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Background: Identification of the opportunistic fungus Pneumocystis jirovecii in respiratory specimens presents challenges, particularly in differentiating between colonization and active infection. The present study assessed a probe-based real time PCR (qPCR) diagnostic effectiveness in patients with diverse underlying conditions, particularly those with COVID-19 and pulmonary insufficiency. Methods: To set up the qPCR, clinical samples from 281 patients with respiratory ailments were tested. Subsequently, a descriptive study was conducted on 112 patients with pulmonary insufficiency with and without COVID-19 suspected of P. jirovecii infection. All specimens were subjected to DNA extraction followed by nested PCR and qPCR targeting the mitochondrial large subunit (mtLSU)- rRNA gene. Results: Based on nested PCR and qPCR, P. jirovecii was identified in 40 out of 281 patients, with slight variations in positive samples observed across dilutions. Three patients who tested positive in nested PCR yielded negative results in probe-based qPCR. Conversely, three patients who tested positive in probebased qPCR yielded negative results in nested PCR. Considering nested PCR as the golden standard, probe-based qPCR demonstrated good diagnostic performance, with 92.5% sensitivity and 98.7% specificity. Based on cycle threshold (Ct) values, the positive cases were categorized: =32 as infection, >35 as colonization, and a grey zone between these values (32 < X = 35). The analysis of 112 PCP-suspected patients revealed a prevalence ranging from 6.25% (nested PCR) to 7% (probe-based qPCR). Conclusions: This study suggested Ct values to differentiate Pneumocystis pneumonia/colonization in immunocompromised patients. To further augment the diagnostic sensitivity, it is recommended to integrate qPCR results with clinical parameters and biomarkers to offer a more precise understanding of Pneumocystis-related conditions. [ABSTRACT FROM AUTHOR]

Leprosy is a chronic infectious disease that mainly affects the skin and peripheral nerves, it can also invade deeper tissues and organs, including mucous membranes, lymph nodes, testes, eyes, and internal organs. Severe cases can result in deformities and disabilities. We encountered the case of a 39-year-old male with unexplained fever, headache and rash. The patient's lesions were taken for histopathological examination and slit skin smear analysis. Further, the patient was detected of Mycobacterium leprae (M.leprae) nucleic acid sequences in the cerebrospinal fluid (CSF) and plasma, and M.leprae gene targets in the skin lesion tissue and blood. The patient was eventually diagnosed with multibacillary leprosy and type II leprosy reaction. These results suggest the possibility of bacteremia in patients with leprosy to some extent, and observation implies the potential invasion of CSF by M.leprae or its genetic material. [ABSTRACT FROM AUTHOR]

Abstract Background Rotavirus-associated gastroenteritis is a common health problem in children, different variations of rotavirus genotypes differ according to geographic locations and the practice of wide-scale vaccination. Therefore, the present study aimed to detect both the G and P genotypes of rotavirus in children ≤ 5 years old in one center in Egypt as a cross-sectional study, to correlate the genotypes with various demographic and clinical data in infected children and to evaluate the common mixed genotypes G and P in infected children. Method The cross-sectional study included children with acute gastroenteritis ≤ 5 years old from January 2023 till March 2024 recruited from Mansoura University Children’s Hospital, Egypt based upon laboratory diagnosis by exclusion of bacterial and protozoa pathogens. The stool samples were obtained from each child and subjected to detection of rotavirus antigen by enzyme-linked immunosorbent assay (ELISA) followed by genotypes identification of G and P genotypes by nested polymerase chain reaction (PCR). Result A nested PCR study for rotavirus genotypes revealed that G1 was the most common genotype (24.7%) followed by G2 (21.1%), G3 (20%), G9 (20%), and G4 (14.1%). The genotyping of the P genotype revealed that P9 was the commonest genotype (24.7%), followed by P4 (21.2%), P10 (20%), P8 (17.6%) and P6 (16.5%). The commonest combined genotypes of G and P were G1P4 (85.7%), G3P8(88.2%), followed by G2P6 (77.8%) and G9P9(76.5%) and G4P9 (66.7%) followed by G4P10 (33.3%), G9P10(23.5%), G2P10(22.2%), G1P10 (14.3%), G3P10(11.8%). The distribution was significant (P = 0.001). The positive rotavirus antigen was more frequently detected in females (55.3%) than males (44.7%, Odd ratio 0.2, 95% CI 0.22–0.71, P = 0.001). There was a significant association between the summer season and positive rotavirus antigen (P = 0.001) and rural residence of the patients (Odd ratio 6,9 95%CI 3,5-13.5, P = 0.001). The significant associated clinical sign with positive rotavirus antigen was fever (Odd ratio 3,3, 95%CI 1,8-6.05, P = 0.001). The genotypes G and P were significantly associated with positive rotavirus antigen as all cases positive by antigen had been detected by nested PCR with the commonest genotypes G4 (24.7%, P = 0.001) and genotype P9 (24.7%, P = 0.001). Conclusion The present study highlights the common genotypes of rotavirus at one center in Egypt, G1, G2, and G3 were the commonest G genotypes. As regard genotype P the commonest genotypes were P9, P4, and P10. The commonest combined genotypes were G1P4, G3P8, G2P6. There was no effect of the practice of rotavirus vaccination at limited rates at private health sections as the rotavirus is still a major pathogen of acute gastroenteritis in children. There is a need for the inclusion of rotavirus vaccination in the national program of children vaccination in Egypt.

Objective: This study was conducted to molecularly identify and classify Cryptosporidium spp. in fecal samples (n=150) from patients with diarrhea received at the microbiology laboratory of a private hospital in Denizli. Methods: In this study, the positivity of Cryptosporidium spp. in fecal samples was investigated using direct microscopy, Kinyoun’s acid-fast staining method, and Nested polymerase chain reaction (PCR) techniques. Positive PCR products were sequenced. Results: In the examined fecal samples of patients with diarrhea, no parasites were detected through direct microscopic examination. Using the Kinyoun acid-fast staining method, Cryptosporidium spp. was identified in 2.7% (n=4) of the samples, while Nested PCR detected it in 4.67% (n=7) of the samples. The four positive samples were sequenced using primers that amplify the 18S rRNA gene region. The sequencing results identified the isolates as C. parvum. Conclusion: Cryptosporidiosis is an important public health issue as it is a zoonotic disease caused by the Cryptosporidium parasite that can be transmitted from animals to humans. This study focuses on the molecular characterization of Cryptosporidium species detected in human fecal samples, which is significant for understanding which specific strains or species are involved in human infections. According to the findings, it is recommended that control measures be implemented to reduce the risk of exposure to Cryptosporidium in both humans and animals in Türkiye.

Leptospirosis is considered to be the most widespread, yet neglected, re-emerging zoonotic disease caused by infection with a pathogenic species of the genus Leptospira. Although this disease is prevalent in Bangladesh, the recent epidemiological status has not yet been well documented. In this study, we aimed to determine the prevalence of leptospirosis among febrile patients using different diagnostic methods and to characterize the epidemiological features and species of Leptospira in Mymensingh, north–central Bangladesh. Among the blood samples of 186 patients with suspected leptospirosis who met the inclusion criteria, including having a fever for more than 5 days (November 2021–June 2022), 88 samples (47%) were Leptospira-positive according to IgM LAT, IgM ELISA, or nested PCR (positivity rates: 38%, 37%, and 42%, respectively). Nested PCR showed a significantly higher positivity rate (54%) in patients with a short fever (5–10 day) than the other methods did, with lower rates among those with a longer fever. Leptospirosis cases were more common in males (68%), those 16–45 years of age (70%), residents of rural areas (81%), and farmers (41%). In addition to a fever, myalgia and jaundice were found in more than 70% of the patients, while variable symptoms were observed. The 16S rRNA sequencing analysis revealed that the Leptospira species in all the 22 samples tested were L. wolffii, belonging to the pathogenic subclade P2. This study showed the recent epidemiological features of leptospirosis in Bangladesh, indicating the presumptive predominance of L. wolffii since 2019.

