Nested Polymerase Chain Reaction Assay Research Articles

Malaria infection among adults residing in a highly endemic region from the Democratic Republic of the Congo.

Adults infected with Plasmodium spp. in endemic areas need to be re-evaluated in light of global malaria elimination goals. They potentially undermine malaria interventions but remain an overlooked aspect of public health strategies. This study aimed to estimate the prevalence of Plasmodium spp. infections, to identify underlying parasite species, and to assess predicting factors among adults residing in an endemic area from the Democratic Republic of Congo (DRC). A community-based cross-sectional survey in subjects aged 18years and above was therefore carried out. Study participants were interviewed using a standard questionnaire and tested for Plasmodium spp. using a rapid diagnostic test and a nested polymerase chain reaction assay. Logistic regression models were fitted to assess the effect of potential predictive factors for infections with different Plasmodium spp. Overall, 420 adults with an estimated prevalence of Plasmodium spp. infections of 60.2% [95% CI 55.5; 64.8] were included. Non-falciparum species infected 26.2% [95% CI 22.2; 30.5] of the study population. Among infected participants, three parasite species were identified, including Plasmodium falciparum (88.5%), Plasmodium malariae (39.9%), and Plasmodium ovale (7.5%) but no Plasmodium vivax. Mixed species accounted for 42.3% of infections while single-species infections predominated with P. falciparum (56.5%) among infected participants. All infected participants were asymptomatic at the time of the survey. Adults belonging to the "most economically disadvantaged" households had increased risks of infections with any Plasmodium spp. (adjusted odds ratio, aOR = 2.87 [95% CI 1.66, 20.07]; p < 0.001), compared to those from the "less economically disadvantaged" households. Conversely, each 1year increase in age reduced the risk of infections with any Plasmodium spp. (aOR = 0.99 [95% CI 0.97, 0.99]; p = 0.048). Specifically for non-falciparum spp., males had increased risks of infection than females (aOR = 1.83 [95% CI 1.13, 2.96]; p = 0.014). Adults infected with malaria constitute a potentially important latent reservoir for the transmission of the disease in the study setting. They should specifically be taken into account in public health measures and translational research.

High positivity rate of caprine arthritis encephalitis virus in Luzon, the Philippines revealed by nested-polymerase chain reaction assay.

Caprine arthritis encephalitis (CAE) is a worldwide economically important disease of small ruminants particularly goats. CAE has been considered to be an emerging/re-emerging disease of goats and a notifiable disease in the Philippines. In this study, a nested-PCR method to detect CAE virus (CAEv) infection was conducted between January 2021 to December 2022. A total of 1334 goat blood samples were collected from 24 goat farms throughout Luzon, the Philippines. The over-all prevalence rate was 31.41% (419/1334) in goats and 91.67% (22/24) of goat farms. These results showed high positivity rate of CAEv and the disease may be widespread in Luzon, the Philippines.

Detection of Plasmodium in small ruminants in Yogyakarta, Indonesia, using a nested PCR assay

Abstract. Priyowidodo D, Wardhana AH, Sawitri DH, Prastowo J, Nurcahyo W, Awaludin A, Nugraheni YR. 2023. Detection of Plasmodium in small ruminants in Yogyakarta, Indonesia, using a nested PCR assay. Biodiversitas 24: 6722-6726. Limited investigation of natural malaria infection in small ruminants in Yogyakarta needs to be explored. Plasmodium sp., as the causative agent of malaria, has been proven to infect ungulates, including small ruminants. This study aimed to investigate the presence of natural malaria infection in goats and sheep in Yogyakarta. A total of 303 blood samples were collected from goats and sheep in this study from four selected areas by a cross-sectional study. A microscopic examination detected the Plasmodium stage in a thin blood smear. Nested Polymerase Chain Reaction (PCR) performed molecular detection, targeting cytochrome b (cytb) as a gene target. It was found that no Plasmodium stage was detected in thin blood smears. However, three out of 70 blood samples from Girimulyo were Plasmodium-positive based on nested PCR assay. The results highlight that molecular assay by nested PCR is more sensitive than microscopic examination. Molecular analysis revealed natural Plasmodium infections in Ettawa cross-bread goats, with a proportion of 0.0429 (95% CI: 0.0089-0.1202). There was no statistical difference between sex and natural malaria infection in goats. This finding suggests that molecular analysis is necessary to detect natural malaria infection with extremely low parasitemia levels. Therefore, further study with larger sample sizes and broader geographical representation is needed to fully understand these malaria infections in goats and sheep.

