Nested PCR Technique Research Articles

The incident and Molecular Identification of the Cryptosporidium Parvum in Cultivated Carp Fish (Cyprinus carpio) in Baghdad

The current study aimed to investigate the occurrence of Cryptosporidium species by traditional and molecular methods in freshwater fish in Baghdad city. A convenient number of stomach and intestinal continent samples were collected from three types of freshwater fish from different areas of Baghdad city from November, 2023 to April, 2024. All samples were examined phenotypically using a modified Ziehl-Neelsen stain and genotypically (nested PCR technique) based on a partial sequence of 18SrRNA genes with sequencing and phylogenetic tree analysis. The overall infection rate of fish with C. parvum was 5.5% (10/180). The common carp (Cyprinus carpio) was the only fish type infected, with a rate of 16.66% (10 60). January and February had the highest infection rate, 3.33%. The molecular identification and sequencing confirmed the infections with C. parvum. Molecular speciation of the parasite confirmed microscopical primary identification results. This study suggests that fish became a new source for transmission to humans and livestock.

Molecular Identification of Cryptosporidiosis in Diabetes and Cancer patients

Cryptosporidium spp., a primary intestinal parasite and obligate intracellular parasite, infects a wide range of vertebrates, including human. The current study aimed to determine the infection rate of Cryptosporidium spp. among immunocompromised patients attending the Diabetes and Endocrinology Centre and Oncology Department at Al-Diwaniya Teaching Hospitals in Al-Diwaniya Providence, Iraq. The research period extended from the first of October 2023 until the end of February 2024. Eighty-seven stool samples were obtained from immunocompromised patients of various ages and both genders who attended the hospital. Stool samples were examined using the nested PCR technique. The results showed an overall infection rate of 22.98%. Researchers found that the infection rate was 20.83% among diabetic patients and 25.64% among cancer patients. Male patients had a recorded infection rate of 23.07%, whereas females recorded 22.85%. A high infection rate (60%) was observed among patients under the age of 15 and among those coming from rural areas, reaching 34.88%. Additionally, it was found that 42.42% of the infected individuals had contact with animals.

The Prevalence of Cryptosporidium spp. and Giardia duodenalis in Marmota himalayana (Rodentia: Sciuridae) in the Qinghai Tibetan Plateau area, China.

Cryptosporidium and Giardia are well-known important intestinal zoonotic pathogens that can infect various hosts and cause diarrhoeal diseases. We aimed to determine the epidemiological prevalence and molecular characterization of Cryptosporidium and Giardia species in Himalayan marmot (Marmota himalayana, class Marmota) in the Qinghai Tibetan Plateau Area of Qinghai Province, Northwest China. Overall, 243 Himalayan marmot fecal samples were collected in 2017 and in 2019 and a two-step nested PCR technique was performed to amplify the fragments of the SSU rRNA gene of Cryptosporidium and 18S ribosomal RNA gene of Giardia. Molecular characterization of Cryptosporidium was performed with the primary primers NDIAGF2 and N-DIAGR2, the secondary primers CPB-DIAGF and CPB-DIAGR. Similarly, molecular characterization of Giardia was used the first primers Gia2029 and Gia2150c, the secondary primers RH11 and RH4. The positive PCR products were sequenced and the sequences were processed by Clustal Omega and BLAST. Phylogenetic analysis was achieved by NJ method in MEGA. The infection rate of Cryptosporidium spp. and G. duodenalis was 4.9% (12/243) and 0.8% (2/243) in M. himalayana, respectively. Cryptosporidium spp. are characterized as novel genotypes Cryptosporidium marmot genotype I (n=3) and Cryptosporidium marmot genotype II (n=9); G. duodenalis assemblage A (n=2) was found in M. himalayana. This is the first report of Cryptosporidium spp. and G. duodenalis infections in M. himalayana in Qinghai of Northwest China. The results indicate the existence of Cryptosporidium species and G. duodenalis infections that may have a potential public health significance.

