Statens Serum Institut Research Articles

Group B Streptococcal Colonization Among Pregnant Women in Delhi, India.

Little is known regarding maternal group B streptococcal (GBS) colonization prevalence and capsular (CPS) serotype distribution among pregnant women in India. The objective of this prospective cohort study was to determine GBS recto-vaginal colonization prevalence in pregnant women at Dr. Ram Manohar Lohia Hospital in Delhi, India. Literature review identified reports from India assessing GBS colonization prevalence in pregnant women. Rectal and vaginal swabs were inoculated into Strep B Carrot Broth (Hardy Diagnostics, Santa Maria, CA) and subcultured onto GBS Detect plates (Hardy Diagnostics, Santa Maria, CA). Isolates were serotyped using ImmuLex Strep-B latex kits (Statens Serum Institut, Copenhagen, Denmark). Thirteen studies were identified citing GBS colonization prevalence during pregnancy as 0.47%-16%. Among 300 pregnant women (mean age: 26.9 years; mean gestation: 34 weeks) enrolled (August 2015 to April 2016), GBS colonization prevalence was 15%. Fifteen percent of women had vaginal only, 29% had rectal only and 56% had both sites colonized. CPS types were Ia (13.3%), Ib (4.4%), II (20%), III (22.2%), V (20%) and VII (6.7%); 13.3% were nontypable. Fetal loss in a prior pregnancy at ≥20-weeks gestation was more common in colonized than noncolonized women (15.6% vs. 3.5%; P = 0.004). Employing recent census data for the birth cohort and estimating that 1%-2% of neonates born to colonized women develop early-onset disease, at least 39,000 cases of early-onset disease may occur yearly in India. Using optimal methods, 15% of third trimester pregnant women in India are GBS colonized. A multivalent vaccine containing 6 CPS types (Ia, Ib, II, III, V and VII) would encompass ~87% of GBS carried by pregnant women in India.

The Follicular Skin Microbiome in Patients With Hidradenitis Suppurativa and Healthy Controls

Although the pathogenesis of hidradenitis suppurativa (HS) remains enigmatic, several factors point to potential involvement of the cutaneous microbiome. Insight into the cutaneous microbiome in HS using next-generation sequencing may provide novel data on the microbiological diversity of the skin. To investigate the follicular skin microbiome in patients with HS and in healthy controls. This case-control study obtained punch biopsy specimens from patients with HS (lesional and nonlesional) and healthy controls between October 1, 2014, and August 1, 2016. Data were analyzed from March to November 2016. Patients with HS were recruited from the Department of Dermatology, Zealand University Hospital, Roskilde, Denmark. Biopsy specimens were analyzed at the Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark. None of the participants received any antibiotics (systemic or topical therapy) within 1 month before the study. In patients with HS, biopsy specimens were obtained from lesional skin (axilla or groin) and nonlesional skin. Only nodules containing at least 1 visible hair follicle were biopsied. Biopsy specimens from healthy controls were obtained from the axilla only. The different microbiomes were investigated using next-generation sequencing targeting 16S and 18S ribosomal RNA. The skin microbiome was characterized in 30 patients with HS (mean [SD] age, 46.9 [14.0] years; 19 [63% female]) and 24 healthy controls (mean [SD] age, 32.2 [12.0] years; 13 [54% female]). The next-generation sequencing data provided a previously unreported (to our knowledge) characterization of the skin microbiome in HS. The study demonstrated that the microbiome in HS differs significantly from that in healthy controls in lesional and nonlesional skin. Overall, the following 5 microbiome types were identified: Corynebacterium species (type I), Acinetobacter and Moraxella species (type II), Staphylococcus epidermidis (type III), Porphyromonas and Peptoniphilus species (type IV), and Propionibacterium acnes (type V). In lesional skin, microbiome types consisted predominantly of type I or type IV. Microbiome type IV was not detected in healthy controls. Several taxa, including Propionibacterium, showed a significantly higher relative abundance in healthy controls vs HS skin, indicating that Propionibacterium may be part of the pathogenesis in HS. The study findings suggest a link between a dysbiotic cutaneous microbiome and HS.

