Polymerase Chain Reaction Examination Research Articles

Desain Primer untuk Deteksi Gen Diphtheria Toxin Repressor (dtxR) sebagai Biomarker Bakteri Corynebacterium diphtheriae Menggunakan In Silico PCR

Corynebacterium diphtheriae is the bacteria that causes diphtheria. The virulence factor of C. diphtheriae comes from the bacteria's ability to produce bacterial toxins. Toxin production is regulated by a set of genes called tox/dtx genes and is regulated by the dtxR gene. The aim of this study was to design primers used to evaluate the dtxR gene using bacterial DNA sequences. This research is experimental research with a literature study approach using the In silico Polymerase Chain Reaction (PCR), NCBI (National Center for Biotechnology Information), Primer3Plus, and Oligo Calculator applications. The sample obtained from genbank NCBI was C. diphtheriae dtxR gene M80337.1. In silico PCR examination was carried out using newly designed primers from Primer3Plus with 50 genomic DNA of Corynebacterium spp. taken from the In silico PCR database. The dtxR primer pair: '5-ACAGTTAGCCAAACCGTTGC-3' and 5'-TGCGTTCAACTTCGTCACTC-3' can produce a single DNA amplicon measuring 226 bp specifically for C. diphtheria types and no amplicon bands were generated from other Corynebacterium genomes. Based on the study results, this pair of specific primers can be used for in vitro PCR testing and can be used to develop rapid detection of diphtheria.

Clinical, Haemato-biochemical Evaluation and Comparison of Polymerase chain reaction with Conventional Blood Smear Examination for the Detection of Babesia gibsoni Infection in Dogs

The objective of the present study was to detect Babesia gibsoni infection in dogs which are presented to Veterinary Clinical Complex, Rajiv Gandhi Institute of Veterinary Education and Research, Puducherry. A total of 85 dogs with clinical signs such as inappetence, fever, enlarged lymph nodes and tick infestation were screened. The diagnostic efficacy of polymerase chain reaction (PCR) technique has been compared with blood smear examination to detect B. gibsoni infection in dogs. By blood smear and PCR examination, six (7.1%) dogs and 28 (42.4%) dogs were positive for B. gibsoni infection respectively. Clinical examination of dogs with B. gibsoni showed tick infestation, anorexia, dullness, pyrexia, pale/congested conjunctival mucus membrane, tachypnoea and swollen lymph nodes. In addition, few dogs had icterus and haemoglobinuria. Laboratory examination of blood samples of B. gibsoni dogs revealed significant reduction in haemoglobin, PCV, erythrocyte count, platelet count and serum albumin levels. Significant increase in levels of ALP, globulin, bilirubin, BUN and creatinine were recorded.

A comprehensive study of Helicobacter pylori infection: molecular analysis, antibacterial susceptibility, and histopathological examination.

Helicobacter pylori is a pathogen associated with gastroduodenal diseases. This study aimed; (i) to investigate H. pylori presence by invasive tests in adult dyspeptic patients, (ii) to determine antibiotic susceptibility and genotypic characteristics of the H. pylori isolates, and (iii) to investigate the relationship between the H. pylori genotypes and the histopathological findings. In this cross-sectional study, gastric biopsy samples from 208 adult dyspeptic patients were used for culture, tissue Polymerase Chain Reaction (PCR), and histopathological analysis. Antibiotic susceptibility of the H. pylori isolates was analyzed by gradient method. Analysis of the virulence genes was performed by monoplex PCR. Genetic profiles (from A to H) were created based on the virulence genes presence. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) was used for the genotyping of the H. pylori isolates. The mean age of the patients was 46 (± 15) years and 128 (61.5%) of them were female. H. pylori positivity was detected by culture, tissue PCR and histopathological examination in 59 (28.4%), 114 (54.8%) and 81 (38.9%) patients, respectively. The overall prevalence of H. pylori was found to be 63% (131/208). All H. pylori isolates were susceptible to tetracycline and amoxicillin. The resistance rates for metronidazole, clarithromycin, levofloxacin, and rifampicin were 67.2%, 27.9%, 34.4% and 13.11%, respectively. Multi drug resistance (MDR) was detected at the rate of 45.9% (28/61). While the most common virulence gene was cagA (93.44%), the least common was vacAm1 (23%). The predominant genetic profile was profile A (47.5%). ERIC-PCR results revealed a total of 26 different patterns. A high prevalence of H. pylori was detected in adult dyspeptic patients as in developing countries. It was observed significant genotypic heterogeneity and virulence gene diversity within the isolates. A considerable resistance rate detected against antibiotics such as clarithromycin, metronidazole, and levofloxacin, which are frequently used in the eradication of H. pylori, should be taken into consideration when creating regional empirical treatment regimens.