Molecular Identification of the Gag and Tax Gene of Bovine Leukemia Virus (BLV) in Women with Breast Cancer

Background: Breast cancer is one of the most common cancers in the world particularly among Iranian women. Although the mortality rate decreased in developed countries, there were increased changes in breast cancer incidences. The study aimed at evaluating the correlation between BLV infection and breast cancer in Iran. Methods: In the cross-sectional study, a total of 85 breast cancer tissue samples and adjacent tumor samples (normal tissue) were collected during the year 2022 from women referred to general hospitals in Qom Province, Iran. The nested PCR technique was performed to determine the presence of tax and gag genes of BLV in the collected samples. To confirm the presence of the tax gene of BLV, PCR products of some BLV-positive samples were subjected to direct sequencing. Results: Based on nested PCR technique, tax and gag genes of BLV were detected in 12.9% (11.85) and 3.4% (3.85) of breast cancer tissue samples, respectively. Also, the tax gene was present in 4.7% of the control samples, but the presence of the gag gene was not observed. Most malignant samples of BLV were III grade. The constructed phylogenetic trees for the tax gene of BLV showed most BLV isolates had similar sequences to Iranian isolates. Conclusion: The results of the study by using nested PCR technique, demonstrate a possible relationship between human breast cancer and bovine leukemia virus infection in women of Iran. BLV is one of the main risk factors for breast cancer.

Molecular Identification of Babesia and Theileria Infections in Livestock in the Qinghai–Tibetan Plateau Area, China

The northwestern region of China, known as the Qinghai–Tibet Plateau Area (QTPA), is characterized by unique climate conditions that support the breeding of various highly-adapted livestock species. Tick vectors play a significant role in transmitting Babesia and Theileria species, posing serious risks to animal health as well as the economy of animal husbandry in QTPA. A total of 366 blood samples were collected from Tibetan sheep (n = 51), goats (n = 67), yaks (n = 43), cattle (n = 49), Bactrian camels (n = 50), horses (n = 65), and donkeys (n = 40). These samples were examined using conventional and nested PCR techniques to detect Theileria and Babesia species. The overall infection rates were 0.3% (1/366) for Babesia spp. and 38.2% (140/366) for Theileria spp. Notably, neither Babesia nor Theileria species were detected in donkeys and yaks. The infection rates of Babesia and Theileria species among animals in different prefectures were significantly different (p < 0.05). Furthermore, Babesia bovis, B. bigemina, B. caballi, and B. ovis were not detected in the current study. To our knowledge, this is the first documented detection of Theileria luwenshuni infection in Bactrian camels and goats, as well as T. sinesis in cattle and T. equi in horses on the Qinghai plateau. These novel findings shed light on the distribution of Babesia and Theileria species among livestock species in QTPA.

Molecular detection of Paracoccidioides spp. in environmental samples from the Southwestern Amazon.

Paracoccidioidomycosis is an infection with the potential for environmental dissemination, especially in regions of hot and humid climate, where human cases have been recorded in the Southwestern Amazon of Brazil, specifically in the state of Acre. Despite studies providing information about the presence of these fungi in soil and animal samples, such as armadillos, further investigations are still needed to determine the epidemiological distribution of the genus Paracoccidioides. The aim of this study was to detect the occurrence of Paracoccidioides fungi in the Southwestern Amazon. To achieve this, 60 soil samples were collected from armadillo burrows on rural properties in the in the municipalities of Acrelândia, Bujari, Plácido de Castro, Rio Branco, Sena Madureira, and Senador Guiomard, located in the state of Acre, Brazil. Fungal DNA was extracted from these samples using the DNEASY® PowerSoil kit-Quiagen, followed by Nested PCR technique with ITS4 and ITS5 as external primers, and PBITS-E and PBITS-R as internal primers. DNA amplification products of about 380 bp compatible with Paracoccidioides spp. were detected in six samples (10%), being sequenced and identified as P. brasiliensis. These findings indicate that the soils of the Acre state could be considered a potential source for Paracoccidioides spp., suggesting that local infections are likely.

