Diagnostic Polymerase Chain Reaction Research Articles

РЕЗУЛЬТАТЫ СРАВНИТЕЛЬНЫХ ДИАГНОСТИЧЕСКИХ ИССЛЕДОВАНИЙ КРОВИ И МОЛОКА НА ЛЕЙКОЗ, С ИСПОЛЬЗОВАНИЕМ РИД И ПЦР

The purpose of the study was to substantiate the use of PCR for blood and milk analysis of cows in a farm with poor bovine enzootic leukemia. When comparing the effectiveness of serological and molecular genetic methods of laboratory diagnostics, it was found that polymerase chain reaction (PCR) is more sensitive to the detection of bovine leukemia virus infected by the method in comparison with the immune diffusion reaction (RID). A total of 271 blood samples and 271 milk samples from cows were examined. In the RID with blood serum samples obtained from 271 heads of cattle, positive results (the presence of antibodies against the leukemia virus) were detected in 43 samples, which also showed the presence of proviral DNA in PCR. The results of REED and PCR studies of blood serum were identical in 38.1% of cases. Parallel studies of PCR diagnostics additionally revealed 70 heads of cattle infected with the virus. As a result of the study of blood samples in PCR using the Leukosis test systems, the presence of proviral DNA of the virus was detected in 113 of the 271 samples studied. The DNA of the pathogen was found in both whole blood and serum, as well as in a blood clot resuspended with buffered saline solution and in whole milk from sick animals. For the study of milk in REED, lactoserum was previously obtained according to the original (developed by university staff) methodology. Of the 271 milk samples studied, 40 cows were found to be lactoseropositive. Studies in RID of whey confirmed the results obtained in RID with blood serum in 93% of cows. The results of REED and PCR studies of blood serum demonstrate a sensitivity of PCR significantly exceeding the sensitivity of REED, however, when analyzing the results of studies, the age and physiological parameters of the tested animals should be taken into account, since the level of antibodies and the amount of proviral DNA in their lifetime can vary significantly.

The public health response to an outbreak of border-spill malaria along China-Myanmar border.

Malaria importation can be caused by cross-border movement either of both people and anopheline mosquitoes. However, there still lacks robust evidence of imported malaria caused by Plasmodium spp. infected anopheles along international border areas (border-spill malaria). The objectives of this study were to confirm whether an outbreak of Plasmodium vivax malaria is border-spill malaria and assess the effects of China's public health response along China-Myanmar border. Epidemiological, parasitological and entomological investigations were conducted to investigate the outbreak of border-spill malaria. Meanwhile, comprehensive interventions were carried out to prevent further transmission and reintroduction of malaria. Rapid diagnostic testing, microscopy and polymerase chain reaction were performed and the infections were confirmed as P. vivax. A total of 22 (9.21%) of 239 workers contracted P. vivax during the outbreak. Multivariate logistic regression analysis identified that the distance of worker shelters in China within 300 meters to the internally displaced person (IDP) camps in Myanmar was a risk factors associated with malaria infection (adjusted odds ratio 7.5920; 95% confidence interval, 2.6079-22.1013; P = 0.0002). After comprehensive interventions, malaria transmission was successfully interpreted and prevented at the project site till the completion of project on 14 January 2020, and recurrence of P. vivax malaria was not detected by the end of 2020. This study provided robust evidence of border-spill malaria along China-Myanmar border. Malaria parasite reservoir and distance travelled by female anopheline mosquitoes are two determinants for border-spill malaria. The public health response to the outbreak indicates that the malaria surveillance and response system works well in preventing reintroduction of malaria. However, prevention of border-spill malaria is still a major challenge in the Yunnan border area, China.

Loop-Mediated Isothermal Amplification: From Theory to Practice.