Morphological and molecular characteristics of Plasmodium juxtanucleare in layer chicken from three districts of Yogyakarta, Indonesia.

Blood parasite infections in poultry, such as Plasmodium, are a serious threat to the poultry industry due to their potential to cause economic losses. To date, there has been inadequate research on the morphological and molecular detection of the different Plasmodium species that infect poultry in Indonesia. Therefore, this study aimed to analyze the morphological and molecular characteristics of Plasmodium spp. and the several predisposing factors for Plasmodium infection in layer chickens from three districts of Yogyakarta, Indonesia. One hundred and five blood samples from layer chickens were collected from 13 farms located in three districts of Yogyakarta (Sleman, Bantul, and Kulon Progo) between September and November 2022. Blood samples were subjected to microscopic and polymerase chain reaction (PCR) analyses. Sequencing was performed using basic local alignment search tools to identify the nucleotide structure of cytochrome b. Phylogenetic analysis of Plasmodium was performed using the MEGA-X software. Microscopic examination revealed that 17/105 positives (16.19%) were positive for blood parasite infection. Trophozoites, erythrocytic meronts, and microgametocytes of Plasmodium were found in blood samples. Based on the morphological examination, the species found in the samples was close to Plasmodium juxtanucleare. Polymerase chain reaction examination revealed that 21/60 samples were positive for Plasmodium (35%). The Plasmodium species identified from the sequenced samples were proven to be P. juxtanucleare. The P. juxtanucleare from Thailand was closely related to samples (99.64%-100%) with a genetic distance of 0%-1%. In addition, age, population, and cage type were not significantly associated with Plasmodium infection. Based on microscopic and PCR examinations, the Plasmodium species found in the three districts of Yogyakarta was P. juxtanucleare. The genetic distance between samples from the three districts of Yogyakarta was closely related (0%-1%) to P. juxtanucleare from Thailand and Japan. There was no correlation between Plasmodium infection and age, cage type, or population.

Application of polymerase chain reaction (PCR) methods in distinguishing pathogenic and saprophytic Leptospira from the traditional market environment in Denpasar City

Background: Leptospirosis disease is still a health problem in Indonesia. The number of leptospirosis cases and deaths in Indonesia tends to increase. Indirect transmission of Leptospira bacteria can occur through poor environmental sanitation polluted by rat urine has the potential to cause leptospirosis. Traditional markets are good places for rats to breed. The purpose of this study was to detect the presence of contamination by pathogenic and saprophytic Leptospira bacteria in the environment around traditional markets in Denpasar City by polymerase chain reaction (PCR) method using three specific primers. Methods: The sample is water from the market environment, taken at 19 location points from eight traditional markets in Denpasar City. After being homogenized, the sample water is filtered. Isolation of specific genes from samples by the PCR method was performed to differentiate pathogenic and saprophytic leptospira using three specific primers designed from the 16S rRNA gene. Starting from the DNA extraction stage, amplification by PCR, and detection of PCR product DNA by electrophoresis. Results: This study found that from 19 water sampling locations, 5/19 (26.3%) point locations found specific DNA genes for pathogenic Leptospira bacteria. 10/19 (52.6%) point locations found specific genes for saprophytic Leptospira bacteria DNA, and 4/19 (21.1%) point locations found no specific genes for pathogenic or saprophytic Leptospira bacteria. Conclusion: PCR examination using a combination of three primers could distinguish the presence of pathogenic Leptospira, saprophytic Leptospira, and the absence of pathogenic and saprophytic Leptospira simultaneously from traditional market environments in Denpasar City.