South Sulawesi is one of the largest rice production centers in Indonesia. Several important diseases of rice plants, such as those caused by viruses and phytoplasmas, can be transmitted by insect vectors, especially leafhoppers and stem plant. Symptoms of diseases caused by viruses and phytoplasmas are quite diverse but visually similar and difficult to distinguish. This study aims to analyze the presence of phytoplasma associated with white-backed planthopper which are commonly found in rice plantations. The research method used is by conducting surveys and explorations of insect samples in six villages in Sidrap District. White-back planthoppers found on rice plantations showing symptoms of yellowing and stunted leaves were sampled for further analysis, including total DNA isolation of insects, standard PCR amplification for insect and Nested-PCR for phytoplasma identification, gene sequencing for both amplicons, and nucleotide analysis using BLAST method and Mega X program. The PCR with CO1 primer successfully amplified a 700 bp amplicon from insects, whereas nested-PCR using fP1/rP7 primers followed by m23SR/R16F2n amplified phytoplasma supposedly around 1800 bp and 1250 bp of 16S RNA gene, respectively. The DNA sequencing analysis results indicate that the insect samples were identified as 83% Sogatella vibix species based on homology percentage analysis using BLAST and Mega X Program. As for the phytoplasma, it leans more towards the 16SrI group or Candidatus phytoplasma asteris (Aster yellows phytoplasma) with a homology percentage of 99%.

Simple Summary: Feline calicivirus (FCV) is a major cause of upper respiratory tract (URT) disease in cats that can lead to acute or subacute illness. It is highly contagious and widespread in the feline population, with many cats being asymptomatic carriers. However, FCV infection can also cause various clinical problems, resulting in high morbidity rates. Unfortunately, existing diagnostic techniques are not always sufficiently quick or specific enough to detect the virus, making it difficult to diagnose and treat. The current study compared the diagnostic abilities of different methods—polymerase chain reaction (PCR), nested PCR, and reverse transcription loop-mediated isothermal amplification (RT-LAMP)—to detect FCV in clinical samples from cats. The development of rapid and sensitive diagnostic methods is crucial for timely healing, reducing the risk of severe symptoms, and preventing the spread of viruses. Feline calicivirus (FCV) is a highly contagious virus that causes upper respiratory tract disease, commonly known as cat flu. It is widely distributed worldwide and poses a major threat to feline health. Therefore, it is essential to find an efficient and rapid method for detecting FCV. In this study, the colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, using neutral red as an indicator, was developed and validated to target the ORF2 gene of FCV for the first time. Additionally, the study compared the diagnostic abilities of polymerase chain reaction (PCR), nested PCR, and RT-LAMP assays for detecting FCV in clinical samples. The optimized RT-LAMP amplification was carried out at 56.3 °C. The technique visually detected FCV within 70 min, with a limit of detection of 14.3 × 101 copies/µL, and showed no cross-reactivity with other feline pathogens. Out of 54 oropharyngeal swab samples, 17 tested positive for FCV using both nested PCR and RT-LAMP, while only one tested positive using conventional PCR. The positivity rate was higher with nested PCR and RT-LAMP (31.48%) compared to conventional PCR (1.85%). Consequently, these results demonstrated the effectiveness of the colorimetric RT-LAMP assay developed in this study as an alternative for diagnosing FCV in cats. [ABSTRACT FROM AUTHOR]

Tick‐borne pathogens (TBPs) are an emerging concern to humans and animals in the Republic of Korea, especially within the military zones of Gangwon Province, a mountainous region abundant with wild fauna and ticks. This study aimed to detect six TBPs in military working dogs (MWDs) and ticks in this region using Nested polymerase chain reaction (nested PCR) and sequencing. The targeted pathogens included Anaplasma phagocytophilum, Bartonella henselae, Borrelia spp., Orientia tsutsugamushi, Severe fever with thrombocytopenia syndrome virus (SFTSV) and tick‐borne encephalitis virus (TBEV). A total of 164 serum samples from MWDs were collected at the Korean Army Military working dog training center and 1418 ticks were collected at various training sites between June 2021 and August 2022. Haemaphysalis longicornis was identified as the predominant species (98.3%, n = 1394), followed by Haemaphysalis flava (1.6%, n = 22) and Ixodes nipponensis (0.1%, n = 2). This study detected A. phagocytophilum and Borrelia theileri in 1.4% (1/72) of the tick pools and detected SFTSV in 0.6% (1/164) of MWD serum samples. The MWD found to be infected with SFTS was a German Shepherd, but showed no significant clinical signs, with a normal complete blood count (CBC). In the phylogenetic analysis, three sequences were acquired. One sequence (OR865211) exhibited 100% homology with 16S rRNA of A. phagocytophilum identified from animals and human patients in the Republic of Korea. Another sequence (OR865152) displayed 99.34%–100% sequence similarity with 16S rRNA of B. theileri fragments. The third sequence (OR865115), which belongs to the SFTS B‐1 genotype, showed 94.7% sequence similarity to a strain identified in the Republic of Korea (KY789441). [ABSTRACT FROM AUTHOR]

Leptospirosis is considered to be the most widespread, yet neglected, re-emerging zoonotic disease caused by infection with a pathogenic species of the genus Leptospira. Although this disease is prevalent in Bangladesh, the recent epidemiological status has not yet been well documented. In this study, we aimed to determine the prevalence of leptospirosis among febrile patients using different diagnostic methods and to characterize the epidemiological features and species of Leptospira in Mymensingh, north–central Bangladesh. Among the blood samples of 186 patients with suspected leptospirosis who met the inclusion criteria, including having a fever for more than 5 days (November 2021–June 2022), 88 samples (47%) were Leptospira-positive according to IgM LAT, IgM ELISA, or nested PCR (positivity rates: 38%, 37%, and 42%, respectively). Nested PCR showed a significantly higher positivity rate (54%) in patients with a short fever (5–10 day) than the other methods did, with lower rates among those with a longer fever. Leptospirosis cases were more common in males (68%), those 16–45 years of age (70%), residents of rural areas (81%), and farmers (41%). In addition to a fever, myalgia and jaundice were found in more than 70% of the patients, while variable symptoms were observed. The 16S rRNA sequencing analysis revealed that the Leptospira species in all the 22 samples tested were L. wolffii, belonging to the pathogenic subclade P2. This study showed the recent epidemiological features of leptospirosis in Bangladesh, indicating the presumptive predominance of L. wolffii since 2019. [ABSTRACT FROM AUTHOR]