Serum proteomic profile of wild stump-tailed macaques (Macaca arctoides) infected with malaria parasites in Thailand.

The number of patients infected with simian malaria is gradually increasing in many countries of Southeast Asia and South America. The most important risk factor for a zoonotic spillover event of malarial infection is mostly influenced by the interaction between humans, monkeys, and vectors. In this study, we determine the protein expression profile of a wild stump-tailed macaque (Macaca arctoides) from a total of 32 blood samples collected from Prachuap Kiri Khan Province, Thailand. The malarial parasite was analyzed using nested polymerase chain reaction (PCR) assays by dividing the samples into three groups: non-infected, mono-infected, and multiple-infected. The identification and differential proteomic expression profiles were determined using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and bioinformatics tools. A total of 9,532 proteins (total proteins) were identified with the filter-based selection methods analysis, and a subset of 440 proteins were found to be different between each group. Within these proteins, the GhostKOALA functional enrichment analysis indicated that 142 important proteins were associated with either of the organismal system (28.87%), genetic information processing (23.24%), environmental information processing (16.20%), metabolism (13.38%), cellular processes (11.97%), or causing human disease (6.34%). Additionally, using interaction network analysis, nine potential reporter proteins were identified. Here, we report the first study on the protein profiles differentially expressed in the serum of wild stump-tailed macaques between non, mono, and multiple malarial infected living in a natural transmission environment. Our findings demonstrate that differentially expressed proteins implicated in host defense through lipid metabolism, involved with TGF pathway were suppressed, while those with the apoptosis pathway, such as cytokines and proinflammation signals were increased. Including the parasite's response via induced hemolysis and disruption of myeloid cells. A greater understanding of the fundamental processes involved in a malarial infection and host response can be crucial for developing diagnostic tools, medication development, and therapies to improve the health of those affected by the disease.

Human parvovirus B19 infection in malignant and benign tissue specimens of different head and neck anatomical subsites

BackgroundThe role of human parvovirus B19 (B19V) infection in malignant and benign lesions such as head and neck squamous cell carcinomas (HNSCCs) and oral mucocele lesions has not been established. Herein, we examined, for the first time, the presence of B19V in HNSCCs from Iranian subjects.MethodsOne hundred and eight HNSCC specimens were analyzed for the presence of B19V using nested polymerase chain reaction (nPCR) and TaqMan quantitative PCR assays. Immunohistochemistry procedures were performed to evaluate the expression of B19V VP1/VP2 proteins, p16INK4a, and NF-κB in tumor tissues and their adjacent non-tumor tissues. In addition, 40 oral mucocele, 30 oral buccal mucosa swabs, and 30 nasopharyngeal swabs obtained from healthy adults were analyzed as controls.ResultsB19V DNA was detected in 36.1% of HNSCCs. Further, 23.3% of HNSCC specimens showed immunoreactivity against B19V VP1/VP2 proteins. There was a significant difference in the frequency of B19V DNA-positive cases between the patient and control groups (p < 0.0001). Moreover, comparing tumoral tissues and their adjacent non-tumor tissues in terms of immunoreactivity against B19V structural proteins, a significant association was found between tumor tissues and B19V infection (p < 0.0001). Finally, investigating the simultaneous presence of B19V and high-risk human papillomaviruses (HPV) DNA, we found a significant association between these two viral infections in HNSCCs (p = 0.031).ConclusionsTo sum up, B19V was frequently present in HNSCC tissues of Iranian patients but mostly absent in the adjacent non-tumor tissues as well as oral mucocele lesions, buccal, and nasopharyngeal swabs of healthy subjects. HPV possibly contributes to B19V persistence in HNSCC tissues. Additional research is required to investigate potential etiological or cofactor roles of B19V in the development of HNSCCs.