The completeness of chest X-ray procedure codes in the Danish National Patient Registry.

ObjectiveThe aim of this validation study was to assess the completeness of the registrations of chest X-rays (CXR) in two different versions of the Danish National Patient Registry (DNPR).Material and methodsWe included electronic record data on CXR performed on patients aged 40 to 99 years from nine radiology departments covering 20 Danish hospitals. From each department, we included data from three randomly selected weeks between 2004 and 2011 (reference standard). In two versions of the DNPR from the State Serum Institute (SSI) and Statistics Denmark, respectively, we investigated the proportion of registered CXR compared to the reference standard. Furthermore, we compared the completeness of the recorded data according to the responsible department (main department).ResultsWe identified 11,235 patients and 12,513 CXR in the reference standard. The data from the SSI contained 12,265 (98%) CXR, whereas the data from Statistics Denmark comprised 9,151 (73.1%) CXR. The completeness of the SSI data was fairly constant across years, radiology departments, medical specialties, and age groups. The data from Statistics Denmark was almost complete in 2011 (95.8%). However, for the remaining study period, the data with radiology departments registered as the main department were lacking in the version from Statistics Denmark, and so the overall completeness was 73.1%.ConclusionThe completeness of CXR registrations varied between 98% and 73% depending on the information source, and this should be considered when investigating radiology services in the basis of DNPR.

Safety and efficacy of the C-Tb skin test to diagnose Mycobacterium tuberculosis infection, compared with an interferon γ release assay and the tuberculin skin test: a phase 3, double-blind, randomised, controlled trial

Targeted screening and treatment of Mycobacterium tuberculosis infection substantially reduces the risk of developing active tuberculosis. C-Tb (Statens Serum Institute, Copenhagen, Denmark) is a novel specific skin test based on ESAT-6 and CFP10 antigens. We investigated the safety and diagnostic potential of C-Tb compared with established tests in the contact-tracing setting. Negative controls, close contacts, occasional contacts, and patients with active pulmonary tuberculosis were enrolled at 13 centres in Spain. We compared C-Tb with the QuantiFERON-TB Gold In-Tube ([QFT] Qiagen, Hilden, Germany) interferon γ release assay (IGRA) and the purified protein derivative (PPD) RT 23 tuberculin skin test ([TST] Statens Serum Institute). All participants older than 5 years were tested with QFT. Some participants in the negative control group received C-Tb without the TST to test for potential interactions between C-Tb and PPD RT 23. The rest were randomly assigned in blocks of ten and tested with both C-Tb and TST, with five in each block receiving injection of C-Tb in the right arm and the TST in the left arm and five vice versa. The primary and safety analyses were done in all participants randomly assigned to a group who received any test. This trial is registered with ClinicalTrials.gov, number NCT01631266, and with EudraCT, number 2011-005617-36. From July 24, 2012, to Oct 2, 2014, 979 participants were enrolled, of whom 263 were negative controls, 299 were occasional contacts, 316 were close contacts, and 101 were patients with tuberculosis. 970 (99%) participants completed the trial. Induration sizes were similar for C-Tb and TST, but TST positivity was affected by BCG vaccination status. We found a strong positive trend towards C-Tb test positivity with increasing risk of infection, from 3% in negative controls to 16% in occasional contacts, to 43% in close contacts. C-Tb and QFT results were concordant in 785 (94%) of 834 participants aged 5 years and older, and results did not differ significantly between exposure groups. The safety profile of C-Tb was similar to that for the TST. C-Tb delivered IGRA-like results in a field-friendly format. Being unaffected by BCG vaccination status, the C-Tb skin test might provide more accurate treatment guidance in settings where the TST is commonly used. Statens Serum Institut.