Discrepancy of PCR and serologic test on genital herpes: a case report

Background: Laboratory investigation for genital herpes (GH) includes serologic tests and polymerase chain reaction (PCR) with different sensitivity and specificity; occasionally, the results are not conclusive. Clinical symptoms are often atypical. We report a case of GH in HIV-infected patients with a non-reactive serologic test but positive PCR.
 Case Description: A 29-years old HIV-positive man presented with multiple painful shallow ulcers with pus on his penis in the last five days. PCR examination showed a positive result, whereas anti-HSV 1 IgM, anti-HSV 2 IgM, anti-HSV 2 IgG, VDRL, and TPHA were negative. The anti-HSV- 1 IgG result was reactive with a CD4 value of 122 cells/μL. The patient was given Acyclovir 3x400 mg for ten days and Co-amoxiclav 3x625 mg for seven days. Significant improvement was obtained, and the pain subsided.
 Conclusion: Clinical features of GH in infected HIV patients are often atypical and resemble other genital ulcers, thus requiring additional investigations. The serologic test may sometimes present a false negative, whereas PCR is much more sensitive and specific, nearing 100%. In patients with HIV infection, GH diagnosis at an early stage can shorten the course of the disease and prevent complications.

First Molecular Genotyping of Cryptosporidium felis in Cattle, Iraq.

To the best of our knowledge, this is the first Iraqi study to detect C. felis in cattle by the polymerase chain reaction (PCR) assay and to confirm the local isolates in the National Center for Biotechnology Information (NCBI). Overall, 130 diarrheic calves of different ages and sexes were selected randomly from rural and sub-urban areas in Wasit province (Iraq) from February to April (2021) and subjected to direct collection of fresh fecal samples for DNA extraction and PCR examination. Targeting the heat shock protein 70 (HSP70) gene of C. felis showed that 17.69% of the animals were positive. Findings from clinical examination revealed no significant differences in values of temperature, pulse, or respiratory rates between the positive and negative calves. The association between the positive results and demographic risk factors showed no significant differences in the prevalence rate of infection and risk of exposure to C. felis between the rural and sub-urban areas; however, higher significant values were reported in calves aged 6 months than in calves aged 12 months, as well as in females than in males. Five of the local C. felis isolates were documented under the accession numbers MZ964600.1, MZ964601.1, MZ964602.1, MZ964603.1, and MZ964604.1. Finally, the data presented here provide epidemiological and molecular evidence that the range of C. felis in cattle is wider than expected globally, with a high probability of infection transmission from cattle to humans.

A Pilot Study on the Evaluation of Cryptosporidium Infection in Patients with Lung Cancer: Respiratory Cryptosporidiosis.

Lung carcinoma is one of the most common cancers and the leading cause of cancer-related death worldwide. Increasing evidence has shown that Cryptosporidium spp., an opportunistic parasite, is associated with cancers, causing life-threatening infections. The most common clinical form of Cryptosporidium is intestinal infections. However, respiratory cryptosporidiosis has rarely been documented, although the parasite infects respiratory epithelial cells and gastrointestinal (GIS) epithelial cells. To evaluate respiratory cryptosporidiosis in patients with lung cancer, we investigated Cryptosporidium spp. in patients with lung cancer (n = 69) in comparison with healthy groups (n = 40). Sputum and stool samples were examined microscopically and by polymerase chain reaction (PCR). Two cancer patients were diagnosed with respiratory cryptosporidiosis (2.9%), on PCR examination of the sputum samples. Cryptosporidium spp. was detected in the stool samples of one patient (1.5%) and 2 healthy individuals (5.4%) by PCR and microscopy. First, respiratory cryptosporidiosis was documented in 2 patients with lung cancer. Cryptosporidium is an important agent of the respiratory tract and GIS infections in cancer patients. These new findings highlight the molecular prevalence of Cryptosporidium spp., an opportunistic infection, in patients with lung cancer. Respiratory cryptosporidiosis should also be considered when patients have respiratory symptoms.