Predominance of K1 Allele in Erythrocytic form of Plasmodium falciparum MSP-1 Gene Signifies Severe Malaria in Jos, Nigeria

Aim: The study explored the diversity of Plasmodium falciparum Merozoite Surface Protein 1 (msp-1) gene sequence and delineated the K1 allele occurrence and profile. The Plasmodium falciparum msp-1 gene encodes an antigen that is being proposed as a major vaccine candidate against the parasite infections.
 Study Design: The design of the study was experimental.
 Place and Duration of Study: The study was undertaken, between October 2018 and June 2019, in the Department of Biochemistry, Faculty of Basic Medical Sciences, College of Health Sciences, University of Jos, Nigeria.
 Methodology: The DNA of Plasmodium falciparum was extracted from 117 blood samples of malaria patients confirmed by microscopy in three different hospitals in Jos. The msp-1 (block 2) allelic family’s genotyping was carried out using PCR and nested PCR techniques. Sequencing and Bioinformatics of the K1 alleles were done to further identify the K1 alleles.
 Results: Out of the 117 DNA extracted, 13 samples were positive for Plasmodium falciparum and each was genotyped for msp-1. K1 was the most predominant allele (6/13) compared to MAD20 (4/13) and R033 (2/13). The allelic frequency of K1 was calculated to be 46.15%. The 225 bp Plasmodium falciparum K1 allele of the msp-1 gene studied displayed polymorphism. However, the sequence was very similar to those of Plasmodium falciparum already characterized.
 Conclusion: Predominance of K1 allele is an indication that Jos is endemic to severe Plasmodium falciparum malaria.

In Planta Detection of Beauveria bassiana (Ascomycota: Hypocreales) Strains as Endophytes in Bean (Phaseolus vulgaris L.).

Beauveria bassiana (B. bassiana) is a significant entomopathogenic fungus (EPF) in agriculture as a sprayable biocontrol agent. It has the potential to be established as an endophyte (ENP) in various crops, resulting in beneficial effects for the host plants, including resistance to pest insects and increased growth and yield. However, it is not known whether a B. bassiana strain has such a favorable impact on the plant, since it is a common soil microorganism. Therefore, techniques that allow strain monitoring will be advantageous. To date, methods for detecting or monitoring a specific EPF strain after external application are scarce. In the present study, an in planta nested PCR technique was standardized to differentiate between three B. bassiana strains (GHA, PTG4, and BB37) established as endophytes in bean plants under laboratory conditions by detecting the insertion profile of four group I introns located in the 28S gene of B. bassiana ribosomal DNA. This technique recognized a distinct pattern of bands of different sizes for each strain, with a sensitivity of 1 pg per 10 ng of plant DNA. This molecular approach may be more effective monitoring B. bassiana strains after application to evaluate their significance on crops.

First molecular detection of Francisella tularensis in turtle (Testudo graeca) and ticks (Hyalomma aegyptium) in Northwest of Iran

Francisella tularensis, causative agent of tularemia, is a contagious zoonotic ailment. This study was aimed to molecularly detect F. tularensis in tortoise blood (n = 100) and ticks (n = 100) collected in the West Azerbaijan province, Iran suing a 16SrRNA gene of the Francisella genus through employment of the Nested-PCR technique. The identified ticks were s Hyalomma aegyptium by morphological analysis. Seven percent (with a 95% CI: 3.5%–13.75%) of animal blood samples yielded positive results for the presence of the Francisella. Meanwhile, the Francisella was identified in tick samples at a rate of fifteen percent (15%) (with a 95% CI: 9%–23%). The samples containing positive results were specifically classified as F. tularensis subsp. holarctica. The samples were taken from ticks belonging to the H. aegyptium species that were gathered in Oshnavieh, southern part of West Azerbaijan province, Iran. This research was aimed to validate the existence of F. tularensis in ticks found within the West Azerbaijan province. Consequently, it is vital to acknowledge the potential of these ticks to transmit the bacteria to both livestock and humans through tick bites in this specific area.

