nested polymerase chain reaction
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2022 ◽  
Vol 43 (2) ◽  
pp. 713-726
Flávio Gomes Barcelos ◽  
Taís Ramalho dos Anjos ◽  
Leila Auxiliadora de Arruda Alencar ◽  
Vinicius Silva Castro ◽  

Mycobacterium bovis is a bacterium belonging to the Mycobacterium tuberculosis complex that causes tuberculosis in cattle and in other domestic and wild animals, as well as in humans. Disease control measures are carried out by slaughtering animals tested positive in the intradermal tuberculinization test and sanitation of their original living spaces, in addition to epidemiological surveillance carried out through the sanitary inspection of bovine carcasses in slaughterhouses. In the latter, official inspection services collect samples from macroscopic lesions suspected of bovine tuberculosis, which are then sent for laboratory analysis. Knowledge concerning the variables associated with the occurrence of M. bovis can aid in decision-making regarding control and disease eradication efforts. In this context, the aim of this study was to identify the risk factors for a positive M. bovis diagnosis in suspected bovine tuberculosis lesions obtained during epidemiological surveillance activities in the state of Mato Grosso, Brazil. A total of 105 suspicious lesions were analyzed using the Nested Polymerase Chain Reaction (nested q-PCR) method, of which 14 (13.33%) tested positive for M. bovis. Univariate and bivariate statistical analyses indicated that the variable “animal slaughter” was the only risk factor presenting statistical significance associated with the diagnosis of M. bovis (p < 0.05), demonstrating that macroscopic lesions suspected as being caused by bovine tuberculosis from animals with an in vivo diagnosis were 2.82 - fold more likely to result in a positive M. bovis diagnosis by molecular tests.

2023 ◽  
Vol 83 ◽  
M. F. Nadeem ◽  
A. A. Khattak ◽  
N. Zeeshan ◽  
U. A. Awan ◽  
S. Alam ◽  

Abstract Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistan’s war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria-related education locally, targeting children and parents alike.

Zahra Sadeghian ◽  
Hossein Bannazadeh Baghi ◽  
Vahdat Poortahmasebi ◽  
Javid Sadeghi ◽  
Alka Hasani ◽  

Background. Human papillomavirus (HPV) is one of the most important viral agents associated with several classes of cancers in humans. The aim of this study was to investigate HPV in esophageal cancer in the East Azerbaijan province, northwest of Iran. Methods. 140 paraffin-embedded specimens of esophageal tissues were investigated using nested-polymerase chain reaction (nested-PCR) with primer designing for the L1 region of HPV genome. According to the pathological diagnosis, the samples were divided into two groups: 70 patients with esophageal cancer EADC (n = 35) and ESCC (n = 35) as the case group and those without tumour in esophagus tissue as a control (n = 70). Results. HPV DNA was isolated from 20 (28.57%) of the 70 paraffin-embedded tissue specimens of esophagus cancer. Of these, 6 cases (17.14%) of EADC and 14 cases (40%) of ESCC were positive. In contrast, all cases of the control group were negative for the HPV genome. Sequence analysis revealed that HPV types 16 and 18 are the most frequent ones identified in this study. Conclusion. The prevalence of HPV in esophageal cancer can vary depending on the geographical location and other factors. Based on the findings of this study, HPV infection may possibly have contributed to an increased risk of esophageal cancer in a group of patients in Tabriz.

