Corrigendum to “Primary and Nested PCR Amplification of B1 Gene to Confirm Seropositivity of Toxoplasmosis Among Cancer Patients in Sri Lanka”

To explore epidermal growth factor receptor (EGFR) and HER2 gene status, to assess the correlation between EGFR and HER2 gene status, and to investigate the role of copy number increase and amplification of EGFR gene and HER2 gene in the tumorigenesis and disease progression of non-small-cell lung cancer. Using Path Vysion kit and LSI EGFR SpectrumOrange/CEP7 Spectrum Green probes, EGFR gene and HER2 gene status were evaluated by fluorescence insitu hybridization (FISH) using formalin-fixed, paraffin-embedded samples from 31 patients with non-small-cell lung cancer, including 20 adenocarcinomas, 2 squamous cell carcinomas, 2 large cell carcinoma, 4 bronchoalveolar carcinomas and 3 adenosquamous carcinomas. The correlation between EGFR and HER2 gene status was analyzed. Six of thirty-one carcinomas showed EGFR gene amplification. Of 25 cases without EGFR gene non-amplification, four tetrasomy and 5 polysomy were detected. Overall, 15 out of 31 carcinomas demonstrated either EGFR gene copy number increase or gene amplification (15/31). HER2 gene amplification was seen in 2 of the 31 cases. Four trisomy, one tetrasomy and nine polysomy cases were found in 29 tumors that had no HER2 gene amplification. Overall, 16 of 31 cases showed either HER2 gene copy number increase and/or amplification (16/31). Synchronous EGFR and HER2 gene numerical changes, i.e. gene copy number increase and gene amplification, were found in 12 of 31 cases (12/31), and almost all such patients had either clinical stage III or IV tumor. EGFR gene numerical changes significantly correlated with HER2 gene abnormality (chi(2)(Adj) = 7.3045, P = 0.0069). EGFR or HER2 copy number increase is much more frequent than gene amplification in no-small-cell lung cancer. Our data based on gene alterations indicate, for the first time, that there is a significant correlation between EGFR alterations and HER2 abnormalities. Both genes are involved in the tumorigenesis and development of lung cancer. EGFR/HER2 dimer is one of the predominant heterodimerization types in lung cancer. The interactions between EGFR and HER2 may play a rule in the progression of non-small-cell lung cancer.

The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene.

In the present study, Mycoplasma gallinaceum was detected by PCR amplification of 16S rRNA gene from chronic respiratory disease in village chickens of Cauvery delta region of Tamil Nadu. Necropsy was performed to find out the etiological agent in desi birds mortality. At necropsy, airsacculitis with caseous exudate were found in the thoracic and abdominal cavity. Caseous material from airsacs was collected aseptically from dead birds for detection of Mycoplasma species. DNA was extracted from caseous material by using tissue DNA extraction kit. PCR was carried out using primers to amplify 16S rRNA gene belonging to Mycoplasma species. The amplified product yielded approximately 700-bp length (703 to 713 bp) of the 16S rRNA gene specific for Mycoplasma species. Further, it was subjected to sequence analysis and confirmed as Mycoplasma gallinaceum by NCBI blast analysis. In the present communication, detection of M. gallinaceum by PCR amplification of 16S rRNA gene provides a powerful tool for rapid diagnosis.

To investigate the clinicopathological features of patients with lung cancers associated with epidermal growth factor receptor (EGFR) gene amplification and/or mutations. Mutations and amplification status of EGFR gene were detected by PCR-DNA sequencing and FISH, respectively, followed by subsequent clinicopathological correlative studies. Among 454 patients, the overall mutation rate of EGFR was 48.2% (219/454). The EGFR mutation rate in females was significantly higher than that of males, 59.6% (118/198) vs. 39.5% (101/256), P<0.001. The mutation rate of EGFR gene of non-smokers was higher than that of smokers, 52.7% (147/279) vs. 41.4% (72/175), P=0.017. The mutation rate in patients with adenocarcinoma was higher than that in patients with other cancer types, 56.8% (193/340) vs. 22.8% (26/114), P<0.05. Moreover, a significant difference of mutation rates among different subtypes of adenocarcinomas was found (P=0.001). Among 134 patients with available FISH analysis, no statistical significance of EGFR gene amplification was found in age, gender, histopathological types and subtypes of adenocarcinomas (P>0.05). There was a significant correlation between EGFR mutation and its gene amplification (P=0.0005), although with poor consistency (P=0.275). EGFR gene mutations occur more frequently in females, non-smokers and patients with adenocarcinoma subtype. A significant variation of the mutation types exits among the subtypes of adenocarcinoma. The presence of EGFR amplification appears not related to age, gender, histopathological types of lung cancer and subtypes of adenocarcinoma. There is a significant correlation between EGFR mutations and its gene amplification (P=0.0005), although with poor consistency.

PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.

The aim of this work was to test the feasibility of introducing an anaerobic microbial reductive dechlorination activity into non sterile soil slurry microcosms by inoculation with the pure anaerobic bacterial strain Desulfomonile tiedjei, which is capable of dechlorinating 3-chlorobenzoate to benzoate. To show that the bacterium was established in the microcosms we followed the expression of the reductive dechlorination activity and a molecular probe based on PCR amplification of the 16S rDNA gene was developed. However, the success of PCR amplification of the 16S rDNA gene depends on the DNA extraction and purification methodologies applied, as shown through the use of several protocols. In this study we report a DNA extraction and purification method which generates sufficient and very clean DNA suitable for PCR amplification of the D. tiedjei 16S rDNA gene. The threshold of detection was about 5.10(3) bacteria per gram of soil slurry. Introduction of D. tiedjei in soil slurry microcosms proved successful since 3-chlorobenzoate dechlorination activity was established with this bacterium in microcosms normally devoid of this dechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetate plus formate as cosubstrate, at their respective concentrations of 5 and 6 mM, led to a total biotransformation of 2.5 mM of 3-chlorobenzoate within 12 days. After complete 3-chlorobenzoate dechlorination, the 16S rDNA gene of this bacterium was specifically detected only in the inoculated microcosms as shown by PCR amplification followed by restriction mapping confirmation.

Isolation and identification of mycobacteria in New World primates maintained in captivity

Multiple different biologically and clinically relevant genes are often amplified in invasive breast cancer, including HER2, ESR1, CCND1, and MYC. So far, little is known about their role in tumor progression. To investigate their significance for tumor invasion, we compared pure ductal carcinoma in situ (DCIS) and DCIS associated with invasive cancer with regard to the amplification of these genes. Fluorescence in situ hybridization (FISH) was performed on a tissue microarray containing samples from 130 pure DCIS and 159 DCIS associated with invasive breast cancer. Of the latter patients, we analyzed the intraductal and invasive components separately. In addition, lymph node metastases of 23 patients with invasive carcinoma were included. Amplification rates of pure DCIS and DCIS associated with invasive cancer did not differ significantly (pure DCIS vs. DCIS associated with invasive cancer: HER2 22.7 vs. 24.2%, ESR1 19.0 vs. 24.1%, CCND1 10.0 vs. 14.8%, MYC 11.8 vs. 6.5%; P > 0.05). Furthermore, we observed a high concordance of the amplification status for all genes if in situ and invasive carcinoma of individual patients were compared. This applied also to the corresponding lymph node metastases. Our results indicate no significant differences between the gene amplification status of DCIS and invasive breast cancer concerning HER2, ESR1, CCND1, and MYC. Therefore, our data suggest an early role of all analyzed gene amplifications in breast cancer development but not in the initiation of invasive tumor growth.