Molecular detection and viability discrimination of zoonotic protozoan pathogens in oysters and seawater

The presence of foodborne protozoan pathogens including Cryptosporidium parvum, Giardia duodenalis, Toxoplasma gondii, and Cyclospora cayetanensis in commercial shellfish has been reported across diverse geographical regions. In the present study, a novel multiplex nested polymerase chain reaction (PCR) assay was validated to simultaneously detect and discriminate these four targeted parasites in oyster tissues including whole tissue homogenate, digestive gland, gills, and hemolymph, as well as seawater where shellfish grow. To differentiate viable and non-viable protozoan (oo)cysts, we further evaluated reverse transcription quantitative PCR (RT-qPCR) assays through systematic laboratory spiking experiments by spiking not only dilutions of viable parasites but also mixtures of viable and non-viable parasites in the oyster tissues and seawater. Results demonstrate that multiplex PCR can detect as few as 5–10 (oo)cysts in at least one oyster matrix, as well as in 10 L of seawater. All parasites were detected at the lowest spiking dilution (5 (oo)cysts per extract) in hemolymph, however the probability of detection varied across the difference matrices tested for each parasite. RT-qPCR further discriminated viable from non-viable (heat-inactivated) C. parvum and T. gondii in seawater and hemolymph but did not perform well in other oyster matrices. This systematic spiking study demonstrates that a molecular approach combining multiplex PCR for sensitive and affordable screening of protozoan DNA and subsequent RT-qPCR assay for viability discrimination presents an important advance for accurately determining the risk of protozoal illness in humans due to consumption of contaminated shellfish.

First report of 'Candidatus Phytoplasma rubi' associated with Rubus stunt disease of raspberry and blackberry in the Czech Republic.

Raspberries (Rubus idaeus L.), occurring in the temperate zone of the northern hemisphere and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrolled by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera: Cicadellidae) (de Fluiter and van der Meer, 1953; Linck and Reineke 2019b). During a survey in commercial field in June 2021, over 200 raspberry bushes cv Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing/reddening, stunted growth, severe phyllody and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cv Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of expected size, while no product was amplified in asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from different location) were cloned and bi-directionally Sanger sequenced (GenBank Accession Nos.OQ520100-2). The sequences spanned nearly full-length of 16S rRNA gene, 16S-23S rRNA intergenic spacer, tRNA-Ile gene, and a partial 23S rRNA gene. BLASTn search revealed the highest sequence identity (99.8-99.9%, query coverage 100%) to 'Candidatus Phytoplasma rubi' strain RS (GenBank Accession No. CP114006). To further characterize the 'Ca. P. rubi' strains, all these three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (Acc. Nos. OQ506112-26) were obtained as described previously (Fránová et al. 2016). Comparison to GenBank sequences confirmed their highest identity (99.6-100%, query coverage 100%) with 'Ca. P. rubi' RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98,65 % 'Ca. Phytoplasma' strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity of the analysed 16S rRNA gene sequences and the high identity in the other genes with the reference 'Ca. P. rubi' RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of 'Ca. P. rubi' from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.

High Prevalence of Occult Hepatitis B Virus Infection among Iranian Hemodialysis Patients.

