Rapid Detection of Orthopoxvirus by Semi‐Nested PCR Directly From Clinical Specimens: A Useful Alternative for Routine Laboratories

AIM: The aim of this study was to evaluate the prevalence of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in clinical specimens hospitalized to “Mother Theresa” Hospital Center for 2 years. METHODS: We isolated and identified S. aureus on 356 clinical specimens using standard tests. Furthermore, for further accurate microbial identification, we have to use the VITEK® 2 system. The samples were tested to detect the presence of MRSA by a slide latex agglutination kit for the rapid detection of PBP2. RESULTS: The overall prevalence of S. aureus in patients was 34.2%. The prevalence of MRSA was 20.5% of cases. Of the MRSA isolates identified in this study, 28% were susceptible to antibiotics, 24% demonstrated intermediate resistance, and 48% were multi-drug resistant with resistance to nineteen antibiotics involved in the examination. In addition, seven of the 25 MRSA cases showed 100% resistance to norfloxacin, imipenem, meropenem, levofloxacin, etc. CONCLUSIONS: The rate of S. aureus in hospitalized patients on this study was 34.2% and the MRSA 20.5%. These results indicated that this type of infection is a significant concern for health services and patients included. A screening of all hospitalized cases can lead to reduce the incidence of this infection in the hospital environment.

Download Full-text

Direct Identification of Vibrio vulnificus in Clinical Specimens by Nested PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.36.10.2887-2892.1998 ◽

1998 ◽

Vol 36 (10) ◽

pp. 2887-2892 ◽

Cited By ~ 40

Author(s):

Shee Eun Lee ◽

Soo Young Kim ◽

Sei Jong Kim ◽

Hyun Soo Kim ◽

Jong Hee Shin ◽

...

Keyword(s):

Dna Extraction ◽

Nested Pcr ◽

Extraction Method ◽

Vibrio Vulnificus ◽

High Yield ◽

Chromosomal Dna ◽

Direct Identification ◽

Clinical Specimens ◽

Dna Extraction Method ◽

Dna Fragment

This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification ofVibrio vulnificus in clinical specimens. We designed two sets of primers targeting the V. vulnificushemolysin/cytolysin gene. The target of the first primer set (P1-P2; sense, 5′-GAC-TAT-CGC-ATC-AAC-AAC-CG-3′, and antisense, 5′-AGG-TAG-CGA-GTA-TTA-CTG-CC-3′, respectively) is a 704-bp DNA fragment. The second set (P3-P4; sense, 5′-GCT-ATT-TCA-CCG-CCG-CTC-AC-3′, and antisense, 5′-CCG-CAG-AGC-CGT-AAA-CCG-AA-3′, respectively) amplifies an internal 222-bp DNA fragment. We developed a direct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA–0.5% Triton X-100 solution. The new DNA extraction method was more sensitive and reproducible than other conventional methods. The DNA extraction method guaranteed sensitivity as well, even when V. vulnificus cells were mixed with other bacteria such asEscherichia coli or Staphylococcus aureus. The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU of V. vulnificus. We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients. Seventeen (94.4%) of the 18V. vulnificus culture-positive specimens were positive by the nested PCR. Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.

Download Full-text