Enzyme-Linked Immunosorbent Assay Method for Detection of Cytomegalovirus Strain-Specific Antibody Responses

Reliable methods for the detection of cytomegalovirus (CMV) strain-specific serological responses are lacking. We describe a simple and reliable enzyme-linked immunosorbent assay method developed to detect antibodies against the polymorphic epitopes within the two envelope glycoproteins of CMV, glycoproteins H and B. This assay is useful for the detection of serologic responses to CMV strains and the identification of CMV reinfections.Cytomegalovirus (CMV) is an important pathogen in immunocompromised hosts and a frequent cause of congenital infection. CMV isolated from clinical samples exhibits extensive genetic variation (7, 12, 13), and CMV reinfections have been demonstrated to occur in seropositive individuals. However, it is thought that these reinfections have little untoward consequences with respect to congenital infections. Recent studies documenting higher rates of congenital CMV infection in populations with nearly universal seroreactivity to CMV suggest that infection with new or different virus strains could be responsible for the intrauterine transmission of CMV in immune mothers (5, 17, 18). The frequency and consequences of infection with multiple CMV strains are unclear because of the lack of reliable methods for the accurate identification of CMV strain-specific antibody responses. By utilizing the defined heterogeneity within the antibody binding epitopes on envelope glycoprotein H (gH) and gB of the AD169 and Towne strains of CMV, an enzyme-linked immunosorbent assay (ELISA) method was developed to distinguish serological responses against infection with different CMV strains.Serum samples from 96 CMV-seropositive women participating in an ongoing study and 51 seronegative individuals were tested for anti-CMV strain-specific antibodies. Informed consent was obtained from the study participants, and the study was conducted in accordance with the guidelines of the Institutional Review Board for Human Use of the University of Alabama at Birmingham.Purified recombinant antigens based on polymorphic antibody binding sites defined on gH (antigen gpUL75) and gB (antigen gpUL55) were used as antigens (Fig. ​(Fig.1).1). The gH antigens were constructed as β-galactosidase fusion proteins containing the coding region for amino acids (aa) 15 to 142 of gpUL75 from the AD169 strain (the AP86 antigen) and aa 14 to 42 of the Towne strain (the TO86 antigen) of CMV (16). The recombinant peptides were expressed in Escherichia coli and were purified as described previously (8). gB antigens were prepared as six-His-tag-labeled peptides by cloning the coding region (aa 1 to 116) from strains AD169 (the AD55 antigen) and Towne (the TO55 antigen) (9) into expression vector pET21a (EMD, Gibbstown, NJ) by using the HindIII and BamHI endonuclease restriction sites. The peptides were expressed in E. coli Rosetta cells and were purified by using Talon Superflow metal affinity columns (Clonetech, Mountain View, CA). A positive control antigen was constructed by cloning the antigen domain 1 (AD-1) region of the gene coding gB, which has been shown to be highly conserved among clinical isolates of CMV, as described previously (2), into each vector (AD-1). The reactivity against an empty vector expressing fusion protein alone or nonantigenic proteins of mouse origin was used as a negative control.Open in a separate windowFIG. 1.Amino acid sequence alignment of the amino-terminal regions of gpUL75 (gH) and gpUL55 (gB) from the AD169 and Towne strains of CMV depicting the differences betweens the two strains.Strain-specific ELISA was performed on PolySorp microtiter plates (Nunc, Roskilde, Denmark). The wells of the plates were coated overnight with 50 μl of purified gH antigens (antigens AP86 and TO86) (1) or gB antigens (antigens AD55 and TO55) diluted in carbonate buffer and blocked with 3% goat serum in borate buffer (BB) for 2 h at 37°C. Serum samples diluted 1:100 in BB were added to the wells, and the plates were incubated at 37°C for 1 h. After the plates were washed three times with BB containing 0.05% Tween 20, goat anti-human immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated antibody (Pierce, Rockford, IL) diluted 1:10,000 was added and the plates were incubated for 1 h at 37°C. The plates were developed by the addition of 50 μl of one-step Ultra TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Pierce) for 10 min at room temperature (RT), and the reaction was stopped by the addition of 2 N sulfuric acid. The optical density (OD) values were determined with a spectrophotometer. A positive result was defined as an OD value more than three times the mean result obtained for each antigen with seronegative samples.Western blot assays were performed with a subset of 12 samples. Appropriate antigens were run on a 12.5% sodium dodecyl sulfate-polyacrylamide gel and then blotted onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Billerica, MA), according to the manufacturer''s recommendations. The membranes were blocked for 2 h in 3% goat serum in SuperBlock buffer (Pierce, Rockford, IL) and 0.05% Tween 20 at RT. Human sera were diluted 1:5,000 in blocking buffer and applied to the membrane, and the membrane was shaken at RT for 2 h. The membranes were washed four times in wash buffer (BB with 0.05% Tween 20), and goat anti-human IgG HRP-conjugated antibody (Pierce) diluted 1:100,000 in blocking buffer was added. After incubation at RT for 2 h, the membranes were washed four times in wash buffer and soaked into the substrate Luminol West Femmto (Pierce) for 10 min. The membranes were placed on X-ray film, and images were developed and acquired by using a VersaDoc imaging system (Bio-Rad, Hercules, CA).Of the 96 baseline serum samples from CMV-seropositive women participating in an ongoing study testing for strain-specific antibodies, 58 (60%) samples were positive for at least one of the four antigens and 18 samples were positive for two or more antigens. The OD values (mean ± standard deviation) for each antigen for the group of 51 CMV-seronegative individuals and the samples that were considered positive and negative from the 96 CMV IgG antibody-positive women are shown in the Table ​Table1.1. Forty-five percent (43/96) of the samples had reactivity to the AP86 antigen, with a mean OD value of 1.240 ± 0.498, whereas the OD values were 0.291 ± 0.134 and 0.196 ± 0.052 for the negative samples and the CMV IgG antibody-negative control serum samples, respectively. Fifteen percent (14/96) of the study samples were positive for the TO86 antigen, with an average OD reading of 1.069 ± 0.317. Responses against the AD55 and TO55 antigens were seen in 8% and 20% of the samples, respectively, with corresponding OD values of 0.681 ± 0.103 and 0.708 ± 0.278 (Table ​(Table11).