Considering the potential risks associated with occult hepatitis B virus (HBV) infection, this study was designed to investigate the magnitude and genotypic pattern of occult HBV infection among hemodialysis patients. All patients on regular hemodialysis attending the dialysis centers located in southern Iran and 277 nonhemodialysis controls were invited to participate in this study. Serum samples were tested for detection of hepatitis B core antibody (HBcAb) and hepatitis B surface antigen (HBsAg) by competitive enzyme immunoassay and sandwich ELISA, respectively. The molecular evaluation of HBV infection was conducted by two nested polymerase chain reaction (PCR) assays, targeting S, X, and precore regions of HBV genome, and sequencing by Sanger dideoxy sequencing technology. Moreover, HBV viremic samples were tested for hepatitis C virus (HCV) coinfection by HCV Ab ELISA and seminested reverse transcriptase PCR. Of 279 hemodialysis patients, five (1.8%) were positive for HBsAg, 66 (23.7%) were positive for HBcAb, and 32 (11.5%) had HBV viremia with HBV genotype D, sub-genotype D3 and subtype ayw2. Moreover, 90.6% of the hemodialysis patients with HBV viremia had occult HBV infection. Hemodialysis patients (11.5%) had significantly higher prevalence of HBV viremia than nonhemodialysis controls (1.08%; P = 0.0001). The prevalence of HBV viremia among hemodialysis patients was not statistically associated with duration of hemodialysis, age and gender distribution. In contrast, HBV viremia was significantly associated with place of residency and ethnicity, with residents of Dashtestan and Arab having had significantly higher prevalence of HBV viremia compared with the residents of other cities and Fars patients. Notably, 27.6% and 6.9% of hemodialysis patients infected with occult HBV infection were also positive for anti-HCV antibodies and HCV viremia, respectively. High prevalence of occult HBV infection was detected in hemodialysis patients, whereas 62% of patients with occult HBV infection were negative for HBcAb. Therefore, screening of all hemodialysis patients by sensitive molecular tests, regardless of the pattern of HBV serological markers, is recommended to increase the diagnosis rate of HBV infection.

Comparative Evaluation of LAMP and Nested PCR for the Rapid Diagnosis of Mycobacterium marinum Infection.

Culture of Mycobacterium marinum is very time-consuming, taking several weeks to produce positive results. Seeking rapid and sensitive diagnostic methods for diagnosis can greatly improve patient treatment. Our study aimed to compare the rapid diagnostic abilities of polymerase chain reaction (PCR), nested PCR and loop mediated isothermal amplification (LAMP) of detecting M. marinum in skin samples from patients with M. marinum infection. A total of 6 M. marinum strains and 6 skin samples with definite diagnosis of M. marinum infection were included in the study. We optimized LAMP performance for detection of M. marinum genomic DNA and confirmed the specificity of the primers. Then, the sensitivity of the LAMP and nested PCR assays were assessed by M. marinum strains and clinical samples. Nested PCR was 10-fold more sensitive than the LAMP assay by serial dilution of M. marinum DNA. PCR positive samples were all positive by LAMP detection of 6 clinical M. marinum strains. Out of 6 clinical skin specimens confirmed as M. marinum infection, 0 (0%), 3 (50%), 3 (50%), and 4 (66.6%) were positive by PCR, nested PCR, LAMP and culture. The LAMP shared the same sensitivity than nested PCR in M. marinum strains and clinical samples, but it was easy to perform and faster than nested PCR assay. Compared with conventional PCR, LAMP and nested PCR are more sensitive and have a higher detection rate of M. marinum in clinical skin specimens. The LAMP assay proved to be more suitable for rapid diagnosis of M. marinum infection in a shorter time, especially in resource-limited settings.

Molecular detection and genotype analysis of Kudoa septempunctata from food poisoning outbreaks in Korea.

Concerns about foodborne illnesses caused by Kudoa septempunctata are steadily growing, but reports of K. septempunctata in clinical and food specimens related to food poisoning in Korea are limited. This study aimed to genetically identify K. septempunctata in patients with acute diarrhea and in clinical and food samples related to food poisoning caused by sashimi consumption. Both real-time and nested polymerase chain reaction assays were performed to detect K. septempunctata 18S and 28S rDNA genes in the stools of 348 patients with acute diarrhea, 11 samples (6 stool and 5 rectal swab samples) from patients with food poisoning, and 2 raw Paralichthys olivaceus samples collected from a restaurant where a food poisoning incident occurred. K. septempunctata was identified in 5 clinical specimens (4 stools and 1 rectal swab) and 1 P. olivaceus sashimi sample. All detected K. septempunctata were of genotype ST3. This is the first study to identify K. septempunctata in both patients and food samples with epidemiological relevance in Korea, providing evidence that it is a pathogen that causes food poisoning. Also, this is the first study to confirm the presence of K. septempunctata genes in rectal swabs. Despite continuing suspected occurrences of Kudoa foodborne outbreaks, the rate of identification of K. septempunctata is very low. One reason for this is the limitation in obtaining stool and vomit samples for the diagnosis of Kudoa infection. We strongly suggest the inclusion of rectal swabs among the diagnostic specimens for Kudoa food poisoning.