Detection of Malaria Parasites and Other Haemosporidia in Migratory and Native Birds in Mazandaran and Golestan Provinces, Iran.

A variety of haemoprotozoa including Plasmodium, Haemoproteus and Leucocytozoon cause infections in birds and are transmitted by some known vectors. These parasites cause anemia, low appetite, weakness and ultimately death in birds. The present study was aimed to determine these parasites, in birds of Mazandaran and Golestan provinces in Iran. The project was performed on 340 live birds in 2016. The samples were collected from February to September 2016, from each bird, two thin and thick blood smears were prepared and the remaining blood about 1ml was kept in EDTA-containing tubes for molecular studies. The slides were stained with 10% Giemsa, then examined microscopically. About ten percent of the negative samples were considered for Polymerase Chain Reaction (PCR) technique, using specific primers to diagnose Plasmodium and Haemoproteus spp. Electrophoresis was done for PCR products and relevant bands to the parasites were identified based on the size. The considered birds belonged to ducks, chickens, roosters, and pigeons. From 340 microscopically examined blood samples 32 (9.5%) samples were positive. Twenty-five (7.35%) of them were infected with the genus Haemoproteus. Seven samples (14%) out of 50 microscopically negative samples were found as Haemoproteus or Plasmodium spp when PCR technique was employed. This study revealed the existence of malaria parasites and other haemosporidia in birds in Iran. Employing molecular methods (PCR examination) could detect more infections.

Difference between Microscopic and PCR Examination Result for Malaria Diagnosis and Treatment Evaluation in Sumba Barat Daya, Indonesia.

Microscopic examination is the backbone of malaria diagnosis and treatment evaluation in Indonesia. This test has limited ability to detect malaria at low parasite density. Inversely, nested polymerase chain reaction (PCR) can detect parasites at a density below the microscopic examination’s detection limit. The objective of this study is to compare microscopic and PCR results when being used to identify malaria in suspected patients and patients who underwent dihydroartemisinin–piperaquine (DHP) therapy in the last 3–8 weeks with or without symptoms in Sumba Barat Daya, Nusa Tenggara Timur, Indonesia. Recruitment was conducted between April 2019 and February 2020. Blood samples were then taken for microscopic and PCR examinations. Participants (n = 409) were divided into three groups: suspected malaria (42.5%), post-DHP therapy with fever (4.9%), and post-DHP therapy without fever (52.6%). Microscopic examination found five cases of P. falciparum + P. vivax infection, while PCR found 346 cases. All microscopic examinations turned negative in the post-DHP-therapy group. Conversely, PCR result from the same group yielded 29 negative results. Overall, our study showed that microscopic examination and PCR generated different results in detecting Plasmodium species, especially in patients with mixed infection and in patients who recently underwent DHP therapy.

Presentation of Human Cytomegalovirus (HCMV) in Liver Tissues of Cholestatic Infants with Extrahepatic and Non-Extrahepatic Biliary Atresia

