Conventional PCR Research Articles

Genotypic Characterisation of Carbapenemases in Clinical Isolates of Acinetobacter baumannii in a Tertiary Care Teaching Hospital of Kerala

Background: The multidrug-resistant and carbapenem-resistant Acinetobacter baumannii strains are an increasing global concern. The primary cause of carbapenem resistance in A. baumannii is production of various beta-lactamases with versatile hydrolytic capacities. The present study investigates the prevalence of different carbapenemases among clinical isolates of carbapenem-resistant A. baumannii from a tertiary care teaching hospital in Kerala, India. Methods: Non-duplicate isolates from sputum, endotracheal aspirate, pus, urine, and blood samples were included in the study. Isolate identification and antibiotic susceptibility testing of the isolates were done using vitek 2 system. A conventional polymerase chain reaction was used to detect the carbapenemase-encoding genes in the isolates. Results: A total of the 126 isolates studied, among these 105 (83.3%) isolates were from intensive care unit patients and 21(16.6%) were from non-ICU inpatients. Most of the isolates were obtained from respiratory specimens-78(61.9%) followed by pus-33(26.2%), urine-12(9.5%) and blood- 3(2.4%). The prevalence of carbapenemase genes were as follows:blaOXA-51(n=126, 100%) blaOXA-23 (n=98, 77.7%), blaOXA-58 (n=7, 5.6%), blaNDM (n=66, 52.4%), blaIMP (n=26, 20.6%), and blaVIM (n=20, 15.9%). Among the total isolates 78 (61.9%) isolates harbored metallo-beta-lactamase genes, 47(37.3%) isolates harboured a single carbapenemase gene, while 69(54.8%) isolates harbored two or more genes. Conclusion: This study reveals a higher prevalence of metallo-beta lactamases and the co-occurrence of multiple carbapenemase genes in Carbapenem resistant A. baumannii isolates.

Molecular typing of Protoparvovirus carnivoran 1 in Egyptian cats diagnosed with feline panleukopenia

Feline panleukopenia (FPL) in cats is caused by either feline parvovirus (FPV) or canine parvovirus (CPV-2), which belong to the same species “Protoparvovirus carnivoran 1”. While FPV is widely recognized as the principal cause of FPL, CPV-2 has been detected at a higher rate than FPV in sick cats in a recent Egyptian study. To assess this conflict, the present study aimed to determine which Protoparvovirus carnivoran 1 is commonly associated with FPL in Egyptian cats. From Dec-2022 to Jan-2024, 43 cats presenting with acute gastroenteritis and testing positive for FPL using in-clinic assay, SNAP® parvo, were tested for Protoparvovirus carnivoran 1 DNA using conventional PCR. Typing of Protoparvovirus carnivoran 1 was conducted by partial VP2 gene sequencing. Additional epidemiological aspects of the disease were investigated, including seasonal pattern, case-fatality rate, median survival time to death, and the association between FPL outcomes and selected factors like age, sex, vaccination status, and clinical signs (vomiting and diarrhea). All cats tested positive for Protoparvovirus carnivoran 1 DNA and FPV was detected in all cats with strong PCR amplicons (n=39). The following seasonal pattern was recorded: cases emerging in autumn, peaking during winter, declining in spring, and disappearing in summer. The case-fatality rate was 41.6%, and the median time to death was two days. None of the studied factors affected FPL outcomes. In conclusion, FPL in Egyptian cat populations is primarily caused by FPV, not CPV-2, and is particularly prevalent in winter.