Prevalence and Molecular Identification of Cryptosporidium Species among Human Population in Zakho District, Duhok Province, Kurdistan Region, Iraq

This cross-sectional study was carried out to determine the prevalence and molecular identifications of Cryptosporidium species among the human population in Zakho district, Duhok Province, Kurdistan Region, Iraq. The study was performed during the period from August 2021 to March 2022 and involved 700 fecal specimens of both genders and different ages collected from Zakho General Hospital and pri¬mary schools located in the suburban area of Zakho City. The data were analyzed using statistical soft¬ware version SPSS (25). Cryptosporidium species oocyst was found in 87.57% (613/700) of the examined specimens using the Modified Ziehl Neelsen technique. Males showed a slightly higher infection rate than females (88.4% vs 85.5%) with a non-significant difference (P> 0.05). The highest infection rate (91.7%) was present in individuals between the ages of >30 to 40 years, with a statistically significant difference (P<0.05). Molecular identification of Cryptosporidium species was estimated using 18S-rRNA gene for Cryptosporidium genus using Nested PCR technique and genus-specific primers (Cry18 AS1 and Cry18 S2) that yielded 347 bps product indicating Cryptosporidium genus, then species-specific (Cry18 AS1 and Cry18 S1) were used for the detection of Cryptosporidium parvum and Cryptosporidium hominis. 45/50 samples were positive and revealed 240 bps. The acquired sequences were deposited into the GeneBank database under the accession numbers OP107010 for C. hominis, OP104444, OP153870, OP163188, OP163414, and OP164631 for C. parvum.

Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile.

Respiratory infections are the most serious condition in cystic fibrosis (CF) patients; therefore, a thorough comprehension of the diversity and dominant microbial species in CF airways has a crucial role in treatment. Our objective was to determine the antibiotic resistance profile of CF airways microbiota and compare culture methods and PCR-DGGE to evaluate bacterial diversity. Pharyngeal swabs from 121 CF patients were collected. The samples were then cultured, identified and antibiotic resistance testing was performed. Thirty samples were subjected to further molecular surveys. DNA contents of these samples were extracted and amplified using nested-PCR technique and their bacterial diversity was assessed by DGGE. The DGGE patterns were visualized and certain bands were excised and purified. Next, the DNA was amplified by another round of PCR and sent out for sequencing. Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the most prevalent species isolated using culture methods. S. aureus was the most common bacteria among 6 years and younger patients; while, P. aeruginosa had more prevalence among older ones. The PCR-DGGE results showed more diversity than culture methods, particularly in younger patients who exhibited more bacterial diversity than the older groups. Sequencing results unveiled the presence of certain bacterial species including Haemophilus parainfluenzae and Stenotrophomonas maltophilia which were completely missed in culture. Even though culture-dependent methods are cost-effective, PCR-DGGE appeared to be more efficient to determine bacterial diversity. PCR-DGGE detects less abundant species, though their viability could not be determined using this method.

A Novel Multiplex LAMP Assay for the Rapid and Accurate Diagnosis of Visceral Leishmaniasis Caused by Leishmania infantum from Iran.

