Prospective Laboratory-based Study Research Articles

Development and Validation of a Rodent Model of Amniotic Fluid Exchange: A Prospective Laboratory-Based Study

Development and Validation of a Rodent Model of Amniotic Fluid Exchange: A Prospective Laboratory-Based Study

Epidemiology and emm types among group A streptococcal pharyngitis in Finland: a prospective laboratory-based study

PurposeStreptococcus pyogenes (mostly termed group A Streptococcus - GAS) is the most important bacterial causative of pharyngitis. However, epidemiology of GAS pharyngitis is not widely established. This study describes GAS pharyngitis cases and emm-type distribution in a prospective study covering over 2 years in two Hospital Districts in Finland.MethodsA prospective, systematic collection of GAS pharyngitis isolates was conducted between March 2018 and December 2020 in two large Hospital Districts in Finland. Patient characteristics (age, gender) were included if available. All GAS isolates collected were emm typed.ResultsAltogether 1320 GAS pharyngitis strains were collected, 904 in the Hospital District 1 (HD1) and 416 in Hospital District 2 (HD2). In HD1, age and gender data were available. Females were overrepresented (58% of all cases). In addition, the age and gender distributions were noted to be significantly different (p < 0.0001) with females having a more uniform distribution until age of 40. emm28 was common among the age group of 20–29-year-olds and emm89 in children under 10 years of age, respectively. In HD1, most of the isolates were collected during winter and autumn months. Significant differences by season in the frequency of emm12, emm89, emm75 and group of “others” were observed.ConclusionAge distribution among GAS pharyngitis cases was significantly different between genders (p < 0.0001). In addition, age group specific and seasonal variations in emm GAS types causing the disease were observed. These findings warrant further investigation, especially for understanding population-based spread of GAS even in more detail.

Application of Nested PCR and Loop-Mediated Isothermal Amplification (LAMP) to Target 56kDa gene in Scrub Typhus Patients and Phylogenetic Analysis to Identify Orientia tsutsugamushi Strains Circulating In and Around Puducherry

Scrub typhus (ST) is a re-emerging zoonotic disease caused by Orientia tsutsugamushi and is transmitted by chiggers. Serological tests targeting IgM and/or IgG antibodies play a major role in the detection of ST cases. Orientia 56kDa genome is common target for the molecular diagosis of ST to identify the prevalence of specific serotypes of O. tsutsugamushi in and around Puducherry by targeting 56kDa gene with the application of phylogenetic analysis. This prospective laboratory-based study was conducted in a tertiary care teaching hospital, from November 2105 to March 2018. Blood samples were collected from out-patients/in-patients, and those tested positive for ST IgM ELISA (n=140) and an equal number of negative samples were archived and anonymized. Genomic DNA was extracted and analyzed by using Nested PCR and LAMP assay. The positive products were purified and sequenced. Phylogenetic tree was constructed based on the sequences. Among 280 samples, 45 (16.1%) N-PCR and 102 LAMP (36.43%) positivity was observed for 56kDa gene. Forty-one N-PCR positive products were sequenced and accession numbers were obtained (MG601875 to MG601917) for the isolates. Phylogenetic analysis was done by Maximum Likelihood methods and this study has showed that 32.3% are similar to the Karp isolates. Molecular diagnosis of Scrub typhus has become essential in case of doubtful serology and early acute phase of illness. Gene sequencing result indicates that most of them were different from the existing ones, which may belong to the newer strains. The identification of newer strains will be helpful in future for development of scrub typhus vaccine.

Predicting Tumour Progression of ID-8 Syngeneic Mouse Ovarian Cancer with Blood Biomarkers of CA-125, IL-6 and VEGF: A Prospective Laboratory-Based Study

Predicting Tumour Progression of ID-8 Syngeneic Mouse Ovarian Cancer with Blood Biomarkers of CA-125, IL-6 and VEGF: A Prospective Laboratory-Based Study

Pre-extensively drug-resistant tuberculosis among pulmonary multidrug-resistant tuberculosis patients in Eastern Nigeria.

