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Background & objectives: The highly sensitive method for a true understanding of malaria prevalence is of utmost importance for India's elimination strategy. The PCR reaction type with rapid detection, cost-effectiveness, and less workforce should be preferable. Multiplex PCR type accomplishes the present requirement by saving time and resources to find true surveillance data for malaria, especially in low-parasitemia/asymptomatic groups or populations. Methods: The present study focuses on designing multiplex PCR (mPCR) to detect simultaneously Plasmodium genus (PAN) and two common Plasmodium species found in India. It is compared to standard nested PCR on 195 clinical samples to diagnose malaria. The mPCR was designed with a minimum number of primers, leading to less clogging and effective and enhanced detection. It contains one common reverse primer and three forward primers amplifying three targeted genes corresponding to P. falciparum, P. vivax, and Plasmodium genus. Results: The sensitivity and specificity for mPCR were 94.06 and 95.74, respectively. The limit of detection for mPCR was 0.1 parasites/µl. The study has shown a ROC curve area for the mPCR of 0.949 for Plasmodium genus and P. falciparum and 0.897 for P. vivax with standard nPCR. Interpretation & conclusion: The mPCR is rapid in detecting species together, cost-effective, and requires fewer human resources than the standard nPCR. Therefore, the mPCR can be used as an alternative technique for the higher sensitive detection of the malaria parasite. It could also become a vital tool for determining malaria prevalence, facilitating the application of the most effective measures.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen’s kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Three molecular assays were used to detect and quantify white spot syndrome virus (WSSV) in DNA extracted from seston size-fractioned (0.02, 0.2, 1.2, and 20 μm) samples collected from a coastal lagoon and an adjacent shrimp farm. From 107 DNA extracts, only two from one sample tested positive for WSSV with nested PCR in the 1.2 and 20 μm fractions. These results were confirmed by a semi-quantitative (IQ2000TM WSSV Detection and Prevention System) and a quantitative (IQREALTM WSSV Quantitative System) detection system based, based, respectively, on nested PCR and real-time PCR. A first viral load reference value (6.54 × 104 WSSV copies/mL) was established in a seston size fraction (1.2−20 μm). The results suggest that WSSV could be associated with both resuspension of fine clays and silts, and nanoplankton and organic colloids during infectious events.

Background Johne's disease, also known as paratuberculosis, is a chronic granulomatous enteritis disease that affects ruminants worldwide. Objective The objective of this study was to assess the effectiveness of the immunomagnetic bead separation‐immunosensor (IMB‐IS) detection method compared to Nested‐PCR for identifying Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle feces samples. Methods Ninety rectal fecal samples were collected from selected cattle, comprising 59 serum‐positive and 31 serum‐negative cases based on serum ELISA. Following DNA extraction, nested‐PCR was conducted using the IS900 primer sequence targeting the MAP‐specific gene. Immunomagnetic bead (IMB) nanoparticles were synthesized by purifying hyperimmune donkey IgG through affinity chromatography and then conjugating it to Fe nanoparticles. Rhodamine‐B hydrazone immunosensor (IS) was synthesized and conjugated to hyperimmune rabbit IgG. The synthesized IMB and IS were used to identify MAP in cattle fecal samples. Results The results of this study revealed that of the 90 stool samples tested using the nested‐PCR method, 62 samples (68.88%) were positive, while 28 samples (31.12%) were negative. In the IMB‐IS test based on optical density (OD), 64 samples were positive (71.1%), while 26 samples were negative (28.8%). This test exhibited a sensitivity of 100%, specificity of 92.85%, and an overall test accuracy of 97.77%. Conclusion Given the considerations of cost, time, positive and negative predictive values, and acceptable accuracy of the IMB‐IS test, it is recommended for evaluation in screening and epidemiological studies. Rhodamine B hydrazone immunosensor for the detection of Mycobacterium subspecies paratuberculosis in fecal samples from cattle yeilds results comparable to those obtained using nested PCR.

Downy mildew is a major disease of boysenberries in New Zealand, caused by Peronospora sparsa. Most boysenberry plant material, including tissue culture propagated plants are systemically infected and this pathogen also presents as a latent infection. The current nested PCR method to detect latent infection of P. sparsa in asymptomatic boysenberry plants is time consuming as it employs two separate PCR, with potential contamination producing false positive and/or false negative results. To overcome these issues a one step nested PCR method was developed. The method was optimised for primer concentrations, PCR cycle number and DNA concentration. DNA was extracted using a CTAB method. The one step nested PCR method could detect latent infection of P. sparsa in both dormant and active plant growth at 0.4 pg genomic DNA. The most reliable detection was achieved from crown or root tissues. For surety of the infection status, replicate plant tissues should be assessed by PCR as inconsistency between the one step nested PCR and fluorescence microscopy indicated that P. sparsa colonisation is discontinuous through the plant. This method can be recommended for screening P. sparsa latent infection in boysenberry mother plants and daughter plants in nurseries, due to high sensitivity, improved throughput, low cost, and low contamination risk.