Visceral leishmaniosis (VL) is one of the neglected tropical diseases despite being responsible for serious clinical symptoms, some of which lead to fatal outcomes. Thus, there is a need to apply accurate, rapid, and specific diagnostic measurements in order to control the disease and reduce the mortality rate. We aimed to develop and validate a multiplex LAMP assay for the diagnosis of VL caused by Leishmania infantum (L. infantum). Moreover, a thorough assessment was conducted to determine the effectiveness of multiplex LAMP in identifying various Leishmania species, such as Leishmania tropica (L. tropica) and Leishmania major (L. major) in comparison to Leishmania infantum (L. infantum). The diagnostic performance of the multiplex LAMP method for VL was compared to each LAMP assay, real-time polymerase chain reaction (RT-qPCR), and nested PCR technique. Two separated primers were set and used in a multiplex LAMP assay which was designed based on the ITS2 (internal transcribed spacer II) and were selected on the basis of conserved and high copy number region. Multiplex LAMP primers were designed using an online tool available at https://www.primerexplorer.jp/e. The alignment was performed using MEGA5, and the primers were further adjusted utilizing GENE Runner software. All molecular methods were tested on the serial dilution of cloned plasmid containing ITS region from standard strains of L. infantum, L. tropica, and L. major. Moreover, multiplex LAMP assay was evaluated and compared based on both standard strains and 55 clinical samples from humans as well as dogs. Various approaches were applied to interpret the multiplex LAMP reaction which deciphered a higher sensitivity when compared to the RT-qPCR for L. infantum (one copy number of plasmid, equal to 0.85 femtograms (fg) of plasmid concentration, and 0.004 parasite DNA per μL) detection while these three standard strains of Leishmania were confirmed to contain 40 DNA copies using RT-qPCR. Additionally, the multiplex LAMP detection limit was approximately equivalent to RT-qPCR for L. major and L. tropica, which included 0.342 picograms (pg) and 342 femtograms (fg) of plasmid concentration, 4 × 103 and 4 × 102 copy number of plasmid, and 17.1 and 1.71 parasite DNA per μL for L. major and L. tropica, respectively. Nested PCR exhibited a lower detection limit for L. infantum of 4 × 106 plasmid copy number compared to multiplex LAMP and RT-qPCR. Multiplex LAMP has the potential for accurate and rapid detection of infectious disease, successful treatment, and finding and monitoring asymptomatic cases, especially in low-income countries.

Absence of Asymptomatic Malaria Reservoirs in an Area with a Previous History of Local Malaria Transmission: A Successful Experience in Line with the Malaria Elimination Program in Iran.

Asymptomatic malaria is a major challenge to be addressed in the implementation of the malaria elimination program. The main goal of the malaria surveillance system in the elimination phase is to identify reliably all the positive cases of malaria reliably (symptomatic and asymptomatic) in the shortest possible time. This study focused on the monitoring of asymptomatic malaria reservoirs in areas where local transmission had been previously established. It was a case-study approach that was conducted in the Anarestan area. A total of 246 residents and immigrants living in the area at the age range of 4-60 years old were randomly selected to be tested for malaria by microscope, RDT, and nested-PCR techniques. The inclusion criterion for participants to be entered into the study was the absence of specific symptoms of malaria. Moreover, participants who have been taking antimalarials for the last month were excluded from the study. The results indicated no positive cases of asymptomatic malaria among the participants tested by all methods. The results of this study have shown that, without concerns for asymptomatic parasitic patients, a malaria elimination program has been successfully implemented within the studies area. In addition, the findings emphasized the existence of a strong malaria surveillance system in this area.

Molecular investigation of Haemoproteus and Plasmodium species of some raptors in Hatay province: new CytB lineages for raptors of Accipitriformes in Turkey.

Accipitriform raptors are significant indicators of biodiversity and environmental health. Currently, most of the studies on avian haemosporidian parasites are on passerine birds, and data on raptors is constricted, with similarities both around the world and in Turkey. This study aimed to investigate the presence of Haemoproteus and Plasmodium spp. in raptors by microscopy and nested PCR technique. The study material consisted of 47 accipitriform raptors (Buteo buteo: 14, Buteo rufinus: 7, Clanga pomarina: 8, Circaetus gallicus: 12, Milvus migrans: 6). The prevalence of haemosporidian infection was 12.8% (6/47, 1 from Buteo buteo, 4 from Clanga pomarina, 1 from Milvus migrans) microscopically and 14.9% (7/47) molecularly. One Circaetus gallicus, microscopically found to be negative, probably due to low parasitemia, was molecularly found to be positive. All PCR-positive amplicons were bidirectionally sequenced, and the identification of lineages of the isolates and phylogenetic analysis were performed using the MalAvi and GenBank databases. The study revealed H-MILANS02 lineage in Buteo buteo, H-MILANS02 and P-CIAE1 lineages in Clanga pomarina, P-GRW06 lineage in Circaetus gallicus, and P-RTSR1 lineage in Milvus migrans, respectively. While this study removes the uncertainty regarding the reporting of the H-MILANS02 lineage in Turkey, it is also the first report to reveal 3 different Plasmodium spp. CytB lineages in raptors. Moreover, the fact that the P-GRW06 lineage (Plasmodium elongatum) detected in passerine birds was detected in a raptor, Circaetus gallicus, draws attention to the need for further investigations on host-parasite interaction and gives clues about the host-shifting ability of this parasite.