364. Diagnosis of Amebic Liver Abscess by a Commercial Multiplex Stool Amplification Assay. A Potentially Useful Alternative in Limited-Resources Settings.

Abstract Background Amoebic liver abscess (ALA) is caused by the protozoan parasite Entamoeba histolytica. Clinical and radiological features of ALA often mimic those of pyogenic liver abscess (PLA). Currently, differential diagnosis between ALA and PLA relys on microscopic examination, anti-amoebic IgG serology, and culture of aspirate for pyogenic organisms. Therefore, there is a need for new, rapid and more accurate microbiological methods for diagnosing ALA. Unfortunately, with few exceptions, available reports involved the use of inhouse or laboratory-tailored polymerase chain reaction (PCR) assays. We herein report our recent experience with the successful use of a commercial multiplex, nested PCR assay designed for stool, in the diagnosis of two cases of ALA. Methods Two severely ill adult Latin American migrants presented with fever, malaise, non-bloody diarrhea, abdominal pain and leukocytosis. CT scans revealed single large hypodense, inflammatory, liver cystic lesions. Results Necrotic material obtained by needle aspiration under guidance and stool samples were processed by FilmArray Gastrointestinal Panel (BioFire Diagnostics, Salt Lake City), designed to detect 13 bacterial, 5 viral and 4 parasitic pathogens, both amplified only for E. hystolitica DNA. ELISA serology for amoeba was also positive. Bacterial cultures were sterile. Treatment with Metronidazole and paromomycin resulted in complete recovery. Conclusion Analysis of liver abscess content using a commercially available, multiplex, nested PCR assay designed for stool, may represent a convenient alternative to currently available diagnostic methods, providing rapid results and allowing for immediate choosing of specific therapy targeting single pathogens on more rational basis. Since there are several commercially available multiplex PCR panels containing primers for E. histolytica, their use for evaluation of liver abscess samples has some theoretical advantages for the diagnosis of ALA, such as simplicity, ample availability, rapid processing time and capacity to exclude simultaneously several potential pathogens. Nevertheless, further testing is necessary to validate their use in liver abscess diagnosis and define their false positivity and negativity rates. Disclosures All Authors: No reported disclosures.

Application of a nested polymerase chain reaction assay to detect Anaplasma bovis and Anaplasma phagocytophilium in cattle in Lorestan province, Iran

Anaplasmosis is an important tick-borne disease caused by Anaplasma species in cattle and other ruminants. This study aimed to determine the leukocytotropic species of bovine anaplasmosis (Anaplasma bovis and A. phagocytophilium) among cattle of Lorestan province, Iran using nested polymerase chain reaction (nPCR). Two hundred blood samples were taken from the jugular vein from healthy cattle, randomly. The extracted from DNA blood cells was screened using the universal primers, which amplify a DNA fragment (1468bp) from the 16S rRNA gene of the genus Anaplasma. The PCR products from positive samples were examined for A. bovis and A. phagocytophilium 16S rRNA gene by species-specific nPCR. Out of the 200 cattle examined, 10 (5%) and 4 (2%) were found nPCR positive for A. bovis and A. phagocytophilium, respectively. No morulae were observed in the blood smears and clinical signs were not seen in infected cattle. Findings in the present study corroborate the low infection rate of A. bovis and A. phagocytophilum in Lorestan province, Iran. Identifying interaction among animal reservoirs, vectors, and pathogens in different climatic zones in Iran are necessary to be performed in future investigations.