Increasing the accuracy of pathogen identification and reducing the duration of analysis remain relevant for modern molecular diagnostics up to this day. In laboratory and clinical practice, detection of pathogens mostly relies on methods of nucleic acid amplification, among which the polymerase chain reaction (PCR) is considered the "gold standard." Nevertheless, in some cases, isothermal amplification methods act as an alternative to PCR diagnostics. Upon more than thirty years of the development of isothermal DNA synthesis, the appearance of loop-mediated isothermal amplification (LAMP) has enabled new directions of in-field diagnostics of bacterial and viral infections. This review examines the key characteristics of the LAMP method and corresponding features in practice. We discuss the structure of LAMP amplicons with single-stranded loops, which have the sites for primer annealing under isothermal conditions. The latest achievements in the modification of the LAMP method are analyzed, which allow considering it as a unique platform for creating the next-generation diagnostic assays.

Accompanying a semi-nested PCR assay to support histopathology findings of fungal keratitis in formalin-fixed paraffin-embedded corneal samples.

Fungal species are responsible for 40%-50% of all microbial keratitis cases. Due to the low amount of extracted DNA in ocular Formalin-fixed Paraffin-embedded (FFPE) samples, selecting a reliable molecular method is a substantial issue in this field. Sixty-six samples were collected via the penetrating keratoplasty (PK) technique. Histopathology assays were performed using hematoxylin-eosin (H&E) and periodic acid Schiff (PAS) staining methods. The ITS1/ITS4 and ITS1/ITS2 primer pairs were used in a semi-nested polymerase chain reaction (PCR) to target the universal internal transcribed spacer (ITS) region. Some PCR results were validated through sequencing. Fungal DNA was detected in 44 of 66 samples (66.7%), and histopathology was positive for 41 of 66 samples (62.1%). Of 41 histopathologically proven fungal-positive cases, 39 were PCR-positive (95%). Moreover, of 44 PCR-positive samples, 39 (88.6%) were histopathology-positive, and 5 (11.3%) were histopathology-negative. Totally in 39 cases (59%), both histopathology and PCR yielded positive results. The Kappa agreement rate between the two diagnostic methods, including histopathology and PCR, was 0.77. Sensitivity, specificity, positive predictive value, and false predictive value were reported as 88.64%, 90.9%, 95.12%, and 80%, respectively. As we reached the acceptable Kappa agreement rate, we concluded that applying the semi-nested PCR assay is a promising method for supporting the evidence by histopathology. Finally, we suggest targeting more specific gene regions using primer pairs that amplify smaller amplicon sizes and surveying novel molecular methods such as NGS to achieve higher sensitivity and Kappa agreement rates.

Lithographic SERS Aptasensor for Ultrasensitive Detection of SARS-CoV-2 in Biological Fluids.

In this paper, we propose a technology for the rapid and sensitive detection of the whole viral particles of SARS-CoV-2 using double-labeled DNA aptamers as recognition elements together with the SERS method for detecting the optical response. We report on the development of a SERS-aptasensor based on a reproducible lithographic SERS substrate, featuring the combination of high speed, specificity, and ultrasensitive quantitative detection of SARS-CoV-2 virions. The sensor makes it possible to identify SARS-CoV-2 in very low concentrations (the limit of detection was 100 copies/mL), demonstrating a sensitivity level comparable to the existing diagnostic golden standard-the reverse transcription polymerase chain reaction.

Обнаружение особо опасных вирусных болезней рыб семейства лососёвых (Salmo salar) с применением полимеразной цепной реакции

Significant economic damage to aquaculture facilities and marine aquatic organisms is caused by viral diseases of fish, almost a quarter of the detected viruses cause diseases. Pathogens mainly fall during the transportation of infected fish from disadvantaged farms to "clean" aquatic farms, as well as from "escaped" fish from cages to the sea, where contamination of natural salmon and other aquatic organisms occurs. When delivering fish planting material to Russia from countries with different epizootological situations, constant monitoring and forecasting of possible risks is necessary. To prevent the spread of viral infections of fish, diagnostics and prevention occupy a leading place. Laboratory diagnostics of fish diseases of viral etiology is based on the isolation of the pathogen and its identification by serological methods that require a long time and research in specialized specialized laboratories of large research institutes. More effective and highly sensitive are molecular diagnostic methods, in particular, polymerase chain reaction (PCR) with reverse transcription to detect a number of particularly dangerous viral diseases of fish. Infectious necrosis of hematopoietic tissue, infectious pancreatic necrosis, viral hemorrhagic septicemia and infectious anemia of salmon were identified by highly specific and sensitive diagnostic PCR method. The improved technique of PCR with reverse transcription makes it possible to detect pathogens in salmon and other fish both with obvious clinical signs and with hidden virus carrier.

