee pain and swelling, fever, and rash. Knee aspirate showed the presence of monosodium urate crystals, and the synovial culture grew gram-negative diplococci, requiring multiple joint washouts. The urine nucleic acid amplification test (NAAT) was indeterminate. She was treated with high-dose intravenous ceftriaxone for one week post-joint washout with rapid improvement in her condition and resolution of the rash.

Neglected SARS-CoV-2 variants and potential concerns for molecular diagnostics: a framework for nucleic acid amplification test target site quality assurance.

IMPORTANCE: Molecular tests like polymerase chain reaction were widely used during the COVID-19 pandemic but as the pandemic evolved, so did SARS-CoV-2. This virus acquired mutations, prompting concerns that mutations could compromise molecular test results and be falsely negative. While some manufacturers may have in-house programs for monitoring mutations that could impact their assay performance, it is important to promptly report mutations in circulating viral strains that could adversely impact a diagnostic test result. However, commercial test target sites are proprietary, making independent monitoring difficult. In this study, SARS-CoV-2 test target sites were sequenced to monitor and assess mutations impact, and 29 novel mutations impacting SARS-CoV-2 detection were identified. This framework for molecular test target site quality assurance could be adapted to any molecular test, ensuring accurate diagnostic test results and disease diagnoses.

Treponema pallidum PCR screening at mucosal sites of asymptomatic men who have sex with men taking HIV pre-exposure prophylaxis.

Early detection and treatment of syphilis will reduce the infectious period and transmission. We aimed to determine whether screening men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP) for syphilis using Treponema pallidum polymerase chain reaction (PCR) could detect syphilis before the appearance of syphilis antibodies in serology. MSM attending 3-monthly PrEP clinic visits in Melbourne, Australia, were screened with a PCR assay targeting the polA gene of T. pallidum from an anal swab and an oral rinse between November 2019 and March 2020. Participants were serologically screened for syphilis using chemiluminescence immunoassay. A total of 309 asymptomatic participants provided an anal swab and oral rinse sample for T. pallidum PCR screening. Two syphilis cases (0.6%) were detected: one man had a positive serology only; another man had T. pallidum detected by PCR from an anal swab and a positive serology. PCR positivity was 0.3% (n = 1) for anal swabs and 0% (n = 0) for oral rinse. In this study, T. pallidum PCR screening at routine PrEP clinic visits did not identify additional cases of early syphilis over serological screening performed at these visits. IMPORTANCE With the ongoing syphilis epidemic in men who have sex with men (MSM), we investigated the role of using Treponema pallidum polymerase chain reaction (PCR) testing at the oral cavity and anus in MSM taking pre-exposure prophylaxis for the early detection of syphilis. We evaluated whether the PCR tests from these mucosal sites can detect syphilis infection early, before the development of syphilis antibodies in serology. Our study found two syphilis cases among 309 MSM, and only one syphilis case had a positive anal PCR swab, although serology was positive. We conclude that additional PCR testing is likely to be expensive and would not be cost effective for individuals who regularly screen for syphilis. However, future studies with a larger sample size are required.

A rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for detection of Entamoeba histolytica and Giardia duodenalis.

We developed a rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for two common intestinal parasites-Entamoeba histolytica and Giardia duodenalis, where early detection may be helpful. The mLAMP assay was optimized for the detection of DNA of E. histolytica (18S rRNA gene) and G. duodenalis (Elongation factor 1 alpha gene) from standard strains by using six specific primers FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer), B3 (backward outer primer), loopF (forward loop primer), and loopB (backward loop primer) for each gene target. The amplification time was 16-26 min for E. histolytica and 10-15 min for G. duodenalis, and the parasites could be distinguished based on melting-curve analysis for specific annealing temperatures (Tm) of 84°C-86°C and 88°C-90°C for E. histolytica and G. duodenalis, respectively. The analytical sensitivity was one fg, and no cross-reactivity with other intestinal pathogens was observed. Thus, the mLAMP assay could detect and clearly distinguish E. histolytica and G. duodenalis with a rapid turnaround time and excellent analytical sensitivity and specificity.

Treponema pallidum Detection at Asymptomatic Oral, Anal, and Vaginal Sites in Adults Reporting Sexual Contact with Persons with Syphilis.

We investigated Treponema pallidum PCR positivity at mucosal sites (oral, anal, and vaginal sites) among adults who had sexual contact with a person with syphilis (syphilis contacts). All syphilis contacts had oral rinse and swab samples collected for testing. Men who have sex with men had anal swab and women had vaginal swab samples collected for testing, regardless of the presence of lesions. Of 407 persons tested, 42 (10%) had early syphilis diagnosed; of those, 19 (45%) tested positive by PCR from any anatomic site and had a positive serologic test. T. pallidum was positive from vaginal samples in 3 women, anal samples in 3 men, and oral cavity samples in 2 women and 3 men, without symptoms at those sites. Three women had no prior syphilis serologic test. T. pallidum detection at asymptomatic mucosal sites suggests early syphilis infections, particularly in cases that would conventionally be staged as latent syphilis of unknown duration.

A portable system for economical nucleic acid amplification testing.

Introduction: Regular and rapid large-scale screening for pathogens is crucial for controlling pandemics like Coronavirus Disease 2019 (COVID-19). In this study, we present the development of a digital point-of-care testing (POCT) system utilizing microfluidic paper-based analytical devices (µPADs) for the detection of SARS-CoV-2 gene fragments. The system incorporates temperature tuning and fluorescent detection components, along with intelligent and autonomous image acquisition and self-recognition programs. Methods: The developed POCT system is based on the nucleic acid amplification test (NAAT), a well-established molecular biology technique for detecting and amplifying nucleic acids. We successfully detected artificially synthesized SARS-CoV-2 gene fragments, namely ORF1ab gene, N gene, and E gene, with minimal reagent consumption of only 2.2 µL per readout, representing a mere 11% of the requirements of conventional in-tube methods. The power dissipation of the system was low, at 6.4 W. Results: Our testing results demonstrated that the proposed approach achieved a limit of detection of 1000 copies/mL, which is equivalent to detecting 1 copy or a single RNA template per reaction. By employing standard curve analysis, the quantity of the target templates can be accurately determined. Conclusion: The developed digital POCT system shows great promise for rapid and reliable detection of SARS-CoV-2 gene fragments, offering a cost-effective and efficient solution for controlling pandemics. Its compatibility with other diagnostic techniques and low reagent consumption make it a viable option to enhance healthcare in resource-limited areas.