Molecular Detection of Francisella tularensis Isolated from Ticks of Livestock in Kurdistan Region, Iraq.

Background: Francisella tularensis is a Gram-negative bacterium that causes tularemia in both human and animals. Tularemia is a potential serious zoonotic disease that is transmitted by different routes, including tick bites. Materials and Methods: This study deals with investigating the prevalence of F. tularensis in the ticks of local animal farms in Kurdistan region since the farmers are normally in close contact with livestock. We used molecular methods for this purpose. A total of 412 tick and 126 blood samples were gathered from goat, sheep, and cow flocks. The existence of F. tularensis 16Sr RNA gene was examined in the samples using nested-PCR technique. Results: In the animal blood specimens, no F. tularensis was found. The incidence of F. tularensis was 1.7% (7 out of 412) in the tick samples, representing a very lower possibility of tuleremia infection. Moreover, the two subspecies of F. tularensis novicida and holarctica were identified based on the sequencing of pdpD and RD genes, respectively. The F. tularensis subsp. novicida was isolated from four species of ticks, Hyalomma anatolicum, Rhipicephalus annulatus, Rhipicephalus sanguineus, and Ornithodoros spp., whereas the F. tularensis subsp. holarctica was isolated from Haemaphysalis parva and Hyalomma dromedarii species of ticks. Conclusion: Although its prevalence is very low, the isolation of F. tularensis subsp. holarctica from the ticks of farm animals suggests possible transmission of Tularemia through tick bite in Kurdistan region of Iraq. Ref: IR-UU-AEC-3/22.

Characteristics of molecular markers associated with chloroquine resistance in Plasmodium vivax strains from vivax malaria cases in Yunnan Province, China

BackgroundChloroquine (CQ) has been the preferred clinical treatment for vivax malaria in Yunnan Province since 1958, with over 300,000 patients. This study aimed to help make trend predictions regarding variations the in anti-malarial drug susceptibility of Plasmodium vivax distributed in Yunnan Province and effectively implement monitoring measures on the efficacy of anti-malarial drugs for vivax malaria.MethodsBlood samples collected from patients with mono-P. vivax infections were employed in this study based on the principle of cluster sampling. The whole gene of P. vivax multidrug resistance 1 protein gene (pvmdr1) was amplified by nested-PCR techniques and the PCR amplification produce were sequenced by Sanger bidirectional sequencing. The mutant loci and haplotypes of coding DNA sequence (CDS) were identified by comparison with the reference sequence (NC_009915.1) of the P. vivax Sal I isolate. Parameters such as Ka/Ks ratio were calculated using MEGA 5.04 software.ResultsA total of 753 blood samples from patients infected with mono-P. vivax were collected, of which 624 blood samples yielded the full gene sequence (4392 bp) of the pvmdr1 gene, with 283, 140, 119, and 82 sequences from 2014, 2020, 2021 and 2022, respectively. A total of 52 single nucleotide polymorphic (SNP) loci were detected for the 624 CDSs, of which 92.3% (48/52), 34.6% (18/52), 42.3% (22/52), and 36.5% (19/52) SNPs were detected in 2014, 2020, 2021 and 2022, respectively. All of 624 CDSs were defined for a total of 105 mutant haplotypes, with CDSs of 2014, 2020, 2021, and 2022 containing 88, 15, 21, and 13 haplotypes, respectively. Of the 105 haplotypes, the threefold mutant haplotype (Hap_87) was the starting point for stepwise evolution, and the most drastic tenfold mutations were Hap_14 and Hap_78, and the fivefold, sixfold, sevenfold, and eightfold mutations.ConclusionsIn the majority of vivax malaria cases in Yunnan Province, most of them were infected with strains carrying demonstrating highly mutated in pvmdr1 genes. However, the dominant mutation strains types varied from year to year, which warrants further exploration in order to confirm the correlation between with phenotypic changes in P. vivax strains and their susceptibility to anti-malarial drugs such as chloroquine.