The 2023 National Proficiency Test for molecular detection of shrimp infectious myonecrosis virus (IMNV) in China

Infectious myonecrosis virus (IMNV), an emerging virus that can cause severe viral disease in penaeid shrimp, was first confirmed to have spread to China in 2020. In order to standardize diagnostic procedures and reliable detection capability, and to evaluate the performances of aquatic clinical diagnostic laboratories, the competent authority has launched a proficiency test (PT) scheme for the molecular detection of IMNV in China from the year of 2023. In the ring test of IMNV-PT 2023, five tissue samples containing different concentrations of IMNV were provided as a panel of test samples. A total of 68 laboratories participated in the PT test of qualitative and quantitative analysis of the five samples using standard molecular assays. The results showed that 92.65 % (63/68) of the participants correctly reported all samples in the panel using Real-time reverse transcription PCR and 62 participants achieved satisfactory results, one participant using conventional PCR and four participants had not completed the test. Out of the 62 participants in the z-scores analysis, 88.71 % (55/62) achieved acceptable ZB-scores, with two outlier laboratories and five laboratories being questionable. Additionally, 98.39 % (61/62) achieved ZW-scores, with only one laboratory being questionable. Furthermore, the national PT conducted in the present study proved to be an effective tool for evaluating laboratory performance in IMNV molecular testing. This assessment can aid in improving the capabilities of IMNV molecular testing of the participating laboratories, and also provided technical supporting to enhance the national response to IMN outbreaks.

Evaluation of the Prevalence, Antimicrobial Resistance Trait, and Virulence Determinants in Staphylococcus aureus Isolates from the Anogenital Area of 35-37 Weeks Pregnant Women

Staphylococcus aureus is a major pathogen in the postpartum period being frequently implicated as a cause of mastitis, and nursery outbreaks, among others as well as its increasing resistance to common antibiotics. This study therefore aimed at evaluating the presence of S. aureus in pregnant women attend¬ing clinics in rural towns in Eastern Cape, South Africa. A total of 82 pregnant women at 35‒37 weeks of gestation were sampled for the presence of S. aureus. Using standard microbiological methods, presump¬tive S. aureus isolates were obtained from vaginal and rectal swab samples. The isolates were confirmed by conventional polymerase chain reaction and then tested for antimicrobial susceptibility using the disc diffusion technique. Presence of resistance genes and virulence determinants were equally profiled. A to¬tal of 47 (57.3%) out of 82 pregnant women were colonized with S. aureus. Only two virulence genes PVL- 37/47 (78.7%) and eta-10/47 (21.3%) were detected in the isolates. The isolates were all resistant to penicillin G and clindamycin (100%), while resistances to tetracycline, vancomycin, rifampicin were 71% and resistance to oxacillin and erythromycin were above 80% while resistance to other antibiotics tested were below 40%. The isolates showed multiple resistance to 5-6 antimicrobials with indices ranging from 0.5-0.6. Genes encoding resistance to erythromycin (ermB), tetracycline (tetM), and rifampicin (rPOB) were found in 72.7% (34/47) of the isolates, while 15% (7/47) possessed the Bla-Z (penicillin). The high antibiotics resistance traits found in the isolates analysed indicates limited therapeutic options and portents a major threat to public health.

Prevalence of Metallo β-Lactamase Genes Among MDR Pseudomonas <i>Aeruginosa</i> Isolated from Different Sources in Baghdad