Continuous Pneumococcal Invasive Disease Reduction, High Impact on Pneumococcal Empyemas and Meningitis, and Serotype 19A Disappearance Following PCV-13 Vaccination in Children: Eleven and a Half Years of Active Surveillance in a Mexican Hospital on the U.S. Border

BackgroundThe Tijuana, Mexico and San Diego, California, is the world’s most transited frontier. We have previously published pneumococcal serotype replacement following PCV-7 introduction, especially by serotypes 19-A, 3 and 6A/C. Since May/2012 universal vaccination with PCV-13 was introduced for all children. This study analyzes the effectiveness of PCV-13 after its implementation in the region.MethodsSince October/2005 until March/2017 (11.5 years) we underwent an active surveillance for all Invasive Pneumococcal Diseases (IPD) in children < 16 years admitted at the Tijuana General Hospital. Only culture-confirmed cases were included. Following identification, serotyping was performed using the Quellung Reaction (Statens Serum Institute® Copenhagen, Denmark). Descriptive analysis was performed using Excel®.ResultsFifty-seven cases of confirmed IPD were found. Clinical diagnosis were pleural empyemas (PPE) (45.6%), meningitis (PM) (28%), otomastoiditis (15.7%), only sepsis (7%), and bacteremic pneumonia (3.5%). Median age was of 3.9 years (2 days – 15.9 years), median hospitalization days was of 14 (1–90). Overall mortality was of 6 (10.5%). Following PCV13 introduction there was a decreased on overall IPD mortality by 73.29%, overall IPD by 68%, PPE by 68.1%, and PM by 55.4%. However, during 2015–16, we had three PM cases, two of which were immunocompromised patients, and none vaccinated with PCV13. From an average of 1.75 yearly cases due to pneumococcal serotype 19-A before PCV13 implementation, there had been no patients with IPD’s caused by this serotype. (See Figure 1). After PCV13 vaccination, serotypes associated with IPD cases had been as follow: Two 22F, and one each by 6A, 18C, 3, 7F, 15, 7B, 35B, 24F and 33F.ConclusionThis is the first Mexican study based on active surveillance that shows consistent effectiveness of PCV-13 on reduction of overall IPD, disappearance of pneumococcal serotype 19-A, and strong impact on PPE in children. Effectiveness on PM is still above 50%, however, during 2015–16 there had been three cases, but with particular conditions (immunodeficiencies and no vaccination). Continuous active surveillance is mandatory.Disclosures All authors: No reported disclosures.

First-in-human safety and immunogenicity investigations of three adjuvanted reduced dose inactivated poliovirus vaccines (IPV-Al SSI) compared to full dose IPV Vaccine SSI when given as a booster vaccination to adolescents with a history of IPV vaccination at 3, 5, 12 months and 5 years of age

BackgroundThere is a demand of affordable IPV in the World. Statens Serum Institut (SSI) has developed three reduced dose IPV formulations adsorbed to aluminium hydroxide; 1/3 IPV-Al, 1/5 IPV-Al and 1/10 IPV-Al SSI, and now report the results of the first investigations in humans. Methods240 Danish adolescents, aged 10–15years, and childhood vaccinated with IPV were booster vaccinated with 1/3 IPV-Al, 1/5 IPV-Al, 1/10 IPV-Al or IPV Vaccine SSI. The booster effects (GMTRs) of the three IPV-Al SSI were compared to IPV Vaccine SSI, and evaluated for non-inferiority. Immunogenicity resultsThe pre-vaccination GMTs were similar across the groups; 926 (type 1), 969 (type 2) and 846 (type 3) in the total trial population.The GMTRs by poliovirus type and IPV formulation were:Type 1: 17.0 (1/3 IPV-Al), 13.0 (1/5 IPV-Al), 7.1 (1/10 IPV-Al) and 42.2 (IPV Vaccine SSI).Type 2: 12.5 (1/3 IPV-Al), 13.1 (1/5 IPV-Al), 7.6 (1/10 IPV-Al) and 47.8 (IPV Vaccine SSI).Type 3: 14.5 (1/3 IPV-Al), 16.2 (1/5 IPV-Al), 8.9 (1/10 IPV-Al) and 62.4 (IPV Vaccine SSI)Thus, the three IPV-Al formulations were highly immunogenic, but inferior to IPV Vaccine SSI, in this booster vaccination trial. Safety resultsNo SAE and no AE of severe intensity occurred. 59.2% of the subjects reported at least one AE. Injection site pain was the most frequent AE in all groups; from 24.6% to 43.3%. Injection site redness and swelling frequencies were<5% in most and<10% in all groups. The most frequent systemic AEs were fatigue (from 8.2% to 15.0%) and headache (from 15.0% to 28.3%). Most AEs were of mild intensity. In conclusion, the three IPV-Al SSI were safe in adolescents and the booster effects were satisfactory.ClinicalTrials.gov registration number: NCT02280447.