Screening of West Nile Virus, Herpesvirus, and Parvovirus in Rattus spp. in Klang Valley, Malaysia

Free-roaming and scavenging lifestyles of Rattus spp. in densely populated urban areas expose them to multiple pathogens that facilitate the transmission of infection to the human population more rapidly, raising public health concerns. There is limited information on the status of rat susceptibility to virus infection, particularly West Nile virus (WNV), herpesvirus, and parvovirus, to prepare for emerging zoonosis. A total of 23 (n = 23) blood samples collected from Rattus spp. in the wet market areas of Klang Valley, Malaysia, were subjected to molecular assay using a one-step reverse transcription-polymerase chain reaction (RT-PCR) to detect the highly conserved region of the WNV capsid and pre-membrane protein via nested polymerase chain reaction (PCR) assay targeting highly conserved amino acid motifs within the herpesviral DNA-directed DNA polymerase gene (DPOL) and polymerase chain reaction (PCR) assay targeting the parvovirus non-structural (NS) protein. As a result, 4 out of 23 (17.39%) rats were positive for herpesvirus DNA, but none were positive for WNV RNA and parvovirus DNA. The positive PCR amplicons of herpesvirus DNA were subjected to partial DNA sequencing analysis, 100% identical to Acomys herpesvirus SVMS 226,222 from Betaherpesvirinae, which is highly suggestive of rat cytomegalovirus (RCMV). This study has successfully demonstrated the presence of RCMV from Rattus spp. in the Klang Valley. The RCMV potentially crosses species barriers and establishes infection, raising public health concerns. The non-viraemic state of WNV or parvovirus infection, low sample size, and limited niche distribution emphasise the need for the expansion of this study in the future.

Development of an ultra-sensitive single-tube nested PCR assay for rapid detection of Campylobacter jejuni in ground chicken

Traditional culture-based detection methods for Campylobacteri jejuni, a leading cause of human bacterial gastroenteritis worldwide, are time-consuming, cumbersome, and lacking in reliability. While polymerase chain reaction (PCR) has been frequently used for pathogen testing, it might generate false-negative results due to inadequate sensitivity. This study was the first to explore novel single-tube nested PCR (STN-PCR) to detect pathogens in food. Two pairs of nested PCR primers were designed based on the hippuricase gene of C. jejuni. The annealing temperatures and concentrations of nested primers were optimized to ensure the sequential use of outer and inner pairs of primers during amplification. Efficacy of the developed STN-PCR assay was compared with standard culture-based methods and conventional PCR in artificially contaminated ground chicken homogenate. Limit of detection of the STN-PCR assay was determined to be 3.6 × 101 CFU/ml of C. jejuni in the homogenate without enrichment, which was 100 times lower than conventional PCR. Moreover, 0.1 CFU/g of C. jejuni in ground chicken homogenate was identified by STN-real time PCR (rtPCR) after 24 h of enrichment, while a 48-h enrichment was required for culture-based methods and conventional rtPCR. This developed assay provides a powerful tool for rapid, highly specific, and ultra-sensitive detection of C. jejuni and may potentially be used to identify contaminated chicken and other foods.

Detection of Coxiella burnetii in raw milk samples collected from dairy farms in Mazandaran province, north of Iran