Background: Pseudomonas aeruginosa is a gram-negative apportunistic pathogen that has ability to couse different type of infections and charecterised by their high resistance to commonly used antibiotics via different stratiges. Objective: The objective of this study is to detect the prevalence rate of MβL gene among MDR Pseudomonas aeruginosa. Methods: Forty clinical isolates of Pseudomonas aeruginosa were obtained from different hospitials in Baghdad/Iraq. Their identification was confirmed by using 16s rDNA as a housekeeping gene (reference gene). Specific primers were used to detect 3 types of Metallo β-lactamase (MβL) genes vim, spm1, and imp1 genes followed by sequencing the amplified fragment which was analyzed by Geneious software. Antibiotic sensitivity test for 15 antimicrobial agents was done using the Kirby-Bauer disc diffusion method. Results: The revealed resistance pattern was as following: 100% for the combination of Trimethoprim/Sulphamethoxazole and Nitrofurantoin, 95% for Tigecycline, 82.5% for Ciprofloxacin, 67.5% for Cefepime, 60% for Levofloxacin, 57.5% for Carbenicillin, 55% for Piperacillin, 50% for Amikacin, 47.5% for Tobramycin, 45% for both Imipenem and Ceftazidime, 27.5% for piperacillin/tazobactam, 22.5% for Aztreonam and 7.5% for Colistin. More than half (55%) of isolates were positive for MβL enzymes production during phenotypically assessed by combined disc test. The MIC of meropenem ranged from 16µg/ml to 32 µg/ml. The percentage of MβL genes among the total isolates during conventional PCR was as follows: 95% for vim, 25% of the isolates harbored both vim and imp1, while spm 1 are negative in all isolates. The result of the study explained that 10 isolates from the antibiotic susceptibility test were resistant to 10-14 antimicrobial agents that harbored both vim and imp1 genes. Conclusions: MβL gene could be used as a genetic marker to determine the degree of resistance in MDR P. aeruginosa which are more resistant than other isolates that have one gene of MβL responsible for break the β-lactam ring then inactivating the β-lactam antibiotic.

Investigation of Babesia species using molecular methods

Aims: To determine the presence of Babesia species in ticks in the Ankara region and neighboring provinces; the aim of this study was to detect and distribute Babesia species using molecular methods in ticks collected from nature and hosts. Methods: Babesia was detected at the species level by conventional PCR method by DNA extraction of 191 tick samples collected from Ankara and neighboring provinces, which applied to the Parasitology National Reference Laboratory of the Ministry of Health, General Directorate of Public Health, between 2020-2021. Results: A total of 191 tick samples were examined in the study, and as a result of the conventional PCR study, 4 Babesia agents were found positive, 1 of them was Babesia bigemina (B. bigemina), 2 were Babesia ovis, 1 was Babesia spp. were determined. Conclusion: In tick-borne diseases such as Babesiosis; it should not be forgotten that ticks continue to focus some infections in nature and serve as reservoirs for disease agents in nature, and the necessary strategies for tick control should be followed.

Biochemical and Molecular Detection of Aeromonas Hydrophila Isolated from Infected Cyprinus Carpio in Salah Al-Din Governorate

This study aimed to identify the presence of Aeromonas hydrophila bacteria in diseased common carp (Cyprinus carpio) fish collected from Salah al-Din Governorate in Iraq. For this purpose, 128 swabs were obtained from skin, gill and fin lesions of 70 fish showing symptoms of hemorrhagic sepsis, skin ulcers and boils, and fin and gill rot. Through different biochemical tests, the isolates showed negative results for Gram stain, methyl red, lactose fermentation, H2S, and string test, while positive results were recorded for glucose fermentation, oxidase, motility test, catalase, Voges-Proskauer, indole, citrate utilization, and gelatin hydrolysis. Eleven isolates were confirmed as A. hydrophila by amplification of the hemolysin gene fragment (hlyA) by conventional PCR. This study represents the first comprehensive study to characterize A. hydrophila bacteria from infected common carp fish in Salah al-Din Governorate. The findings of this study provide valuable insights for developing genetic diagnostic strategies for A. hydrophila infections in this region.

Serological and Molecular Detection of Cytomegalovirus in Aborted Women in Diyala Province, Iraq