Introduction: Human cytomegalovirus (HCMV) is associated with cholestasis in infants. Diagnosis of HCMV infection is most often based on serological anti-HCMV. Identification of HCMV in liver tissue has been rarely reported. The aims of this study were to determine the presentation of HCMV in liver tissues and to analyze its association with serological anti-HCMV of cholestatic infants with extrahepatic and non-extrahepatic biliary atresia. Methods: This observational study was performed during December 2017- December 2018 with ethics from our institutions. The parents or guardians of subjects signed the informed consent. Anti-HCMV serological data were collected from patient medical records. Histopathological diagnosis and polymerase chain reaction (PCR) for HCMV were performed from liver biopsy tissues. The data were analyzed by Chi-square. Results: There were 47 cholestatic infants, 38.3% EBA and 61.7% non-EBA. Anti-HCMV IgM was positive in 38.3% patients and IgG was positive in 91.5% patients. Acute infection or recent infection were 38.3%, past or not acute infection were 53.1%, and uninfected or early infection were 8.5% patients. The presentation of HCMV in liver tissues was 68.1% patients, consisting of 11/18 EBA and 21/29 non-EBA and negative in 31.9% patients, consisting of 7/18 EBA and 8/29 non-EBA. There was no association between serological anti-HCMV and PCR HCMV with histopathological features. Conclusion: It suggests that PCR can be used as a routine tool to detect the presentation of HCMV DNA in liver tissue. Type of cholestasis in infants, both EBA and non-EBA, cannot be determined based on the serological and PCR examination, but based on histopathological features.

Polymorphisms of dtxR Gene of Corynebacterium diphtheriae Isolated from Diphtheria Outbreak in Indonesia

Background: In Corynebacterium diphtheriae, the dtxR gene plays a role in regulating diphtheria toxin synthesis. The dtxR gene is often used as a marker for identifying C. diphtheriae by the polymerase chain reaction (PCR) method because it is present in all strains of this bacterium. Mutations in the dtxR gene can cause the over-synthesis of diphtheria toxin and reduce PCR assays' sensitivity. Objectives: This study aimed to describe the polymorphisms in the dtxR gene of C. diphtheriae isolated from a diphtheria outbreak in Indonesia. Methods: Forty-eight isolates of C. diphtheriae were obtained from clinical samples (throat/nasopharyngeal swabs) of diphtheria cases and close contacts. The isolates were revived on a blood agar plate (BAP), bacterial colonies were harvested, and deoxyribonucleic acid (DNA) was extracted. The DNA sequencing was carried out using a Whole-genome Sequencing (WGS) approach. The data were converted and analyzed with U-gene software. The dtxR gene analysis was performed with C. diphtheriae PW8 as references. Results: There were 59-point mutation locations in 48 isolates examined. None of these single nucleotide polymorphisms (SNPs) coded for amino acid changes. Based on the mutation pattern, seven clades/groups of the dtxR gene of 48 C. diphtheriae isolates were examined. Conclusions: At least seven types of DNA sequences and more than 50 SNPs of the dtxR gene were identified in 48 C. diphtheriae isolates from a diphtheria outbreak in Indonesia. Although all of them are silent mutations, they must be considered in the design of PCR examination in diphtheria laboratories.

Molecular and histopathological detection of lumpy skin disease in Buffaloes, Iraq

The study aim is to confirm infection of Iraqi buffaloes with the lumpy skin disease virus (LSDV) using of molecular conventional polymerase chain reaction (PCR) and histopathology. Totally, 150 buffaloes of different ages and sexes at three Iraqi provinces (Wasit, Dhi-Qar and Maysan) were subjected for collection of 5 ml blood samples and for clinical examination for detection of skin lesions and collection of tick samples during 2021 to March 2022. The total positive results for conventional PCR examination of 150 buffaloes was 8 (5.33%) using the blood samples. Among totally 13 suspected LSD skin lesions, only 1 (7.69%) sample was found to be positive. Among 29 tick samples collected from the 29 infested buffaloes, the findings revealed no positive samples in these samples. Histopathological analysis of nodular skin lesion showed that there were edema, hyperemia, acanthosis, severe hydropic degeneration, and hyperkeratosis in the epidermis; whereas in the dermis, there were mononuclear cell infiltration, inclusion bodies, and vasculitis. Conclusion: In Iraq, despite vaccination programs applied since the first outbreak in 2013, LSDV remains large in persistence and distribution among most areas of the country, resulting in apparent morbidities and mortalities.