TABLE 1.

Reactivities against the gH (AP86 and TO86) and gB (AD55 and TO55) polymorphic epitopes from CMV strains AD169 and Towne of serum samples from 96 CMV-seropositive women and 51 seronegative individuals and two serum samples with known reactivity against AP86, TO55, or TO86

Unusual inferior dislocation of shoulder: reduction by two-step maneuver: a case report

Dislocation of the shoulder is the commonest of all large joint dislocations. Inferior dislocation constitutes 0.5% of all shoulder dislocations. It characteristically presents with overhead abduction of the arm, the humerus being parallel to the spine of scapula. We present an unusual case of recurrent luxatio erecta in which the arm transformed later into an adducted position resembling the more common anterior shoulder dislocation. Such a case has not been described before in English literature. Closed reduction by the two-step maneuver was successful with a single attempt. MRI revealed posterior labral tear and a Hill-Sachs variant lesion on the superolateral aspect of humeral head. Immobilisation in a chest-arm bandage followed by physiotherapy yielded excellent results. The case is first of its kind; the unusual mechanism, unique radiological findings and alternate method of treatment are discussed.     

Correlation of tunnel widening and tunnel positioning with short-term functional outcomes in single-bundle anterior cruciate ligament reconstruction using patellar tendon versus hamstring graft: a prospective study

Objective

To study the correlation between tunnel widening and tunnel position with short-term functional outcomes post-ACL reconstruction with patellar tendon and hamstring autografts in young adults.

Materials and methods

A total of 33 patients who underwent ACL reconstruction between October 2013 and February 2015 were included and followed up for 6 months. A standardized surgical technique was used for each graft type. Intra-op arthroscopy findings and drilled tunnel diameters were noted. They were followed up for 3 and 6 months. Radiological assessment was done at 3 and 6 months with clinical score assessment at 6 months.

Results

At 6 months, clinical scores were comparable in both groups. Tunnel widening in both femoral and tibial tunnel at 3 and 6 months were significantly higher in STG group (p values <0.05). The rate of widening was higher in 0–3 months and reduced in 3–6 months. There was statistically significant negative correlation between femoral tunnel widening by CT and IKDC score at 6 months (p value 0.049). We found a positive correlation between posterior positioning of femoral tunnel and Lysholm and IKDC scores. The correlation with Lysholm scores was statistically significant (p value 0.046).

Conclusion

To conclude, tunnel widening is more with hamstrings graft. Femoral tunnel widening has significant negative correlation with that of IKDC scores at 6 months. Posterior femoral tunnel positioning and Lysholm scores at 6 months had significant correlation.