Background: Cytomegalovirus (CMV) is a common virus that can infect individuals of all ages. In pregnant women, CMV infection can lead to complications such as congenital CMV infection, which can cause miscarriage, stillbirth, or significant fetal abnormalities. Objective: To evaluate the prevalence of CMV infection in women with and without a history of miscarriage in Diyala Province, Iraq. Methods: A total of 120 women were recruited and divided into three groups: 40 pregnant women with a history of miscarriage, 40 non-pregnant women with a history of miscarriage, and 40 healthy women without a history of miscarriage (control group). Blood samples were collected from each participant and analyzed for the presence of CMV-specific IgG and IgM antibodies using enzyme-linked immunosorbent assay (ELISA). Some positive results were retested to detect the MIE gene region IE-2 (UL122) by real-time PCR and the IE1 (UL123) gene by conventional PCR. Demographic data of study groups were recorded, and statistical analysis was performed using SPSS Version 29. Results: The serological analysis revealed the following prevalence of CMV IgG antibodies: 100% in both pregnant and non-pregnant women with a history of miscarriage, and 95% in the control group of healthy women without a history of miscarriage. For CMV IgM antibodies, the prevalence was 47.5% in pregnant women with a history of miscarriage, 40% in non-pregnant women with a history of miscarriage, and 7.5% in the control group of healthy women without a history of miscarriage. The PCR results showed that 9 of the samples were positive for both the UL122 and UL123 genes. Conclusion: The study indicates a high prevalence of CMV infection among women with a history of miscarriage in Diyala Province, Iraq. Routine screening for CMV in pregnant women, especially those with a history of miscarriage, is recommended to reduce potential risks and improve pregnancy outcomes.

Histidine-rich protein (hrp) 2-based RDT false-negatives and Plasmodium falciparum hrp 2 and 3 gene deletions in low, seasonal and intense perennial transmission zones in Cameroon: a cross - sectional study.

False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.

Enhancing Conventional PCR for Detection of Erwinia amylovora

Polymerase chain reaction (PCR) methods, including conventional PCR (cPCR) and quantitative real-time PCR (qRT-PCR), with both plasmid- and chromosome-targeting primers, are currently the most reliable methods for detecting <i>Erwinia amylovora</i> due to their high sensitivity and specificity. Despite qRT-PCR’s quantitative advantage, cPCR remains an attractive method to detect this bacterium in initial screenings of suspected host plants, as it is cost-effective and does not require skilled personnel in well-equipped laboratories. This study aimed to significantly improve cPCR robustness via application of bovine serum albumin (BSA) as a PCR facilitator, with a modified EaF/R primer pair, as previously reported. Experiments have shown that simple supplementation with BSA (10 mg/ml) enhances cPCR reactions using templates such as genomic DNA, bacterial cells, and infected symptomless host organs, including immature apple fruits and seedlings, with EaF/R primers. The cPCR method described in this study is simple, specific, and reliable, and can be applied in routine assays to diagnose fire blight.

Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a/13a Coupled with Isothermal Amplification.

The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.

Virulence factors associated with hyper virulent Klebsiella pneumoniae isolated from Different Clinical Sources

Klebsiella pneumonia is an opportunity pathogen causing several diseases including sepsis, pneumonia and wound infections. There are two pathotypes of Klebsiella pneumonia: classical K. pneumoniae (cKp) and hypervirulent K. pneumonia (hvkp). In this study, we attempted to discover the extent occurrence of hypervirulent K. pneumoniae strains among patients in Kirkuk city and their virulence factors. A total of 150 samples were collected from different hospitals in Kirkuk city (Kirkuk General Hospital, Azadi Teaching Hospital, Gynecological and Pediatric (Al-Nasr) Hospital and Children Hospital) during the period between November 2021 to June 2022. The age of patients ranged between 20- 60 years with both sexes. The results showed that 23.33% of K. pneumoniae isolates were positive for string test. Similar results were obtained by using sedimentation assay. The capsule of hvKp is a key virulence factor aiding in the bacterial dissemination and infection. For this reason, in current study, the capsular polysaccharide (CPS) was extracted and quantified. The results showed excessive production of capsular polysaccharide as seen in Klebsiella isolates 16,22,23,24,25,26,28 with an amount 49,41,50,74,49,49,73 μg/ml of CPS respectively. Siderophore production was also examined by using M9 minimal salts media. The results revealed that all K. pneumoniae isolates (100%) were able to produce siderophore. In addition, resistance to tellurite was also identified by using tellurite containing selective media and the results showed that 50% of isolates were resistant to tellurite and the remaining 50% were susceptible. A molecular study using conventional PCR revealed that 21 out of 30 isolates (70%) harbored the rmpA gene while the remaining 9 isolates (30%) lacked this gene. The presence of the iucA virulence gene was also detected and found to be present in all the isolates. Additionally, our findings indicated that 7 out of the 30 isolates (23%) were classified as hvkp.