Comparison of direct and indirect methods to maximise the detection of Babesia caballi and Theileria equi infections in Central Southern Italy

Equine piroplasmosis is a disease of equids, caused by tick-borne apicomplexan protozoan pathogens Babesia caballi and Theileria equi, which, according to the World Organisation for Animal Health (OIE), can be diagnosed by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR). The present study was conducted to evaluate and compare the assays available for the diagnosis of equine piroplasmosis. Data employed were obtained from 1300 blood samples collected between 2012–2014 from asymptomatic and symptomatic equines (horses and donkeys) of central-southern regions of Italy and analyzed by ELISA, IFAT, PCR (one commercial and one from literature) and blood smear microscopic examination. Statistical differences of the proportions of positivity for each parasite and group (asymptomatic and symptomatic) among the methods were verified by the z test to identify the most sensitive. The concordance between each pair of methods – for each parasite and within the groups – and trends in detection of suspect samples of four hypothetical diagnostic algorithms using serological and biomolecular assays were evaluated to identify the most suitable laboratory diagnostic workflow.The results of this study highlighted a lower capacity to detect suspect samples of commercial ELISA for B. caballi in all groups when compared to biomolecular methods and IFAT; and of the commercial PCRs in asymptomatic animals, identifying a PCR from literature and IFAT as the best choice for a combined diagnosis. For T. equi, IFAT detected more suspect samples than ELISA, even if the latter showed good performance and some samples were positive only by the ELISA and PCR, indicating that their simultaneous employment is still advantageous. Host-parasite interaction, amino-acid/genetic diversity and differences in detection limits among the assays could be among the reasons in explaining the present results.In view of further studies, ELISA should be used in combination with PCR, that should regularly be included in the laboratory diagnosis to maximise the detection of early infections and support the evaluation of pharmacological treatment.

Recent advances in the diagnosis of intestinal tuberculosis

BackgroundIntestinal tuberculosis still has a high incidence, especially in developing countries. The biggest challenge of this disease is the establishment of the diagnosis because the clinical features are not typical. Investigations such as culture, acid-fast bacilli (AFB) staining, and histopathology have low sensitivity, so other investigations are needed. Latest molecular-based diagnostic modalities such as GeneXpert, interferon-gamma (IFN-γ) release assays (IGRA), polymerase chain reaction (PCR), multiplex-PCR, and immunological markers are expected to help diagnose intestinal tuberculosis. This article review will examine the latest diagnostic modalities that can be used as a tool in establishing the diagnosis of intestinal tuberculosis.ResultsThrough a literature search, we were able to review the diagnostic values of various available diagnostic modalities as the appropriate additional test in intestinal tuberculosis. Culture as a gold standard has a sensitivity and specificity value of 9.3% and 100% with the MGIT BACTEC system as the most recommended medium. The sensitivity values of AFB staining, histopathology examination, GeneXpert, IGRA, PCR, multiplex-PCR and, immunological markers were ranged between 17.3 and 31%; 68%; 81–95.7%; 74–88%; 21.6–65%; 75.7–93.1%; and 52–87%, respectively. Meanwhile the specificity values were 100%; 77.1%; 91–100%; 74–87%; 93–100%; 96.4–100%; and 70–95%, respectively.ConclusionThe combination of clinical examination, conventional examination, and the latest molecular-based examination is the best choice for establishing the diagnosis of intestinal tuberculosis. Most recent modalities such as multiplex PCR and immunological marker examinations are diagnostic tools that deserve to be used in diagnosing intestinal tuberculosis as their sensitivity and specificity values are quite high and more evidences are expected to support the application of these examinations shortly soon.

