Species identification and antibiotic susceptibilities of coagulase o negative staphylococci isolated from urinary tract infection specimens

Objective: To determine the incidence of Coagulase - negative S. aureus in urinary tract infections and sensitivities of these isolates to antimicrobial agents

Study Design: Cohort study

Place and Duration of Study: Dr. Essa Laboratory and Immunology and Infectious Disease Research Laboratory [IIDRL], Microbiology Department, University of Karachi, from January 2009 to January 2010

Methodology: Urine specimens, suggestive of urinary tract infection [UTI], were identified. Speciation of isolates was done using API-20 Staph. system. Screening of extracellular products was done using SDS-PAGE electrophoresis and Hemolysin on blood-agar plates. Minimum inhibitory concentration [MICs] of antibiotics was estimated by microtiter well plate method. Frequency and percentages were determined and chi-square test was used for comparing proportions with significance at p < 0.05

Results: Coagulase - negative S. aureus [CONS] were the cause of urinary tract infection in 56 out of 1866 outpatient [3%] and 164 of 1261 inpatient [13%], urinary tract infections [p < 0.001]. Two hundred and twenty CONS isolates were identified. The most common CONS identified was S. saprophyticus [31%, 68 strains]. The relative frequency of Coagulase - negative S. aureus was 6% [13 strains]. All isolates were sensitive to Vancomycin and Linezolid. Resistance was 69% to Ampicillin, 53% to Methicillin, and 37.5% to Ciprofloxacin

Conclusion: CONS are a potential uropathogens, with capability of slime production and resistance to common empirical prescriptions. This also warrants formulation of an appropriate antibiotic policy that covers CONS

Cervical cancer screening and HPV genotype distribution among asymptomatic patients of Karachi Pakistan

Background: Cervical cancer is the second most prevalent cancer in females worldwide. Humanpapillomavirus [HPV] infection is a sexually transmitted infection. However, in addition to HPV infection, other factors exist that influence the risk of developing cervical cancer. In Pakistan most women who developed cervical cancer have been infrequently or never screened. Objective: To determine the prevalence of HPV infection and its subtype profile among asymptomatic patients with pre cancerous cervical intraepithelial lesion. Methods: In this hospital-based descriptive study, 160 asymptomatic females attending gynecology clinics were subjected to HPV screening after obtaining informed consent. Cervical Scrapings were examined by cytopathology and colposcopic directed biopsies taken. High-grade intraepithelial lesion [HSIL] CIN-2, and Low-grade intraepithelial lesion [LSIL] CIN-1 were selected. Samples were analyzed for the presence of HPV-DNA general and type specific genotype 16 and 18. HPV- DNA was extracted by QIA amp DNA kit protocol and amplification was done by polymerase chain reaction [PCR] and genotyped by type specific primers. Results: Out of 160, 17 Pap smear tests were positive, 6 [35.3%] with abnormal results [HSIL] CIN-2 were HPV-DNA positive. Among them, 5 [83.3%] had subtype 16 and in 1 [16.7%] case the genotype was undetectable. The remaining 11[6.9%] with pre cancer minimal abnormal [LSIL] CIN-1 presented. Out of them 3 [27.3%] were HPV-DNA positive with subtype 16. Five [45.4%] were followed by repeated pap smear every six months for two years, and the rest of 3 [27.3%] patients refused for the test. Conclusion: A high incidence of Human papillomavirus [HPV] infection is found in women with pre cancerous lesion of cervix in Pakistani women

Study on alternative therapeutic agents on methicillin-resistant staphylococcus aureus from clinical and environmental isolates

