Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies. The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.     

Large-scale validation of a latex agglutination test for diagnosis of tuberculosis

Large-scale validation of a simple latex agglutination test for the diagnosis of tuberculosis is described. Soluble antigens extracted from a non-pathogenic saprophytic mycobacterium, Mycobacterium w, which shares antigenic determinants with Mycobacterium tuberculosis, were covalently linked to carboxylated polystyrene latex beads. Batch to batch reproducibility of coated latex was ensured. Latex reagents were standardized to overcome non-specific agglutination. Reagents of the test are stable for 1 year at 4 degrees C. A total of 1,058 serum samples of pulmonary and extrapulmonary tuberculosis patients or patients with other pulmonary diseases and healthy controls living in endemic areas were tested. Sensitivity of 94% for pulmonary tuberculosis and 87% for extrapulmonary tuberculosis was obtained. Specificity is 92.2% for healthy controls and patients with other respiratory diseases. We conclude that the latex agglutination test can be utilized for mass screening for both pulmonary and extrapulmonary tuberculosis where diagnosis by existing methods is much more difficult.     

Use of antiserum-coated latex particles for serotyping Streptococcus pneumoniae

The Quellung reaction provides a standard means for serotyping Streptococcus pneumoniae, but it requires microscopic examination with skillful technique. We have developed an improved agglutination method with anti-rabbit IgG-coated latex particles, which are sensitized with pooled antisera for serotyping/serogrouping S. pneumoniae. Our method is as specific and sensitive as the Quellung test, and much easier to perform.     

Latex agglutination test for diagnosing pneumococcal pneumonia in children in developing countries.

OBJECTIVE--To prepare and assess the sensitivity and specificity of a latex agglutination test specific for the serotype of antigen in diagnosing pneumococcal pneumonia in Gambian children. DESIGN--Comparison of agglutination test specific for serotype with culture of blood and lung aspirates, countercurrent immunoelectrophoresis, and commercial latex agglutination tests in diagnosing pneumococcal pneumonia. Cross reaction studies and investigation of 102 control children to determine specificity of agglutination test specific for serotype. SETTING--General medical ward of Medical Research Council laboratories, The Gambia. PATIENTS--101 Gambian children aged between 2 months and 10 years admitted with severe pneumonia. INTERVENTIONS--Serum samples were boiled and treated with edetic acid, and urine samples were boiled and concentrated 25 times before testing. END POINT--A latex agglutination test specific for the serotype of pneumococcal antigen that is sensitive and highly specific for detecting pneumococcus in the urine of patients with pneumococcal pneumonia. MEASUREMENTS AND MAIN RESULTS--Concentrated urine samples from 16 of the 21 children (76%) with pneumococcal pneumonia established by results of culture of blood or lung aspirates gave a positive result with the agglutination test specific for serotype, whereas only four of the 102 urine samples obtained from control children without pneumonia gave positive results. The serotypes of antigens detected in the urine of children with pneumococcal pneumonia and the serotypes of pneumococci isolated from cultures of blood or lung aspirates were the same in all cases. CONCLUSIONS--When performed on urine samples the agglutination test specific for serotype has a high specificity and is more sensitive than culture of blood or lung aspirates, commercial agglutination tests, or countercurrent immunoelectrophoresis in identifying pneumococcal pneumonia. It is easy to use and should be especially useful in communities with limited laboratory facilities.     

Nitrile versus Latex for Glove Juice Sampling

The objective of this study was to explore the utility of nitrile gloves as a replacement for latex surgical gloves in recovering bacteria from the hands. Two types of nitrile gloves were compared to latex gloves using the parallel streak method. Streaks of Klebsiella pneumoniae and Staphylococcus aureus were made on tryptic soy agar plates, and the zones of inhibition were measured around pieces of glove material placed on the plates. Latex gloves produced a mean zone of inhibition of 0.28 mm, compared to 0.002 mm for nitrile gloves (p<.001). While the parallel streak method is not intended as a quantitative estimate of antimicrobial properties, these results suggest that nitrile may be a viable alternative to latex in glove juice sampling methods, since nitrile avoids the risk of latex exposure.     

The PneuCarriage Project: A Multi-Centre Comparative Study to Identify the Best Serotyping Methods for Examining Pneumococcal Carriage in Vaccine Evaluation Studies

Background

The pneumococcus is a diverse pathogen whose primary niche is the nasopharynx. Over 90 different serotypes exist, and nasopharyngeal carriage of multiple serotypes is common. Understanding pneumococcal carriage is essential for evaluating the impact of pneumococcal vaccines. Traditional serotyping methods are cumbersome and insufficient for detecting multiple serotype carriage, and there are few data comparing the new methods that have been developed over the past decade. We established the PneuCarriage project, a large, international multi-centre study dedicated to the identification of the best pneumococcal serotyping methods for carriage studies.

