Search Results (44)

Rotavirus A is a leading cause of acute gastroenteritis in infants and young children under the age of five worldwide. The introduction of two live-attenuated oral vaccines, Rotarix and RotaTeq, has significantly decreased illness and death associated with rotavirus in countries where they are included in childhood immunization schedules. In Thailand, these two vaccines have been part of the national childhood immunization program since 2020. To monitor the changing patterns of rotavirus genotype distribution in the post-vaccination era, a molecular epidemiological study of rotavirus A was conducted in pediatric patients with acute diarrhea in Chiang Mai from 2020 to 2023, which was the period after the rotavirus vaccine was implemented in Thailand. A total of 1192 stool specimens collected from children with acute gastroenteritis were screened for rotavirus A by real-time PCR. The G- and P-genotypes were determined by using semi-nested PCR and nucleotide sequencing. A total of 60 out of 1192 (5.0%) samples were positive for rotavirus A. Among these, G3P[8] (55.0%) was identified as the most prevalent genotype, followed by G8P[8] (15.0%), G1P[8] (13.2%), G9P[8] (3.3%), G2P[4] (3.3%), G1P[6] (1.7%), G9P[4] (1.7%), and G8P[X] (1.7%). Additionally, the unusual rotavirus strains G3P[9] (1.7%), G3P[23] (1.7%), and G5P[23] (1.7%) were detected in this study. Phylogenetic analysis of the VP7 and VP4 genes revealed that the G3P[9] strain was closely related to those of feline rotaviruses, while the G3P[23] and G5P[23] strains showed high similarity to those of the porcine rotavirus strains detected previously in Thailand. This study demonstrated a significant decline in the prevalence of rotavirus A infection in pediatric patients in Chiang Mai, Thailand, during the post-vaccination period. The findings from this study contribute to a better understanding of rotavirus epidemiology in Thailand following the implementation of the rotavirus vaccines. Full article

Wild animals often serve as reservoirs for vector-borne zoonoses, which are on the rise worldwide but have not yet been sufficiently researched. Vector-borne zoonoses, such as those caused by Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Dirofilaria immitis, are a growing public health concern due to their increasing incidence and broad host range. The aim of this study was to determine the prevalence and risk factors for vector-borne bacterial (borreliosis, anaplasmosis, ehrlichiosis) and parasitic (dirofilariasis) pathogens and to detect some of these pathogens in the red fox (Vulpes vulpes) population in Croatia. A total of 179 blood samples from foxes from nine districts were analysed. The SNAP ^® 4Dx ^® Plus rapid test was used to detect circulating D. immitis antigen and antibodies against B. burgdorferi, A. phagocytophilum/Anaplasma platys, and Ehrlichia canis/Ehrlichia ewingii. Circulating D. immitis antigen was detected in 6.70% of the samples (95% CI: 3.20–10.19%), while antibodies against A. phagocytophilum/A. platys were found in 10.06% (95% CI: 5.8–14.25%). Only one sample was positive for B. burgdorferi, while no antibodies were detected for E. canis/E. ewingii. Spatial analysis revealed statistically significant differences in prevalence by geographical region (district) and age, while no significant correlations were found. In the standard PCR analysis, DNA of D. immitis was not detected in any of the eight positive and eight negative SNAP ^® 4Dx ^® Plus samples. D. repens, A. reconditum, or co-infections were also not detected by PCR. Of the nine samples that tested positive for A. phagocytophilum/A. platys antibodies, four were confirmed to be positive for A. phagocytophilum by nested and semi-nested PCR targeting the 16S rRNA and GroEL genes. Phylogenetic analysis revealed similarities with various European strains, including zoonotic strains. This study is the first molecular detection of A. phagocytophilum from blood samples of red foxes in Croatia. The results show that red foxes are not free from infections such as anaplasmosis and dirofilariasis, emphasising their possible role in the maintenance and transmission of these pathogens in certain regions of Croatia. These results underline the need for further research to better understand the epidemiological importance of red foxes in the spread of vector-borne diseases. Full article

