Journal of Medical Microbiology and Infectious Diseases
Volume:2 Issue: 3, Summer 2014

Some snails play an important role in the transmission of helminthes, mainly trematodes of medical and veterinary importance. There seems to be no information on the freshwater snails of Gahar Lake in Lorestan province of Iran. The present study aimed to identify medically important snails of this lake. Samples were collected from ten localities around Gahar Lake in April 2015 by hand. The snails were classified according to shells morphology. The data were then analyzed using descriptive statistics. A total of 6 snail species were collected from all localities. Four medically important snail species including: Lymnaea truncatula, Lymnaea peregra, Melanoides tuberculata and Melanopsis spp. were detected. M. tuberculata was seen in all sampling sites. Physa acuta and Melanopsis spp. were observed in five sampling sites. Planorbis intermixtus, L. peregra and L. truncatula were found in four, three and two sampling sites, respectively. Presence of medically important snails in Gahar Lake could be a source of trematode infections for visitors. Therefore control measures, especially biological ones should be applied to the lake.

Toxigenic Clostridium difficile is the major cause of antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficile is related to toxins A&B. Children with cancer are at risk of developing C. difficile infection (CDI) due to increased exposure to antibiotics, immunosuppression, and longer hospital stays. Recently, due to higher sensitivity and specificity of nucleic acid amplification test (NAATs) compared to toxin enzyme immunoassays (EIAs), many laboratories are transitioning to NAATs for detection of CDI. We aimed to use a multiplex PCR to detect the C. difficile toxin genes tcdA, tcdB and tpi in stool of cancerous children. We also aimed to show the effects of chemotherapy regimens on the prevalence of C. difficile in these children. 105 fecal samples were collected from cancerous children who were hospitalized and undergoing chemotherapy. The presence of tcdA, tcdB, and tpi genes were tested. C. difficile was identified in 17.14% of children and the detection rate of A-B+ strains was higher than A+B+ and A+B- strains. C. difficile was found in 17.8% of children who received single antibiotic (5/28 cases virulence genes were detected in 4 cases) and in 41.4% of patients who received more than one antibiotics (12/29 cases virulence genes were detected in 8 cases﴿. Multiplex PCR is a powerful technique for preliminary screening and rapid detection of C. difficile and its virulence genes in the stool of cancerous children. The prevalence of C. difficile in cases receiving several antibiotics was higher than those receiving single antibiotics.

Methicillin-Resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious hospital- and community-acquired infections. USA300 is known to be the most common cause of community-acquired infections, but recently there have been some reports on hospital-acquired infections caused by this strain. Totally 171 isolates of S. aureus were collected from different clinical samples in selected university hospitals in Mashhad, Tehran, and Isfahan cities. Then, they were assessed by agar screening and disk diffusion methods to determine their resistance to Methicillin. The isolated MRSA strains were confirmed by detection of mecA gene. The staphylococcal cassette chromosome mec (SCCmec), agr, and spa typing and also detection of Panton-Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) genes were performed on mecA harboring isolates. Multilocus sequence typing was performed on PVL/ACME positive MRSA strains. We found a PVL/ACME positive MRSA isolate. Genetic evaluation results for this isolate produced the following profile: positive for mecA, pvl, arcA, and hla genes, negative for vanA, sec, and tst1, and belonged to agr I, SCCmec IV, sequence type 8 (ST8), and spa t008. Our results suggest a finding of USA300CA-MRSA isolate in Mashhad, Iran. This is an uncommon finding, because USA300 is routinely found in areas other than Middle East. A notable point about these isolates is that they belong to American Endemic clones.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause serious and life-threatening hospital- and community-acquired infections. Colonized and infected patients represent the most important reservoir of MRSA in health care facilities. Therefore, in this study, MRSA isolates collected from Shohada Hospital in Tabriz were evaluated for the frequency of mecA gene and their antimicrobial susceptibility in a period of three years, from 2010 to 2012. A total of 182 S. aureus isolates were collected from clinical specimens and first genotypically identified by detection of nuc gene. Antimicrobial susceptibility test was performed by disc agar diffusion method using cefazolin, methicillin, tetracycline, and cefoxitin according to clinical and laboratory standards institute (CLSI) recommendation. Phenotypic (cefoxitin 30 µg/disc) and genotypic (mecA gene detection by PCR) methods were used for detecting methicillin sensitivity. All isolates expressed S. aureus specific sequence gene (nuc) in their PCR products. Eighty-one (44.5%) isolates were confirmed as MRSA by cefoxitin disc screening test and 97 (53.3%) isolates by showing the presence of mecA gene. All the methicillin sensitive S. aureus (MSSA) isolates and 64 (66%) MRSA isolates were found to be susceptible to cefazolin, but 25 (25.8%) MRSA were resistant to tetracycline and cefazolin. The results of this study showed high frequency (53.3%) of MRSA with no significant differences in rates within the three years of study, indicating the inefficiency of control programs to care for patients with MRSA.