Methods and Findings

Reference sample sets were distributed to 15 research groups for blinded testing. Twenty pneumococcal serotyping methods were used to test 81 laboratory-prepared (spiked) samples. The five top-performing methods were used to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. Sensitivity and positive predictive value (PPV) were determined for the test methods and the reference method (traditional serotyping of >100 colonies from each sample).For the alternate serotyping methods, the overall sensitivity ranged from 1% to 99% (reference method 98%), and PPV from 8% to 100% (reference method 100%), when testing the spiked samples. Fifteen methods had ≥70% sensitivity to detect the dominant (major) serotype, whilst only eight methods had ≥70% sensitivity to detect minor serotypes. For the field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method 93.8%), and PPV from 82.2% to 96.4% (reference method 99.6%). The microarray had the highest sensitivity (95.8%) and high PPV (93.7%). The major limitation of this study is that not all of the available alternative serotyping methods were included.

Conclusions

Most methods were able to detect the dominant serotype in a sample, but many performed poorly in detecting the minor serotype populations. Microarray with a culture amplification step was the top-performing method. Results from this comprehensive evaluation will inform future vaccine evaluation and impact studies, particularly in low-income settings, where pneumococcal disease burden remains high.     

Multi-Serotype Pneumococcal Nasopharyngeal Carriage Prevalence in Vaccine Na?ve Nepalese Children,Assessed Using Molecular Serotyping

Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49·5%) of samples with more than one serotype being found in 67/297 (20·2%) of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44·4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.     

Body muscles of the lamprey. Some structural features of the T system and sarcolemma

Electron microscopic studies confirm and extend the conclusions derived previously from a quantitative biochemical study of the phagocytosis of polystyrene and polyvinyltoluene latex beads by Acanthamoeba (1). Latex beads 1.305, 1.90, and 2.68 µ in diameter are ingested individually, with each bead tightly surrounded by a membrane derived from the plasma membrane. Latex beads 0.557, 0.264, 0.126, and 0.088 µ in diameter are accumulated at the surface of the ameba and then phagocytosed, with many beads tightly packed within one membrane-bounded vesicle.     

One-Step Multiplex PCR Assay for Detecting Streptococcus pneumoniae Serogroups/Types Covered by 13-Valent Pneumococcal Conjugate Vaccine (PCV13)

The life-threatening illnesses caused by Streptococcus pneumoniae have been declined significantly after the use of pneumococcal conjugate vaccines. Continuous monitoring of the vaccine serogroups/types is necessary to follow the changing epidemiology of invasive pneumococcal diseases. Recently, the sequential multiplex PCR approach, which uses several different sets of reactions, has been commonly adopted for determining capsular serogroups/types of S. pneumoniae isolates. In our study, we focused on development of a one-step multiplex PCR assay detecting all 1, 3, 4, 5, 6A/B, 7F, 9V, 14, 18C, 19A, 19F and 23F serogroups/types targeted by PCV13. The content of multiplex PCR mix and the cycling conditions were optimized in a manner that allowed rapid and accurate serotyping of a pneumococcal isolate by performing only a single amplification reaction. In our study of 182 clinical isolates, the one-step multiplex PCR assay exhibited 100% sensitivity and specificity, suggesting that its utilization can significantly reduce the use of traditional antiserum method requiring expensive reagents.     

Updated Campylobacter jejuni Capsule PCR Multiplex Typing System and Its Application to Clinical Isolates from South and Southeast Asia

Campylobacter jejuni produces a polysaccharide capsule that is the major determinant of the Penner serotyping scheme. This passive slide agglutination typing system was developed in the early 1980’s and was recognized for over two decades as the gold standard for C. jejuni typing. A preliminary multiplex PCR technique covering 17 serotypes was previously developed in order to replace this classic serotyping scheme. Here we report the completion of the multiplex PCR technology that is able to identify all the 47 Penner serotypes types known for C. jejuni. The number of capsule types represented within the 47 serotypes is 35. We have applied this method to a collection of 996 clinical isolates from Thailand, Cambodia and Nepal and were able to successfully determine capsule types of 98% of these.     

Population-Based Analysis of Invasive Nontypeable Pneumococci Reveals That Most Have Defective Capsule Synthesis Genes

Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the cps locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006–2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the cps locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained cps loci. Twenty-two nontypeables exhibited serotype 8 cps loci with defects, primarily within wchA. Six of the remaining nine isolates contained previously-described aliB homologs in place of cps loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking cps loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective cps locus genes, with an over-representation of defective serotype 8 cps variants.     