Evaluation of molecular typing of foodborne pathogens in European reference laboratories from 2012 to 2013.

In 2012, the European Centre for Disease Prevention and Control (ECDC) initiated external quality assessment (EQA) schemes for molecular typing including the National Public Health Reference Laboratories in Europe. The overall aim for these EQA schemes was to enhance the European surveillance of food-borne pathogens by evaluating and improving the quality and comparability of molecular typing. The EQAs were organised by Statens Serum Institut (SSI) and included Salmonella enterica subsp. enterica, verocytotoxin-producing Escherichia coli (VTEC) and Listeria monocytogenes. Inter-laboratory comparable pulsed-field gel electrophoresis (PFGE) images were obtained from 10 of 17 of the participating laboratories for Listeria, 15 of 25 for Salmonella, but only nine of 20 for VTEC. Most problems were related to PFGE running conditions and/or incorrect use of image acquisition. Analysis of the gels was done in good accordance with the provided guidelines. Furthermore, we assessed the multilocus variable-number tandem repeat analysis (MLVA) scheme for S. Typhimurium. Of 15 laboratories, nine submitted correct results for all analysed strains, and four had difficulties with one strain only. In conclusion, both PFGE and MLVA are prone to variation in quality, and there is therefore a continuous need for standardisation and validation of laboratory performance for molecular typing methods of food-borne pathogens in the human public health sector.

Comparative Evaluation of Genomic and Laboratory Approaches for Determination of Shiga Toxin Subtypes in Escherichia coli

The determination of Shiga toxin (ST) subtypes can be an important element in the risk characterization of foodborne ST-producing Escherichia coli (STEC) isolates for making risk management decisions. ST subtyping methods include PCR techniques based on electrophoretic or pyrosequencing analysis of amplicons and in silico techniques based on whole genome sequence analysis using algorithms that can be readily incorporated into bioinformatics analysis pipelines for characterization of isolates by their genetic composition. The choice of technique will depend on the performance characteristics of the method and an individual laboratory's access to specialized equipment or personnel. We developed two whole genome sequence-based ST subtyping tools: (i) an in silico PCR algorithm requiring genome assembly to replicate a reference PCR-based method developed by the Statens Serum Institut (SSI) and (ii) an assembly-independent routine in which raw sequencing results are mapped to a database of known ST subtype sequence variants (V-Typer). These tools were evaluated alongside the SSI reference PCR method and a recently described PCR-based pyrosequencing technique. The V-Typer method results corresponded closely with the reference method in the analysis of 67 STEC cultures obtained from a World Health Organization National Reference Laboratory. In contrast, the in silico PCR method failed to detect ST subtypes in several cases, a result which we attribute to assembly-induced errors typically encountered with repetitive gene sequences. The V-Typer can be readily integrated into bioinformatics protocols used in the identification and characterization of foodborne STEC isolates.

Antiviral activity of hemolymph of Podalia against rubella virus.

