Detection of antibody responses against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis proteins in children with community-acquired pneumonia: effects of combining pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness.

We evaluated the effects of combining different numbers of pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness on the detection of IgG responses against eight Streptococcus pneumoniae proteins, three Haemophilus influenzae proteins, and five Moraxella catarrhalis proteins in 690 children aged <5 years with pneumonia. Serological tests were performed on acute and convalescent serum samples with a multiplexed bead-based immunoassay. The median sampling interval was 19 days, the median age was 26.7 months, and the median duration of illness was 5 days. The rate of antibody responses was 15.4 % for at least one pneumococcal antigen, 5.8 % for H. influenzae, and 2.3 % for M. catarrhalis. The rate of antibody responses against each pneumococcal antigen varied from 3.5 to 7.1 %. By multivariate analysis, pre-existing antibody levels showed a negative association with the detection of antibody responses against pneumococcal and H. influenzae antigens; the sampling interval was positively associated with the detection of antibody responses against pneumococcal and H. influenzae antigens. A sampling interval of 3 weeks was the optimal cut-off for the detection of antibody responses against pneumococcal and H. influenzae proteins. Duration of illness was negatively associated with antibody responses against PspA. Age did not influence antibody responses against the investigated antigens. In conclusion, serological assays using combinations of different pneumococcal proteins detect a higher rate of antibody responses against S. pneumoniae compared to assays using a single pneumococcal protein. Pre-existing antibody levels and sampling interval influence the detection of antibody responses against pneumococcal and H. influenzae proteins. These factors should be considered when determining pneumonia etiology by serological methods in children.

Use of a rapid test of pneumococcal colonization density to diagnose pneumococcal pneumonia.

BACKGROUND: There is major need for a more sensitive assay for the diagnosis of pneumococcal community-acquired pneumonia (CAP). We hypothesized that pneumococcal nasopharyngeal (NP) proliferation may lead to microaspiration followed by pneumonia. We therefore tested a quantitative lytA real-time polymerase chain reaction (rtPCR) on NP swab samples from patients with pneumonia and controls. METHODS: In the absence of a sensitive reference standard, a composite diagnostic standard for pneumococcal pneumonia was considered positive in South African human immunodeficiency virus (HIV)-infected adults hospitalized with radiographically confirmed CAP, if blood culture, induced good-quality sputum culture, Gram stain, or urinary Binax demonstrated pneumococci. Results of quantitative lytA rtPCR in NP swab samples were compared with quantitative colony counts in patients with CAP and 300 HIV-infected asymptomatic controls. RESULTS: Pneumococci were the leading pathogen identified in 76 of 280 patients with CAP (27.1%) using the composite diagnostic standard. NP colonization density measured by lytA rtPCR correlated with quantitative cultures (r = 0.67; P < .001). The mean lytA rtPCR copy number in patients with pneumococcal pneumonia was 6.0 log(10) copies/mL, compared with patients with CAP outside the composite standard (2.7 log(10) copies/mL; P < .001) and asymptomatic controls (0.8 log(10) copies/mL; P < .001). A lytA rtPCR density ≥8000 copies/mL had a sensitivity of 82.2% and a specificity of 92.0% for distinguishing pneumococcal CAP from asymptomatic colonization. The proportion of CAP cases attributable to pneumococcus increased from 27.1% to 52.5% using that cutoff. CONCLUSIONS: A rapid molecular assay of NP pneumococcal density performed on an easily available specimen may significantly increase pneumococcal pneumonia diagnoses in adults.

Natural acquired humoral immunity against serotype-specific group B Streptococcus rectovaginal colonization acquisition in pregnant women.