Bio-ecological study of Culex quinquefasciatus as a potential vector of Japanese encephalitis in some provinces in Indonesia

Culex quinquefasciatus is a mosquito known as Japanese encephalitis (JE) vector in several regions in Indonesia. The bioecological study is essential to optimize the vector control of JE. The purpose of the study was to obtain the ecological information of Cx. quinquefasciatus in 15 provinces in Indonesia: Aceh, West Sumatra, Lampung, Bangka Belitung, Banten, West and East Java, West and South Kalimantan, North and Southeast Sulawesi, East and West Nusa Tenggara, Maluku, and North Maluku. Mosquitoes were collected using the human landing catches (HLCs), light trap, and morning resting collection. The larva survey was conducted in potential habitats of Cx. quinquefasciatus. The mosquito was detected for the JE virus using the polymerase chain reaction (PCR) method. The distribution of Cx. quinquefasciatus was recorded using a GPS tool and visualized using Global Mapper. The results showed that Cx. quinquefasciatus in 15 provinces had similar behavior. The Cx. quinquefasciatus sucked blood indoors and was found throughout the night. Peak density of Cx. quinquefasciatus varies by province. Cx. quinquefasciatus breeding places are found in most ecosystems in various altitudes. Based on PCR examination, the JE virus has not been detected, thus lowering the potential for JE transmission in some provinces in Indonesia.

DIAGNOSA VIBRIO CHOLERAE DENGAN METODE KULTUR DAN PCR PADA SAMPEL SUMBER AIR MINUM

Acute diarrhea due to infection can be caused by a bacterial, viral or parasitic infection. One of the bacteria that causes diarrhea is Vibrio cholerae and usually the diarrhea caused is called cholera diarrhea. Cholera diarrhea is caused by enterotoxins produced by V. cholerae bacteria and forms colonies inside the small intestine. Symptoms include vomiting, defecation such as large amounts of rice water resulting in dehydration, electrolyte loss and increased blood acidity. In severe cases, the sufferer continuously defecates accompanied by vomiting, so that the sufferer will lose fluids and electrolytes quickly from the gastrointestinal tract. This leads to a rationing of metabolic acidity and when left untreated can lead to death. V. cholerae bacteria are not invasive, do not enter the bloodstream but remain in the intestinal tract. At the time of infection through contaminated food and beverages ingested, then after passing through the stomach acid defense V. cholerae produces two virulence factors that cause cholera, namely coregulated pilus toxin (TCP) and cholera toxin (CT). The existence of specific enterotoxin cholera only found in V. cholerae pathogens can be targeted in laboratory tests for the diagnosis of pathogenic V. cholerae bacteria using biomolecular techniques such as polymerase chain reaction (PCR) methods. From the results of the examination of drinking water samples at the drinking water depot around the bottom of the pakam, obtained the results of the PCR examination confirmed by electrophorensis is 302 bp, which means that in the sample there are bacteria that are identic with Vibrio cholera.

Estimation of limit of detection of Salmonella typhimurium in artificially contaminated chicken meat by cultured-based and polymerase chain reaction techniques

The objective of this study was to develop Polymerase Chain Reaction (PCR) procedure for detection of Salmonella Typhimurium in artificially contaminated chicken meat. The experiments were conducted with various dilutions of Salmonella Typhimurium reference the American Type Culture Collection ATCC (ATCC13311TM 4.4*107) High concentration 4.4*103 Colony Forming Units (CFU)/ml, low concentration 4.4*102 CFU/ml, very low concentration 4.4*101 CFU/ml inoculated in chicken meat, in order to determine limits of detection (LOD), optimum incubation times 18 to 20 hours of pre-enrichment in Buffered Peptone Water (BPW 1%). Hence, cultural methods and DNA extraction were performed according to kits instruction. The microbiological cultural test was capable to detect 1.76 CFU/mL, whereas PCR examination was able to detect 0.18 CFU/ml of initial dilution of Salmonella Typhimurium inoculated in chicken meat. Interestingly, the results were achieved in a less time period than that of classical culture. The PCR technique is beneficial in the methodology for detection of Salmonella in chicken meat.