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology. Rubella virus (genus Rubivirus, family Togaviridae) is a single stranded RNA virus of positive genome polarity. Rubella virus infection of susceptible women during the first trimester of pregnancy often results in long-term virus persistence in the fetus causing multiple organ abnormalities. Potent antiviral activity against rubella virus (RV) has been observed in the hemolymph of Podalia sp. (Lepidoptera: Megalopygidae). This study evaluated the effect of hemolymph on RV infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Results of cell viability and cell proliferation assays indicated that hemolymph was not toxic to cultured SIRC cells. Viral binding assay, antiviral assay, PCR, real-time PCR, and transmission electron microscopy were used to demonstrate that hemolymph in post-treatment could inhibit the production of infectious RV particles. Specifically, hemolymph was found to inhibit RV adsorption to the SIRC cells.

European shortage of purified protein derivative and its impact on tuberculosis screening practices.

In June 2014, we became aware that shortages of purified protein derivative (PPD), the test substance used for the tuberculin skin test (TST), had occurred in several European health care institutions providing care for children with tuberculosis (TB). To establish the extent of the shortage, a survey was performed. Survey conducted over a 1-month period (June-July 2014) among members of the Paediatric Tuberculosis Network European Trials Group (ptbnet). Thirty-five physicians from 23 European countries contributed data. The most commonly used PPD product was RT23 (Statens Serum Institut; n = 22, 63%). Twenty-one (60%) participants reported that their institution was experiencing a PPD shortage. The majority (n = 17, 81%) of those reporting a shortage were using RT23. Thirteen (37%) participants reported changes in screening practices resulting from the shortage, including sourcing PPD from alternative manufacturers, restricting remaining supplies to patients at greatest risk or replacing TST by an interferon-gamma release assay. The data show that a PPD shortage occurred in 2014, affecting multiple European countries. The shortage resulted in changes in TB screening capabilities and practices, potentially compromising both patient care as well as public health efforts. Appropriate actions to prevent future PPD shortages should be explored urgently by public health agencies and key stakeholders.

A Danish Survey of Antihistamine Use and Poisoning Patterns.

The first-generation antihistamine, promethazine, became a prescription-only drug in Denmark as of December 2014. First-generation antihistamines are known to have a higher toxic potential than second-generation antihistamines. The aim of this study was to provide a nationwide description of the antihistamine use and poisoning pattern from 2007 to 2013 in Denmark based on two independent databases. There were 1049 antihistamine exposures in the national, advisory telephone service specialized in poisonings, the Danish Poison and Information Centre (DPIC), and 456 exposures in the three registers used within the State Serum Institute of Denmark (SSI), a department under the Danish Ministry of Health dealing with research-based health surveillance in Denmark. First-generation antihistamines constitute 61% and 73% of antihistamine registrations in DPIC and SSI, respectively. Antihistamine exposures increased by 7 exposures/10,000 enquiries per year in DPIC and six admissions per year in SSI - this increase is not significant due to a sudden decrease in 2012. Intentional exposures constituted 65% in DPIC of which 82% was due to suicide attempts, and 78% of the involved antihistamines were first-generation antihistamines. Accidental exposures constituted 33% of which 61% were due to play and 29% involved first-generation antihistamines. Single antihistamine exposures constituted 65% of DPIC exposures of which 98% involved only one brand of antihistamine. Multidrug exposures constituted 35% of DPIC exposures with equally distributed coingestants. Hospitalization was recommended in 78% of DPIC exposures. Admissions required only 1-day of treatment in 91% of the SSI exposures. One of the 14 identified deaths in the SSI study population was directly related to antihistamine poisoning. Results support the limited disclosure of promethazine in Denmark and illustrate a generation-specific pattern indicating a suspected replacement of promethazine with other first-generation antihistamines.