Comparative evaluation of BinaxNOW™ Malaria rapid diagnostic test, microscopy and polymerase chain reaction for Plasmodium falciparum detection among individuals suspected with malaria in Lagos, southwest Nigeria

Malaria is a major public health problem in the tropics, especially in resource-limited settings where presumptive malaria treatment is well practised. Prompt malaria diagnosis is essential in ensuring definitive antimalarial therapy. Malaria rapid diagnostic tests (mRDTs) are a suitable option in resource-limited settings. The study evaluated and compared the diagnostic performance of BinaxNOW™ mRDT, microscopy and quantitative polymerase chain reaction (qPCR) in suspected malaria cases in Lagos, south-western Nigeria. The cross-sectional study was conducted in four Primary Health Centres and Ikorodu General Hospital in Lagos. A total of 146 suspected malaria patients aged 2 to 67years were screened between August 2013 and November 2013. Venous blood samples were collected after informed consent was obtained from participants or guardians for mRDT, thick blood film for microscopy and dried blood spots on filter paper for PCR assays. Sociodemographic information of each patient was collected with a structured case report form. The sensitivity, specificity, positive and negative predictive values, diagnostic accuracy, and kappa’s level of agreement were analysed. The level of statistical significance was set at ρ ≤ 0.05. Falciparum malaria positive rate and prevalence were temperature (56.8%) and age-dependent (42.5%). The sensitivity and specificity of mRDT and microscopy were 94.7%, 91.7% and 85.4%, 96.5% respectively. While the positive predictive value (PPV) and negative predictive (NPV) were 79.9%, 94.2% and 96.4%, 95.0%. The diagnostic accuracy and kappa’s level of agreement for mRDT and microscopy were 89.0%, 94.5% and 0.78 and 0.89, respectively. The performance of BinaxNOW™ malaria RDT was comparable with microscopy and can be used to guide malaria treatment in resource-limited settings to enhance parasite-based treatment of all suspected malaria cases when microscopy is unavailable.

Evaluation of a haemozoin-based rapid diagnostic test for diagnosis of imported malaria during the phase of prevention of reestablishment in Sri Lanka

BackgroundSri Lanka, an island nation, has eliminated endemic malaria transmission. Maintaining elimination in the continued presence of vectors requires vigilance in screening people travelling from high malaria-risk areas and a rapid response with focal screening for infections identified in the community. Such screening requires accurate and very rapid assays that enable an immediate response. Both microscopy and rapid diagnostic tests (RDTs) have limitations including sensitivity and speed in screening large numbers, while polymerase chain reaction (PCR) is practical only as laboratory confirmation. This study assessed the utility of ‘Gazelle’, a novel rapid malaria assay based on magneto-optical detection of haemozoin, a by-product of malaria parasite metabolism.MethodsBetween October 2020 and March 2021, two groups of individuals were screened for malaria by four methods, namely, microscopy, Rapid Diagnostic Test (RDT), Gazelle and PCR. Passive case detection was carried out for confirmation of diagnosis amongst individuals suspected of having malaria. Individuals at high-risk of acquiring malaria, namely persons returning from malaria endemic countries, were screened by active case detection.ResultsOf the 440 individuals screened for malaria, nine malaria positives were diagnosed by PCR, microscopy and the HRP2 band of RDT, which included five Plasmodium falciparum infections, two Plasmodium ovale, and one each of Plasmodium vivax and Plasmodium malariae. Gazelle correctly detected the P. vivax, P. ovale and P. malariae infections within the 2 min test time, but did not detect two P. falciparum infections giving a sensitivity of 77.8%. Specificity was 100%.DiscussionThe Gazelle, a portable bench top device proved useful to screen a large number of blood samples for non-falciparum parasites within 5 minutes of sample input. Species differentiation, and improvement in P. falciparum detection, will be important to broaden utility.