Group B Streptococcus (GBS) rectovaginal colonization in pregnant women is associated with invasive GBS disease in newborns, preterm delivery and stillbirths. We studied the association of GBS serotype-specific capsular polysaccharide (CPS) antibody on new acquisition and clearance of rectovaginal GBS colonization in pregnant women from 20 weeks until 37 to 40 weeks' gestation. Serum serotype-specific CPS IgG antibody concentration was measured by multiplex enzyme-linked immunosorbent assay and opsonophagocytic activity (OPA) titres. Rectovaginal swabs were evaluated for GBS colonization, using standard culture methods and serotyping by latex agglutination, at five to six weekly intervals. Higher serotype III CPS antibody concentration was associated with lower risk of rectovaginal acquisition of serotype III during pregnancy (p 0.009). Furthermore, serotype-specific OPA titres to Ia and III were higher in women who remained free of GBS colonization throughout the study compared to those who acquired the homotypic serotype (p <0.001 for both serotypes). Serum CPS IgG values of ≥1µg/mL for serotype V and ≥3µg/mL for serotypes Ia and III were significantly associated with protection against rectovaginal acquisition of the homotypic serotype. A GBS vaccine that induces sufficient capsular antibody in pregnant women, including high OPA titres, could protect against rectovaginal colonization during the latter half of pregnancy.

Association of Streptococcus pneumoniae common protein antigen (CPA) antibodies and pneumococcal nasopharyngeal colonization in HIV-infected and HIV-uninfected African children.

Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), pneumococcal common protein antigens (CPAs) are being investigated as potential vaccine candidates. CPAs are likely to be immunogenic in infants and could confer serotype-independent protection. There are limited data on natural antibody kinetics against CPAs in African populations. We aimed to determine the prevalence of naturally acquired antibody titres to 15 CPAs and explore their association to concurrent pneumococcal nasopharyngeal colonization in children aged 4-7 years with and without underlying HIV-infection and/or previous PCV-vaccination. A 15-plex Luminex assay was established to measure serum IgG titres against "cell-wall associated or surface-exposed" proteins (PspA, PspC, LytB, IgA1-proteinase, SP0082, PdB and PcsB), "membrane-associated" proteins (PsaA, SP0609, SP0749, PpmA, SlrA, StkP and SP2194) as well as the hypothetical protein, SP2027. Archived serum samples from HIV-uninfected (n=212) and HIV-infected (n=74) children were analyzed. Concurrent pneumococcal nasopharyngeal colonization was determined with standard microbiological methods. HIV-uninfected children had significantly higher antibody titres against PspA, PspC, PdB, SP0082, LytB, IgA1 proteinase and PcsB compared to HIV-infected children. In contrast, antibody titres against membrane associated proteins (PsaA, SP2027, PpmA and SlrA) were significantly lower in HIV-uninfected compared to HIV-infected children. Higher antibody titres against PdB, and PcsB were associated with the absence of pneumococcal colonization. There was no association between anti-CPA titres and PCV vaccination. In conclusion PdB and PcsB antigens are potential vaccine-candidates which may protect against pneumococcal colonization and consequently pneumococcal disease.

QuantiFERON-TB Gold In-Tube for the detection of Mycobacterium tuberculosis infection in children with household tuberculosis contact.

SETTING: Improved strategies are needed for detecting Mycobacterium tuberculosis infection in children in TB-endemic settings. OBJECTIVE: To determine the prevalence of M. tuberculosis infection by tuberculin skin testing (TST) and by the QuantiFERON-TB Gold In-Tube (QFT-GIT) test in children with an adult household contact with pulmonary TB in South Africa. DESIGN: Cross-sectional study. RESULTS: A total of 167 adult pulmonary TB cases (153/167, 92% human immunodeficiency virus [HIV] infected) and 270 pediatric contacts (median age 6 years, 14/270, 5% HIV-infected) were enrolled. All children completed QFT-GIT testing and 254 (94.1%) completed TST testing. Prevalence of M. tuberculosis infection was 28% (71/254, 95%CI 23-34) using TST (5 mm cut-off) and 29% (79/270, 95%CI 24-35) using QFT-GIT (P = 0.49). Agreement between TST and QFT-GIT was 81% (kappa 0.58). Nineteen (7%) QFT-GIT results were indeterminate. Children aged <2 years were more likely than older children to have indeterminate QFT-GIT results (aOR 5.7, 95%CI 1.5-22, P = 0.01) and discordant QFT-GIT and TST results (aOR 3.5, 95%CI 1.7-7.6, P = 0.001). CONCLUSION: Prevalence of M. tuberculosis infection in pediatric contacts was high regardless of the diagnostic method used. TST should not be excluded for the detection of pediatric M. tuberculosis infection in this setting, but QFT-GIT may be a feasible alternative in children aged ≥ 2 years.