Objective To investigate the antibiotic resistance and resistance genes of carbapenem-resistant Enterobacteriaceaes (CRE) isolated from 5 hospitals in Northeast China. Methods This study collected 85 CRE isolates during January 2013 to June 2015 from five hospitals in Northeast China. Drug sensitivities of 14 antimicrobial agents were determined by the broth microdilution method. The phenotypes of carbapenemases were screened by modified Hodge test and EDTA test respectively. The genotypes of carbapenemases and other extended spectrum β-lactamases (ESBL) were detected by PCR gene amplification and DNA sequencing method. Using the PCR result as gold standard, the performances of other two carbapenemase detection methods were evaluated. Results Among the 85 CRE strains collected in this study, Klebsiella pneumoniae was the most frequently isolated species (61/85, 71.8%). The results of antimicrobial agent sensitivity showed that the 85 CRE strains had resistance rate of cephalosporin and β-lactams/enzyme inhibitor (piperacillin-tazobactam) over 80.0%. The resistance rate of carbapenem was high, with ertapenem 100.0% (85/85), meropenem 65.9% (56/85), imipenem 71.8% (61/85). There were 36 isolates resistant to both meropenem and imipenem. For fluoroquinolones, the resistance rates of levofloxacin and ciprofloxacin were 72.9% (62/85) and 65.9% (56/85), respectively. The resistance rate to fosfomycin and amikacin were 65.0% (55/85) and 54.1% (46/85), respectively. The resistance rate of colistin (21.2%, 18/85) and tigecycline (20.5%, 17/85) were low. Forty-nine strains were modified Hodge test positive and 12 strains were EDTA test positive. By PCR gene amplification and DNA sequencing method, 64 strains carried carbapenemase-encoding genes, of which KPC-2 was the main type (53/85, 62.4%), followed by IMP-4 (10/85, 11.8%), NDM-5 (7/85, 8.2%) and NDM-6 (1/85, 1.2%). At the same time, 85 CRE isolates had the ESBL gene detection and 47 isolates were CTX-M type ESBLs (47/85, 55.3%), with no TEM or SHV type. Conclusions Klebsiella pneumoniae is the majority of CRE strains from 5 large hospitals in Northeastern China. The CRE strains are resistant to most of antimicrobials. Most carbapenemases-producing isolates have the KPC-2 type. Nearly half of the carbapenemase-producing strains also carry ESBL genes, which makes the resistance mechanisms more complicated. Key words: Enterobacteriaceae; Carbapenemase; Extended-spectrum β-lactamases

Objective To compare HER-2 state in breast cancer tissue deteced by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) and analyze their correlation. Methods HER-2/neu protein expression and gene amplification were detected by FISH and IHC in 56 newly-diagnosed cases of female breast cancer from July 2008 to July 2009. Results Of the 56 patients, HER-2 protein expression (-), (+), (++), (+++) was 9 cases (16.1%), 29 cases (51.8%), 11 cases (19.6%) and 7cases (12.5%) respectively; 26 cases (46.4%) had HER-2 gene amplification while 30 cases (53.6%) didnt have. Type of HER-2 gene amplification was mainly HER-2(++) and HER-2(+++), and according gene amplification rate was 72 7% and 100%. HER-2 (+) gene amplification rate was 37.9 %(11cases) and no gene amplification was found in HER-2(-) tissue. The HER-2 positive rate using two methods had significant difference(χ2=19.778,P<0.01). HER-2(-) and HER-2(+++) had good consistency with the FISH results(Kappa=0.969),but HER-2(+) and HER-2(+ +) were poorly consistent with the FISH results(Kappa=0.271). Conclusions IHC is the preliminary screening method for detection of HER-2 expression. HER-2(-) and HER-2(+++) have good consistency with the gene amplification, and can guide clinical treatment. Some patients with HER-2(+) and HER-2(++) have HER-2 gene amplification. FISH is needed for targeted therapy. Key words: Breast cancer; HER-2/neu gene; Immuno-histochemistry; Fluorescent in situ hybridization

Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed.

Arcanobacterium canis is a novel species of the Arcanobacterium most closely related to A. haemolyticum. This study aims to characterize two A. canis isolates recovered from companion animals, specifically the claw of a cat and a vaginal swab from a dog. This study used real-time PCR to characterize A. canis isolated from companion animals. Two isolates of A. canis were recovered from purulent material from the claw of an 11-year-old cat in Germany and a vaginal swab of a dog in the United Kingdom. The samples were characterized phenotypically and genotypically. Both isolates were analyzed using culture methods, biochemical analysis, MALDI-TOF MS, real-time PCR amplification and sequencing of the 16S rRNA gene, and rpoB, gap, and tuf genes. The findings showed that the isolates P5197-15 and M214-96-1 obtained from companion animals were successfully characterized and confirmed to species level by real-time PCR amplification and sequencing of the 16S rRNA gene, as well as the genes of rpoB, gap, and tuf. This study seeks to comprehensively understand the characteristics of A. canis isolates obtained from companion animals. Such knowledge is essential for accurate diagnosis, treatment, and control of infections caused by this pathogen in veterinary medicine. Additionally, it contributes to the broader understanding of the genetic diversity and characteristics of A. canis, which can have implications for public health and animal well-being.