Enteroaggregative Escherichia coli in Daycare—A 1-Year Dynamic Cohort Study

Enteroaggregative Escherichia coli (EAEC) has been associated with persistent diarrhea, reduced growth acceleration, and failure to thrive in children living in developing countries and with childhood diarrhea in general in industrialized countries. The clinical implications of an EAEC carrier-status in children in industrialized countries warrants clarification. To investigate the pathological significance of an EAEC carrier-state in the industrialized countries, we designed a 1-year dynamic cohort study and performed follow-up every second month, where the study participants submitted a stool sample and answered a questionnaire regarding gastrointestinal symptoms and exposures. Exposures included foreign travel, consumption of antibiotics, and contact with a diseased animal. In the capital area of Denmark, a total of 179 children aged 0–6 years were followed in a cohort study, in the period between 2009 and 2013. This is the first investigation of the incidence and pathological significance of EAEC in Danish children attending daycare facilities. Conventional microbiological detection of enteric pathogens was performed at Statens Serum Institute, Copenhagen, Denmark, and at Hvidovre Hospital, Copenhagen, Denmark. Parents completed questionnaires regarding gastrointestinal symptoms. The EAEC strains were further characterized by serotyping, phylogenetic analysis, and susceptibility testing. EAEC was detected in 25 (14%) of the children during the observational period of 1 year. One or more gastrointestinal symptoms were reported from 56% of the EAEC-positive children. Diarrhea was reported in six (24%) of the EAEC positive children, but no cases of weight loss, and general failure to thrive were observed. The EAEC strains detected comprised a large number of different serotypes, confirming the genetic heterogeneity of this pathotype. EAEC was highly prevalent (n = 25, 14%) in Danish children in daycare centers and was accompanied by gastrointestinal symptoms in 56% of the infected children. No serotype or phylogenetic group was specifically linked to children with disease.

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

Adverse reactions to the Bacillus Calmette–Guérin (BCG) vaccine in new-born infants—an evaluation of the Danish strain 1331 SSI in a randomized clinical trial

ObjectiveTo evaluate adverse reactions of the Bacillus Calmette–Guérin (BCG) Statens Serum Institut (SSI) (Danish strain 1331) used as intervention in a randomized clinical trial. DesignA randomized clinical multicenter trial, The Danish Calmette Study, randomizing newborns to BCG or no intervention. Follow-up until 13 months of age. SettingPediatric and maternity wards at three Danish university hospitals. ParticipantsAll women planning to give birth at the three study sites (n=16,521) during the recruitment period were invited to participate in the study. Four thousand one hundred and eighty four families consented to participate and 4262 children, gestational age 32 weeks and above, were randomized: 2129 to BCG vaccine and 2133 to no vaccine. None of the participants withdrew because of adverse reactions. Main outcome and measureTrial-registered adverse reactions after BCG vaccination at birth. Follow-up at 3 and 13 months by telephone interviews and clinical examinations. ResultsAmong the 2118 BCG-vaccinated children we registered no cases of severe unexpected adverse reaction related to BCG vaccination and no cases of disseminated BCG disease. Two cases of regional lymphadenitis were hospitalized and thus classified as serious adverse reactions related to BCG. The most severe adverse reactions were 10 cases of suppurative lymphadenitis. This was nearly a fivefold increase compared to what was expected based on the summary of product characteristics of the vaccine. All cases were treated conservatively and recovered. Six of 10 (60%) families of children experiencing suppurative lymphadenitis compared to 117/2071 (6%) of those with no lymphadenitis indicated that the vaccine had more adverse effects than expected (p-value <0.001). Conclusions and relevanceBCG vaccination was associated with only mild morbidity and no mortality. A higher incidence of suppurative lymphadenitis than expected was observed. All children were treated conservatively without sequelae or complications. Trial registrationTrial registration number NCT01694108 at www.clinicaltrials.gov