Staphyloxanthin and Meropenem Effects on Some Capsular Genes Expressions of Klebsiella pneumoniae Isolated from UTIs

Klebsiella pneumoniae has become one of the most common etiologic agents in the context of recurrent UTI. Capsule is the earliest recognized factor determining bacterial pathogenicity. This study aimed to test the effects of staphyloxanthin, meropenem and a combination of them on capsule genes of Klebsiella pneumoniae. Eighty-eight gram negative bacterial isolates were collected from 150 urine specimens between September and November of 2022.after cultivation on MacConkey agar medium. Oxidase (negative) and catalase (positive) tests were performed. In addition, all isolates were found to be non-motile when their motility was further examined. The results presented that 77 (51.3%) could ferment lactose (lactose fermenter), while 30 isolates (20%) of them were confirmed as K. pneumonia. The antibiotics sensitivity test of (30) K. pneumoniae revealed that 28 (93%) of isolates were sensitive to meropenem, and about 20 (67%), 16 (53%), 8 (27%), 7 (23%), 5 (17%) and 4 (13%) were sensitive toward amikacin, ciprofloxacin, trimethoprim, cefepime, tetracycline and Augmentin respectively. S. aureus (producers of staphyloxanthin) isolates were taken; the staphyloxanthin pigment was extracted, purified by Millipore filter (0.22 mm). Conventional PCR results revealed the presence of galF gene (141 bp) in all eight tested isolates (100%). In addition, the wzi gene (177bp) was found in seven isolates (87.5%). According to the quantitative real-time PCR results, most treatments resulted in downregulation of galF and wzi genes expression, whereas some treatments resulted in upregulation.

Evaluation of the Expression of SIM and NDM Genes in Pseudomonas Aeruginosa Isolated from Clinical Sources

Pseudomonas aeruginosa has recently been labeled a major threat to public health due to its resistance to almost all commonly used antibiotics. Many factors have been suggested for P. aeruginosa's antibiotic resistance. The goal of this study is to find out what role SIM and NDM, which are related to carbapenem resistance, play in P. aeruginosa isolates from local clinical sources. In this study, out of 110 different clinical specimens, 50 were identified as P. aeruginosa from hospitalized patients. All of the isolates were characterized based on the biochemical test and confirmed using the VitekII compact system. P. aeruginosa isolates were tested for antibiotic susceptibility using 8 antibiotics, including: amikacin, tobramycin, ciprofloxacin, levofloxacin, imipenem, meropenem, piperacillin/tazobactam, ticarcillin, and clavulanate. Susceptibility testing results revealed that every isolate was highly resistant to Piperacillin/Tazobactam, with lower resistance to Ticarcillin/clavulanate (TCC). Using the broth dilution method, the minimum inhibitory concentration (MIC) of P. aeruginosa isolates resistant to meropenem ranged from 32µg/100µl to 128 µg/100µl. The EDTA combined disc test was used to detect the ability of P. aeruginosa isolates to produce carbapenemase, and the results showed that all isolates were carbapenemase producers. Additionally, conventional PCR confirmed the identification of P. aeruginosa using 16S. Real-time PCR was adopted to assess the expression of the NDM and SIM genes in 25 of the identified P. aeruginosa isolates. The mean of gene expression results for NDM showed increased expression compared to the control sample of 1.74, while the SIM gene showed expression of 0.95. These genes, SIM and NDM in class B (which are important for resistance in Pseudomonas aeruginosa), result from chromosomal changes that mutate the membrane permeability flow pump, causing excessive expression.

Molecular identification of EhCRT gene Calreticulin isolated from children infected with Entamoeba histolytica.