Acute febrile illness among outpatients seeking health care in Bangladeshi hospitals prior to the COVID-19 pandemic.

Understanding the distribution of pathogens causing acute febrile illness (AFI) is important for clinical management of patients in resource-poor settings. We evaluated the proportion of AFI caused by specific pathogens among outpatients in Bangladesh. During May 2019-March 2020, physicians screened patients aged ≥2 years in outpatient departments of four tertiary level public hospitals. We randomly enrolled patients having measured fever (≥100.4°F) during assessment with onset within the past 14 days. Blood and urine samples were tested at icddr,b through rapid diagnostic tests, bacterial culture, and polymerase chain reaction (PCR). Acute and convalescent samples were sent to the Centers for Disease Control and Prevention (USA) for Rickettsia and Orientia (R/O) and Leptospira tests. Among 690 patients, 69 (10%) had enteric fever (Salmonella enterica serotype Typhi orSalmonella enterica serotype Paratyphi), 51 (7.4%) Escherichia coli, and 28 (4.1%) dengue detected. Of the 441 patients tested for R/O, 39 (8.8%) had rickettsioses. We found 7 (2%) Leptospira cases among the 403 AFI patients tested. Nine patients (1%) were hospitalized, and none died. The highest proportion of enteric fever (15%, 36/231) and rickettsioses (14%, 25/182) was in Rajshahi. Dhaka had the most dengue cases (68%, 19/28). R/O affected older children and young adults (IQR 8-23 years) and was detected more frequently in the 21-25 years age-group (17%, 12/70). R/O was more likely to be found in patients in Rajshahi region than in Sylhet (aOR 2.49, 95% CI 0.85-7.32) between July and December (aOR 2.01, 1.01-5.23), and who had a history of recent animal entry inside their house than not (aOR 2.0, 0.93-4.3). Gram-negative Enterobacteriaceae were the most common bacterial infections, and dengue was the most common viral infection among AFI patients in Bangladeshi hospitals, though there was geographic variability. These results can help guide empiric outpatient AFI management.

SARS-CoV-2 Mutations and Variants May Muddle the Sensitivity of COVID-19 Diagnostic Assays.

The performance of diagnostic polymerase chain reaction (PCR) assays can be impacted by SARS-CoV-2 variability as this is dependent on the full complementarity between PCR primers/probes and viral target templates. Here, we investigate the genetic variability of SARS-CoV-2 regions recognized by primers/probes utilized by PCR diagnostic assays based on nucleotide mismatching analysis. We evaluated the genetic variation in the binding regions of 73 primers/probes targeting the Nucleocapsid (N, N = 36), Spike (S, N = 22), and RNA-dependent RNA-polymerase/Helicase (RdRp/Hel, N = 15) of the publicly available PCR-based assays. Over 4.9 million high-quality SARS-CoV-2 genome sequences were retrieved from GISAID and were divided into group-A (all except Omicron, >4.2 million) and group-B (only Omicron, >558 thousand). In group-A sequences, a large range of variability in primers/probes binding regions in most PCR assays was observed. Particularly, 87.7% (64/73) of primers/probes displayed ≥1 mismatch with their viral targets, while 8.2% (6/73) contained ≥2 mismatches and 2.7% (2/73) contained ≥3 mismatches. In group-B sequences, 32.9% (24/73) of primers/probes were characterized by ≥1 mismatch, 13.7% (10/73) by ≥2 mismatches, and 5.5% (4/73) by ≥3 mismatches. The high rate of single and multiple mismatches- found in the target regions of molecular assays used worldwide for SARS-CoV-2 diagnosis reinforces the need to optimize and constantly update these assays according to SARS-CoV-2 genetic evolution and the future emergence of novel variants.

Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction for the Detection of Plasmodium falciparum Malaria in a Low-Transmission Area, Jazan Region, Southwestern Saudi Arabia.