2022 ◽  
Mostafa Sayyadi ◽  
Saeid Hosseinzadeh ◽  
Gholamreza Abdollahpour ◽  
Seyed Shahram Shekarforoush ◽  
Azadeh Samiei ◽  

Abstract Leptospirosis is a relatively rare bacterial infection that affects people and animals caused by pathogenic species of Leptospira. The present study was conducted using nested polymerase chain reaction (PCR), microscopic agglutination test (MAT) and hematological and biochemical tests on 200 blood samples of renal disorders patients in Shiraz, Iran. Also nested-PCR assay and Warthin-Starry (WS) silver staining method was performed on 30 nephrectomised kidney sample. The frequency of pathogenic species of Leptospira infection in patients with renal disorders was 20 % and this infection was significantly correlated with BUN, anemia, RDW, MCV, MCH and hemoglobin levels (P < 0.01). MAT analysis showed that serum samples had positive titers against L. Grippotyphosa (13 samples), L. Ballum (6 sample), L. Pomona (3 samples), L. Canicola (2 samples), L. Icterohaemorrhagiae (1 sample) and L. Hardjo (1 sample) serovars. Twenty-three percent of the kidney samples from the patients with pyelonephritis were infected with the pathogenic species of Leptospira. This study showed that pathogenic Leptospira serovars are present in this area and in patients with renal disorders more attention should be paid to this zoonotic disease.

2022 ◽  
Vol 21 (10) ◽  
Mitra Nasiri ◽  
Zohreh Yousefi Ghalejoogh ◽  
Angila Ataei-Pirkooh ◽  
Farah Bokharaei-Salim ◽  
Seyed Hamidreza Monavari ◽  

Background: Limited sources of fresh water necessitate the application of health policies for treatment and decontamination of human sewage for further use. A wide variety of infectious agents, including bacteria, fungi, parasites, and viruses, can be found in sewage. Enteric viruses such as hepatitis A virus (HAV) can survive the current treatments and infect susceptible hosts. Objectives: This study aimed to evaluate the HAV contamination in human sewage before and after treatment in the wastewater treatment plant of Ekbatan town in Tehran, Iran, and analyze the phylogenetic properties of the identified viruses. Methods: Over a 12-month period, we collected the wastewater samples including influent, before chlorination, and effluent, from the wastewater treatment plant of Ekbatan town in Tehran, Iran. Ribonucleic acid (RNA) extraction, complementary deoxyribonucleic acid (cDNA) synthesis, and semi-nested polymerase chain reaction (PCR) were performed to identify HAV contamination. Phylogenetic analysis was performed to investigate subgenotypes of the virus. Results: HAV was detected in all influents and samples before chlorination, while the virus was detected in 50% of the effluent samples. All detected viruses belonged to subgenotype IB. Conclusions: Investigating the presence of HAV in sewage provides a general picture of the virus spread in the population of interest. HAV was detected in all influent samples, indicating that the infection is endemic in this area all year round. This also indicates the inability of the current treatment protocols in virus removal, which can be a threat to the public health.

2021 ◽  
Vol 15 (12) ◽  
pp. e0009993
Tetsuya Kakita ◽  
Sho Okano ◽  
Hisako Kyan ◽  
Masato Miyahira ◽  
Katsuya Taira ◽  

Background Leptospirosis is considered an endemic disease among agricultural workers in Okinawa Prefecture, which is the southernmost part of Japan and has a subtropical climate, but data on the current status and trend of this disease are scarce. Methodology/principal findings We conducted a retrospective study of clinically suspected leptospirosis patients whose sample and information were sent to the Okinawa Prefectural Institute of Health and Environment from November 2003 to December 2020. Laboratory diagnosis was established using culture, nested polymerase chain reaction (PCR), and/or microscopic agglutination test (MAT) with blood, cerebrospinal fluid, and/or urine samples. Statistical analyses were performed to compare the epidemiological information, clinical features, and sensitivities of diagnostic methods among laboratory-confirmed cases. Serogroups and the species of Leptospira isolates were determined by MAT using 13 antisera and flaB sequencing. A total of 531 clinically suspected patients were recruited, among whom 246 (46.3%) were laboratory confirmed to have leptospirosis. Among the confirmed cases, patients aged 20–29 years (22.4%) and male patients (85.7%) were the most common. The most common estimated sources of infection were recreation (44.5%) and labor (27.8%) in rivers. Approximately half of the isolates were of the L. interrogans serogroup Hebdomadis. The main clinical symptoms were fever (97.1%), myalgia (56.3%), and conjunctival hyperemia (52.2%). Headache occurred significantly more often in patients with Hebdomadis serogroup infections than those with other serogroup infections. The sensitivities of culture and PCR exceeded 65% during the first 6 days, while the sensitivity of MAT surpassed that of culture and PCR in the second week after onset. PCR using blood samples was a preferable method for the early diagnosis of leptospirosis. Conclusions/significance The results of this study will support clinicians in the diagnosis and treatment of undifferentiated febrile patients in Okinawa Prefecture as well as patients returning from Okinawa Prefecture.