Pre-extensively drug-resistant tuberculosis (Pre-XDR-TB), an emerging form of drug-resistant tuberculosis, is challenging efforts at tuberculosis control, leading to treatment failure among multidrug-resistant tuberculosis (MDR-TB) patients and progression to extensively drug-resistant tuberculosis (XDR-TB). We determined the rate of Pre-XDR-TB among multidrug-resistant patients in Southeast, Nigeria. A prospective laboratory-based study was carried out at the South East Zonal Tuberculosis Reference Laboratory from January 2021 to December 2021. Second-line drug (SLD) resistance was performed on 225 sputum samples of multidrug-resistant patients prior to treatment initiation using GenoType MTBDRsl genotypic drug susceptibility testing (DST) method. The rate of Pre-XDR-TB among 225 MDR-TB cases was 3.1%. Fluoroquinolone-resistant Pre-XDR-TB was observed (100%) in previously treated tuberculosis cases. Only one (0.4%) case showed resistance to both fluoroquinolone (FQ) and one second-line injectable drug (XDR-TB). The extensively drug-resistant case observed was a de-novo resistance. Exactly 0.9% of the multidrug-resistant cases showed resistance to second-line injectables. The prevalence of Pre-XDR-TB among MDR-TB cases was high. There is need for rapid detection of Pre-XDR-TB among MDR-TB cases before treatment initiation.

Time of incubation of agar-plate culture for the diagnosis of Strongyloides stercoralis infection

PurposeAgar-plate culture (APC) remains the most sensitive parasitological technique for S. stercoralis diagnosis. Although it was first described three decades ago, the time of incubation of the plates is neither a commonly described feature nor usually standardized. The aim of the study was to analyze the required time to detect S. stercoralis larvae in APC. MethodsA prospective laboratory-based study including all patients with at least one positive APC was performed. The plates were incubated at room temperature for 7 days. Clinical, analytical and parasitological features including results of the direct visualization of the stool (DV) after formalin-ether concentration and time-to-detection (TTD) of the larvae in APC were recorded. ResultsA total of 141 samples from 75 patients had a positive APC. In 49 of them (65.3%) three or more stool samples were processed for direct visualization (DV) and APC. Of these 49 patients, 8 (16.3%) were also diagnosed with DV and 41 (83.7%) were diagnosed only with APC. In 38 samples from 23 (30.7%) patients, the TTD was below 2 days, while in 27 samples from 13 (17.3%) patients, the larvae were detected on the 6th and 7th day. ConclusionDirect visualization failed to detect S. stercoralis in most of the patients that were diagnosed with APC. Incubation periods below 2 and 5 days would miss an important percentage of infections. At least 7 days of incubation of the APC are required to detect presumably low-burden chronic infections in non-endemic countries.

MUPIROCIN RESISTANCE IN STAPHYLOCOCCUS AUREUS IN A TERTIARY CARE HOSPITAL OF SOUTH INDIA – A PROSPECTIVE STUDY

Objective: Mupirocin is a topical antibiotic used for the treatment of skin and soft tissue infections caused by Staphylococcus aureus and for the nasal decolonization of methicillin-resistant S. aureus (MRSA). The increasing reports of resistance to mupirocin are a matter of concern. We undertook this study to detect and differentiate the mupirocin resistance pattern and to analyze the susceptibility pattern among S. aureus isolates of our hospital.Methods: This is a prospective laboratory-based study conducted during the period May–September 2014. Clinical samples that grew S. aureus during the study period were tested for mupirocin resistance using the 5 μg and 200 μg discs. Minimum inhibitory concentration (MIC) detection of resistant strains was performed using the E-test.Results: Mupirocin resistance was seen in 4.81% of our S. aureus isolates; all of which exhibited high-level resistance with MIC ≥1024 μg/ml.Conclusions: The resistance is bound to rise with the increased usage of mupirocin; regular testing will help in tackling this upcoming problem and in preserving this important antibiotic against MRSA.

MUPIROCIN RESISTANCE IN STAPHYLOCOCCUS AUREUS IN A TERTIARY CARE HOSPITAL OF SOUTH INDIA – A PROSPECTIVE STUDY

Objective: Mupirocin is a topical antibiotic used for the treatment of skin and soft tissue infections caused by Staphylococcus aureus and for the nasal decolonization of methicillin-resistant S. aureus (MRSA). The increasing reports of resistance to mupirocin are a matter of concern. We undertook this study to detect and differentiate the mupirocin resistance pattern and to analyze the susceptibility pattern among S. aureus isolates of our hospital.Methods: This is a prospective laboratory-based study conducted during the period May–September 2014. Clinical samples that grew S. aureus during the study period were tested for mupirocin resistance using the 5 μg and 200 μg discs. Minimum inhibitory concentration (MIC) detection of resistant strains was performed using the E-test.Results: Mupirocin resistance was seen in 4.81% of our S. aureus isolates; all of which exhibited high-level resistance with MIC ≥1024 μg/ml.Conclusions: The resistance is bound to rise with the increased usage of mupirocin; regular testing will help in tackling this upcoming problem and in preserving this important antibiotic against MRSA.