This cross-sectional study aimed to assess the performances of a rapid diagnostic test (RDT)—the AllTest Malaria p.f./p.v., microscopy, and nested polymerase chain reaction (PCR) for diagnosing Plasmodium falciparum malaria in 400 febrile patients from a low-transmission region (Jazan) in southwestern Saudi Arabia. Diagnostic performance of all three methods was compared using microscopy and nested PCR as reference methods. Overall, 42 (10.5%), 48 (12.0%), and 57 (14.3%) samples were found positive by microscopy, RDT, and PCR, respectively. With PCR as reference method, the RDT showed higher sensitivity (79% vs. 71.9%), similar specificity (99.1% vs. 99.7%), and better NLR (0.20 vs. 0.27) and area under the curve (89.0% vs. 85.8%) than microscopy. The sensitivity of RDT and microscopy decreased as age increased, and false negatives were associated with low parasite density. In addition, the sensitivity of RDT and microscopy was higher in non-Saudi than in Saudi participants. Against microscopy, both RDT and PCR showed high sensitivity (83.3% vs. 97.6%), specificity (96.4% vs. 95.5%), and NPVs (98.0% vs. 99.7%), but reduced PPVs (72.9% vs. 71.9%), respectively. The results showed that the performance of the AllTest Malaria p.f./p.v RDT was better than that of microscopy in diagnosing P. falciparum malaria among febrile patients in the Jazan region when nested PCR was used as the reference. However, further studies are required to assess malaria diagnostic methods among asymptomatic individuals in the region.

SARS-CoV-2 Continuous Genetic Divergence and Changes in Multiplex RT-PCR Detection Pattern on Positive Retesting Median 150 Days after Initial Infection.

Being in the epicenter of the COVID-19 pandemic, our lab tested 193,054 specimens for SARS-CoV-2 RNA by diagnostic multiplex reverse transcription polymerase chain reaction (mRT-PCR) starting in March 2020, of which 17,196 specimens resulted positive. To investigate the dynamics of virus molecular evolution and epidemiology, whole genome amplification (WGA) and Next Generation Sequencing (NGS) were performed on 9516 isolates. 7586 isolates with a high quality were further analyzed for the mutation frequency and spectrum. Lastly, we evaluated the utility of the mRT-PCR detection pattern among 26 reinfected patients with repeat positive testing three months after testing negative from the initial infection. Our results show a continuation of the genetic divergence in viral genomes. Furthermore, our results indicate that independent mutations in the primer and probe regions of the nucleocapsid gene amplicon and envelope gene amplicon accumulate over time. Some of these mutations correlate with the changes of detection pattern of viral targets of mRT-PCR. Our data highlight the significance of a continuous genetic divergence on a gene amplification-based assay, the value of the mRT-PCR detection pattern for complementing the clinical diagnosis of reinfection, and the potential for WGA and NGS to identify mutation hotspots throughout the entire viral genome to optimize the design of the PCR-based gene amplification assay.

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules.

Ultrafast, portable, and inexpensive molecular diagnostic platforms are critical for clinical diagnosis and on-site detection. There are currently no available real-time polymerase chain reaction (PCR) devices able to meet the demands of point-of-care testing, as the heating and cooling processes cannot be avoided. In this study, the dual temperature modules were first designed to process microfluidic chips automatically circulating between them. Thus, a novel ultrafast molecular diagnostic real-time PCR device (approximately 18 and 23 min for DNA and RNA detection, respectively) with two channels (FAM and Cy5) for the detection of 12 targets was developed. The device contained three core functional components, including temperature control, optics, and motion, which were integrated into a portable compact box. The temperature modules accurately control temperature in rapid thermal cycles with less than ±0.1 °C, ±1 °C and ±0.5 °C for the temperature fluctuation, uniformity, and error of indication, respectively. The average coefficient of variation (CV) of the fluorescence intensity (FI) for all 12 wells was 2.3% for FAM and 2.7% for Cy5. There was a good linear relationship between the concentrations of fluorescent dye and the FIs of FAM and Cy5(R 2 = 0.9990 and 0.9937), and the average CVs of the Ct values calculated by the embedded software were 1.4% for FAM and Cy5, respectively. The 100 double-blind mocked sputum and 249 clinical stool samples were analyzed by the ultrafast real-time PCR device in comparison with the DAAN Gene SARS-CoV-2 kit run on the ABI 7500 instrument and Xpert C. difficile/Epi, respectively. Among the 249 stool samples, the ultrafast real-time PCR device detected toxigenic C. difficile in 54 samples (54/249, 21.7%) with a specificity and positive predictive values of 99.0 and 96.3%, which were higher than the Xpert C. difficile/Epi values of 94.4 and 88.1% (p > 0.05). The ultrafast real-time PCR device detected 15 SARS-CoV-2 positive samples, which has a 100% concordance with that obtained by the DAAN Gene SARS-CoV-2 kit. This study demonstrated that the ultrafast real-time PCR device integrated with microfluidic chips and dual temperature modules is an ultrafast, reliable, easy-to-use, and cost-effective molecular diagnostic platform for clinical diagnosis and on-site testing, especially in resource-limited settings.