Objective: Molecular identification and antibiotic susceptibility evaluation of Vibrio cholerae from marine fish available in local fish market Thanjavur, Tamil nadu, India.Methods: inoculation was done by using nutrient agar as general media and TCBS agar as selective media and confirmed as V.cholerae by Gram stain (Microscopic Observation), Growth characteristics of different media, Biochemical tests like Methyl Red Test, Nitrate Reduction Tests, Indole Test etc. Sensitivity (drug sensitivity) was done in Muller Hinton Agar (MH Agar) using disc diffusion method ten different antibiotics were used to evaluate the antibiogram profile, molecular detection was done by targeting 16S rRNA gene by using a universal primer.Results: V.cholerae is present in marine fish samples, as showed by culture method and microscopic observation as well biochemical tests. PCR amplification of 16S rRNA gene showed the amplification of targeted gene and antibiogram profile showed that isolates are more sensitive to Ampicillin in comparison with others antibiotics used in this study. Ampicillin can be used for V.cholerae infection by the physicians and amoxicillin must be avoided which is resistant.Conclusion: Molecular detection is safe and rapid methods for bacteria identification as revealed by PCR amplification of 16S rRNA gene. As the isolates are more sensitive to Ampicillin in comparison with others antibiotics used in this study. Ampicillin can be used for V.cholerae infection by the physicians and amoxicillin and nitrofurantoin must be avoided.

Methicillin-resistant Staphylococcus aureus (MRSA) once confined to hospitals (HA-MRSA), in the last two decades, have emerged in the community in people with no exposure to hospital and also in livestock animals, referred to as community-associated (CA-) and livestock-associated (LA-) MRSA, respectively. Prevention of the dissemination of MRSA requires better knowledge of its distribution and transmission. The study aims to investigate samples from different animal farms and veterinary clinics for identification and typing of MRSA circulating among different farm animals. Phenotypic and genetic characteristics were evaluated based on morpho-cultural and biochemical properties and PCR amplification of nuc genes. Methicillin resistance was assessed on ORSAB media and PCR amplification of methicillin resistance mecA gene. SCCmec typing was performed by multiplex PCR to assess the epidemiological relationship between the isolates. Of the 406 samples, 72 confirmed the presence of S. aureus. Among S. aureus isolates, based on PCR amplification of mecA gene, 15 were identified as LA-MRSA. Majority of the LA-MRSA were found to be SCCmec type III with two isolates each from cattle and pig showed presence of both type I and III. The finding suggests the spillover of the SCCmec type III into livestock and emergence of new clones possessing both type I and III elements.

Diversity analyses of the eukaryotic microorganisms in the gut of marine animals is hampered by the presence of host DNA in the samples. PCR amplification of rRNA genes of eukaryotic microorganisms is inefficient with universal primers targeting 18S rRNA gene when the host DNA is dominant. In this study, we designed several blocking primers to inhibit PCR amplification of rRNA genes of the shrimp Litopenaeus vannamei, and tested their efficacy on the oyster Crassostrea hongkongensis. We first compared the intensity of PCR product bands obtained with and without the blocking primers. Then, one primer was selected for further verification using high-throughput sequencing. Our results showed that X-BP2-DPO was the most effective blocking primer in suppressing the host 18S amplification compared to nine other candidates. The inhibition rate was 99% for the amplification of shrimp rDNA, and 17% for the amplification of oyster rDNA. The concentration of the blocking primer in the PCR mixture was an important factor to be considered in the experimental design. The development of blocking primers provided a valid method to study the composition and characteristics of eukaryotic microorganisms in shrimp gut for a better understanding of its diets.