Simple Summary: Cassava witches' broom disease (CWBD) is a major threat to cassava production, and it is believed to be caused by phytoplasma, an unculturable bacterial pathogen. However, recent findings suggest that other pathogens, such as viruses or fungi, may cause witches' broom disease in other crops. This study aims to investigate CWBD in the Philippines, specifically its status, and identify its potential pathogen(s). Currently, CWBD has spread nationwide and causes severe symptoms, such as leaf clustering and drying, as well as the browning of stems and roots. Polymerase chain reaction (PCR) and next-generation sequencing (NGS) showed consistent detection and a high abundance of Ceratobasidium sp., but an inconsistent and low detection rate of phytoplasma (Candidatus Phytoplasma luffae) in Philippine cassava samples. These findings challenge existing notions about CWBD and its causal agent. Confirming the causality and understanding Ceratobasidium sp. involvement can lead to better disease diagnosis and control strategies for ensuring food security. Cassava witches' broom disease (CWBD) is one of the most devastating diseases of cassava (Manihot esculenta Crantz), and it threatens global production of the crop. In 2017, a phytoplasma, Candidatus Phytoplasma luffae (Ca. P. luffae), was reported in the Philippines, and it has been considered as the causal agent, despite unknown etiology and transmission of CWBD. In this study, the nationwide occurrence of CWBD was assessed, and detection of CWBD's pathogen was attempted using polymerase chain reaction (PCR) and next-generation sequencing (NGS) techniques. The results showed that CWBD has spread and become severe, exhibiting symptoms such as small leaf proliferation, shortened internodes, and vascular necrosis. PCR analysis revealed a low phytoplasma detection rate, possibly due to low titer, uneven distribution, or absence in the CWBD-symptomatic cassava. In addition, NGS techniques confirm the PCR results, revealing the absence or extremely low phytoplasma read counts, but a surprisingly high abundance of fastidious and xylem-limited fungus, Ceratobasidium sp. in CWBD-symptomatic plants. These findings cast doubt over the involvement of phytoplasma in CWBD and instead highlight the potential association of Ceratobasidium sp., strongly supporting the recent findings in mainland Southeast Asia. Further investigations are needed to verify the etiology of CWBD and identify infection mechanisms of Ceratobasidium sp. to develop effective diagnostic and control methods for disease management. [ABSTRACT FROM AUTHOR]

This study was conducted with the aim of the molecular identification of the protozoan parasite Cryptosporidium spp. in calves in the early stage of their development on a dairy farm in Eastern Slovakia. Twenty-five Holstein and Holstein cross calves were included in the study and monitored from their birth to the fifth week of life (1–5 weeks). Fresh fecal samples were collected from the same group of calves each week, except during the fourth week, and with the exception of Sample 8. All samples were analyzed using the Ziehl–Neelsen staining method and coproantigen was tested using the ELISA test as the screening method. Using the ELISA method, the highest incidence of cryptosporidiosis was observed in the second week of life of the calves, while the antigen was detected in 21 (91.6%) calves. Using the Ziehl–Neelsen staining method, the highest incidence was also observed in the second week, with an incidence rate of 62.5%. Positive isolates confirmed by the ELISA test were molecularly characterized. The species and subtypes of Cryptosporidium in the positive isolates were identified using PCR and the sequence analysis of the small subunit of the ribosomal 18S RNA (ssu rRNA) and the 60 kDa glycoprotein (gp60) genes of the parasite. The sequence analysis of 29 isolates at the 18S rRNA loci confirmed the presence of two species—Cryptosporidium parvum and Cryptosporidium ryanae. Out of 29 isolates, 25 were assigned to the species C. parvum, with the gp60 locus identified as genotype IIaA17G1R1. Among the individual animal groups, calves are the most common reservoirs of the C. parvum zoonotic species. This disease has significant public health implications as contact with livestock and their feces and working with barn manure are major sources of infection, not only for other animals but also for humans. [ABSTRACT FROM AUTHOR]

The gold standard diagnosis of sporotrichosis is the isolation of Sporothrix sp. in culture media, but this is a time-consuming test that is susceptible to contamination and can be affected by the fungal load. Molecular methods such as nested PCR are gaining more ground in the management of several infections as they are tools for the rapid and accurate identification of microorganisms from pure cultures or directly from biological samples. This study aimed to apply a nested PCR molecular protocol for the rapid detection of Sporothrix spp. directly from clinical samples. Thirteen samples—six from skin biopsies, five from skin exudates, and two from conjunctival secretions—were obtained from patients diagnosed with sporotrichosis due to S. brasiliensis. Calmodulin gene sequencing identified all the isolates as S. brasiliensis. Nested PCR was able to detect all the Sporothrix sensu lato directly from clinical samples as well as the CBS 120339 reference strain. The nested PCR protocol stands out as a diagnostic alternative, as it allows the identification of Sporothrix spp. directly from clinical samples without the need for fungal isolation. [ABSTRACT FROM AUTHOR]

The investigation into the potential association between retroviruses and breast cancer (BC) presents a fascinating area of research. In this study, the focus was on assessing the presence of two retroviruses, Mouse Mammary Tumor Virus (MMTV) and Bovine Leukemia Virus (BLV), in BC samples and exploring their relationship with relevant clinicopathological variables. The study involved analyzing BC samples from 103 Jordanian female patients diagnosed with BC, as well as breast tissue samples from 25 control patients without evidence of breast malignancy. Real-time PCR was used to investigate the evidence of MMTV and BLV infection in these samples, and the findings were then correlated with various clinicopathological characteristics of BC. The results showed that BLV was detected in 19 (18.4%) of the BC samples, while MMTV was detected in only seven (6.8%). Importantly, none of the control samples tested positive for MMTV or BLV. Additionally, MMTV/BLV co-infections were reported in 1.9% of the BC cases. However, the analysis did not reveal any statistically significant associations between the presence of these retroviruses and various clinicopathological variables, such as age, molecular subtypes of BC, stage, grade, lymph node involvement, tumor size, smoking status, or family history. Despite these findings, it is crucial to acknowledge that further investigation with a larger cohort is necessary to establish a clearer association and elucidate the underlying mechanisms that may explain the exact role of retroviruses in breast carcinogenesis. This study provides insights into the potential infection by MMTV and BLV of BC and lays the groundwork for future research in this area. [ABSTRACT FROM AUTHOR]

IntroductionTrichomoniasis remains one of the most significant sexually transmitted disease (STDs) for public health. The disease is caused by parasitic protozoa, Trichomonas vaginalis (T. vaginalis), which is often underestimated in tropical medicine. Despite its public health importance, the epidemiology and molecular characteristics of trichomoniasis in Egypt remains poorly understood, particularly in the southern part of the country (Upper Egypt). This study targeted exploring the genetic variability of T. vaginalis infections in Egyptian women living in Upper Egypt using restriction fragment length polymorphism (RFLP).Patient and techniquesThis cross-sectional study included 150 female patients, who visited the gynaecology and obstetrics outpatient clinics at Sohag General Hospital between 2019 and 2022, exhibiting symptoms of trichomoniasis. Vaginal washout samples were collected from each patient and analyzed using three diagnostic techniques: direct wet mount microscopy, culture on TYM Diamond’s medium, and PCR amplification and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) targeting the actin gene, which was applied to all 16 samples that tested positive in culture. The PCR-RFLP results were then visualized through agarose gels electrophoresis to detect DNA fragments.ResultsOut of 150 vaginal washout samples, 12 cases (8%) tested positive for T. vaginalis trophozoites via direct wet mount microscopy, while 16 samples (10.6%) were positive in culture. Additionally, PCR-RFLP analysis of the 16 culture-positive samples revealed that 13 samples were confirmed positive using this molecular method. The amplified products were digested with the restriction enzyme Hind II, yielding three DNA fragments of 60, 213, and 827 bp, which were then detected by agarose gel electrophoresis. Digestion with RsaI produced five fragments measuring 87, 103/106, 236, and 568 bp, while MseI digestion resulted in three distinct fragments of 204, 315, and 581 bp.ConclusionThis study provides robust baseline data on the prevalence and microscopic characteristics of T. vaginalis in Upper Egypt, while also presenting, for the first time, molecular detection and genotyping and revealed that genotype E is the only prevalent genotype in the region.

