Histopathological and Molecular Investigation of Natural Cases of Bovine Tuberculosis Infection in Cattle

Bovine tuberculosis (bTB), a chronic infection in cattle caused by Mycobacterium tuberculosis/bovis, that impacts productivity and represents a major public health threat. Although the considerable economic costs and zoonotic risk consequences associated with the disease, accurate estimates of bTB prevalence are lacking in many countries, including India. Therefore, in the current study for collection of tubercular lesions the postmortem examination of 100 cattle was conducted. All major viscera and regional lymph nodes were examined and incised. Histopathology was performed in the cases where gross lesions were suggestive of tuberculosis. PCR was performed on the tissue and faecal samples by using IS6110 insertion sequence, Mycobacterium tuberculosis/bovis complex PCR kit. In 12 animals, nodular lesions with casseating mass suggestive of tuberculosis were observed in the lung tissue. All the 12 lung impression smear and only five faecal smear showed acid fast bacilli stained by Ziehl-Neelsen staining. Histologic features comprised a classic granuloma as a characteristic lesion of tuberculosis composed of a central caseous necrosis with mantle of macrophages, lymphocytes, plasma cells, epithelioid macrophages and Langhan’s giant cells and were observed in all 12 cases. All the tissue samples and 11 faecal samples were positive for the Mycobacterium tuberculosis complex using IS6110 sequence. 8 tissue samples and 4 faecal samples were positive by using Mycobacterium tuberculosis/bovis complex PCR kit. It can be concluded that there was good agreement between histopathology, acid fast staining and PCR. It can also be concluded that faecal samples which are easier to collect should be preferred for diagnosis of TB by PCR in cattle

Detection of integrons and ISCR1 in and genotyping of NDM-1-producing Citrobacter freundii / 中国感染与化疗杂志

Objective To investigate the distribution of integrons and ISCR1 elements in NDM-l-producing Citrobacterfreundii isolates,and analyze the genotypes of these strains to understand their homology.Methods A total of 18 strains of NDM-1-producing Citrobacterfreundii were collected from the First Affiliated Hospital of Kunming Medical University during the period from June 2012 and October 2014.The isolates were identified and subjected to antimicrobial susceptibility testing with VITEK 2 System.Class Ⅰ,Ⅱ,and 1Ⅲ integrons and ISCR1 elements were detected by PCR.Clonal relatedness was assessed by pulsed field gel electrophoresis (PFGE).Results Most (77.8％,14/18) strains were positive for class Ⅰ integron conserved region,27.8％ (5/18) isolates were positive for ISCR1 conserved region.No class Ⅱ or Ⅲ integron was detected.Most (72.2％,13/18) isolates were positive for class Ⅰ integron variable region.None of the strains harbored class Ⅱ integron or ISCR1 variable region.Integron variable regions included gene cassette encoding resistance to aminoglycosides (aadA1,aadA5,aac(6')-Ib-cr) and trimethoprim-sulfamethoxazole (dfrA,dfrA15,dfrA17).PFGE revealed 17 clusters among 18 NDM-l-producing Citrobacter freundii isolates.Conclusions The clonal dissemination of NDM-l-producing Citrobacterfreundii isolates is not significant.Class I integron is prevalent in NDM-l-producing Citrobacter freundii.The presence of ISCR1 is relatively rare.The two mobile elements are not related to the spread of NDM-1 gene in this hospital.

ISAba15 Inserted into Outer Membrane Protein Gene carO in Acinetobacter baumannii

We identified ISAba15 inserted into the carO gene in an Acinetobacter baumannii isolate. The insert disrupted the lpxD gene, resulting in colistin resistance in A. baumannii. Persistence in carbapenem resistance in A. baumannii isolates with an intact carO gene indicates that loss of the encoded CarO may play a minor role in carbapenem resistance.

Presencia de Bordetella holmesii en brote epidémico de coqueluche en Chile / Presence of Bordetella holmesii in an outbreak of pertussis in Chile

The incidence of whooping cough in Chile ranges from 4.1 and 7.5 per hundred thousand inhabitants. B. pertussis detection is performed by Real Time PCR (Q-PCR) directed to the insertion sequence IS481. However, this sequence is also found in the genome of B. bronchiseptica and B. holmesii. The latter is also a respiratory pathogen whose clinical features are similar to B. pertussis. However, it is important to differentiate between these species because in immunosuppressed patients B. holmesii is more likely to cause bacteremia and is less susceptible to erythromycin. The goal of this work is to measure prospectively and retrospectively the presence of B. holmesii in samples reported positive for B. pertussis in the period 2010-2011. During this period, 1994 nasopharyngeal specimens entered the laboratory for Bordetella sp. PCR, of which 224 were positive. The analysis by Q-PCR directed to the recA gene of B. holmesii of all 224 positive samples determined a prevalence of B. holmesii of 0.6% (12/1994). Because of its more aggressive behavior in immunosupressed patients and its different resistance pattern, routine screening of B. pertussis and B. holmesii is currently performed for all samples in which Bordetella sp PCR is initially detected.