Query fever is an important zoonotic disease caused by the obligate intracellular microorganism of Coxiella burnetii. Moreover, the present study aimed to determine the prevalence of C. burnetii in bulk milk samples from dairy bovine herds using a nested polymerase chain reaction assay. During four seasons, a total of 100 samples of bulk milk from traditional, semi-industrial, and industrial dairy bovine herds located in six cities of Mazandaran province, northern Iran from May 2019 to February 2020 were collected. The samples were subjected to detect C. burnetii using a nested-PCR assay. Twenty - seven out of 100 bulk milk samples (27%, 95% CI: 18.3-35), 7% were infected with C. burnetii. The prevalence was 54.3% (19 samples) in traditional dairy herds and 12.3 % (8 samples) in semi-industrial industrial dairy herds. Furthermore, our results revealed that the chance of milk being infected with C. burnetii bacteria in traditional milk is 8.5 times higher than the milk of semi-industrial and industrial dairy herds. Based on the obtained results of the current study, the highest and lowest prevalence of C. burnetii were seen in Ghaemshar (66.7%) and Babol (0%), respectively (p&lt;0.05). Spring season (37%) had the highest contamination of C. burnetii in bulk milk samples. According to the findings of this study, the clinically healthy dairy cows are important sources of C. burnetii infection in this area, amplified by the traditional herds of these animals. Because these animals can transmit the infection to humans, this common zoonotic bacterium can be a potential health problem in Mazandaran province, Northern Iran.

Molecular detection of H1N1 virus by conventional reverse transcription PCR coupled with nested PCR

Influenza A pdm09 virus has been spreading worldwide and creates a serious public health threat. Among different subtypes/lineages of swine influenza A viruses, the H1N1 subtype is more prevalent in all-over world infections followed by H3N2 which is present in the northern hemisphere. Influenza A pdm09 is dominant for centuries but the last pandemic was in 2009 and is responsible for 151,700–575,400 deaths within one year worldwide (Centres for Disease Control and Prevention). Rapid diagnosis for H1N1 is essential which must be highly sensitive and specific and can differentiate between influenza A subtypes and other respiratory pathogens. In this study, a conventional polymerase chain reaction assay and nested polymerase chain reaction assay were developed which can detect H1N1 pdm09. One outer set of primer is designed and one inner set of primer is designed specifically for hemagglutinin gene (H1). Specificity was checked using different pathogenic genomes for amplification of the H3N2 subtype, Neisseria meningitides, Salmonella typhi, etc. The sensitivity of the nested polymerase chain reaction is 0.001ng/6 ​μL of PCR amplified product of outer primer product of HA1 gene as compared to 50 ​ng/μL of two-step conventional polymerase chain reaction. The nested PCR and conventional PCR are performed on 80 samples which are consistent with the Real-Time PCR results. Real-Time is a standard test for H1N1 detection. But it is very expensive but current method is less expensive and provides comparable results with Real-Time PCR.

Prevalence and genotype of Toxoplasma gondii in stranded Hawaiian cetaceans.

Toxoplasma gondii is a significant threat to endangered Hawaiian wildlife including birds and marine mammals. To estimate the prevalence of T. gondii in stranded cetaceans from 1997 to 2021 in Hawai'i, we tested tissues from 37 stranded spinner dolphins Stenella longirostris and 51 stranded individuals that represented 18 other cetacean species. DNA from cetacean tissue extracts were screened using a nested polymerase chain reaction (PCR) assay targeting the Toxoplasmatinae internal transcribed spacer 1 of the nuclear ribosomal DNA. A positive result was obtained in 9 tissues examined for each of 2 spinner dolphins out of 525 tissue samples analyzed by PCR. The PCR-positive spinner dolphins had disseminated acute toxoplasmosis with necrosis, inflammation, and intralesional protozoal cysts and tachyzoites in multiple organs. Discrete positive immunostaining for T. gondii was observed in all tissues tested including the adrenal gland, brain, liver, and lung. Both positive spinner dolphins were negative for cetacean morbillivirus. The T. gondii genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) based on 10 genetic markers. The PCR-RFLP analysis revealed the T. gondii belonged to PCR-RFLP-ToxoDB genotype #24, previously detected in wild pig Sus scrofa in O'ahu, bobcats Lynx rufus from Mississippi, USA, and chickens Gallus gallus from Costa Rica and Brazil. These cases represent the first report of this genotype in aquatic mammals and the second and third reports of fatal disseminated T. gondii infection in stranded spinner dolphins from Hawai'i. Nearshore species, like spinner dolphins, may be at increased risk of mortality from this parasite in marine coastal waterways via sewage systems, storm water drainage, and freshwater runoff.