Emergence Klebsiella pneumoniae resistant to quinolone antibiotics due to mutations in gyrA and parC genes created problem for treatment of patients in different hospitals in Iran. The objective of this study was to determine the amino acid substitutions of GyrA and ParC proteins in certain clonal lineages of the K. pneumoniae conferring high level quinolone resistance. One hundred and eleven isolates of K. pneumoniae were recovered from clinical specimens in a teaching hospital in Kerman, Iran. The antibiotic susceptibility and MIC of quinolones were determined according to CLSI guidelines. Clonal lineages of the isolates were determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR amplification using ERIC specific primer sequences. Amino acid mutation profile of gyrA and parC amplicons of six high quinolone resistant isolates was also investigated by DNA sequencing. Twenty two isolates were resistant to nalidixic acid (MIC 256µg/ml), ciprofloxacin (MIC 32µg/ml), levofloxacin (MIC 32µg/ml), and ofloxacin (MIC 32 µg/ml). Typing by ERIC-PCR identified 4 clusters and six singleton, the largest one was belonged to cluster-3 obtained from urine samples. Sequencing of gyrA gene showed three amino acid substitutions (Ser83→Ile Lys154→Arg Ser171→Ala) in the strains 18, 20, 33, two mutations (Lysine154→Arg Ser171→Ala) in the strains 27, 65 and six substitutions in the strain 66, of which, three (Ser83→Phe Asp87→Ala Val190→Gly) were unique for this strain. Sequencing of parC gene revealed double substitutions (Ser129→Ala Ala141→Val) in the strains 18, 27, 66 and three aminoacid changes (Ser80→Ile Ser129→Ala Ala141→Val) in the strains 20 and 33 respectively. Alignment and phylogenetic tree analysis of the gyrA sequence from highly quinolone resistant isolate 66 with homologs sequences obtained from the NCBI database confirmed 99.8% similarity to gyrA gene of the K. pneumoniae ha10 (GenBank: JX123017.1). The results of present study suggest that acquisition of mutations in certain positions of gyrA and parC genes confer high level resistance to quinolones.

Spirochetes of Borrelia can be visualized directly in infected ticks by dark-field microscopy. Inoculation of in phosphate buffered saline (PBS) suspension of ground Argasid soft ticks to susceptible animals or allowing the ticks to feed on the same species followed by microscopic examination of the animals’ blood have also been practiced. With the advent of molecular methods and introduction of various gene markers, Borrelia persica DNA was detected in Ornithodoros tholozani ticks by using several gene markers, but the data on transovarial transmission of Borrelia in this tick by these methods is very scarce. In this study we tried to detect Borrelia in field collected O. tholozani ticks by allowing them to feed on guinea pigs and then to examine the animals’ blood for spirochetes by microscopy. We also used two PCR methods targeting highly repetitive regions of rrs gene to detect Borrelia DNA in adult ticks, larvae, and eggs. All the guinea pig blood samples were negative for spirochetes by microscopy. However, out of the 17 adult ticks, 2 males and 5 females were positive for Borrelia DNA. None of the larvae was positive, but two batches of eggs yielded the expected 540 bp amplicon by nested PCR. Presence of Borrelia DNA in adult O. tholozani ticks and their eggs is an indication for transovarial transmission of relapsing fever agent in this tick.

Most B-cell malignancies are diagnosed based on morphologic and immunohistochemical criteria. Some cases, however, still present a challenge for the pathologist to discriminate between reactive hyperplasia and neoplastic disorders. Molecular techniques can be used as a helpful diagnostic tool in these cases. In this study, we assessed the value of polymerase chain reaction (PCR) technique in determination of monoclonality of immunoglobulin heavy chain gene rearrangements for the diagnosis of large B-cell non-Hodgkin's lymphoma (NHL) in paraffin embedded tissue samples. DNA was extracted from paraffin embedded tissues of 44 diffuse large B-cell lymphoma (DLBCL) cases and 20 samples of reactive lymphoid tissues from appendix and tonsils as control. Framework 3 and the joining region (FR3/JH) of the variable segment of the immunoglobulin heavy chain gene were amplified using degenerated primers. PCR products from each sample were analyzed on 8% polyacrylamide gels following AgNO3 staining. Monoclonal rearrangements were identified in 33 of 44 cases (75%) of DLBCL using FR3/JH primers. Monoclonal IgH gene rearrangements were not detected in any of the reactive lymphoid hyperplasic samples. All these control cases showed polyclonal pattern. Through PCR analysis, using degenerated primers, monoclonality was demonstrated in 75% of DLBCL cases. This technique can thus be used as a sensitive, reliable and valuable diagnostic supplement to conventional morphologic examination and immunohistocytochemical evaluation of lymphoproliferative disorders, particularly in cases with restrictions in amount or type of analytic material.

Acanthamoeba is an opportunistic protist, which is ubiquitously distributed in the environment. Infection with Acanthamoeba spp. poses threat to human health, such as, Acanthamoeba keratitis (AK) that is a vision-threatening infection of the cornea. This study aimed to identify the species of Acanthamoeba strains isolated from cornea of keratitis patients by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Acanthamoeba isolates investigated in this cross-sectional study, were collected from patients referring to Farabi Eye Reference Center. All 10 isolates were subjected to species identification using DNA based method. BspLI (NlaIV) and HpyCH4IV restriction enzymes were used to categorize the PCR amplified DNA by PCR-RFLP method. Six samples were identified as Acanthamoeba palestinensis and 4 isolates as Acanthamoeba culbertsoni, which implies that all the isolates belong to pathogenic strains of Acanthamoeba. Acanthamoeba can enter the corneal tissue and survive in the eye, which results in AK. To the author's knowledge, no study is available on species identification of this genus using these enzymes and technique. This is the first time in Iran that Acanthamoeba isolates are subjected to species identification using PCR-RFLP method.