BackgroundIdentification of the opportunistic fungus Pneumocystis jirovecii in respiratory specimens presents challenges, particularly in differentiating between colonization and active infection. The present study assessed a probe-based real time PCR (qPCR) diagnostic effectiveness in patients with diverse underlying conditions, particularly those with COVID-19 and pulmonary insufficiency.MethodsTo set up the qPCR, clinical samples from 281 patients with respiratory ailments were tested. Subsequently, a descriptive study was conducted on 112 patients with pulmonary insufficiency with and without COVID-19 suspected of P. jirovecii infection. All specimens were subjected to DNA extraction followed by nested PCR and qPCR targeting the mitochondrial large subunit (mtLSU)-rRNA gene.ResultsBased on nested PCR and qPCR, P. jirovecii was identified in 40 out of 281 patients, with slight variations in positive samples observed across dilutions. Three patients who tested positive in nested PCR yielded negative results in probe-based qPCR. Conversely, three patients who tested positive in probe-based qPCR yielded negative results in nested PCR. Considering nested PCR as the golden standard, probe-based qPCR demonstrated good diagnostic performance, with 92.5% sensitivity and 98.7% specificity. Based on cycle threshold (Ct) values, the positive cases were categorized: ≤32 as infection, >35 as colonization, and a grey zone between these values (32 < X ≤ 35). The analysis of 112 PCP-suspected patients revealed a prevalence ranging from 6.25% (nested PCR) to 7% (probe-based qPCR).ConclusionsThis study suggested Ct values to differentiate Pneumocystis pneumonia/colonization in immunocompromised patients. To further augment the diagnostic sensitivity, it is recommended to integrate qPCR results with clinical parameters and biomarkers to offer a more precise understanding of Pneumocystis-related conditions.

The investigation into the potential association between retroviruses and breast cancer (BC) presents a fascinating area of research. In this study, the focus was on assessing the presence of two retroviruses, Mouse Mammary Tumor Virus (MMTV) and Bovine Leukemia Virus (BLV), in BC samples and exploring their relationship with relevant clinicopathological variables. The study involved analyzing BC samples from 103 Jordanian female patients diagnosed with BC, as well as breast tissue samples from 25 control patients without evidence of breast malignancy. Real-time PCR was used to investigate the evidence of MMTV and BLV infection in these samples, and the findings were then correlated with various clinicopathological characteristics of BC. The results showed that BLV was detected in 19 (18.4%) of the BC samples, while MMTV was detected in only seven (6.8%). Importantly, none of the control samples tested positive for MMTV or BLV. Additionally, MMTV/BLV co-infections were reported in 1.9% of the BC cases. However, the analysis did not reveal any statistically significant associations between the presence of these retroviruses and various clinicopathological variables, such as age, molecular subtypes of BC, stage, grade, lymph node involvement, tumor size, smoking status, or family history. Despite these findings, it is crucial to acknowledge that further investigation with a larger cohort is necessary to establish a clearer association and elucidate the underlying mechanisms that may explain the exact role of retroviruses in breast carcinogenesis. This study provides insights into the potential infection by MMTV and BLV of BC and lays the groundwork for future research in this area.

The purpose of the research is to monitor farms located in the European part of the Russian Federation to identify resistance to effects of benzimidazole anthelmintics in populations of nematodes Haemonchus contortus dwelling in the gastrointestinal tract of small cattle.Materials and methods. The studies were conducted in slaughterhouses located in the Moscow Region in 2023–2024. At the first stage, taxonomic identification of parasitic nematodes and larvae (L3) was made, and Strongylata species was determined from sheep. The study material was the abomasum with duodenum fragments and a distal rectum fragment with feces. For molecular studies, we used mature nematodes and H. contortus L3 larvae isolated from the abomasum and feces of small cattle brought to slaughterhouses in the Moscow Region from 8 regions of the European part of the Russian Federation: Moscow, Astrakhan, Oryol, Lipetsk, Tula, Bryansk regions, Stavropol and Dagestan. The studies were conducted at the premises of the Laboratory of Molecular Biology, the VNIIP – FSC VIEV. Statistical processing of the obtained data was made, and mean infection rates of parasitic nematodes (infection intensity and prevalence) were determined. Fifty-six DNA samples of nematodes H. contortus were examined using nested isothermal amplification (PCR) to identify gene alleles that determine resistance to benzimidazole drugs.Results and discussion. Molecular genetic studies of H. contortus DNA sampled from sheep brought from different Regions only detected homozygous individuals (100%) resistant to benzimidazole in the parasitic nematode population from the Oryol Region. Other regions identified only homozygous and heterozygous individuals susceptible to benzimidazole.

In Iraq, one of the endemic illnesses is cutaneous Leishmaniasis. This study aimed to characterize local isolates of Leishmania tropica and Leishmania major molecularly and determine how closely related they were to reference isolates from nearby nations. A total of 140 and 60 skin lesion samples were collected from patients and dogs, respectively, from September 2021 to March 2022; molecular methods carried out to achieve the prevalence of cutaneous Leishmaniasis in humans and dogs, Nested PCR was done using the kDNA gene for phylogenetic analyses. The overall prevalence of cutaneous Leishmaniasis was 35% and 88.33% in humans and dogs, respectively; the findings showed the total prevalence of Leishmania major significant in dogs was 71.69% compared to Leishmania tropica was 28.35% with significant differences. Fifteen positive samples (Ten human and five dogs) were sequencing to Gen-bank database for phylogenetic analyses, which detected that seven of local isolates skin lesion human samples belongs to Leishmania major isolates IQ Kut isolates, Iraq and three isolates belongs to Leishmania tropica isolates IQ3, Iraq. Four of the local isolates skin lesion dog samples belong to Leishmania major isolates IQ Kut, Iraq, and one isolate belong to Leishmania tropica isolates IQ-7 Iraq. Determining many Leishmania major in humans and dogs indicates that dogs are key parasite reservoirs and significant zoonotic contributes to disease transmission to humans in the Middle Euphrates, Iraq.

The risk of P. equorum infection in horses remains critical. Little studies were conducted to investigate prevalence and molecular analysis of P. equorum in Baghdad city, Iraq. In this study traditional detection followed by molecular technique depending on ITS-2 region were used as an attempt to evaluate this nuclear region as a genetic marker to diagnose the parasitic infection. One hundred and thirty-eight fecal samples of horses were collected and examined by direct wet mount smears, floatation method by NaCl. Extraction of genomic material, nested PCR was done followed by phylogenic analysis depending on ITS-2 region performed for the first time in Iraq and genetic substitutions to analyze Iraqi horses. Nested PCR were done after determining the total infection rate 6.52% by conventional technique, including 3.84% in males and 10% in females with significant difference. Highly infection rate 11.42% was recorded in the age group under 2 years and the lower infection rate 4% was found in the age group between 2-4 years with significant difference. The Iraqi isolates were recorded in the Gen Bank under the accession numbers MZ400507.1, MZ400508.1, MZ400509.1, and MZ4005010.1; while, phylogenetic analysis recorded an identity range between 97-100% with China, Australia and USA isolates. P. equorum is more distributed in younger horses than elderly in Baghdad city and ITS-2 region is a certain molecular marker for detection P. equorum isolates in Iraqi horses.