La incidencia de coqueluche en Chile varía entre 4,1 y 7,5 por 100.000 habitantes. La detección de Bordetella pertussis se realiza por RPC-tiempo real (Q-RPC) dirigida a la secuencia de inserción IS481. Sin embargo, esta secuencia se encuentra también en el genoma de B. bronchiseptica y B. holmesii. Este último es también un patógeno respiratorio que produce un cuadro similar a B. pertussis. Sin embargo, es importante diferenciar entre estas especies porque en pacientes inmunosuprimidos B. holmesii tiene mayor tendencia a causar bacteriemia y además es menos susceptible a eritromicina. El objetivo de este trabajo es determinar, prospectiva y retrospectivamente, la presencia de B. holmesii en muestras informadas positivas para B. pertussis en el período 2010-2011. Durante ese período ingresaron al laboratorio 1. 994 muestras de hisopado nasofaríngeo para RPC de Bordetella sp., de las cuales 224 fueron positivas. El análisis por Q-RPC dirigido al gen recA de B. holmesii de las 224 muestras positivas determinó una prevalencia de B. holmesii de 0,6% (12/1994). Debido al comportamiento más agresivo en inmunosuprimidos y al patrón de resistencia de B. holmesi, se decide incorporar la detección de rutina de B. pertussis y B. holmesii en todas las muestras en que se detecta inicialmente la presencia de Bordetella sp.

Multiplex polymerase chain reaction using insertion sequence 6110 (IS6110) and mycobacterial protein fraction from BCG of Rm 0.64 in electrophoresis target genes for diagnosis of tuberculous lymphadenitis.

Purpose: Tubercular lymphadenitis (TBLA) is a common manifestations of extrapulmonary tuberculosis (EPTB) accounting for 30-40% of cases. Prompt diagnosis and timely initiation of anti-tubercular therapy (ATT) is the key for successful clinical outcome. This study was carried out to evaluate multiplex polymerase chain reaction (MPCR) using MPB64 and IS6110, and compare with the conventional methods for rapid diagnosis of TBLA. Materials and Methods: In our study, lymph node fine-needle aspirates of patients were evaluated for TBLA. They were classified as Group I: TBLA group, divided into (a) Confirmed TBLA cases (n0 = 80): Culture/smear-positive or cytological examination showing presence of epithelioid cell granuloma with or without multinucleate giant cell and caseation necrosis with presence of AFB, and (b) suspected TBLA cases ( n = 30): Culture/smear-negative and cytological examination showing presence of epithelioid cell granuloma and response to ATT and Group II (Control) (n = 25): Patients of lymphadenopathy confirmed to be caused by other diseases such as sarcoidosis, lymphoma, etc., All samples were subjected to conventional tests and MPCR. For MPCR we used Mycobacterium tuberculosis-specific deoxyribonucleic acid sequences specific for the MPB64 and IS6110 region. Results: In the confirmed TBLA group, Ziehl-Neelsen (ZN) smear, cytology, culture, and MPCR positivity was 30%, 70%, 26.3%, and 91.3% respectively. In the suspected TBLA group, smear and culture were negative, and sensitivity of cytology and MPCR was 73.3% and 86.6%, respectively. In the control group all tests were found to be negative, thus giving a specificity of 100% to all the tests in the study. Conclusion: In conclusion, techniques like MPCR with high sensitivity and specificity can play an important role in rapid diagnosis of TBLA.

A Study on the Distribution and Structure of ClassⅠIntegron and ISCR1 in Clinical Isolates of Acinetobacter Baumannii / 天津医药

Objective To investigate the distribution and the antibiotic resistance genes carried by classⅠintegron and insertion sequence common region(ISCR1)among clinical isolates of Acinetobacter baumannii. Methods Fifty-one clinical isolates of multidrug-resistant Acinetobacter baumannii were collected. Polymerase chain reaction method was used to detect the classⅠintegrase gene, variable region of classⅠintegron, ISCR1 and genes related to antibiotics resistance lo-cated downstream of ISCR1 in 51 multidrug-resistant Acinetobacter baumannii. Antibiotics resistance genes carried by classⅠintegron and ISCR1 were performed by DNA sequencing. The relationship between classⅠintegron and ISCR1 was detected by PCR-mapping. Results Among 51 multidrug-resistant Acinetobacter baumannii isolates, 45 strains were found con-taining classⅠintegrase genes, 32 strains were found containing variable regions. Sequencing results showed that the gene cassette arrays were aacA4-catB8-aadA1, aacC1-orfA-orf-B-aadA1 and blaPSE-1-aadA2-cmlA1-aadA1. Twenty-two strains were found containing ISCR1 and 5 strains were found containing resistance genes located downstream of ISCR1. DNA sequencing results showed the resistant gene of qnrA1-ampR. The results of PCR-mapping showed that ISCR1 located directly downstream of 3' conserved segment of classⅠintegron in 20 Acinetobacter baumannii strains. Conclusion ClassⅠintegron and ISCR1 play an important role in mechanisms of drug resistance of Acinetobacter baumannii. ClassⅠintegron and ISCR1 could connect in series in Acinetobacter baumannii.