     

Nutritional interventions or exposures in infants and children aged up to 3 years and their effects on subsequent risk of overweight,obesity and body fat: a systematic review of systematic reviews

This study, performed as part of the international EarlyNutrition research project ( http://www.project‐earlynutrition.eu ), provides a systematic review of systematic reviews on the effects of nutritional interventions or exposures in children (up to 3 years of age) on the subsequent risk of obesity, overweight and adiposity. Electronic databases (including MEDLINE, Embase and Cochrane Library) were searched up until September 2015. Forty systematic reviews were included. A consistent association of breastfeeding with a modest reduction in the risk of later overweight and obesity in childhood and adulthood was found (the odds decreased by 13% based on high‐quality studies), but residual confounding cannot be excluded. Lowering the protein content of infant formula is a promising intervention to reduce the risk of later overweight and obesity in children. There is no consistent evidence of an association of the age of introducing complementary foods, sugar‐sweetened beverage or energy intake in early childhood with later overweight/obesity, but there are some indications of an association of protein intake during the complementary feeding period with later overweight/obesity. There was inadequate evidence to determine the effects of other nutritional interventions or exposures, including modifications of infant formula composition, fat intake or consumption of different food groups.     

Pathway to care of epilepsy patients: Exploratory study from an urban slum in Northern India

Introduction:

Epilepsy is a chronic neurological disorder with major psychosocial correlates. Most epilepsy patients in developing countries are untreated or inadequately treated. It is essential to understand the pathway, to care taken by epilepsy patients in a community, to be able to target appropriate services to them.

Materials and Methods:

A community based study was conducted on all epilepsy patients in an urban slum in Northern India to study their pathways to care. A list of persons suffering from epilepsy was generated by house to house visits, snowballing, and key informant contacts. In-depth interview and Medical Record Review were used to document their pathway to care.

Results:

Thirteen of the twenty two patients had contacted a health-care provider for their first episode. The most common first link of care for the patients was secondary level Government hospitals. The next common was private practitioners, followed by Tertiary Care Hospitals, and registered medical practitioners. Eleven out of twenty two patients had to contact a Tertiary Level Center for seeking care. The number of health-care facilities consulted before arriving at their latest point of care ranged from 0 to 5. Traditional or faith healers were consulted at some point of time for cure.

Conclusion:

There is a need to focus on strengthening and capacity building of the primary care settings for managing epilepsy to enable their better utilization. This shall prevent unnecessary referrals and hence the load on the already burdened higher facilities.     

Tumor-induced osteomalacia—a diagnostic dilemma for an orthopedic surgeon

Tumor-induced osteomalacia is a rare condition caused by excess production of phosphatonins most notably fibroblast growth factor?23 (FGF-23), by the tumor cells, leading to phosphate wasting and consecutive severe hypophosphatemia. This results in patient developing gradually progressive muscle weakness and bone loss resulting in severe osteomalacia, making the patient bedbound. These tumors are mostly benign mesenchymal tumors, which remain hidden in soft tissues or bone and thus are difficult to diagnose. And in the presence of normal serum calcium and parathyroid levels with only mild alteration of vitamin D levels, the diagnosis gets further delayed causing a lot of apathy to the patient. We hereby present a thorough review of this condition along with our experience in diagnosing and treating this patient. This underlines the fact that a high index of suspicion is required for diagnosing this condition in a patient with persistent complains of fatigue and bone pains. Appropriate investigations done at an early stage can help one in identifying and excising these tumors, bringing about a rapid relief of symptoms and saving the patient from a lot of distress.     

Does age, time since injury and meniscal injury affect short term functional outcomes in arthroscopic single bundle anterior cruciate ligament reconstruction?

Purpose

We conducted this study to correlate the short term clinical outcomes after anterior cruciate ligament (ACL) reconstruction with patients' age, time since injury and associated meniscal injury.

Effective Control of Salmonella Infections by Employing Combinations of Recombinant Antimicrobial Human β-Defensins hBD-1 and hBD-2

We successfully produced two human β-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities against Salmonella enterica serovar Typhi, Escherichia coli, and Staphylococcus aureus employing both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of the bacterial population when used individually and up to 90% when used in combination. The 50% lethal doses (LD₅₀) of hBD-1 against S. Typhi, E. coli, and S. aureus were 0.36, 0.40, and 0.69 μg/μl, respectively, while those for hBD-2 against the same bacteria were 0.38, 0.36, and 0.66 μg/μl, respectively. Moreover, we observed that bacterium-derived antimicrobial peptides were also effective in increasing survival time and decreasing bacterial loads in the peritoneal fluid, liver, and spleen of a mouse intraperitoneally infected with S. Typhi. The 1:1 hBD-1/hBD-2 combination showed maximum effectiveness in challenging the Salmonella infection in vitro and in vivo. We also observed less tissue damage and sepsis formation in the livers of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combination. Based on these findings, we conclude that bacterium-derived recombinant β-defensins (hBD-1 and hBD-2) are promising antimicrobial peptide (AMP)-based substances for the development of new therapeutics against typhoid fever.     

Levels of compliance of self-care practices of diabetes mellitus type 2 patients: a study from a tertiary care hospital of North India

International Journal of Diabetes in Developing Countries - Diabetes management strategies are interdependent and comprise of three basic key elements: self-care activities, effective drug...