Coxiella burnetii is an obligate intracellular bacterium that causes Q fever and belongs to the gamma subdivision of Proteobacteria. Several studies discovered incidences of Q fever in various parts of Indonesia, primarily in ruminants. Identification and detection of Q fever in East Java province was reported in 1976. Since then, there has been no report of the presence of this disease in the East Java region. This research aimed to detect the current presence of C. burnetii in ruminants in East Java using samples of the liver, spleen, kidney, lung, and heart of 125 cattle and 156 local goats slaughtered in slaughterhouses in East Java province. Detection of C. burnetii was performed by nested PCR method using outer membrane protein (OMP) and 16S rRNA primers, followed by confirmation using the immunohistochemistry method and hematoxylin-eosin staining. The results showed that C. burnetii bacteria were only detected in 8 out of 80 (10%) local goat samples from Malang by nested PCR using OMP primer. In comparison, nested PCR with 16SrRNA primer could only detect C. burnetii in 3 out of 8 goat's positive samples. Furthermore, immunohistochemistry showed immunoreactivity in the lung, spleen, and kidney. The positive sample of C. burnetii was found only in a small part of Malang local goats but neither in cattle in Malang nor other ruminants in other areas of East Java province. The results of this study strengthen the assumption that Q fever has already been endemic in East Java province.

Chlamydia trachomatis is one among the sexually transmitted diseases causing genital tract infection frequently associated with complications of infertility. The aim of our study is to detect the presence of C. trachomatis infection (CTI) in female infertility by nested Polymerase Chain Reaction (PCR) in a tertiary care center. A cross-sectional study was done with 230 infertile women attending the OPD of Reproductive Medicine and Surgery. CTI was detected among the study participants by screening for momp and cryptic plasmid gene using nested PCR. Based on the history and clinical presentation, the enrolled patients categorized as primary and secondary infertility. The results of the nested PCR for the primary and secondary infertile women were tabulated and compared for the statistical significance using Epi info version 7 and Chi-square test. A p-value of < 0.05 considered significant. In the study, participants 1 (20%) was primary and 4 (80%) belonged to secondary infertility. Of the 230 infertile women screened 2.2% of them had PCR positive for either momp or cryptic plasmid gene. CTI was seen more (80%) in secondary infertile than in primary infertile women. CTI was seen more in the age group 26-30 years (60%) followed by 21-25 years and 31-35 years (20%). The results of our study showed CTI is associated with infertility and recurrent spontaneous abortion. It’s imperative to screen for CTI by molecular method in young females which necessitates early therapy and prevention of long term complications like infertility.

PCR-based diagnostics has revealed the previously largely unknown Cryptosporidium transmission and infections in high-income countries. This study aimed to determine domestic and imported subtypes of Cryptosporidium species in Norway, evaluate their demographic distribution, and identify potential small outbreaks. Cryptosporidium-positive human faecal samples were obtained from six medical microbiology laboratories between February 2022 and January 2024, together with 22 Cryptosporidium-positive animal samples. Species and subtypes were identified by sequencing PCR products from gp60 and SSU rRNA genes. Most cryptosporidiosis cases occurred during late summer/early autumn, primarily in children and young adults. Of 550 human samples, 359 were successfully characterized molecularly (65%), revealing infection with 10 different Cryptosporidium species. C. parvum occurred in 245 (68%) human isolates with IIa and IId being major allele families, with distinct regional distribution patterns of common subtypes. A kindergarten outbreak with 5 cases was due to C. parvum IIaA14G1R1. C. mortiferum was identified in 33 (9.2%) human cases of which 24 were known to be of domestic origin, making it the second most common species in human autochthonous cases in Norway. All C. mortiferum isolates were of the same genotype; XIVaA20G2T1, including 13 cases from a suspected small outbreak in Trøndelag. C. hominis occurred in 68 typed cases (19%), but mostly in infections acquired abroad, with allele families Ib and If occurring most often. In conclusion, this study of recent Cryptosporidium spp. and subtypes in Norway, highlights the predominance of C. parvum and the emergence of C. mortiferum among autochthonous cases.

Simple Summary: In this paper, a three-round nested PCR assay with high specificity and sensitivity (2.62 copies/reaction), employing the Pseudomonas plecoglossicida sctU gene, was established. The detection system was used to detect large yellow croaker samples in the Zhejiang and Fujian areas. It was found that March and April were the peak times of visceral white nodules disease, which was inversely related to temperature. Using this detection method, early detection of visceral white nodules disease in large yellow croakers could be realized. The visceral white nodules disease in the internal organs of Larimichthys crocea has caused significant harm in the aquaculture of this species, with Pseudomonas plecoglossicida considered one of the core pathogens causing this disease. In this study, we designed three pairs of specific nested PCR primers targeting the sctU gene of P. plecoglossicida, a crucial component of the Type III secretion system (T3SS), which is instrumental in bacterial pathogenesis and virulence. Through the optimization of PCR reaction conditions, specificity testing, and sensitivity determination, a method was established for the accurate detection of P. plecoglossicida. This method yielded single amplification products, exhibited a false positive rate of zero for reference bacteria, and achieved a detection sensitivity of a minimum of 2.62 copies/reaction for the target sequence. Using the detection method, we conducted analyses on the diseased populations of L. crocea, involving a total of 64 screened fishes along the southeast coast of China from 2021 to 2023. The results revealed that the infection rate of P. plecoglossicida in diseased L. crocea exceeded over 90% in March and April, while in other months, the maximum recorded infection rate was merely 10%. The detection method developed in this study shows potential for early warning and routine monitoring of visceral white nodules disease in the internal organs of species such as L. crocea. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We reported a case of a 22-year-old male presented with blurry vision in his left eye. He had skin erosion on his testicles for the last year. His presenting visual acuity was 20/200 (left) and 20/200 (right). His right eye funduscopic showed an indistinct margin of papillary cranial nerve II (CN II) and hyperemia. He had reactive VDRL, TPHA, and HIV infection. Nested polymorphism chain reaction (nested PCR) showed T. pallidum A2059G mutation, but results were negative from whole genome sequencing (WGS). He received benzathine penicillin 2.4 million international unit injection (intramuscular) and symptomatic therapy. After three months of treatment, he had clinical improvement. In conclusion, ocular syphilis can occur at any stage of syphilis and may mimic various ocular diseases. Thus, it is often referred to as "the great masquerader". [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is one among the sexually transmitted diseases causing genital tract infection frequently associated with complications of infertility. The aim of our study is to detect the presence of C. trachomatis infection (CTI) in female infertility by nested Polymerase Chain Reaction (PCR) in a tertiary care center. A cross-sectional study was done with 230 infertile women attending the OPD of Reproductive Medicine and Surgery. CTI was detected among the study participants by screening for momp and cryptic plasmid gene using nested PCR. Based on the history and clinical presentation, the enrolled patients categorized as primary and secondary infertility. The results of the nested PCR for the primary and secondary infertile women were tabulated and compared for the statistical significance using Epi info version 7 and Chi-square test. A p-value of < 0.05 considered significant. In the study, participants 1 (20%) was primary and 4 (80%) belonged to secondary infertility. Of the 230 infertile women screened 2.2% of them had PCR positive for either momp or cryptic plasmid gene. CTI was seen more (80%) in secondary infertile than in primary infertile women. CTI was seen more in the age group 26-30 years (60%) followed by 21-25 years and 31-35 years (20%). The results of our study showed CTI is associated with infertility and recurrent spontaneous abortion. It's imperative to screen for CTI by molecular method in young females which necessitates early therapy and prevention of long term complications like infertility. [ABSTRACT FROM AUTHOR]