Objectives: The study aimed to detect Neospora caninum by nested PCR (nPCR) in aborted fetuses of cattle, sheep, and goats in Bangladesh. Materials and Methods: The head portion of each aborted fetus (111) was dissected at each sampling site and transferred to the laboratory in an ice box. Data on risk factors associated with N. caninum infection were simultaneously collected. Deoxyribonucleic acid was extracted from brain tissue to perform nPCR targeting the internal transcribed spacer 1 (ITS1) ribosomal DNA (rDNA) gene of N. caninum and sequencing was performed from the representative positive samples. Results: By nPCR, N. caninum was found in 16.0% of aborted fetuses of cattle, followed by sheep (14.81%) and goats (11.78%). The highest prevalence was found in aborted fetuses of animals during the second trimester (27.78%) of pregnancy aged 2 to 4 years (18.75%). Obtained sequences showed they were completely matched with N. caninum ITS1 rDNA gene deposited in GenBank. Univariate analysis demonstrated that pregnancy stages (trimesters), abortion history of the animals, and access to dogs in animal farms were significantly (p ≤ 0.05) correlated with N. caninum infection. Conclusion: This study represents the first investigation into the molecular detection, phylogenetic characterization, and analysis of risk factors associated with N. caninum in livestock in Bangladesh. According to the research findings, N. caninum infection may have a role in abortion cases and the ensuing financial losses in the nation’s livestock industry.

Background 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. Methods 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. Results 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. Conclusion 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. Following the clinical suspicion for fungal keratitis (FK), the corneal samples were collected by penetrating keratoplasty (PK) from the patients. Specimens were prepared by microtome and were stained by hematoxylin and eosin (H&E), periodic acid schiff (PAS) stainings. For the molecular assay, samples were subjected to DNA extraction and histopathology examination. The amplification of the β‐globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi‐nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1‐5.8 S‐ITS2) to identify causative agents was performed on PCR products. The results were analyzed by researchers.

Objective: The present study aims to investigate molecular confirmation for Cryptosporidium species in fish handlers in Baghdad City, central Iraq. Materials and Methods: Sixty stool samples were collected between early November 2023 and late April 2024. All samples were examined phenotypically using a modified Ziehl-Neelsen stain and genotypically (nested polymerase chain reaction technique) based on a partial sequence of 18S rRNA genes with sequencing and phylogenetic tree analysis. Results: The total molecular results identified Cryptosporidium parvum with an infection rate of 45% (27/60). A higher infection rate of 51.9% (14/27) was found in the age group between 15 and 35 years, and male handlers recorded a lower infection rate (45%) than females (41.6%). April had a higher elevation in the infection rate of 60% (6/10) than other months. Conclusion: The C. parvum was the only species found in fish handlers, and these local isolates have higher similarity with other isolates of China and Iran.

Background Fungal rhinosinusitis (FRS) encompasses a various spectrum of diseases. Histopathology is the “reference method” for diagnosing FRS, but it cannot determine the genus and species. Moreover, in more than 50% of the histopathologically proven cases, the culture elicited no reliable results. This study was an attempt to evaluate the diagnostic efficiency of semi‐nested polymerase chain reaction (PCR) from formalin‐fixed paraffin‐embedded (FFPE) functional endoscopic sinus surgery (FESS) in FRS patients. Methods One hundred ten specimens were subjected to DNA extraction and histopathology examination. The amplification of the β‐globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi‐nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1‐5.8S‐ITS2) to identify causative agents was performed on PCR products. Results Sixty‐four out of 110 samples were positive by histopathology evidence, of which 56 samples (87.5%) were positive by PCR. Out of 46 negative samples by histopathological methods, five samples (10.9%) yielded positive results by PCR. Sensitivity, specificity, positive predictive value, and negative predictive value of the semi‐nested PCR method were reported 87.5%, 89.2%, 92.7%, and 85.2%, respectively. The kappa factor between PCR and histopathological methods was 0.76, indicating substantial agreements between these two tests. Conclusion Due to the acceptable sensitivity and specificity of the present method, it might be used to diagnose fungal sinusitis infections along with microscopic techniques. This method is recommended to confirm the diagnose of suspected fungal sinusitis with negative histopathology results. Following clinical susception, for the fungal rhinosinusitis (FRS), the samples were collected from the rhino‐nasal cavity of 110 patients and were stained by hematoxylin and eosin (H&E), periodic acid‐Schiff (PAS) stains. For the molecular assay, 110 specimens were subjected to DNA extraction and histopathology examination. The amplification of the β‐globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi‐nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1‐5.8S‐ITS2) to identify causative agents was performed on PCR products. Sixty‐four out of 110 samples were positive by histopathology evidence, of which 56 samples (87.5%) were positive by PCR. Out of 46 negative samples by histopathological methods, five samples (10.9%) yielded positive results by PCR. Sensitivity, specificity, positive predictive value, and negative predictive value of the semi‐nested PCR method were reported 87.5%, 89.2%, 92.7%, and 85.2%, respectively. The kappa factor between PCR and histopathological methods was 0.76, indicating substantial agreements between these two tests.

Anaplasma phagocytophilum is a gram-negative obligate intracellular tick-borne rickettsia with veterinary and public health importance worldwide. This organism is an etiologic agent of tick-borne fever (TBF) in domesticated animals and human granulocytic anaplasmosis (HGA) as well. Hard ticks (Ixodida: Ixodidae) are incriminated as the main biologic vectors for Anaplasma spp. Studies represent that Ixodes spp. are the main vectors for A. phago-cytophilum and few reports hinted that other tick species may play this role. So, the goal of the presented work was to investigate the A. phagocytophilum in 2000 hard ticks in Khuzestan province of Iran by specific nested-PCR performing two consecutive amplifications of 16SrRNA gene fragment with highly variable nucleotide region. Each reaction included 10 salivary glands of distinct tick species. Specific nested-PCR on accumulated salivary glands detected specific bands in 15.5% of reactions (31 of 200) in electrophoresis only in Rhipicephalus sanguineous and Hyalomma marginatum ticks. We concluded that the broad distribution of A. phagocytophilum infection is not only is due to the existence of Ixodes spp. but other hard ticks may also play a role in this issue.