The in vitro antimicrobial activity of Sea buckthorn [SBT] [Hippophaerhamnoides], Green tea [Camellia sinensis] and Dandasa [Juglansregia] on selected methicillin resistant Staphylococcus aureus [MRSA] isolates from clinical samples was tested. The in vitro antimicrobial activity of six antiseptics/disinfectants against MRSA isolated from environmental samples was also evaluated. Experimental Observational. This study was conducted in Immunology and Infectious Diseases Research Laboratory [IIDRL] Lab, University of Karachi from January 2011 to July 2011. Minimum Inhibitory Concentration [MICs] of plant extracts was determined by micro- broth dilution method and, susceptibility of MRSA isolates from environmental samples against antiseptics/ disinfectants was estimated by the agar disk diffusion and agar well diffusion methods. None of the plant extracts inhibited the isolates originating from blood samples. SBT offered comparatively more inhibitory zones and among the antiseptics/disinfectants, savion was the most effective. In view of the rising antibiotic resistance, exploring possible natural plant extracts for their antimicrobial action seems like an attractive substitute. The results showed some degree of susceptibility and can be suggested for use in vivo after standard clinical trials

Wound etiology, resistance pattern and incidence of bacteremia in patients with surgical site infections

To determine the wound etiology, antibiotic resistance pattern of pathogens and incidence of bacteremia in patients with surgical site infections [SSI]. Experimental study. Department of Microbiology University of Karachi, from September, 2008 to October, 2012. Pus swab [n=250] and blood samples [n=56] were collected from patients having SSIs. Swabs were inoculated on 5% sheep blood agar, MacConkey's agar and Sabrouad dextrose agar and identified by standard microbiological and quick test strip methods. Blood samples were dispensed into blood culture bottles and incubated at 35[degree]C for 5-7 days. Resistance pattern of isolates were determined by Kirby Bauer disc diffusion technique and results were interpreted as outlined by Clinical Laboratory Standards Institute [CLSI] specifications. E. coli ATCC 25922 and S. aureus ATCC 25923 were used as control strains. Data was analyzed by SPSS 13. In Pus swabs positive growth was obtained in 224 [90%] specimens. Escherichia coli was the commonest pathogen isolated [n=109, 26%] followed by Candida spp. [n=77, 18%], S. aureus [n=73, 17%], Klebsiella spp. [n=66, 16%], Pseudomonas spp. [n=32, 8%], Proteus spp. [n=29, 7%], Morganella spp. [n=17, 4%] and Providencia spp. [n=15, 4%] whereas in blood samples bacteremia was found in 15 [27%] specimens of which E. coli [47%] and S. aureus [33%] were the most common isolates. Gram negative bacteria demonstrated high resistance against commonly prescribed drugs while Polymyxin B showed reasonable activity against these organisms. S. aureus showed high rates of Methicillin resistance with cross-resistance to most antimicrobial agents. Frequency of bacteremia and resistance to antibiotics was high in patients with SSIs

Sensitivity and specificity of phenotypic and genotypic tests for detection of methicillin-resistant staphylococcus aureus in clinical isolates

Methicillin-resistant Staphylococcus aureus [S. aureus] [MRSA] is an important pathogen in hospitals and communities. There is advance in technologies but infections by MRSA are difficult to diagnose. The current study was conducted to compare three phenotypic methods for with a genotypic method. Cross-sectional study. Methicillin resistance was tested in 450 isolates of S. aureus retrieved from various clinical samples received in Dr. Ziauddin Laboratory Department of Molecular Genetics [DMG], Karachi from January to October 2011. Methods used were, oxacillin disc diffusion, oxacillin screen agar, cefoxitin disc diffusion and Polymerase Chain Reaction [PCR] for mecA gene, which was taken as the gold standard. The Kirby Bauer disc diffusion, using cefoxitin discs 30microg, identified a total of 174 [38.6%] S. aureus isolates as methicillin resistant. With oxacillin disk diffusion test, 152 [33.77%] strains were MRSA with sensitivity of 87.35% and specificity of 100%. Oxacillin-agar screen detected 168 [37.33%] strains with sensitivity of 96.5% and specificity of 100%. PCR method detected mec A gene in 169 [37.55%] isolates with sensitivity of 97.12%, specificity of 100%. The cefoxitin disc diffusion, as suggested by the Clinical and Laboratory Standards Institute [CLSI], is a consistent method for MRSA diagnosis but must be augmented with other methods like oxacillin screen agar, so that no MRSA is overlooked. PCR although taken as the gold standard cannot be recommended as routine because of the high cost and time consumption