Entamoeba gingivalis (E. gingivalis) and Trichomonas tenax (T. tenax) are two species of parasitic protozoans that inhabit the human buccal cavity. Poor hygiene increases infection with these parasites. There has recently been a need to use orthodontics, despite its benefits, but the damage caused by orthodontics cannot be overlooked. The current study aims to assess the prevalence of oral parasites among individuals who use orthodontic appliances (fixed and mobile). The study included 200 participants, 100(76 females and 24 males) orthodontics patients, and 100(75 females and 25 males) nonorthodontics participants (control). Gathered one pair of swabs from each participant for microscopic analysis, culture, and implementation of the nested PCR method. Chi-square and Fisher's tests were conducted to identify any statistically significant connection between parasitic infections and orthodontic treatments. The findings indicated that orthodontic patients had elevated infection rates with these parasites. Specifically, the infection rate of E. gingivalis in orthodontic patients was more infected (47%) than in non-orthodontic subjects (25%) which were the control. The prevalence of T. tenax was only 2.0% in the orthodontic patients while it was 1.0% in the control group. The prevalence rates of both parasites (E. gingivalis and T. tenax) in the orthodontic patients exhibited the highest prevalence of 19% and 16% respectively among the control subjects. The study found a significant correlation between infection and orthodontic applications at P= 0.01. The effects of other factors on oral parasite infection such as (sex, age, and orthodontic type) were also studied and compared between the two groups. [ABSTRACT FROM AUTHOR]

Anaplasmosis, a tick-borne disease with worldwide distribution, impacts ruminants, equines, carnivores, and humans. This study aimed to investigate Anaplasma phagocytophilum in horses from Ardabil province and Anaplasma ovis in small ruminants from East Azerbaijan province using the Nested Polymerase Chain Reaction (PCR) method. Blood samples were taken from the jugular vein of 100 healthy horses in the Ardabil province and 156 healthy sheep and goats (116 sheep and 40 goats) in the East Azerbaijan province during the spring and summer seasons of 2016 in northwest Iran. The collected blood samples were stored at -20°C until the molecular experiments were conducted. Nested PCR was employed to detect A. phagocytophilum in horses and A. ovis in small ruminants using extracted DNA and amplifying 16S rRNA and msp4 genes. The Chi-square test of independence was used to determine the relationship between Anaplasma spp., infection, and independent variables, including age, gender, animal species, and sampling location. None of the 100 samples collected from horses in the Ardabil province were positive for A. phagocytophilum. In the East Azerbaijan province, 11 out of the 156 (7.05%) blood samples collected from sheep and goats tested positive for A. ovis. In addition, A. ovis infection was not significantly related to the independent variables. Phylogenetic analysis showed that the sequence obtained in this study (MH790273) had 100% homology with the sequence obtained from sheep infected with Anaplasma in Ahvaz province (JQ621903.1). The findings of this study can contribute to the prevention and control of anaplasmosis in farm animals in northwestern Iran. [ABSTRACT FROM AUTHOR]

Background: Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. Methods: Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. Results and Conclusion: The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination. [ABSTRACT FROM AUTHOR]

Bovine babesiosis has substantial economic implications in the cattle industry, emphasizing the need for a thorough understanding of the genetic diversity of the causative apicomplexan pathogen. Although babesiosis has been extensively studied globally, the genetic diversity of Babesia species in Malaysian and Nigerian cattle remains unreported. This study aims to bridge this gap by detecting and characterizing Babesia species in selected cattle herds. Our investigation explores the genetic diversity of Babesia species in cattle from Selangor, Malaysia, and Ribah, Nigeria. Blood samples revealed a 32.9% infection rate via PCR analysis. Further genetic analysis detected variations in Malaysian Babesia bigemina isolates but genetic similarity among Nigerian isolates. Conversely, all Babesia bovis isolates displayed genetic homogeneity. In summary, this research identifies genetic diversity in Babesia species affecting Malaysian and Nigerian cattle, highlighting regional disparities. [ABSTRACT FROM AUTHOR]

ABSTRACT Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is a traditional non-culture technique that can provide a fingerprint of the microbial community. In the field of gut microbiota analysis, PCR-DGGE still holds potential for development. In the present study, we utilized an improved nested PCR-DGGE approach targeting the V3 region of 16S ribosomal DNA to investigate the impact of whole grain highland hull-less barley (WHLB), a cereal known for its significant hypocholesterolemic effect, on the gut microbiota profiles of high-fat diet rats. Seventy-two male Sprague–Dawley rats were divided into four groups and fed a normal control diet, a high-fat diet, or a high-fat diet supplemented with a low or high dose of WHLB for 4 or 8 weeks. The results revealed that the dominant bands varied among different dose groups and further changed with different treatment times. The compositions of bacterial communities in feces and cecal content were similar, but the dominant bacterial bands differed. After performing double DGGE, extracting the bands, sequencing the DNA, and aligning the sequences, a total of 19 bands were classified under the Firmicutes and Bacteroidetes phyla, while two bands were identified as unclassified uncultured bacteria. The relative abundance of Lactobacillus gasseri, Uncultured Prevotella sp., and Clostridium sp. increased following the administration of WHLB. Illumina-based sequencing was employed to assess the reliability of DGGE, demonstrating its reliability in analyzing the dominant taxonomic composition, although it may have limitations in accurately detecting the alpha diversity of bacterial species.IMPORTANCEWhile next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.

Paratuberculosis (Johne's disease) is a chronic granulomatous small intestine disease caused by MAP. Diagnosing and isolating infected animals is the most important measure for controlling the disease. Therefore, this study aimed to molecularly identify mycobacterium isolated from ELISA-positive cows with Johne's disease by nested PCR from the samples from Markazi Province, Iran. For this purpose, 2938 samples were decontaminated and then cultured on the Herrold egg culture medium containing mycobactin and no mycobactin. After DNA extraction, PCR for 16S rRNA was first performed, followed by nested PCR on positive samples. Of 2938 samples, 87 were positive, and 26 were suspected. All positive isolates were observed in Ziehl-Neelsen staining in microscopic expansion. A 543-bp band was observed in 26 tested samples and mycobacterium strains in PCR for 16S rRNA, indicating the presence of mycobacterium in the above samples. Nested PCR was performed for all isolates and positive and negative control strains. A 398-bp band was obtained in the first stage, and a 298-bp fragment was obtained in the second stage, indicating the presence of MAP in the samples. Accordingly, nested PCR is suggested as a proper method for the quick and definitive diagnosis of disease cases.

Purpose. The routine diagnosis of enterobiasis consists of a microscopic examination of perianal imprints on a transparent scotch tape for the presence of parasitic eggs. However, this method has low sensitivity when the number of eggs is small. The development of highly sensitive and specific methods, such as PCR, will increase the possibilities of detecting the parasite and will enable its genetic characterization. Material/Methods. The study was conducted from May 2022 to September 2022 and comprised 24 patients infected with Enterobius vermicularis. DNA was extracted from parasite eggs/adult worms using PureLink Genomic DNA Mini Kit (INVITROGEN), according to the manufacturer's instructions. Nested polymerase chain reaction (PCR) for detection of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was applied with two sets of primers. Results. The developed and described method for genetic determination of Enterobius vermicularis in samples from infected individuals is suitable and applicable to improve the diagnosis of the disease, as well as for further phylogenetic research of the parasite, which is of great importance for its successful treatment and control. Conclusions. Regardless of existing morphological diagnostic methods, genotyping studies concerning Enterobius vermicularis need to be deepened. Molecular-biological analyses increase diagnostic sensitivity, provide valuable knowledge about the geographic distribution and diversity of helminths, and allow the monitoring of therapy.