In Cabo Verde, Acute Respiratory Infection caused by various pathogens was the most reported condition in children under 5 years old between 2014–2020, and the fourth leading cause of mortality in this age group, with Human Respiratory Syncytial Virus (HRSV) being one of the main etiological agents. However, limited literature on the subject hinders the study of its epidemiology and the evaluation of potential implications for public health. In this work, we developed and validated a primer collection for the amplification and sequencing of the G and F genes of HRSV, using a sequential workflow including conventional and semi-nested PCR, followed by Sanger sequencing. This strategy not only allowed for the identification of HRSV linages but also facilitated the detection of mutants in the HRSV F protein, a critical step towards evaluating and ensuring the continued efficacy of Nirsevimab or Palivizumab as prophylactic therapies. Our analysis revealed the presence of the HRSV lineages A.D.2.2.1, A.D.3, B.D.4.1.1, and B.D.E.1, corresponding to the globally circulating lineages during the study period (years 2019 and 2022). No previously described mutations in the F protein that confer resistance to Palivizumab and Nirsevimab were found. However, continuous monitoring of HRSV genotypes is crucial to promptly identifying resistant viruses, considering their potential impact on public health. Full article

Background/Objectives: Hepatocellular carcinoma (HCC) remains a major cause of cancer-related mortality, with diagnostic limitations of existing biomarkers such as alpha-fetoprotein (AFP). This study evaluates plasma Fibrinogen chain A mRNA (FGA mRNA), alone and combined with AFP, for improving HCC diagnosis. Methods: A semi-nested RT-PCR assay was developed to quantify plasma FGA mRNA in 80 HCC patients and 74 controls (57 chronic liver disease [CLD] and 17 healthy donors [HDs]). Receiver operating characteristic (ROC) analysis was used to assess diagnostic performance, and logistic regression evaluated the combined biomarker model. Results: Plasma FGA mRNA levels were significantly higher in HCC patients than in CLD and HD controls (p < 0.0001). The area under the curve (AUC) for HCC vs. the combined control group (CLD + HD) was 0.721 (95% CI: 0.643–0.790), improving to 0.866 (95% CI: 0.782–0.927) when comparing HCC to HDs alone but declining for HCC vs. CLD (AUC = 0.678, 95% CI: 0.592–0.755). Combining FGA mRNA with AFP significantly enhanced diagnostic accuracy for HCC vs. CLD (AUC = 0.859, 95% CI: 0.790–0.913), with a sensitivity of 87.50% and specificity of 71.93%. In patients with low AFP levels (<20 ng/mL), the combined model identified 68.75% of HCC cases, outperforming AFP alone. Conclusions: FGA mRNA alone provides moderate diagnostic utility but substantially improves accuracy when combined with AFP, especially in low-AFP cases. This multi-biomarker approach holds promise for improving HCC detection and warrants further validation in larger cohorts. Full article

Pinna nobilis, an ecologically significant and critically endangered bivalve endemic to the Mediterranean Sea, has been classified as “Critically Endangered” by IUCN due to habitat degradation, climate change, and mass mortality events caused by the protozoan parasite Haplosporidium pinnae. Effective conservation efforts require robust molecular tools for species identification and genetic monitoring, necessitating the development of optimized DNA extraction and amplification protocols for a non-invasive sampling protocol. In this study, we evaluated multiple DNA extraction methods—Chelex-100, the sodium chloride (NaCl) method, a modified CTAB protocol, and a commercial kit, NucleoSpin Tissue Kit—using minute shell fragments from both ethanol-preserved and air-dried (dead) samples. We optimized key parameters, including incubation times, temperatures, and sample preparation, to determine the most effective protocol for obtaining high-quality DNA suitable for downstream applications. Additionally, we assessed different PCR strategies, including nested and semi-nested approaches targeting the COI gene marker, to enhance species identification. To further refine the methodology, we evaluated novel specific primers for nested PCR, improving sensitivity and specificity in detecting P. nobilis DNA from minute and degraded samples. Our results provide an optimized, cost-effective, and time-efficient workflow for non-invasive molecular identification of P. nobilis, with broad implications for conservation genetics, biodiversity monitoring, and species recovery programs. Full article