Molecular and Immunopathological Investigations of Marek’s Disease in HVT Vaccinated Chicken Flocks

Background: The present investigation was carried out on poultry carcasses brought to the Department of Veterinary Pathology, LUVAS, Hisar from Herpesvirus of turkeys (HVT) vaccinated chicken flocks and suspected for viral neoplastic conditions on the basis of necropsy. Conventional and molecular techniques for the confirmation of the Marek’s disease (MD) were studied. For differential diagnosis from other oncogenic viral infections immunohistochemistry as an important tool was investigated. Methods: The conventional diagnosis of MD) was done on basis of clinical signs, gross lesions and histopathology of proliferate lesions; as lymphomatous lesions consist of pleomorphic lymphocytes. For confirmatory diagnosis molecular techniques were employed to detect MD virus specific Meq gene by Taqman probe real time PCR and nested PCR. For differential diagnosis of mixed oncogenic viral infections, immunohistochemistry (IHC) using B and T cell differentiation markers (CD79 alpha and CD3) was employed. Result: Specific Meq gene by Taqman probe real time PCR was positive in 92.5% suspected cases; while nested PCR technique gave positivity in 55% cases. The immunohistochemical reactivity for CD3 gave positive reactivity in the cytoplasm and cell membrane and negative reaction for CD79 alpha in MD positive cases, while in cases with mixed infections of MDV and other viral neoplastic conditions, cells showed positive reactivity to both CD3 and CD79 alpha in neoplastic proliferates.

New Recording of Toxoplasma gondii in Wild Tortoise Testudo graeca Using Nested PCR Method.

Toxoplasmosis is one of the most widespread zoonotic diseases, especially in warm and humid areas, and affects all mammals, including humans and many herbivores and carnivores. The present study investigated the Toxoplasma gondii (T. gondii) parasite in tortoises for the first time in Iraq using PCR technology. A total of 28 tortoises/Testudo graeca (T. graeca) were collected between October 2018 and March 2019 from the study stations and then sent to the Animal House, which belongs to the Department of Biology, Faculty of Education, University of AL-Qadisiyah, Iraq, to perform the dissection. The body cavity was opened, and all organs were removed. The tortoises' liver, heart, and brain were removed and kept at -20ºC until use. Afterward, the samples were subjected to DNA extraction. The Nested-PCR technique was implemented using two pairs of primers, and then the PCR products were analyzed using 1.5% agarose gel electrophoresis. The amplification of the gene during the first cycle indicated that 10 samples gave positive results with a total percentage of (11.9%), including five liver samples, three heart samples, and two brain samples (17.85%, 10.71%, and 7.14%, respectively). On the other hand, during the second cycle of the reaction, the amplification of the gene was obtained in seven samples (8.33%). The highest percentage of the presence of the gene was recorded in the tortoises' liver (14.28%) and the lowest in their brain (3.57%). This study is among the first to investigate the molecular detection of T. gondii in wild tortoises (T. graeca) in Iraq. The findings imply that tortoises have a role in transmitting T. gondii and are believed to acquire infection by feeding on small invertebrate animals or plants contaminated with the oocysts of the parasite.

Diversity of picornaviruses detected in diarrheal samples from children in Belém, Brazilian Amazon (1982-2019).