Objective: The aim of this study is to detect the prevalence and diagnosis of molecular characteristics of Cryptosporidium infection in handlers of domestic pigeons in Baghdad City. Traditional and molecular diagnostic methods were employed to detect and identify Cryptosporidium species. Materials and methods: Sixty stool samples were collected from the handlers of domestic pigeons, and various techniques, including direct smear, flotation concentration, staining methods, and DNA extraction coupled with nested polymerase chain reaction (PCR), were utilized. Results: The results obtained from traditional methods indicated an overall infection rate of 55% in handlers of domestic pigeons, while significant variations were noted between male and female handlers. Age group 21–40 years were found to have higher infection rates were found to have higher infection rates. The prevalence of Cryptosporidium also displayed temporal variability throughout the study period. Molecular analysis using nested PCR revealed higher infection rates of 86% in handlers of domestic pigeon samples. Genotyping and phylogenetic analysis identified the presence of Cryptosporidium parvam species in handlers of domestic pigeons, indicating zoonotic transmission potential. Conclusion: These findings underscore the high prevalence of Cryptosporidium infection among handlers of domestic pigeons in Baghdad City. [J Adv Vet Anim Res 2023; 10(4.000): 744-750]

Scrub typhus (ST) is a re-emerging zoonotic disease caused by Orientia tsutsugamushi and is transmitted by chiggers. Serological tests targeting IgM and/or IgG antibodies play a major role in the detection of ST cases. Orientia 56kDa genome is common target for the molecular diagosis of ST to identify the prevalence of specific serotypes of O. tsutsugamushi in and around Puducherry by targeting 56kDa gene with the application of phylogenetic analysis. This prospective laboratory-based study was conducted in a tertiary care teaching hospital, from November 2105 to March 2018. Blood samples were collected from out-patients/in-patients, and those tested positive for ST IgM ELISA (n=140) and an equal number of negative samples were archived and anonymized. Genomic DNA was extracted and analyzed by using Nested PCR and LAMP assay. The positive products were purified and sequenced. Phylogenetic tree was constructed based on the sequences. Among 280 samples, 45 (16.1%) N-PCR and 102 LAMP (36.43%) positivity was observed for 56kDa gene. Forty-one N-PCR positive products were sequenced and accession numbers were obtained (MG601875 to MG601917) for the isolates. Phylogenetic analysis was done by Maximum Likelihood methods and this study has showed that 32.3% are similar to the Karp isolates. Molecular diagnosis of Scrub typhus has become essential in case of doubtful serology and early acute phase of illness. Gene sequencing result indicates that most of them were different from the existing ones, which may belong to the newer strains. The identification of newer strains will be helpful in future for development of scrub typhus vaccine.

Scrub typhus is one of the important vector borne illness which is largely underdiagnosed, particularly in children. It causes mild febrile illness to severe complications. More than 20 genotypic clusters are documented from various geographical regions based on sequence variations of 56kDa type specific antigen gene of Orientia tsutsugamushi, the causative agent of scrub typhus. Adequate knowledge about epidemiology and genetic diversity in endemic regions is an important tool for clinical management, development of diagnostic kit and vaccines. Limited studies are available based on genotypic characterization of Orientia tsutsugamushi in children. The present study determined the prevalence and genotypic characterization of Orientia tsutsugamushi in febrile pediatric patients admitted in tertiary care hospital of Chennai, South India. Both serum and blood samples were collected from 239 scrub typhus suspected febrile pediatric patient’s aged between 6 months to 12 years. IgM ELISA and 56kDa nested PCR were performed on all the patient samples. Nested PCR positive samples were sequenced and analyzed for genotypic differences. Among 239 samples, 103 were positive for IgM ELISA and 35 were positive for nPCR analysis. Out of the 108 scrub typhus positive cases, 45.31% (58/128) were male and 45.05% (50/111) were female. Eschar was positive in 56.48% of patients. Pneumonia (4/108), hypotensive shock (3/108), and myocarditis (1/108) were the most common clinical complications associated with scrub typhus positive children. Karp (56.6%) was the most common genotypic cluster found in our study, followed by TA716 (33.33%), TA763 (2/30), and Gilliam (1/30).

[Objective] In order to establish a nested polymerase chain reaction (PCR) for detecting the pathogens of Mycoplasma ovipneumo- nia, and determine the target organs of mycoplasma infection. [Method] According to the 16S rRNA sequence of sheep mycoplasma in Gen- Bank website, two pairs of specific primers were designed and synthesized. PCR reaction conditions were optimized by using the genome DNA of M. ovipneumonia as templates. The nested PCR products were verified by sequencing. A nested PCR method for detecting the pathogens of M. ovipneumonia was established. The established method was used to detect the lung, lung lymph, heart, kidney, liver, spleen, skin, small intestine and peripheral blood of clinical positive samples, and the lung tissue from clinical suspected samples. [Result] 864 bp specific gene fragment was amplified by using nested PCR method. The lung and lung lymph of sheep were the target organs of mycoplasma infection. The coincidence rate between the detection rate of clinical samples by the nested PCR and pathogen culture and identification results was 100%. [Conclusion] The nested PCR method for detection of M. ovipneumonia could be applied for the laboratory diagnosis of clinical samples. [ABSTRACT FROM AUTHOR]

The high incidence of oral carcinomas is due to its multifactorial etiology and the presence of various risk factors. Human Papillomavirus (HPV) has a proven role in the pathogenesis of oral carcinomas, but in the recent times there has been an increasing incidence of oral cancers who are negative for HPV infection. Also, these patients are non-smokers and non-drinkers so it could be speculated that these oral cancers are due to some other etiological factor probably of other viral infections. Therefore, this study examined the prevalence of Epstein Barr Virus (EBV) and Herpes Simplex Virus (HSV) among oral cancer patients. This cross-sectional study was conducted from January 2019 to June 2020. Biopsy samples from 47 newly diagnosed untreated patients with oral malignancies were collected along with their demographic and clinicopathological information. DNA extracted from the biopsies was processed for nested PCR for the detection of EBV and HSV. All the samples tested negative for HPV and HSV infection. Nested PCR detected 29 cases (70.7%) to be positive for EBV. The non-cancerous adjacent tissues also were negative for HPV, EBV and HSV. The prevalence of EBV was found to be more in males (62.1%) and the highest number of cases was of the left buccal mucosa compromising 34% of the total cases. From the present study it can be concluded that EBV but not HSV infection is associated with an increased risk of developing oral cancers. Although, 70.7% of the patients were found to be positive for EBV whether the viral infection played any role in the driving the malignancy needs to be further elucidated. [ABSTRACT FROM AUTHOR]