To evaluate the efficacy and reliability of commonly available tests for the diagnosis of dengue fever in children in Karachi

Diagnosis of dengue fever in financially constraint countries like Pakistan is usually made on immunochromatographic [ICT] based serological tests. To compare the diagnostic accuracy of commercially available dengue rapid immunochromatographic tests [ICT] with dengue ELISA in acute dengue infection in children. This retrospective study was carried out at the National Institute of Child Health, Karachi in the Department of Pathology from October 2010 to January 2011. Hospitalized children who fulfilled the WHO and CDC definition of probable dengue fever i.e. fever of 2 to 7 days with two or more than two of the associated symptoms like headache, retro orbital pain, myalgia, arthralgia, rash, hemorrhagic manifestation, leucopenia, thrombocytopenia and supporting serology were included in the study after taking informed consent from their parents / care givers. Malarial parasite positive cases were excluded from the study. Dengue ICT based IgG, IgM, IgA antibodies were done using rapid devices, dengue NS[1] was done on ICT rapid device and dengue IgM was done using enzyme linked immunosorbent assay [ELISA] by micro titer well plate. Data entered and analyzed on SPSS version 16. Out of 80 suspected cases [being a retrospective study it would be better that we give number of all suspected cases and then delete the numbers that did not have complete tests], 39 fulfilled the criteria of suspected dengue fever patients. Ages of these children ranged from 6 months to 13 years. There were 18 males and 21 females. Mean haemoglobin was 8.7g/dl and mean haematocrit was 26.3%. Elevated haematocrit was seen in 7.5% cases only. Mean WBC count was 6164/cmm, with leucopenia seen in 33.3% and leucocytosis in 5.12% cases. Thrombocytopenia was present in all cases except one. Positivity rate of dengue IgG was 25.64%, IgM 15.4%, IgA 43.59%, rapid antigen by ICT method 20.5% and ELISA IgM [by micro titer well- plate] was 76.9%. Sensitivity / specificity of dengue IgG, IgM, IgA and rapid antigen by ICT were compared with ELISA IgM [gold standard] and these were 33.3/100%, 16.67/88.89%, 50.0/77.78%, 23.3/88.8% respectively. ELISA IgM antibody [by micro titer well-plate] test was most reliable as compared to ICT tests. Among the ICT tests, dengue IgA antibody was superior to other tests

Chemical and antimicrobial studies on the essential oil from Salvia santolinifolia Boiss

In view of the reputation of genus Salvia in folklore medicine and its abundance in our region, the chemical composition and antimicrobial activity of essential oil from S. santolinifolia Boiss was analyzed. Chemical analysis, using gas chromatography and gas chromatography mass spectrometry, retention indices and C-13 nuclear magnetic resonance spectroscopy has resulted in identification of 116 constituents, comprising about 97% of the total constituents. Out of these 116, 78 constituents are hitherto unreported from this source. The species belongs to alpha-pinene chemotype. In antibacterial assay, gram negative gastropathogens [Shigella boydii, S. flexneri, S. dysenteriae. Vibrio cholera]; causative agent of urinary tract infection [Proteus mirabilis and P. vulgaris] and pneumonia [Klebsiella pneumoniae] were found sensitive to this essential oil while Corynebacteria species and Staphylococcus epidermidis were significantly inhibited in antibacterial assay against gram positive bacteria. Clinical and Laboratory Standards Institute protocol was used for determining antimicrobial activity. Thus the essential oil from this species can be utilized as potential chemotherapeutic agent