Mutations in the dihydrofolate reductase gene of trimethoprim-resistant isolates of Streptococcus pneumoniae.

Streptococcus pneumoniae isolates resistant to several antimicrobial agent classes including trimethoprim-sulfamethoxazole have been reported with increasing frequency throughout the world. The MICs of trimethoprim, sulfamethoxazole, and trimethoprim-sulfamethoxazole (1:19) for 259 clinical isolates from South Africa were determined, and 166 of these 259 (64%) isolates were resistant to trimethoprim-sulfamethoxazole (MICs > or =20 mg/liter). Trimethoprim resistance was found to be more strongly correlated with trimethoprim-sulfamethoxazole resistance (correlation coefficient, 0.744) than was sulfamethoxazole resistance (correlation coefficient, 0.441). The dihydrofolate reductase genes from 11 trimethoprim-resistant (MICs, 64 to 512 microg/ml) clinical isolates of Streptococcus pneumoniae were amplified by PCR, and the nucleotide sequences were determined. Two main groups of mutations to the dihydrofolate reductase gene were found. Both groups shared six amino acid changes (Glu20-Asp, Pro70-Ser, Gln81-His, Asp92-Ala, Ile100-Leu, and Leu135-Phe). The first group included two extra changes (Lys60-Gln and Pro111-Ser), and the second group was characterized by six additional amino acid changes (Glu14-Asp, Ile74-Leu, Gln91-His, Glu94-Asp, Phe147-Ser, and Ala149-Thr). Chromosomal DNA from resistant isolates and cloned PCR products of the genes encoding resistant dihydrofolate reductases were capable of transforming a susceptible strain of S. pneumoniae to trimethoprim resistance. The inhibitor profiles of recombinant dihydrofolate reductase from resistant and susceptible isolates revealed that the dihydrofolate reductase from trimethoprim-resistant isolates was 50-fold more resistant (50% inhibitory doses [ID50s], 3.9 to 7.3 microM) than that from susceptible strains (ID50s, 0.15 microM). Site-directed mutagenesis experiments revealed that one mutation, Ile100-Leu, resulted in a 50-fold increase in the ID50 of trimethoprim. The resistant dihydrofolate reductases were characterized by highly conserved redundant changes in the nucleotide sequence, suggesting that the genes encoding resistant dihydrofolate reductases may have evolved as a result of inter- or intraspecies recombination by transformation.

Trimethoprim resistance in South African isolates of aerobic gram-negative faecal flora.

Aerobic gram-negative commensal faecal flora from 362 healthy volunteers was examined for resistance to trimethoprim. Three hundred fifty-seven trimethoprim-resistant organisms were isolated from 272 of the volunteers (297 Escherichia coli, 46 Klebsiella spp., 9 Enterobacter spp. and 7 other species). Trimethoprim resistance was associated with resistance to other antibiotics at the following frequencies: ampicillin 71.4%, tetracycline 88%, cephalosporins 14% and aminoglycosides 4%. High-level resistance to trimethoprim (MIC > or = 1024 mg/l) occurred in 98.6% of the isolates. Trimethoprim resistance was transferable in 51.2% of the isolates. An X+ factor was required to mobilize resistance in a further 3.4%. Resistance to other antibiotics cotransferred with trimethoprim at the following frequencies: ampicillin 55.4%, tetracycline 30%, cephalosporins 1.5% and aminoglycosides 2.6%. Restriction enzyme analysis of 148 plasmids revealed 79 different profiles. Two restriction profiles represented 10.1 and 8.8% of these plasmids, respectively. The large number of different antibiograms and restriction profiles indicates that there is a large gene pool of trimethoprim-resistant organisms in the faecal flora.