Development of the reverse passive latex agglutination method for the detection and quantification of the genus Nitrospira in the wastewater treatment process

This report describes a new immunological method for the detection and quantification of Nitrospira populations using the reverse passive latex agglutination (RPLA). The numbers of the genus Nitrospira have been quantified only by molecular biological techniques such as FISH and quantitative PCR to date. Using high-density latex particles and a specific polyclonal antibody, Nitrospira populations in the wastewater treatment process were quantified in the shortest 4 h of incubation. The minimum detectable number of Nitrospira cells was 7.0x10(5) (log(10) 5.85) cells/ml. It is thought that the RPLA method can quantify Nitrospira populations more simply, economically, and speedily than molecular biological techniques or the culture method, because this procedure has a simple protocol and does not require the use of specialized equipment, expensive reagents, or technical skill. Therefore it is applicable for use in the everyday control and maintenance of water quality in wastewater treatment facilities where equipment is not sufficient or in the field.     

The Eiken Latex test for detection of a cryptococcal antigen in cryptococcosis

A latex agglutination test for cryptococcal antigen, the Eiken Latex test (Eiken, Tokyo, Japan), was compared with a monoclonal antibody-based agglutination assay, Pastorex^® Cryptococcus (Diagnostics Pasteur, Marneur-la-Coquette, France). In a murine model of disseminated cryptococcosis, the kinetics of the antigen titers by the Eiken Latex were similar to those by the Pastorex^® Cryptococcus, but sensitivity was much higher. In HIV-negative patients with pulmonary cryptococcosis, a cryptococcal antigen was detected in 6 of 10 patients by the Eiken Latex test and in only 3 of those patients by the Pastorex^® Cryptococcus. The results indicate that the Eiken Latex is more sensitive for the detection of the cryptococcal antigen, even in non-disseminated cryptococcosis. The sensitivity and specificity of the Eiken Latex were examined using 195 sera from 25 patients with pulmonary cryptococcosis and 170 patients with non-cryptococcosis. The cutoff value of 1:8 showed a sensitivity of 76% (19/25) and a specificity of 98.9% (168/170).     

Determination of antibodies to diphtheria and tetanus toxoid by latex agglutination technique

A micromethod of latex agglutination was worked out for determination of antibodies to diphtheria and tetanus toxoids. It is suitable for pediatrics since a minimum amount of serum (0.05 ml.) is required, which can be used without being inactivated or absorbed. Particles of polystyrene latex of Czechoslovak production were used as antigen carriers thus enabling better standardization of the technique. The particles are well defined both in physical and chemical terms and replace red blood cells in passive hemagglutination. The method was compared with passive hemagglutination in a dynamic study of children inoculated at monthly intervals with the trivalent vaccine Alditepera.     

Screening of aquatic samples for Vibrio cholerae serotype O1 by a dot-blot method and a latex agglutination test.

A dot-blot, enzyme-linked immunosorbent method and a latex agglutination test were studied for their abilities to detect Vibrio cholerae serotype O1 in aquatic samples by testing artificially contaminated water as well as samples from natural potential sources. Water samples were preenriched with alkaline peptone and then enriched with Monsur peptone water. For the dot-blot test, enriched cultures of organisms in a small portion of the Monsur peptone water were transferred to a polyvinylidene difluoride membrane with a microfiltration apparatus. The enzyme-linked immunosorbent assay was performed by using biotin-labeled antibodies and avidin-biotin-peroxidase complex; brown dots developed in the wells that contained serotype O1 vibrios. Latex agglutination tests were performed by mixing 1 drop of the culture in Monsur with 1 drop of reagent coated with monoclonal antibody specific for antigen A. The sensitivities and specificities of the methods were compared with those of the colony-blot method, which identified individual colonies of V. cholerae O1 in mixed bacterial cultures on isolation media. Our results indicate that the dot-blot method is as sensitive as the colony-blot method and is useful for screening for V. cholerae serotype O1 even in specimens that are heavily contaminated with non-O1 vibrios.     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.     

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.     

Antibody-bearing liposomes improve agglutination of latex particles used in clinical diagnostic assays

Ligand-bearing liposomes are used to enhance the agglutination ‘signal’ of a typical latex assay for the detection of human rheumatoid factor. Heat-denatured IgG, the antigen to which rheumatoid factor binds naturally, was covalently attached to latex spheres. The liposomes were covalently coated with a ‘second ligand’ which also recognizes rheumatoid factor, anti-human IgM Fab′ fragments. In the present test configuration, rheumatoid factor present in a patient's serum binds to the IgG attached to the latex particles. The liposomes, in turn, bind rapidly to rheumatoid factor-sensitized latex, via the second ligand, promoting the formation of large, clearly visible latex aggregates. When latex spheres bearing the same type and density of second ligand were used to replace the liposomes they failed to improve agglutination, suggesting that multivalent presentation of the second ligand is not sufficient to insure the improvement. These results suggest that fluidity of the liposomal membrane is a requirement for the effect. Sensitivity as well as ‘readability’ are improved by the liposomes while specificity remains unaffected. The principle of using ligand-bearing liposomes to enhance particle agglutination is applicable to a wide range of other diagnostic assays.