Congenital Toxoplasma gondii (T. gondii) infection, which can be caused by a primary T. gondii infection during pregnancy, results in severe neurological sequelae in affected children. We have been conducting a prospective cohort study since January 2019 on pregnant women who were suspected of having primary T. gondii infection based on serological tests. In this study, congenital infection was diagnosed using semi-nested polymerase chain reaction (PCR) to detect the B1 gene in the body fluids of newborns. Up until December 2023, forty-one newborns born to mothers suspected of having primary T. gondii infection during pregnancy underwent B1 gene semi-nested PCR tests and anti-T. gondii immunoglobulin (Ig) G and IgM measurements of their blood samples. Eight newborns showed no clinical symptoms of congenital T. gondii infection; however, they were diagnosed with congenital T. gondii infection according to positive PCR results. However, none of the eight infants eventually exhibited any sign of congenital infection, as their serum samples tested negative for anti-T. gondii IgM and IgG until 12 months of age. Therefore, clinicians should consider discrepancies in the diagnosis of congenital T. gondii infection between PCR tests using body fluids of newborns and serological tests during their infantile period. Full article

The aim of this study was to investigate the distribution of human papillomavirus (HPV) genotypes among cervical cancer cases in Moroccan women living in the Souss-Massa region. A total of 155 formalin-fixed, paraffin-embedded cervical tissue samples were tested for the presence of HPV DNA using a semi-nested PCR assay. HPV genotypes were identified using a direct Sanger sequencing assay. The prevalence of HPV was 85.8%. HPV DNA was found in 87.5% of high squamous intraepithelial lesions (HSIL) cases and 85.7% of invasive cervical cancer (ICC) cases. Ten distinct HPV genotypes were identified, including seven high-risk HPV (HR-HPV) genotypes and three low-risk HPV (LR-HPV) genotypes. Among HR-HPV genotypes, HPV16 was the most prevalent in both HSIL and ICC, detected, respectively, in 42.9% and 55.6% of cases. In ICC cases, HPV18 was the second most common genotype detected, in 10.3% of cases. In addition, HPV31, 33, 35, 45 and 58 were detected in 10.4% of ICC cases. LR-HPV genotypes, namely HPV62, 70 and 87, were detected in 2.4% of ICC cases. Adenocarcinoma (ADC) accounted for 4.1% of ICC cases, with HPV 16 and HPV 18 identified in 60% and 40% of these cases, respectively. Overall, our findings show that the genotypes covered by the bivalent and nonavalent HPV vaccines account, respectively, for 65.4% and 74.6%. These results highlight the importance of introducing HPV vaccination and primary HPV testing for mass screening in Morocco in order to effectively prevent and manage cervical cancer and ultimately save women’s lives. Full article

Caliciviruses including noro- and sapoviruses of family Caliciviridae are important enteric human and swine pathogens, while others, like valoviruses, are less known. In this study, we developed a detection and typing pipeline for the most prevalent swine enteric caliciviruses—sapovirus GIII (Sw-SaV), norovirus GII (Sw-NoV), and valovirus GI (Sw-VaV). The pipeline integrates triplex RT-qPCR, 3′RACE semi-nested PCR, and next-generation sequencing (NovaSeq, Illumina) techniques. A small-scale epidemiological investigation was conducted on archived enteric and, for the first time, on oral fluid/saliva samples of diarrheic and asymptomatic swine of varying ages from Hungary and Slovakia. In enteric samples, Sw-SaV was the most prevalent, detected in 26.26% of samples, primarily in diarrheic pigs with low Cq values, followed by Sw-NoV (2.53%) in nursery pigs. In oral fluid samples, Sw-NoV predominated (7.46%), followed by Sw-SaV (4.39%). Sw-VaVs were sporadically found in both sample types. A natural, asymptomatic Sw-SaV outbreak was retrospectively detected where the transient shedding of the virus was <2 weeks. Complete capsid sequences (n = 59; 43 Sw-SaV, 13 Sw-NoV, and 3 Sw-VaV) including multiple (up to five) co-infecting variants were identified. Sw-SaV sequences belong to seven genotypes, while Sw-NoV and Sw-VaV strains clustered into distinct sub-clades, highlighting the complex diversity of these enteric caliciviruses in swine. Full article