Enhanced expression of SAM-pointed domain-containing Ets-like factor in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by significant goblet hyperplasia and mucus hypersecretion. However, the molecular mechanism underlying mucin overexpression in CRSwNP has not been well characterized. This study sought to assess the expression of SAM-pointed domain-containing Ets-like factor (SPDEF) and its regulation of mucin production in CRSwNP patients. Prospective laboratory-based study. Polyp and control nasal tissues were collected from 27 CRSwNP patients and 15 control subjects. The expression of SPDEF and MUC5AC in the nasal tissues was examined by immunohistochemistry staining, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. In addition, SPDEF and MUC5AC expression was evaluated in cultured polyp epithelial cells and bronchial epithelial cells (BECs) in the presence of proinflammatory cytokines, corticosteroids and SPDEF small interfering RNA (siRNA) using qRT-PCR and western blot analysis. We found significantly increased SPDEF and MUC5AC expression in CRSwNP patients compared to the controls (P < .05); the mRNA level of SPDEF was positively correlated with MUC5AC expression in CRS patients (P < .05). There was a significant difference in SPDEF and MUC5AC expression in eosinophilic and noneosinophilic CRSwNP patients (P < .05). Interleukin (IL)-13, IL-8, and IL-17A significantly increased SPDEF and MUC5AC expression in vitro (P < .05). SPDEF siRNA significantly inhibited SPDEF and MUC5AC expression in BECs in response to IL-13, IL-8, and IL-17A (P < .05). Our findings suggested that SPDEF may be regarded as a promising therapeutic target for modulating mucus hypersecretion in CRSwNP.

An ex vivo evaluation of efficacy of refrigerated canine plasma.

To determine thawing times of fresh frozen plasma (FFP), and to evaluate the activity of hemostatic proteins (coagulation factors V, VII, VIII, IX, X, and fibrinogen), clotting times (prothrombin time and activated partial thromboplastin time), and sterility of canine plasma stored refrigerated. Prospective laboratory-based study. Veterinary teaching hospital blood bank. Phase 1: Six units of canine FFP were retrieved from the blood bank and thawed individually in a warm water bath. Time for thaw was recorded in minutes and reported as mean ± SD. Phase 2: One unit of fresh whole blood was collected from 9 dogs and processed routinely. Resulting plasma was divided into 2 aliquots, 1 stored as refrigerated plasma (RP) and 1 as frozen plasma. Samples from the RP were taken at 0, 1, 5, 7, and 14 days and from the FFP at days 0 and 14 for determination of clotting factor activity (V, VII, VIII, IX, and X and fibrinogen) and clotting times. Coagulation factors and clotting times were analyzed using a mixed effects linear model for ANOVA, comparing changes over time as well as differences between groups. For all comparisons, a P value of <0.05 was considered significant. Batch bacterial aerobic and anaerobic cultures of the RP samples were submitted on days 7 and 14 and from the frozen plasma on day 14. Time to thaw for FFP units was 34.7 ± 1.38 minutes. Refrigerated storage resulted in significant decreases in the activity of all clotting factors and a subsequent prolongation in clotting times. However, no values were outside of the reference interval. All bacterial cultures yielded no growth. Refrigerated storage results in only minor loss of coagulation factor activity in canine plasma. The use of RP, therefore, may be a viable option in high-volume veterinary hospitals for rapid correction of coagulopathy in critical care patients.