Asymptomatic Malaria in Households and Neighbors of Laboratory Confirmed Cases in Raya Kobo District, Northeast Ethiopia.

Malaria is the leading vector-borne parasitic disease that is causing high morbidity and mortality worldwide. So far huge efforts to control and eliminate malaria are hindered by the occurrence of asymptomatic carriers that are a potential source of infection. Yet, there is a scarcity of data nationally and in the current study area as well. Therefore, this study was aimed to assess the prevalence of asymptomatic malaria in Northeast Ethiopia. A community-based cross-sectional study was conducted in 2019 involving a total of 270 study participants recruited via purposive non-probability sampling technique. A structured questionnaire was used to collect data on sociodemographic characteristics, individual and household factors related to asymptomatic malaria. Data were entered in Epi Data 3.1 version and analyzed by using SPSS version 20, and p< 0.05 was considered statistically significant. The overall prevalence of asymptomatic malaria was 7.0%, with 3.0%, 5.2%, and 12.0%, respectively by Rapid diagnostic tests (RDT), Microscopy and Polymerase chain reaction (PCR). The majority of infections (73.7%) were identified from index households. Previous malaria history (AOR: 4.030, 95% CI: 1.021-15.903), living with index cases (AOR: 3.880, 95% CI: 1.275-11.806) and family size > 6 members (AOR: 4.820, 95% CI: 1.260-18.437) were significant predictors of asymptomatic malaria. Reactive case detection had identified considerably higher asymptomatic malaria cases in the community. Therefore, active case investigation should be established in the community by tracking the symptomatic cases at the health facilities.

Malaria prevalence in Commune 5 in Tumaco (Nariño, Colombia)

Background Urban malaria is a public health problem in Colombia and there is still lack of knowledge about its epidemiological characteristics, which are key to the implementation of control measures. The presence of urban malaria cases and disease diagnosis are some of the challenges faced by malaria elimination programs. The objective of this research was to estimate malaria prevalence, explore associated factors and detect pfhrp 2/3 genes, in the urban area of Tumaco between July and December 2019. Methods A prevalence study was conducted by using a stratified random probability sample. Structured surveys were administered and blood samples were taken and examined through optical microscopy, rapid diagnostic tests (RDT) and polymerase chain reaction (PCR). A logistic regression model was used to explore associated factors. Results 1,504 people living in 526 households were surveyed. The overall prevalence was 2.97% (95% CI: 2.1 - 4.3%). It was higher in males, in the 10-19 age group and in asymptomatic cases. The prevalence of pfhrp2 amplification was 2.16% (95% CI: 1.6 - 2.9%). Households with three or more people had a higher risk of malaria infection (adjusted odds ratio (ORa) 4.05; 95% confidence interval (CI) 1.57-10.43). All cases were due to P. falciparum. Conclusions The prevalence of urban malaria was low. Strategies to eliminate malaria in urban areas should be adjusted considering access to early diagnosis, asymptomatic infection, and the RDTs used to detect the presence of the pfhrp2 gene.

Genetic Variation in Rhipicephalus sanguineus s.l. Ticks across Arizona.