To detect the gene of entamoeba histolytica by polymerase chain reaction and investigate the expression of immunogene entamoeba histolytica calreticulin in stool samples of infected patients. The case control study was conducted at Central Teaching Hospital of Paediatrics and Al Mahmoudia General Hospital, Iraq, from December 30, 2020, to September 1, 2021, and comprised diarrhoeal faecal samples collected from 86 children with age ranging from ˂1 year to 13 years who were suspected of having been infected with entamoeba histolytica. Microscopically positive samples were then subjected to conventional and real-time polymerase chain reaction for the detection of entamoeba histolytica HM1:IMSS strain using Phage shock protein (Psp) gene sequences and detection of entamoeba histolytica calreticulin expression. Of the 86 patients, 71(82.6%) were found to be infected with entamoeba histolytica; 39(54.93%) boys and 32(45.07%) girls. The remaining 15(17.4%) patients were taken as non-amoebic controls; 8(53.3%) boys and 7(46.7%) girls. There were 36(50.70%) cases and 8(53.33%) controls aged 1-4 years. Among the Entamoeba histolytica gene was detected in 44(62%) of the cases using conventional polymerase chain reaction, and immunogene entamoeba histolytica calreticulin was expressed in 36(50.7%) using real-time polymerase chain reaction. Data was analysed using SPSS 24. Polymerase chain reaction was found to be a useful tool for diagnosing entamoeba histolytica infection in children.

Development of point-of-need colourimetric, isothermal diagnostic assays for specific detection of Bacillus subtilis using shikimate dehydrogenase gene.

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500pg/µL. DNA concentration as low as 10pg/µL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100pg/µL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

Genotyping of Hydatid Cyst Isolated from Human and Domestic Animal in Babylon Province

The study involved 200 feces samples of cattle and sheep, in Babylon Governorate in Iraq. Used for diagnosis of hydatid cyst, polymerase chain reaction (PCR), using specific primers and the method was the conventional PCR. Cattle and sheep was classified into subgroups according to age, area and sex in this study association of these risk factors with rate of infection. The diagnosis of hydatid cyst in cattle and sheep was determined by (pcr). study which are collected from human, sheep, cattle The prevalence of Hydatid Cysts infections was highest in Al-Qassim and western hamza districts which were 36.36b % (40) of cattle, 22.5a% (9) of sheep and 17.5a % (7) of cattle , 22.72b% (25) of sheep respectively , while the prevalence of Hydatid Cysts in Al-Hilla District was 18.18ab% (20) of cattle, 37.5c% (15) of sheep and the lower prevalence of was in Al-Musayyab and Al-mahaweel district which was 9.09a % (10) of cattle, 12.5ab % (5) of sheep and 13.63ab % (15) of cattle , 10b % (4) of sheep respectively. The results of present study were exhibited on high prevalence of in cattle and sheep of some districts in Babylon province. The statistical analysis recorded that there was Significant difference (at P<0.05) difference in the prevalence of parasite among geographical areas. In the present study the incidence of hydatid cysts was different according to age of the host. In sheep the incidence of hydatid cysts has a positive proportion to age of sheep; a high prevalence rate (45b%) was observed in age of sheep (1-2 years); while less prevalence rate observed in (>3) years ages (10a%),In cattle a higher rate (58.18b%) was recorded at ages (2-5 years) while a lower rate (3.63a%) was recorded in (>8) years with Highly significant difference at P<0.05 difference in the prevalence of parasite between age intervals. The result of infection with hydatidosis according to sex revealed that Females were exposed to infection more than males, with no significant difference at P<0.05 in sheep and cattle.

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the New Root-lesion Nematode Pratylenchus dakotaensis on Soybean.