In this investigation, fecal specimens from children with diarrhea were collected from four community studies conducted between 1982 and 2019 in Belém, Brazilian Amazon. A total of 234 samples were tested by quantitative reverse transcription polymerase chain reaction (RT-qPCR)to detect infections by picornaviruses of the Enterovirus (EV),Parechovirus (HPeV),Cosavirus (HCoSV),Kobuvirus (Aichivirus - AiV) and Salivirus (SalV) genera. The positive samples were subjected to different amplification protocols of the VP1 region of the genome, such as nested PCR or snPCR, and were subsequently genotyped by sequencing VP1 and VP3 of the viral genome. Positivity was observed in 76.5% (179/234) of the samples tested using RT-qPCRfor at least one virus, and co-infection was observed in 37.4% (67/179) of the cases. EV was detected in 50.8% (119/234), HPeV in 29.9% (70/234), HCoSV in 27.3% (64/234), and AiV/SalV in 2.1% (5/234) of the specimens tested by RT-qPCR.Using nested PCR and/or snPCR techniques, the positivity rates were 94.11% (112/119) for EV, 72.85% (51/70) for HPeV, and 20.31% (13/64) for HCoSV. It was not possible to amplify the samples that were positive for AiV/SalV. Sequencing revealed 67.2% (80/119) EV, 51.4% (36/70) HPeV, and 20.31% (13/64) HCoSV. Forty-five different types of EV were found among species A, B, and C; HCoSV identified five species, including a possible recombinant strain; all HPeV were identified as belonging to species A, in two samples a possible recombination involving three different strains was verified. This study demonstrated the high circulation and diversity of different types of picornaviruses in fecal samples, including those collected more than 30 years ago. This endorsed the evaluation of important points in the epidemiology of these viruses, such as the presence of co-infection and the possibility of knowing more about these agents, considering that some were recently described; therefore, their detection in older samples can provide more data about their ancestry.

Genetic Diversity and Characteristics of Drug Resistance Among Treatment-Naive People Living with HIV in Xi'an, China.

The genetic diversity and genetic predisposition for drug resistance mutations are the primary features of human immunodeficiency virus type 1 (HIV-1), which could cause the incidence of failure of antiretroviral therapy (ART). This study investigates the distribution of various HIV-1 genotypes and the incidence of pretreatment drug resistance (PDR) in the antiretroviral-naive HIV-1 infected participants in Xi'an, China. In this study, a cross-sectional analysis was carried out at the Xi'an Eighth Hospital between January 2020 and December 2021 among newly-diagnosed ART-naive HIV-1 infected participants. A nested PCR technique was used for amplifying the target segment of 1.3 kb present in the pol gene that spanned the reverse transcriptase and the protease regions. HIV-1 genotypes and the PDR-associated mutations were identified using the Stanford HIV Drug Resistance Database. A total of 317 pol gene sequences were retrieved, amplified, and sequenced. The circulating recombinant form (CRF), CRF07_BC (51.7%) was seen to be the most prevalent HIV-1 genotype, followed by other genotypes like CRF01_AE (25.9%), B (14.2%), and CRF55_01B (4.7%). PDR was found in 18.3% of the population. The PDR mutation frequency in the non-nucleoside reverse transcriptase inhibitor (NNRTI) (16.1%) was significantly higher compared to that of the nucleoside reverse transcriptase inhibitor (NRTI) (4.4%) and the protease inhibitor (0.9%). V179D/E (both were 4.4%) was seen to be the most predominant type of NNRTI mutation. K65R and M184V (1.3%) were the most frequent NRTI-associated mutations. About half (48.3%) of the sequenced HIV-1 strains that had mutations could show a potential low-level NNRTI resistance owing to V179D/E. Multivariate regression analysis revealed one PDR mutation associated with subtype CRF01_AE (p=0.002) and CRF55_01B (p<0.001) as a higher risk mutation. Diverse and complex HIV-1 genotypes are distributed in Xi'an, China. Considering new evidence, it is necessary to screen for baseline HIV-1 drug resistance among the newly-diagnosed HIV-1 individuals.