Rhipicephalus sanguineus s.l. (Latreille, 1806), the brown dog tick, is the most widely distributed tick species in the world. The two dominant lineages, a temperate group and a tropical group, are recognized as important disease vectors for both dogs and humans. The temperate and tropical lineages overlap in range in some regions of the world, including the southwestern United States, where recent outbreaks of Rocky Mountain spotted fever are linked to R. sanguineus s.l. While it is unclear to what extent they may differ in their capacity to transmit pathogens, finer-scale resolution of temperate and tropical lineage distribution may provide insight into the ecology of these two tick groups and the epidemiology of R. sanguineus s.l.-vectored diseases. Using diagnostic polymerase chain reaction assays, we examined the geospatial trends in R. sanguineus s.l. lineages throughout Arizona. We found the temperate and tropical lineages were well delineated, with some overlap in the eastern part of the state. In one county, tropical and temperate ticks were collected on the same dog host, demonstrating that the two lineages are living in sympatry in some instances and may co-feed on the same host.

Population Genetic Structure and Hybridization of Schistosoma haematobium in Nigeria.

Background: Schistosomiasis is a major poverty-related disease caused by dioecious parasitic flatworms of the genus Schistosoma with a health impact on both humans and animals. Hybrids of human urogenital schistosome and bovine intestinal schistosome have been reported in humans in several of Nigeria’s neighboring West African countries. No empirical studies have been carried out on the genomic diversity of Schistosoma haematobium in Nigeria. Here, we present novel data on the presence and prevalence of hybrids and the population genetic structure of S. haematobium. Methods: 165 Schistosoma-positive urine samples were obtained from 12 sampling sites in Nigeria. Schistosoma haematobium eggs from each sample were hatched and each individual miracidium was picked and preserved in Whatman® FTA cards for genomic analysis. Approximately 1364 parasites were molecularly characterized by rapid diagnostic multiplex polymerase chain reaction (RD-PCR) for mitochondrial DNA gene (Cox1 mtDNA) and a subset of 1136 miracidia were genotyped using a panel of 18 microsatellite markers. Results: No significant difference was observed in the population genetic diversity (p > 0.05), though a significant difference was observed in the allelic richness of the sites except sites 7, 8, and 9 (p < 0.05). Moreover, we observed two clusters of populations: west (populations 1–4) and east (populations 7–12). Of the 1364 miracidia genotyped, 1212 (89%) showed an S. bovis Cox1 profile and 152 (11%) showed an S. haematobium cox1 profile. All parasites showed an S. bovis Cox1 profile except for some at sites 3 and 4. Schistosoma miracidia full genotyping showed 59.3% of the S. bovis ITS2 allele. Conclusions: This study provides novel insight into hybridization and population genetic structure of S. haematobium in Nigeria. Our findings suggest that S. haematobium x S. bovis hybrids are common in Nigeria. More genomic studies on both human- and animal-infecting parasites are needed to ascertain the role of animals in schistosome transmission.

Effects of Different Centrifugation Protocols on the Detection of EGFR Mutations in Plasma Cell-Free DNA.

ObjectivesVarious preanalytical factors, including the collection tube, storage conditions, and centrifugation, affect the detection results of plasma cell-free DNA (cfDNA). We compared the effect of different centrifugation protocols on the detection of EGFR mutations in cfDNA.MethodsWe analyzed 117 plasma specimens from 110 patients with non–small cell lung cancer using the cobas EGFR Mutation Test v2 (Roche Diagnostics). We compared the identified EGFR mutations and semiquantitative index values from the 1- and 2-step centrifugation groups and confirmed the clinical impact of differences in the results after further high-speed centrifugation.ResultsWe detected EGFR mutations in 44 (37.6%) and 47 (40.2%) samples that were centrifuged once and twice, respectively; the 2 groups showed an 89.7% (105/117) concordance and a strong correlation in their semiquantitative index values (r = 0.929). Among the 12 inconsistent result pairs, 9 samples of 2-step centrifugation (75%) were consistent with the results of a recent tissue biopsy.ConclusionsAdditional high-speed centrifugation has been shown to increase the sensitivity of EGFR mutation detection in a commercial in vitro diagnostic real-time polymerase chain reaction device and is an optimal preanalytical factor for detecting low-allele frequency gene mutations using low concentrations of cfDNA.