Mechanical restraint and staffing level in a psychiatric ward

IntroductionThe use of coercion raises ethical dilemmas, as it deprives patients of their autonomy. In Denmark, the subject has gained political attention, and decreases in the use of coercion are demanded. In the attempt of reducing the use of coercion, it is of interest to obtain more knowledge about the circumstances bound to it.AimThe aim of this study was to investigate a possible correlation between the incidence of mechanical restraint (MR), and staffing level, staff demographics, patient characteristics, type of shift (day/evening/night) and change of shifts.MethodsA naturalistic descriptive method was used to study cases of mechanical restraint in patients admitted to a psychiatric ward. In the study period of one year, 114 cases of MR were carried out. In 66% of cases, the patient was diagnosed with borderline personality disorder. Each case of MR was obtained from Statens Serum Institut's Electronic Reporting System. The care workers from each shift were identified using duty rosters. The statistical package SPSS version 20 was used for data processing, including a logistic regression analysis.ResultsA significant association was found between the use of MR and the presence of male care workers on the ward, OR = 1.639, 95% CI: 1.163–2.311, P = 0.005. The risk of MR is increased during evening shifts, compared to day and night shifts, OR = 1.868, 95% CI: 1.165–2.995, P = 0.009. No significant associations were found between MR and staffing level, age, education, or experience of care workers.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Improvements of ATP Assay as a Substitute for the CFU Method in Estimating Viable Cell Count for BCG/rBCG Vaccine Preparations

Bacillus Calmette–Guerin (BCG) and recombinant BCG (rBCG) vaccines can be genetically traced back to a live attenuated strain of Mycobacterium bovis. As organism viability is essential for the stimulation of a protective immune response, monitoring the count of viable organisms is an integral part of vaccine quality control. The colonyforming unit (CFU) test has been the conventional assay for determining BCG viability, and is a widely accepted surrogate for BCG potency. CFU analysis, however, is problematic and time consuming. The slowness and high variability of CFU test results are the main driving forces for manufacturers and control laboratories to look for a rapid, more reproducible alternative viable count assay. A modified adenosine triphosphate (ATP) luminescence assay was developed by Statens Serum Institut and was promoted by the WHO as an alternative viable count assay. However, certain conditions during the processes of sample preparation and ATP extraction have to be established before the ATP assay can meet the requirements of robustness and reproducibility. This study is focused on identifying the conditions necessary for a reliable process of ATP analysis for BCG/rBCG preparations. Using our improved ATP assay protocol, we demonstrated that the correlation coefficient between CFU count and ATP concentration of BCG/rBCG vaccines was high (R2=0.83 for accelerated stability samples and R2>0.97 for all other preparations). The ATP luminescence assay is a rapid, sensitive, reliable, strain-non-specific method in quantification of the viability of live attenuated mycobacterial vaccine preparations.

Successful Treatment of Disseminated Bacillus Calmette-Guérin Disease in an HIV-Infected Child with a Linezolid-Containing Regimen.

Upon HIV infection diagnosis, an 8-month-old boy was transferred for evaluation of worsening respiratory distress requiring mechanical ventilation. Pneumocystis jirovecii pneumonia (PCP) was diagnosed; the boy also had a nonhealing ulcer at the site of vaccination with Statens Serum Institut (Danish strain) Bacillus Calmette-Guérin (BCG) vaccine and associated axillary lymphadenopathy. PCP treatment resulted in weaning from mechanical ventilation. Antimycobacterial treatment was immediately attempted but was discontinued because of hepatotoxicity. Over several months, he developed splenic lesions and then disseminated skin and cystic bone lesions. M. bovis was repeatedly cultured from both skin and bone lesions despite various multidrug antimycobacterial regimens which included linezolid. Eventually, treatment with a regimen of rifabutin, isoniazid, ethambutol, and linezolid led to definitive cure. Clinicians should consider a linezolid-containing regimen for treatment of severe disseminated BCG infection, especially if other drug regimens have failed. Although drug toxicity is a particular concern for young children, this patient received linezolid for 13 months without serious toxicity. This case also highlights the need for universal screening among pregnant women to prevent vertical transmission of HIV. Finally, routine immunization with BCG vaccine at birth should be questioned in countries with low and declining burden of tuberculosis.