The Active Surveillance of Staphylococcus aureus using Polymerase Chain Reaction-based Identification Method among Hospitalized-patient of Haji Adam Malik General Hospital, Medan, Indonesia

BACKGROUND: Active surveillance of methicillin-resistant Staphylococcus aureus (MRSA) carriers is associated with the lower incidence of bacteremia and lower mortality rates throughout literature; yet, this important step still remains problematic for developing countries, particularly Indonesia. AIM: The study aimed to demonstrate MRSA colonization rate in Haji Adam Malik Hospital, Medan, Indonesia. MATERIALS AND METHODS: The study enrolled 200 mucocutaneous isolates obtained from hospitalized patients during a 1-year period of study (2018). VITEK-2 system in addition to standard bacterial identification, such as gram staining, latex agglutination test, and hemolysis pattern, was performed to select S. aureus colonies in two different laboratories, Microbiology laboratory of Haji Adam Malik General Hospital and Multidisciplinary Laboratory, Faculty of Medicine, Universitas Sumatera Utara, for polymerase chain reaction (PCR) examination. RESULTS: Based on the VITEK-2 system preliminary identification, there were 80 S. aureus colonies which then underwent PCR examination. Through standard PCR assay, there were 32 bacterial isolates contained the mecA gene and it can be determined MRSA colonization rate of the hospital was 16% with consistent results of standard bacterial identification. CONCLUSIONS: Active surveillance of MRSA carriers is mandatory and urged it as a regular program in a hospital setting to decrease MRSA transmission rate.

Coinfection of Chlamydia spp. and herpesvirus in juvenile farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

Background and Aim:For a decade, chlamydial and herpesvirus infections have caused significant morbidity and mortality in farmed crocodiles. In September 2017, a total of 160 juvenile freshwater Siamese crocodiles (Crocodylus siamensis) with conjunctivitis/pharyngitis lesions were admitted at the Veterinary Aquatic Animal Research Health Care Unit, Faculty of Veterinary Science, Mahidol University. All crocodiles did not respond well to antibiotics or supportive treatments and died. This study aimed to detect and identify the causative agents associated with conjunctivitis/pharyngitis and fatal outcomes in juvenile farmed Siamese crocodiles.Materials and Methods:A total of 138 pharyngeal and conjunctival swabs and conjunctival scrapes were collected from live crocodiles. All swab and scrape samples were DNA-extracted and amplified by polymerase chain reaction (PCR) using Chlamydiaceae- and herpesvirus-specific primers. Tissue samples (brain, lung, liver, heart, spleen, and intestine) were collected from two representative postmortem animals. All tissue samples were processed for molecular and pathological analyses.Results:PCR examinations identified chlamydial and herpesvirus DNA in 92% (126/138) and 100% (138/138), respectively, of the tested swab and scrape samples. Of those positive samples, 79% (26/33), 67% (4/6), and 98% (97/99) of the pharyngeal swabs, conjunctival swabs, and conjunctival scrapes, respectively, were positive for both chlamydial and herpesvirus DNA. Histopathological examination indicated necrosis and mononuclear cell infiltration in the liver, kidney, and intestine of the affected animals. The intracytoplasmic accumulation of Chlamydia was randomly observed in the examined tissue sample. Moreover, the presence of chlamydial and herpesvirus DNA was also detected in the tissue samples, including the heart, intestine, brain, lung, liver, and spleen, of the affected animals by PCR. Phylogenetic analyses revealed that Chlamydia spp. detected in the juvenile Siamese crocodiles was notably different from other known species in the Chlamydia genus, while the herpesvirus detected in the crocodiles was closely related to crocodyline herpesvirus 1.Conclusion:Based on histopathological and molecular examinations, this report provided the first evidence of coinfection of Chlamydia spp. and crocodyline herpesvirus 1 in juvenile Siamese crocodiles in Thailand.