Frequency of HBsAg positivity: a laboratory data analysis

The objective of the study was to evaluate the current frequency ratio of HBsAg seropositivity in a laboratory-based setting. It was a retrospective study, conducted during September 2009 and November 2010 at Dr. Essa's Diagnostic Laboratory and its branches in Karachi, Pakistan. Blood samples of 8,648 individuals were screened for HBsAg using the BioRad EVOLIS EIA method. Pertinent questionnaires filled in by 50 practicing physicians were also evaluated. The 551 subjects [6.4%] that tested positive for HBsAg included 360 males [65.3%] and 191 females [34.6%]. The highest frequency of HBsAg was noted in those aged 16-29 years [43.9%]. HBsAg rate in the studied population has steadily increased and is more common in the sexually active age group, with males tending to be more often seropositive than females

Current pattern of antibiotic resistance in stapbylococcus aureus clinical isolates and the emergence of vancomycin resistance

To determine the pattern of antibiotic resistance in the clinical isolates of Staphylococcus [S.] aureus, Methicillin Resistant S. aureus [MRSA] and define the possible emergence of Vancomycin resistant S. aureus [VRSA] in Karachi. An observational study. Essa Laboratories and Department of Molecular Genetics, Ziauddin Hospital, from January to December 2009. Staphylococcal isolates from different clinical specimens, pus, urine, blood, high vaginal swab and other secretions received at Ziauddun laboratories and Dr.Essa laboratories were collected. The specimens were inoculated on blood agar, MacConkey agar and Chrom agar. Antibiotic susceptibility to conventional antibiotics was done by disc diffusion, and E-test. Methicillin resistance was tested by using Oxacillin and Methicillin disks and confirmed by gold standard PCR for presence of mecA gene. All MRSA strains were subjected in addition to Vancomycin screen agar test. Out of the 450 S. aureus isolates 174 [38.6%] were found to be MRSA. In those isolates, high resistance was found to Cefixime [100%] Doxicycline [100%] Oxacillin [96.5%] Gentamicin, [96.3%], Timethoprim/Sulfametoxazole [95.6%] Chloramphenicol [93%] Tobramicin [81.03%], Ofloxacin [72.4%] and Ciprofloxacin [63.7%]. Low resistance was found to Ceftazidine [36%], Amoxicillin/Clavulanate [32.7%], Fosfomycin [31%], Cefroxime [24%], Amikacin [17.2%] and Meropenem [13%]. One isolate was found to be Vancomycin resistant [MIC 32 microg/ml]. Four isolates had intermediate resistance, with two strains having MIC of 16 microgram/ml and two having MIC of 8 microgramg/ml. These strains were also resistant to all the other tested antibiotics except Linezolid to which all isolates were susceptible. Antibiotic resistance to all the conventionally used antibiotics was high in the tested isolates. All the strains were susceptible to Linezolid which is an expensive alternative with adverse side effects. Judicious use of antibiotics focused on the compliance and formation of antibiotic policy guide lines is highly recommended

Use of CHROMagar candida for the presumptive identification of candida species directly from clinical specimens in resource-limited settings

Identification of yeast isolated from clinical specimens to the species level has become increasingly important. Ever-increasing numbers of immuno-suppressed patients, a widening range of recognized pathogens, and the discovery of resistance to antifungal drugs are contributing factors to this necessity. A total of 487 yeast strains were studied for the primary isolation and presumptive identification, directly from clinical specimen. Efficacy of CHROMagar Candida has been evaluated with conventional methods including morphology on Corn meal between 80 agar and biochemical methods by using API 20 C AUX. The result of this study shows that CHROMagar Candida can easily identify three species of Candida on the basis of colonial color and morphology, and accurately differentiate between them i.e. Candida albicans, Candida tropicalis, and Candida krusei. The specificity and sensitivity of CHROMagar Candida for C. albicans calculated as 99%, for C. tropicalis calculated as 98%, and C. krusei it is 100%. The data presented supports the use of CHROMagar Candida for the rapid identification of Candida species directly from clinical specimens in resource-limited settings, which could be very helpful in developing appropriate therapeutic strategy and management of patients