Mahfuja Begam ◽  
Shaira Akter Shampa ◽  
Aleya Farzana ◽  
Abu Taher MD Zabed Hasan ◽  
Shirin Tarafder

Background: Rickettsial infections are re-emerging arthropods born worldwide zoonotic disease caused by Rickettsia, which is responsible for spotted fever and typhus fever. The diagnosis of a rickettsial illness is important for appropriate antibiotic treatment. Aims: The study aimed to determine the diagnostic accuracy and clinical usefulness of using nested polymerase chain reaction (PCR) by comparing nested PCR, ELISA, and Weil-Felix (WF) tests.   Methodology: This was a prospective type of cross-sectional study. A total of 135 clinically suspected rickettsial infection cases were enrolled. Peripheral blood was taken to detect gltA, 17 kDa lipoprotein antigen gene (17 kDa), ompA, and ompB gene of Rickettsia by nested PCR. ELISA and Weil-Felix tests were done to compare with nested PCR. Results: Out of 135 cases, we detected Rickettsia in 70(51.85%) cases by nested PCR assay (p<0.01), 33((24.4%) by Weil- Felix test, 34 (25.18%) by ELISA. Only 26.66% of cases were PCR positive, which were negative by both ELISA and Weil-Felix test. Fifteen (11.11%) cases were positive by all three tests. Among 70 PCR positive rickettsia cases most frequently detected gene was ompB 42(60%), followed by 17kDa 34(48.58%); gltA 21(30%), and ompA 3(4.28%).  Multiple gene combinations (ompB, 17kDa and gltA) detected in 98.57 % cases. Conclusion: Nested PCR assays showed the highest rate of detection of rickettsia cases than ELISA and Weil-Felix test. Multiple gene combinations (ompB, 17kDa, and gltA) showed the highest positivity. Therefore, diagnosis of rickettsial infection can be confirmed by PCR assay, and clinicians can plan appropriate treatment for these patients.

2021 ◽  
Vol 4 (2) ◽  
pp. 14
Abdul Halim Sadikin ◽  
Irene Dian ◽  
Mukharjon Mukharjon ◽  
Rini Puspitaningtum ◽  
Septelia Inawati Wanandi

Background: In some people, acetaldehyde, a toxic product from ethanol oxidation, cannot be oxidized to acetate. The excess of acetaldehyde could cause facial flushing, dizziness, and hypertension when they consume ethanol. This ethanol sensitivity is caused by a deficiency of ALDH2. Objective: This study aims to analyze and count the polymorphism frequency of the ALDH2 gene in Indonesia’s Minang ethnic. Methods: DNA samples were taken randomly from hair bulbous of 60 subjects (male and female, 3rd generation). A nested polymerase chain reaction was conducted to amplify the ALDH2 in the samples. Afterward, restriction fragment length polymorphism (RFLP) was conducted to the amplicons using the EcoRI restriction enzyme. The measured parameters were the distribution of the wildtype, atypical homozygote, and heterozygote. Results: Results showed that out of 60 subjects, 53.33% have an atypical homozygote gene (subjects prone to hypersensitive to alcohol), 28.33% have a heterozygote gene, and 18.33% have a wildtype gene. The frequency of the atypical alleles in Minang ethnic is 0.675. Conclusion: The atypical ALDH2 allele was much higher than the normal ALDH2 allele, in which most participants have atypical homozygote ALDH2, suggesting the samples are sensitive to alcohol.