Usefulness of Nested PCR Assay for the Molecular Diagnosis of Human Rickettsial Infection: A Study in Bangladesh

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.&#x0D; 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. &#x0D; 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.&#x0D; Results: Out of 135 cases, we detected Rickettsia in 70(51.85%) cases by nested PCR assay (p&lt;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.&#x0D; 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.

DNA diagnostics for reliable and universal identification of Helicobacter pylori.

Reliable diagnostics are a major challenge for the detection and treatment of Helicobacter pylori (H. pylori) infection. Currently at the forefront are non-invasive urea breath test (UBT) and stool antigen test (SAT). Polymerase chain reaction (PCR) is not endorsed due to nonspecific primers and the threat of false-positives. The specificity of DNA amplification can be achieved by nested PCR (NPCR), which involves two rounds of PCR. If the primers are properly designed for the variable regions of the 16S rRNA gene, it is not difficult to develop an NPCR assay for the unambiguous identification of H. pylori. Elaborate NPCR for a 454 bp amplicon was validated on 81 clinical biopsy, stool, and saliva samples, each from the same individuals, and compared with available H. pylori assays, namely histology, rapid urease test, SAT, and 13C-UBT. The assay was much more sensitive than simple PCR, and it was equally sensitive in biopsy samples as the 13C-UBT test, which is considered the gold standard. In addition, it is sufficiently specific because sequencing of the PCR products exclusively confirmed the presence of H. pylori-specific DNA. However, due to the threshold and lower abundance, the sensitivity was much lower in amplifications from stool or saliva. Reliable detection in saliva also complicates the ability of H. pylori to survive in the oral cavity aside from and independent of the stomach. The reason for the lower sensitivity in stool is DNA degradation; therefore, a new NPCR assay was developed to obtain a shorter 148 bp 16S rRNA amplicon. The assay was validated on stool samples from 208 gastroenterological patients and compared to SAT results. Surprisingly, this NPCR revealed the presence of H. pylori in twice the number of samples as SAT, indicating that many patients are misdiagnosed, not treated by antibiotics, and their problems are interpreted as chronic. Thus, it is unclear how to properly diagnose H. pylori in practice. In the first approach, SAT or UBT is sufficient. If samples are negative, the 148 bp amplicon NPCR assay should be performed. If problems persist, patients should not be considered negative, but due to threshold H. pylori abundance, they should be periodically tested. The advantage of NPCR over UBT is that it can be used universally, including questionable samples taken from patients with achlorhydria, receiving proton pump inhibitors, antibiotics, bismuth compound, intestinal metaplasia, or gastric ulcer bleeding.

Genetic characterization of Toxoplasma gondii isolates from human spontaneous aborted fetuses in Jahrom, southern Iran

Toxoplasma gondii (T. gondii) is an intracellular protozoan that infects the fetus through the placenta and leads to severe complications in the fetus. One of the complications of congenital toxoplasmosis is spontaneous abortion. The prevalence of toxoplasmosis infection was investigated among spontaneously aborted fetuses (SAFs), and the genotypes of parasite isolates were determined in the present study. Placentas from 330 samples of SAFs were collected in Jahrom (Fars province) from February to September 2018. DNA was extracted from each placental tissue. The T. gondii infection was detected using nested polymerase chain reaction (Nested-PCR) assay based on a 529 bp repeat element (RE) gene. Afterward, Toxoplasma was genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) based on the GRA6 gene. The frequency of T. gondii infection was found to be 14.5% (48 out of 330 samples). Genotyping of nine T. gondii isolates revealed that all belonged to genotype II. Statistically, the prevalence of T. gondii infection was significantly correlated with the education levels of the mothers and the age of the fetus (P < 0.05). The lowest prevalence of Toxoplasma infection belonged to mothers with university education and the highest frequency of infection was observed among the fetuses in the age group of 8–9 weeks. The findings of the present study suggest a significant role for toxoplasmosis in SAFs in Jahrom city.