Localised Fusobacterium necrophorum infections: a prospective laboratory-based Danish study

During a 3-year prospective laboratory-based study in Denmark from 1998 to 2001, all patients who were diagnosed with localised Fusobacterium necrophorum infections were registered with the purpose of describing the variety of localised infections caused by F. necrophorum, especially in the head. We found 267 patients, most of them previously healthy, with localised F. necrophorum infections in the head and neck. In children, F. necrophorum caused otitis media and solitary abscess formation in cervical lymphadenitis. In adolescents, F. necrophorum was found in 21% of peritonsillar abscesses. We also found F. necrophorum in young adults with tonsillitis and in middle-aged adults with sinusitis. F. necrophorum was found in substantial amounts and as the only bacterial pathogen in the majority of patients. All 267 patients recovered without sequelae. We found another 21 localised non-head-and-neck-associated F. necrophorum infections, mainly subcutaneous wound infections in adults. This study shows that F. necrophorum causes a variety of localised infections, especially in the head and neck region, with a characteristic age distribution. We recommend that anaerobic culture is performed on swabs from children with recurrent otitis media and adolescents with non-streptococcal group A tonsillitis.

O-63: Angiogenesis factors in human ovarian follicular development: The presence of vascular endothelial growth factor and placental growth factor in follicular fluid

It has been demonstrated that angiogenesis plays an important role in normal cyclical ovarian function. In particular, follicular growth is dependent upon the proliferation of new capillary vessels, and, it has been proposed that the process of selection of a dominant follicle is likely to be associated with angiogenesis. Vascular endothelial growth factor (VEGF) and its homolog the placental growth factor (PlGF) are vascular cell-specific mitogen factors with a key function in angiogenesis. Their role in human follicular growth and oocyte maturation however, is less well-understood and the presence of PlGF in human follicles has not been previously demonstrated. This study sought to determine the content of both VEGF and PlGF within the follicular fluid (FF) of large and small follicles of women undergoing in vitro fertilization (IVF) and to relate their content to patient age A prospective laboratory-based study at an academic center. Women between the ages of 25 - 43 years old undergoing IVF were enrolled under IRB approval during the time of oocyte retrieval. Follicles were measured by transvaginal ultrasound in 2 dimensions prior to aspiration. FF from lead (>14mm) and small (< 12mm) follicles was collected by transvaginal aspiration. Cellular components and immunoglobulins were removed by centrifugation and protein A/G chromatography. VEGF and PlGF concentration in FF were then analyzed via enzyme-linked immunosorbent assay (ELISA) on lead and small follicles from young (</= 34 years) and old (>/= 40 years) samples and confirmed by western blot with specific antibodies. Results were analyzed with students’t test and statistical significance was set at a p value of < 0.05. The results of the Western Blot analysis showed the presence of both VEGF and PlGF within the FF. The mean concentration of PlGF in lead follicles was 42.41 pg/ml and the mean concentration in small follicles was 55.91 pg/ml (p= 0.10). The mean concentration of VEGF in lead follicles was 5,745.76 pg/ml whereas the mean concentration in small follicles was 3,485.70 pg/ml (p<0.01). The concentration of VEGF and PlGF did not vary with respect to age. This study demonstrated a significantly increased concentration of VEGF in the follicular fluid of lead follicles compared with smaller follicles irrespective of age, lending proof to the idea that angiogenesis is an important part of lead human follicle development. The trend of our results for PlGF supports findings suggesting that PlGF may act as a natural VEGF antagonist. Additionally, our western blot and ELISA results are the first demonstration of the presence of PlGF in human FF. Overall, this study demonstrates that angiogenesis factors are important in follicular development in women undergoing IVF.

An In Vitro Study of Human Lens Epithelial Cell Adhesion to Intraocular Lenses with and without a Fibronectin Coating

To demonstrate differences in human lens epithelial cell adhesion to different intraocular lens biomaterials in vitro and to determine whether these differences can be influenced by coating the intraocular lens surface with commercially available fibronectin. A prospective laboratory-based study comparing human lens epithelial cell adhesion to silicone (n=18), polymethylmethacrylate (PMMA; n=18), and acrylic (n=18) intraocular lenses in vitro. The three types of intraocular lenses were then coated with fibronectin: silicone (n=6), PMMA (n=6), and acrylic (n=6). The main outcome measure was the mean number of lens epithelial cells attached to each lens type after 24 hours of incubation. In the uncoated lens group, there was a significantly higher number of lens epithelial cells attached to the acrylic than to the silicone or PMMA lenses (P<0.001). Coating the lenses with fibronectin caused a significant increase in attachment of lens epithelial cells for all three lens types. There was a significant difference in the degree of lens epithelial cell attachment to the various types of intraocular lenses in vitro. Cell attachment was more prominent in the acrylic lenses, but the fibronectin coating negated differences in lens type and caused a significant increase in cell attachment across all groups.