Simple Summary: Cattle ringworm is a mycotic infection of the bovine skin caused by dermatophytes that are also transmissible to humans. To limit the spread of bovine ringworm, calves at auction markets in Styria, Austria, are visually inspected and excluded from auction if they display skin lesions that are typical of ringworm. To investigate whether these clinical assessments correspond to laboratory diagnoses, samples from skin lesions were examined through microscopy, culture, and nested PCR approaches; the relatedness of the isolated dermatophytes was determined using multi-locus sequence typing (MLST). Overall, the clinical assessments were largely supported through the results of the nested PCR laboratory diagnosis, possessing an analytical sensitivity superior to that of the culture approach. Thus, this represents a fast and sensitive diagnostic test for the detection and identification of dermatophytes. Most of the isolated dermatophytes were assigned to a unique Trichophyton (T.) verrucosum MLST genotype, indicating that ringworm in calves at auction was predominantly caused by a single T. verrucosum strain. To limit the spread of bovine ringworm, control measures such as movement restrictions are highly recommended. In this context, calves at auction markets in Styria, Austria, displaying skin lesions characteristic for bovine ringworm, are excluded from the auctions. To investigate whether these clinical assessments correspond to laboratory diagnosis, a total of 166 samples taken from skin lesions assigned to the three clinical categories 'ringworm very likely (v), likely (l) or unlikely (u)' were mycologically examined using microscopy, culture, and nested PCR followed by amplicon sequencing. Further, the relationships of isolated dermatophytes were determined through multi-locus sequence typing (MLST). Overall, a high agreement between clinical assessment and laboratory results were observed with microscopy and nested PCR, providing more consistent results and molecular detection possessing an analytical sensitivity superior to that of cultural isolation (culture 21.7% vs. nested PCR 48.2%). Phylogenetic analyses revealed that most of the isolated dermatophytes belong to a unique Trichophyton verrucosum MLST genotype. In conclusion, clinical assessments were largely confirmed through laboratory diagnosis with nested PCR and sequencing, providing rapid, sensitive, and species-specific detection of dermatophytes in calves at auction markets displaying skin lesions typical for ringworm; this seems to be predominantly caused by a single Trichophyton verrucosum strain. [ABSTRACT FROM AUTHOR]

A comprehensive study was conducted on the prevalence of grapevine trunk diseases in Greece, focusing specifically on contamination of grapevine propagation material by Phaeomoniella chlamydospora and black foot disease‐related species. Additionally, detection of esca pathogen Fomitiporia mediterranea, causing white rot in grapevine and other woody hosts, was assessed using a new PCR‐based assay that distinguishes F. mediterranea from F. punctata. Development of a nested PCR assay, combined with a cost‐effective DNA extraction protocol, revealed a high percentage of infection by P. chlamydospora (51%) and Ilyonectria species associated with black foot disease (28%) in different types of grapevine propagation material (dormant cuttings, field‐rooted benchgrafts and green‐growing plants). Interestingly, black foot disease‐related pathogens were more prevalent in nursery plants grown in the field for 6 months (57%) than in other types of propagation material and compared with P. chlamydospora (43%), indicating increased infection of propagation material during growth in the nursery field. The cost‐effective molecular method developed in this study could be used in mass inspections of propagation material for phytosanitary purposes. Finally, using primers specific for F. mediterranea and F. puncata developed in this study, combined with the universal ITS4 primer, a collection of Fomitiporia isolates from mature grapevine and other woody hosts from Greece and Italy (southern Europe) were characterized as F. mediterranea, whereas German and Swedish isolates from forest plants (central Europe) were classified as F. punctata. The developed primers can discriminate between the two Fomitiporia species, which are indistinguishable based on culture and morphological characteristics alone. [ABSTRACT FROM AUTHOR]

Introduction: Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic and zoonotic pathogen which is associated with many diseases in humans and animals. However, the pathogenicity of K. pneumoniae has been neglected and the prevalence of K. pneumoniae is poorly studied due to the lack of rapid and sensitive diagnosis techniques. Methods: In this study, we infected mice and pigs with K. pneumoniae strain from a human patient. An indirect ELISA was established using the KHE protein as the coating protein for the detection of K. pneumoniae specific antibody in clinical samples. A nested PCR method to detect nuclei acids of K. pneumoniae was also developed. Results: We showed that infection with K. pneumoniae strain from a human patient led to mild lung injury of pigs. For the ELISA, the optimal coating concentration of KHE protein was 10 μg/mL. The optimal dilutions of serum samples and secondary antibody were 1:100 and 1:2500, respectively. The analytical sensitivity was 1:800, with no cross-reaction between the coated antigen and porcine serum positive for antibodies against other bacteria. The intra-assay and inter-assay reproducibility coefficients of variation are less than 10%. Detection of 920 clinical porcine serum samples revealed a high K. pneumoniae infection rate by established indirect ELISA (27.28%) and nested PCR (19.13%). Moreover, correlation analysis demonstrated infection rate is positively correlated with gross population, Gross Domestic Product (GDP), and domestic tourists. Discussion: In conclusion, K. pneumoniae is highly prevalent among pigs in China. Our study highlights the role of K. pneumoniae in pig health, which provides a reference for the prevention and control of diseases associated with K. pneumoniae. [ABSTRACT FROM AUTHOR]

South Sulawesi is one of the largest rice production centers in Indonesia. Several important diseases of rice plants, such as those caused by viruses and phytoplasmas, can be transmitted by insect vectors, especially leafhoppers and planthoppers. Symptoms of diseases caused by viruses and phytoplasmas are quite diverse but visually similar and difficult to distinguish. This study aimed to analyze the presence of phytoplasma associated with whitebacked planthopper which are commonly found in rice plantations. The research was done by conducting surveys and explorations of insect samples from six villages in Sidrap District. White-back planthoppers found on rice plantations showing symptoms of yellowing and stunted leaves were sampled for further analysis, including total DNA isolation of insects, standard PCR amplification for insect and Nested-PCR for phytoplasma identification, gene sequencing for both amplicons, and nucleotide analysis. The PCR with CO1 primer successfully amplified a 700 bp amplicon from insects, whereas nested-PCR using fP1/rP7 primers followed by m23SR/R16F2n amplified around 1800 bp and 1250 bp of supposedly 16S RNA gene of phytoplasma, respectively. Results of DNA sequencing analysis identified the insect samples as Sogatella vibix based on 83% homology using BLAST and Mega X Program. As for the phytoplasma, it leans towards the 16SrI group or Candidatus phytoplasma asteris (Aster yellows phytoplasma) with a homology percentage of 99%. [ABSTRACT FROM AUTHOR]

Increasing incidences of phytoplasma infestations in Almond trees warrants the better management approach to prevent yield losses. Disease management rely on identification of the pathogen based on molecular profiling. The present study aimed, to identify the phytoplasma agent in almond trees and to measure the biochemical responses it causes in the host. Direct and Nested PCRs performed using phytoplasma specific primer pairs 16S rRNA, detected the presence of phytoplasma agent in symptomatic trees but not in symptomless trees. Phylogeny based on the sequence analysis revealed that the infecting agent was closely related to 'Candidatus Phytoplasma phoenicium' (16SrIX‐B subgroup). Then the study was carried out to determine the responses of physiological and biochemical mechanisms in almond trees infected with phytoplasma. Total chlorophyll and protein contents of infected almond trees were lower compared to healthy control, on the other hand, the levels of catalase and peroxidase activity increased in infected trees. Higher levels of stress‐related metabolites such as proline (20.53–39.23 μmol/g), phenol (3.00–4.44 mg GAE/g), salicylic (94.96–138.22 ng SA/mg protein) and jasmonic acid (965.86–1465.10 ng JA/mg protein) were observed in infected trees compared to asymptomatic trees, respectively in healthy and infected trees. The results showed that the Ca. P. phoenicium, which was detected for the first time in Türkiye, was able to change the physiological and biochemical mechanisms. The pathogenic agent could possess a potential danger in almond production areas. It is crucially important that this agent should be considered in certification programs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]