To validate the prevalence and biodiversity of ticks and tick-borne pathogens in Chongqing, a total of 601 ticks were collected from dogs, cattle, and goats within the Ta-pa Mountain range in Chongqing, China. Five distinct tick species were identified, including Ixodes ovatus (1.66%, 10/601), I. acutitarsus (0.50%, 3/601), Haemaphysalis flava (10.32%, 62/601), Ha. hystricis (9.82%, 59/601), and Ha. longicornis (77.70%, 467/601). A suit of semi-nest PCR and nest PCR primers were custom-synthesized for the detection of tick-borne pathogens. The analysis yielded positive results for 7.15% Rickettsia (Candidatus R. principis, R. japonica, and R. raoultii), 3.49% Anaplasma (A. bovis and A. capra), 1.16% Ehrlichia, 1.83% Coxiella burnetii, and 3.49% protozoa (Theileria. capreoli, T. orientalis, T. luwenshuni, and Babesia sp.) in ticks. Notably, Ca. R. principis was identified for the first time in I. ovatus and Ha. longicornis. These findings underscore the significant prevalence and diversity of ticks and their associated pathogens within the Chongqing Ta-pa Mountain region. This study accordingly provides an extensive dataset that contributes to the epidemiological understanding and disease prevention strategies for tick-borne illnesses in the local area. Full article

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10⁻⁸ of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity. Full article

(1) Background: The detection of methylated SEPT9 (mSEPT9) in plasma is a promising approach to non-invasive colorectal cancer (CRC) screening. Traditional approaches have limitations in sensitivity and cost-effectiveness, particularly in resource-limited settings. (2) Methods: We developed a semi-nested realtime PCR assay utilizing extendable blocking probes (ExBP) to enhance the detection of low-level mSEPT9 based on DNA melting. This assay allows for the discrimination of mSEPT9 in the presence of high concentrations of non-methylated SEPT9 (up to 100,000 times higher). (3) Results: The assay demonstrated a sensitivity of 73.91% and specificity of 80%, showcasing its ability to detect very low levels of methylated DNA effectively. The innovative use of ExBP without costly modified probes simplifies the assay setup and reduces the overall costs, enhancing its applicability in diverse clinical settings. (4) Conclusions: This novel assay significantly improves the detection of mSEPT9, offering a potential advance in CRC screening and monitoring. Its cost-efficiency and high sensitivity make it particularly suitable for the early detection and management of CRC, especially in settings with limited resources. Future studies are encouraged to validate this assay in larger populations to establish its clinical benefits and practical utility. Full article

Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. SHOX2 gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting SHOX2 methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated SHOX2 DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated SHOX2 DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting SHOX2 methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings. Full article