2021 ◽  
Vol 937 (2) ◽  
pp. 022133
N V Girsova ◽  
D Z Bogoutdinov ◽  
I A Yengalycheva ◽  
T B Kastalyeva

Abstract Phytoplasma DNA was detected in 72 samples of vegetable crops collected in eight regions/territories of the Russian Federation, including the Republic of Crimea. The analyzed plants belonged to 13 species (Armoracia rusticana, Artemisia dracunculus, Capsicum annuum, Conundrum sátivum, Cucumis melo, Cucurbita maxima, Daucus carota var.sativus, Melissa officinalis, Petroselinum crispum, Phaseolus vulgaris, Solanum lycopersicum, Solanum melongena and Vicia faba), to 7 families (Apiacea, Asteracea, Brassicaceae, Cucurbitaceae, Fabaceae, Lamiaceae and Solanaceae). The belonging of phytoplasma to a group/subgroup was established by restriction fragment length polymorphism (RFLP) analysis of amplicons obtained in nested polymerase chain reaction (PCR). We identified phytoplasmas of four groups most characteristic of the Russian Federation: Aster yellows - 16SrI, X-disease - 16SrIII, Clover proliferation - 16SrVI and Stolbur - 16SrXII. All phytoplasmas isolated from plants collected in the southern regions of the Russian Federation (Astrakhan and Rostov regions, Krasnodar Territory, and the Republic of Crimea) belonged to stolbur group, subgroup 16SrXII-A, like most phytoplasmas from plants of the Samara region. Phytoplasmas of the 16SrVI group were found in plants from the Moscow, Samara, and Novosibirsk regions, the 16SrIII group - in plants from the Vologda and Moscow regions, and the 16SrI group - only in samples from the Moscow region.

2021 ◽  
Vol 14 (1) ◽  
Yongcai He ◽  
Wangkai Chen ◽  
Ping Ma ◽  
Yaoping Wei ◽  
Ruishan Li ◽  

Abstract Background Anaplasma, Babesia and Theileria are tick-borne pathogens (TBPs) that affect livestock worldwide. However, information on these pathogens in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibet Plateau (QTP), China, is limited. In this study, Anaplasma spp., Babesia spp. and Theileria spp. infections were assessed in yaks and Tibetan sheep from Qinghai Province. Methods A total of 734 blood samples were collected from 425 yaks and 309 Tibetan sheep at nine sampling sites. Standard or nested polymerase chain reaction was employed to screen all the blood samples using species- or genus-specific primers. Results The results showed that 14.1% (60/425) of yaks and 79.9% (247/309) of Tibetan sheep were infected with at least one pathogen. Anaplasma ovis, Anaplasma bovis, Anaplasma capra, Anaplasma phagocytophilum, Babesia bovis and Theileria spp. were detected in this study, with total infection rates for all the assessed animals of 22.1% (162/734), 16.3% (120/734), 23.6% (173/734), 8.2% (60/734), 2.7% (20/734) and 19.3% (142/734), respectively. For yaks, the infection rate of A. bovis was 6.4% (27/425), that of B. bovis was 4.7% (20/425) and that of Theileria spp. was 3.3% (14/425). Moreover, 52.4% (162/309) of the Tibetan sheep samples were infected with A. ovis, 30.1% (93/309) with A. bovis, 56.0% (173/309) with A. capra, 19.4% (60/309) with A. phagocytophilum and 41.4% (128/309) with Theileria spp. Conclusions This study revealed the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in yaks and Tibetan sheep in Qinghai Province, China, and provides new data for a better understanding of the epidemiology of TBPs in these animals in this area of the QTP, China. Graphical Abstract

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