Molecular Diagnosis of Cat Scratch Disease: a 25-Year Retrospective Comparative Analysis of Various Clinical Specimens and Different PCR Assays.

ABSTRACTCat-scratch disease (CSD), caused primarily by Bartonella henselae, is a common etiology of infectious regional lymphadenopathy. Lymphadenopathy is preceded by a primary inoculation lesion and may progress to suppuration. Laboratory diagnosis of CSD is hampered by the limitations of available confirmatory tests. PCR, in general, is highly sensitive and specific; however, clinical sensitivity in CSD varies greatly between studies. We aimed to identify clinical specimens and PCR assays best suited for CSD diagnosis using a national CSD registry and a uniform case definition. Different clinical specimens and PCR assays, including conventional and real-time PCR, were evaluated. PCR was positive in 335/390 (86%) CSD patients and 425/482 (88%) PCR tests. The highest PCR sensitivity was achieved in lymph node pus aspirates (96%; n = 278 tests) followed by primary lesions (88%; n = 50), lymph node fine needle aspirations (85%; n = 46), lymph node biopsy specimens (73%; n = 91) and paraffin-embedded lymph nodes (59%; n = 17), (P < 0.001). Sensitivity was similar in all types of PCR assays studied. PCR negative predictive value of pus aspirate and lymph node biopsy specimen patient groups was 82% and 72%, respectively. Specificity was 100% based on 125 non-CSD patients with negative PCR. In conclusion, the specimen type rather than the PCR assay type has a major impact on CSD molecular diagnosis. We assume that the inadequate sensitivity of the biopsy specimens was due to sampling errors or the presence of inhibitory factors. Primary lesions should be sampled more frequently for CSD diagnosis. Physicians should be aware of the low PCR negative predictive value of lymph node biopsy specimens.IMPORTANCE Polymerase chain reaction (PCR) for the detection of Bartonella henselae is an important tool for the diagnosis of cat scratch disease (CSD); however, clinical sensitivity varies greatly between studies. The current study shows that the specimen type, with pus aspiration, fine needle aspiration, and primary inoculation lesion having significantly higher sensitivity than fresh or formalin-fixed paraffin-embedded lymph node biopsy specimen, rather than the type of the PCR assay, whether a conventional or a real-time assay, has a major impact on the performance of diagnostic PCR for CSD. The new data provide new tools for the clinical microbiologist when interpreting the results of the PCR assays. Primary inoculation lesions, although easily accessible, are often neglected and should be sampled more frequently for molecular diagnosis of CSD. Physicians should be aware that negative PCR, particularly if performed on fresh or paraffin-embedded lymph node biopsy specimens, does not exclude CSD.

Detection and Kinetics of Subgenomic Severe Acute Respiratory Syndrome Coronavirus 2 RNA Viral Load in Longitudinal Diagnostic RNA-Positive Samples.

While detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by diagnostic reverse-transcription polymerase chain reaction (RT-PCR) is highly sensitive for viral RNA, the nucleic acid amplification of subgenomic RNAs (sgRNAs) that are the product of viral replication may more accurately identify replication. We characterized the diagnostic RNA and sgRNA detection by RT-PCR from nasal swab samples collected daily by participants in postexposure prophylaxis or treatment studies for SARS-CoV-2. Among 1932 RT-PCR–positive swab samples with sgRNA tests, 40% (767) had detectable sgRNA. Above a diagnostic RNA viral load (VL) threshold of 5.1 log10 copies/mL, 96% of samples had detectable sgRNA with VLs that followed a linear trend. The trajectories of diagnostic RNA and sgRNA VLs differed, with 80% peaking on the same day but duration of sgRNA detection being shorter (8 vs 14 days). With a large sample of daily swab samples we provide comparative sgRNA kinetics and a diagnostic RNA threshold that correlates with replicating virus independent of symptoms or duration of illness.