Detection of beta-lactamase, porin-coding genes and linkage of KPC-ISKpn6 of the carbapenems-resistant Klebsiella pneumoniae / 中华微生物学和免疫学杂志

Objective To investigate the resistance-mechanism of the carbapenems-resistant Klebsiella pneumoniae isolated from clinical.Methods The clinical isolates of carbapenems-resistant Klebsiella pneumoniae from top three comprehensive hospitals of Nanjing area were examined by 40 beta-lactamase,porin-coding genes and linkage of KPC-ISKpn6 using PCR method,the PCR positive results were picked out for sequencing and sequencing BLAST search for comparison analysis.Results Twenty-four strains of carbapenems-resistant Klebsiella pneumoniae were detected,the positive rate of A beta-lactamase TEM-1 and SHV was 100％ (24/24),KPC-2 and LAP-2 was 95.8％ (23/24),45.8％ (11/24) respectively,and C beta-lactamase DHA was 4.2％ (1/24).Meanwhile,the positive detection rates of KPC-ISKpn6 linkage was 95.8％ (23/24),and the mutation rate of porin-coding genes ompK35 and ompK36 were up to 95.8％ (23/24) and 100％ (24/24).Conclusion High incidence of beta-lactamase TEM-1,SHV,KPC-2 and LAP-2 was found in the group of Klebsiella pneumoniae isolates,and carbapenems-resistant of which was primarily due to the high carrying rate of KPC-2 and the high mutation rate of porin-coding genes ompK35 and ompK36.The Insertion sequence ISKpn6 may be involved in the KPC-2 gene mediated-expression.

Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine samples

The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8 percent and 52 percent in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29 percent and 26.9 percent in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.

Distribution and molecular epidemiologic characterics of insertion sequence IS1301 in Neisseria meningitidis isolated from China / 中华流行病学杂志

Objective To research the distribution and molecular epidemiology of insertion sequence IS1301 in Neisseria (N.) meningitidis strains in China, so as to provide scientific and available evidence for a new method of genotyping in N.meningitidis strains with IS1301. Methods Examined the IS1301 by PCR in 219 N.meningitidis strains from 16 provinces and 3 cities during 2007 and 2008 in China, productions of amplification were sent for sequencing. The positive N.meningitidis strains were analyzed by pulse field gel electrophoresis (PFGE) and nucleic acid blotting hybridization(Southern blot) by electrophoresis. Results The positive rates with IS1301 were 15.53%, 11.11%, 20.75%, 6.17% and 28.57% for four serotypes (A, B, C, N) respectively. The sequence comparability between the amplification productions and No.Z49092.1 N.meningitidis which registered in GenBank was 94%-100%. There were two types of clusters devided by cladogram analysis. There appeared large IS1301 sequence difference between the serotype C and others. The number of IS1301 replica ranged from 6-17 per strain at least. The number of IS1301 replica changed in the same type of PFGE N.meningitidis respectively. Conclusion Typing by IS1301 combined with PFGE could comprehend the homology and genetic polymorphism of N.meningitidis epidemic strains at the molecular level.

Real-time Quantitative Assay of Insertion Sequence 6110 DNA of Mycobacterium Tuberculosis and Its Value for Diagnosis of Tuberculosis / 中国医师杂志

Objective To set up a real-time quantitative assay method of insertion sequence 6110 DNA of mycobacterium tuberculosis, and explore its value for diagnosing tuberculosis. Methods Real-time quantitative assay of insertion sequence 6110 DNA of mycobacterium tuberculosis was performed with Taqman technique and Lightcycler quantitative PCR system. Results 213 clinical samples of different types were detected, 32 cases were positive, and the positive rate was 15 02%. The scope of quantitative results of positive samples was 3 1～7 2?10 6 copies/ml, and the sensitivity and specificity for diagnosis of tuberculosis were 82 76% and 95 65% respectively Conclusion Real-time quantitative assay of insertion sequence 6110 DNA of mycobacterium tuberculosis is a rapid and effective method for diagnosis of tuberculosis.