Evaluation and Optimization of an ELISA Procedure to Quantify Antibodies Against Pneumococcal Polysaccharides Included in the 13-Valent Conjugate Vaccine

The 13-valent pneumococcal conjugate vaccine (PCV-13) is recommended for HIV-infected people, although its effectiveness in this population remains under evaluation. In this study, we describe the development, optimization, and analytical validation of an ELISA procedure to measure specific antibodies for the pneumococcal polysaccharide serotypes included in PCV13 vaccine, testing sera obtained from HIV-infected outpatients (n = 30) who received the vaccine. The protocol followed the last version of WHO guidelines, based on the new standard 007sp, with the modification of employing Statens Serum Institut (SSI) antigens.We supplied the assay performance validation in terms of sensitivity, reproducibility, precision and accuracy. In addition we detailed optimal antigen-coating concentrations and ELISA conditions common to all 13 serotypes, suitable for laboratories performing these assays in order to standardize the method. Our procedure showed reproducibility and reliability, making it a valid alternative for evaluating the response to pneumococcal serotypes included in PCV13 vaccine.

Stereoisomeric Prodrugs to Improve Corneal Absorption of Prednisolone: Synthesis and In Vitro Evaluation.

A series of stereoisomeric prodrugs have been designed to examine efficacy in generating higher corneal absorption relative to prednisolone. Prodrugs have been studied and identified with LC/MS/MS and NMR analyses. Prodrugs have been characterized for aqueous solubility, buffer stability, and cytotoxicity. Cellular uptake and permeability studies have been conducted across MDCK-MDR1 cells to determine prodrug affinity towards P-glycoprotein (P-gp) and peptide transporters. Enzyme-mediated degradation of prodrugs has been determined using Statens Seruminstitut rabbit cornea (SIRC) cell homogenates. Prodrugs exhibited higher aqueous solubility relative to prednisolone. Prodrugs circumvented P-gp-mediated cellular efflux and were recognized by peptide transporters. Prodrugs (DP, DDP) produced with D-isomers (D-valine) were significantly stable against both chemical and enzymatic hydrolyses. The order of degradation rate constants observed in chemical and enzymatic hydrolyses were in the same order, i.e., L-valine-L-valine-prednisolone (LLP) > L-valine-D-valine-prednisolone (LDP) > D-valine-L-valine-prednisolone (DLP) > D-valine-D-valine-prednisolone (DDP). Results obtained from this study clearly suggest that stereoisomeric prodrug approach is an effective strategy to overcome P-gp-mediated efflux and improve transcorneal permeability of prednisolone following topical administration.

Performance of Statens Serum Institut Enteric Medium for Detection of Enteric Pathogens in Stool in Routine Laboratory Diagnostics

Background: We evaluated the performance of Statens Serum Institut enteric medium (SSI) for detection of enteric pathogens in stool in two routine diagnostic laboratories in Nairobi to determine if the detection in stool would be most sensitive and specific in our culture protocol. Methods: Salmonella-Shigella agar (SS), Statens Serum Institut enteric medium (SSI), Deoxycholate Citrate Agar (DCA) and MacConkey media were prepared according to the manufacture's institutions. Diarrheal stool specimens received at the laboratory for routine diagnosis were streaked directly and after enrichment in Selenite F onto the test media and incubated for 24 hours at 37o Centigrade. After incubation, the growth characteristics and chemical reactions was compared with the manual given. Results: SSI medium was a more productive medium in the detection of enteric pathogens in comparison to the other three media (F=2.729, d.f=3 at p≤0.005). It also shows that enrichment of the specimens in Selenite F enhances recovery. Conclusion: SSI enteric medium yields differentiating biochemical reactions that allow direct identification of a range of enteric pathogens thus saving on time and costs. Our revised/modified protocol, where we plated stool samples after enrichment in selenite F broth onto SS and SSI, achieved a high sensitivity of detection and optimal isolation.