Root-lesion nematodes, Pratylenchus spp. are reported to cause serious yield losses in various crops including soybean. A new root-lesion nematode species was recently detected in a soybean field in North Dakota (ND) and named Pratylenchus dakotaensis. Nematode detection and differentiation from other species are critical in management strategies. Thus, a recombinase polymerase amplification (RPA) assay was developed for rapid detection of this nematode from field soils under isothermal conditions. New primers and probes were designed from ITS-rDNA region of the nematode genome and tested for both specificity and sensitivity. The RPA assay was able to detect DNA from a single adult nematode at 39.5°C in 20 minutes using both Basic and Exo kits. The specificity of the primers was initially confirmed through in silico analyses, followed by laboratory tests. The assay successfully amplified DNA from the target species, while no amplification occurred for other Pratylenchus spp. and non-Pratylenchus control species. Sensitivity testing with real-time RPA revealed its ability to detect DNA in dilutions equivalent to 1/32 of a single nematode from DNA extracted from inoculated sterile soil. To further validate the assay, it was tested with 19 field soil samples collected in ND. This assay amplified soil DNA extracts of all P. dakotaensis-infested field samples confirmed through conventional PCR. It did not amplify DNA from 13 other field soils infested with other Pratylenchus spp. This is the first report of RPA develoment for detecting a root-lesion nematode species. The RPA assay developed can help in the rapid detection of this nematode species for effective nematode management.

Molecular Characterization of Human Adenoviruses Associated with Pediatric Respiratory Infections in Omdurman, Sudan

Acute respiratory viral infection (ARVI) is a common cause of morbidity and mortality especially in pediatric patients. Among important respiratory pathogens are adenoviruses, The illnesses can range from the common cold to pneumonia Depending on the type, adenoviruses can cause other illnesses such as gastroenteritis, conjunctivitis, cystitis, and, less commonly, neurological disease. Aim of study: molecular detection of respiratory infection due to adenovirus among children in Khartoum state. Respiratory specimens were collected from children’s patients with RTIs in the emergency department Mohamed Alamin Hamid pediatric hospital Omdurman in Khartoum state during the period of October 2022 to February 2023.the HAdV infection were determined by PCR technique. A total of 50 nasal swap were investigated for HAdV, throughout th PCR technique by using specific primer. The results showed 18 (36.0%) were positive and 32 (64.0%) were negative for HAdVs. The statistical analysis revealed no significant difference between age groups (X2= 0.786 P. value: 0.375). This study summarized the HAdV were the predominant types identified in children less than five years intend to have respiratory tract infection according to clinical symptoms of upper or lower respiratory infections.by using conventional PCR for identify with highly sensitively.

Antibiotic Susceptibility Pattern of Pseudomonas aeruginosa and Detection of Virulence Genes in a Tertiary Care Hospital, Kathmandu, Nepal

Pseudomonas aeruginosa is one of the most common causes of hospital-acquired infections. It threatens worldwide public health because of its intrinsic antibiotic resistance and virulence. In this context, this research aimed to determine the occurrence of P. aeruginosa in a clinical setting, examine its susceptibility to antibiotics, and detect the presence of virulence genes, viz. exoY, oprL and toxA in all isolates. It was a cross-sectional study carried out from June 2022 to December 2022. A total of 1356 clinical specimens were collected and processed in the laboratory for Gram staining, culture techniques, and biochemical tests. The Kirby-Bauer disk diffusion method was applied to examine the antibiotic susceptibility pattern and conventional PCR was used to detect the virulence genes in all confirmed P. aeruginosa isolates. 40.5% of specimens showed bacterial growth, of which only 3.02% were identified as P. aeruginosa. Among them, 61.0% were multidrug resistant and 29.2% were β-lactamase producers. Aztreonam (70.7%) was the most effective antibiotic. Among all P. aeruginosa isolates, 68.3% isolates were exoY positive, 61.0% were oprL positive and 56.1% were toxA positive. Strategic interventions are required to stop the emergence and spread of antimicrobial resistance as a result of the isolation of multidrug resistant (MDR) isolates carrying these virulence genes. This study can benefit the medical staff and the entire community in building a surveillance system and enhancing infection control procedures.