Worldwide, human respiratory syncytial virus (HRSV) is a major cause of severe infections of the lower respiratory system, affecting individuals of all ages. This study investigated the genetic variability of HRSV during the COVID-19 outbreak in Yaoundé; nasopharyngeal samples positive for HRSV were collected from different age groups between July 2020 and October 2021. A semi-nested RT-PCR was performed on the second hypervariable region of the G gene of detected HRSV, followed by sequencing and phylogenetic assessment. Throughout the study, 40 (37.7%) of the 106 HRSV-positive samples successfully underwent G-gene amplification. HRSV A and HRSV B co-circulated at rates of 47.5% and 52.5%, respectively. HRSV A clustered in the GA2.3.5 genetic lineage (ON1) and HRSV B clustered in the GB5.0.5a genetic lineage (BA9). Differences in circulating genotypes were observed between pre- and post-pandemic years for HRSV A. Predictions revealed potential N-glycosylation sites at positions 237-318 of HRSV A and positions 228-232-294 of HRSV B. This study reports the molecular epidemiology of HRSV in Cameroon during the COVID-19 pandemic. It describes the exclusive co-circulation of two genetic lineages. These findings highlight the importance of implementing comprehensive molecular surveillance to prevent the unexpected emergence of other diseases. Full article

Rotavirus A (RVA) causes diarrhea in calves and frequently possesses the G6 and P[5]/P[11] genotypes, whereas G8 is less common. We aimed to compare RVA infections and G/P genotypes in beef and dairy calves from major livestock regions of Argentina, elucidate the evolutionary origin of a G8 strain and analyze the G8 lineages, infer the phylogenetic relationship of RVA field strains, and investigate the evolution and spatio-temporal dynamics of the main G6 lineages in American countries. Fecal samples (n = 422) from diarrheic (beef, 104; dairy, 137) and non-diarrheic (beef, 78; dairy, 103) calves were analyzed by ELISA and semi-nested multiplex RT-PCR. Sequencing, phylogenetic, phylodynamic, and phylogeographic analyses were performed. RVA infections were more frequent in beef (22.0%) than in dairy (14.2%) calves. Prevalent genotypes and G6 lineages were G6(IV)P[5] in beef (90.9%) and G6(III)P[11] (41.2%) or mixed genotypes (23.5%) in dairy calves. The only G8 strain was phylogenetically related to bovine and artiodactyl bovine-like strains. Re-analyses inside the G8 genotype identified G8(I) to G8(VIII) lineages. Of all G6 strains characterized, the G6(IV)P[5](I) strains from “Cuenca del Salado” (Argentina) and Uruguay clustered together. According to farm location, a clustering pattern for G6(IV)P[5] strains of beef farms was observed. Both G6 lineage strains together revealed an evolutionary rate of 1.24 × 10⁻³ substitutions/site/year, and the time to the most recent common ancestor was dated in 1853. The most probable ancestral locations were Argentina in 1981 for G6(III) strains and the USA in 1940 for G6(IV) strains. The highest migration rates for both G6 lineages together were from Argentina to Brazil and Uruguay. Altogether, the epidemiology, genetic diversity, and phylogeny of RVA in calves can differ according to the production system and farm location. We provide novel knowledge about the evolutionary origin of a bovine G8P[11] strain. Finally, bovine G6 strains from American countries would have originated in the USA nearly a century before its first description. Full article

Human non-polio enteroviruses (NPEVs) are the etiological agents involved in most cases of hand-foot-and-mouth disease (HFMD), herpangina and aseptic meningitis. Information on the epidemiology profiles of NPEV in the Ural Federal District and Western Siberia is very limited, with no published data available. The aim of this study is to describe NPEV incidence in the Ural Federal District and Western Siberia among patients with different forms of non-polio enterovirus infections (NPEVIs) during 2022, stratified by age and clinical manifestations. A total of 265 samples that tested positive for NPEV using a polymerase chain reaction (PCR) were genotyped by semi-nested PCR for the VP1 gene. The results showed that 21 genotypes were identified among patients in this study. CVA6 was the most common genotype for HFMD. CVA6, along with CVA10, accounted for the majority of herpangina cases, while CVA9 was implicated in most meningitis cases. Sequence and phylogenetic analysis showed that nearly all of the CVA6 strains identified in this study displayed a close genetic relationship to strains identified in other cities in Russia and strains from China. NPEV surveillance allows for monitoring the circulation of clinically relevant genotypes, resulting in continuous data about NPEV epidemiology. This is important for improving case prevention, diagnosis and guiding clinical management. Full article