Activity of synergistic combination amoxy cassia against salmonella

The object of this study was to formulate new, cost effective anti-microbial combination for Multi Drug Resistant [MDR] Salmonella enterica Serover Typhi [SEST] based on the synergistic activity of amoxicillin with the aqueous fruit solution of Cassia fistula [CFF], a medicinal plant found in Pakistan which when tested alone have weak antimicrobial activity against blood isolates of MDR SEST. MIC of MDR SEST for amoxicillin and CFF alone was 750 micro g/ml and 3750micro g/ml. The MIC of amoxicillin in combination with CFF was 23.4-187.5 micro g/ml and 0.37-1.56 micro g/ml for MDR and MDS SEST respectively. Fractional Inhibitory Concentration Index [FICI] using chequer-board titration suggested synergism for 80% MDR and MDS SEST tested, no antagonism observed. Time kill kinetics showed difference > log2 in CFU of CFF having sub-lethal amoxicillin concentrations. This novel combination is named as Amoxy-cassia. In vivo it is found to be non toxic at 1gm/body weight of mice

Juliflorine susceptibility of animal and human isolates of campylobacter

Juliflorine, the main alkaloid isolated from Prosopis juliflora was tested for its antibacterial activity against both the Gram positive and Gram negative bacteria. Filter paper disks impregnated with juliflorine were placed on streaked agar medium. Zones of growth inhibition for Campy lobacter spp occurred at 10 jig per disk. Other enteropathogens [including the species of Salmonella, Shigella, Vibrio, Escherichia, Proteus, Pseudomonas, Klebsiella and Yersinia] were resistant to at least 30 micro g per disk, with the exception of a strain of Aeromonas hydrophilia, Urhich showed some inhibition at 30 micro g per disk. Most of the 25 Campylobacter strains, which were isolated from human clinical and animal sources, showed zones of inhibition greater than 10 mm with 10 micro g of juliflorine

Pathogenicity of aeromonas Sp for balb/c mice

Clinical strains of Aeromonas hydrophia and Aeromonas sobria were used to develop a mouse model for determining virulence potentials of food isolates. Intraperitoneal injection with these strains caused hemorrhaging and death of 10-12 week old male BALB/c mice within 24 h post challenge. Nonsupplemented injections of Aeromonas sp. suspensions resulted in a median LID 50 value of 4.9 X 107 cells, whereas supplementation with 0.1 ml of 5% iron dextran with the bacterium decreased the LID 50 value to 3.1 X 10 7cells. Immunosuppression of the mice with cadmium chloride did not substantially alter the lethality for mice. Boiled suspensions 109 cells did not cause more deaths, indicating a lack of pathological contribution by the LPS component when using this approach for virulence assessment. Isolates of Aeromonas sp. from retail chicken, beef and pork products were supplemented with iron dextran and injected into BALB/c male mice. Twenty-two of 26 mice injected with 26 A. hydrophila food isolates died; 13 of 14 mice injected with A. sobria isolates died; and 10 of 16 mice injected with A, caviae isolates died. These results indicate that approximately 80% of foodborne aeromonas are lethal for mice via intraperitoneal challenge. This virulence assay may or may not be an appropriate choice to assess the potentials of foodborne isolates of Aeromonas sp. to cause disease in humans

Potential pathogenic mechanisms of animal-passed virulence-enhanced strains of campylobacter jejuni/coli

In order to study the potential pathogenic mechanisms, live cultures, culture-filterates and Poly-myxin B released extracts from original and animal-passed strains of C jejuni/coli were compared by a neonatal mouse infection and in-vitro Vero-Cell cytotoxicity. The cell fractions from mouse-passed strains elaborated strong cytotonic and cytotoxic activity causing elongation, granular appearance and lysis of Vero-Cells. Only weak enterotoxie activity was associated with the original strains. Partial neutralization of the cytotonic activity by mono-clonal anticholera toxin [CT] suggested immunological relationship between the toxins which was also demonstrated by Ouchterlony immunodiffusion using polyclonal anti - CT serum. The in-vivo significance of CT like and other toxic materials produced by Campylobacter strains during animal-passage remains to be assessed