Prevalence of trimethoprim resistant dihydrofolate reductase genes identified with oligonucleotide probes in plasmids from isolates of commensal faecal flora.

In a recent survey of trimethoprim resistance, 357 Gram-negative aerobic organisms were isolated from healthy volunteers from rural and urban populations in South Africa. Trimethoprim resistance was transferable in 184 (52%) of the isolates. A further 12 (3%) transferred in the presence of an X+ actor. The transconjugants were probed with intragenic oligonucleotide probes for the type Ia, Ib, IIIa, VIII, V, VI, VII, IX, X and XII dihydrofolate reductase genes. Contrary to all previous data, the most prevalent dihydrofolate reductase gene was the type Ib (30%) followed by the type VIII (23%), V (13%), Ia (6%), VII (3%), and XII (0.5%). None of the strains hybridised to the type IIIa, XI, X and the VI dihydrofolate reductase probes. Plasmid restriction profiles revealed that the high prevalence of the type Ib and VIII dihydrofolate reductase genes resulted from the presence of ubiquitous plasmids. These results highlight the previous problems associated with the distinction of closely related dihydrofolate reductase genes.

Acquisition of chloramphenicol resistance by the linearization and integration of the entire staphylococcal plasmid pC194 into the chromosome of Streptococcus pneumoniae.

Chloramphenicol resistance in Streptococcus pneumoniae was associated with cat, which has 100% identity with cat(pC194) from Staphylococcus aureus. Inverse PCR with primers specific for pC194 confirmed that in some isolates the entire staphylococcal plasmid was present in the S. pneumoniae chromosome, with linearization having occurred between cat(pC194) and the origin of replication.

Prevalence and genetic location of non-transferable trimethoprim resistant dihydrofolate reductase genes in South African commensal faecal isolates.

In a recent survey of trimethoprim resistance, 357 Gram-negative aerobic organisms were isolated from healthy volunteers from rural and urban populations in South Africa. Trimethoprim resistance did not transfer to an Escherichia coli J62-2 recipient strain by conjugation in a liquid mating in 161 (45.1%) of the isolates. These isolates which did not transfer their resistance were probed with intragenic oligonucleotide probes for the types Ia, Ib, IIIa, V, VI, VII, VIII, IX, X and XII dihydrofolate reductase genes. Contrary to all previous data, the most prevalent dihydrofolate reductase gene in this group of non-transferable isolates which hybridized, was the type VII (38%) followed by the type Ia (25%), Ib (12%), V (1.7%) and VIII (1.2%). None of the strains hybridized to the types IIIa, VI, XI, X and the XII dihydrofolate reductase probes. Southern blots of plasmid and chromosomal DNA from selective isolates revealed that the type VII dihydrofolate reductase genes were located on the chromosome and were associated with the integrase gene of Tn21. However, the type Ib and V dihydrofolate reductase genes were all found on plasmids which could not be mobilized. The type Ia dihydrofolate reductase genes were found on both non-transferable plasmids and on the chromosome. The nature of the genetic structures associated with a dihydrofolate reductase gene strongly affects the means of spread of the gene in a population.

New gene cassettes for trimethoprim resistance, dfr13, and Streptomycin-spectinomycin resistance, aadA4, inserted on a class 1 integron.

In a previous survey of 357 trimethoprim-resistant isolates of aerobic gram-negative bacteria from commensal fecal flora, hybridization experiments showed that 25% (90 of 357) of the isolates failed to hybridize to specific oligonucleotide probes for dihydrofolate reductase types 1, 2b, 3, 5, 6, 7, 8, 9, 10, and 12. Subsequent cloning and sequencing of a plasmid-borne trimethoprim resistance gene from one of these isolates revealed a new dihydrofolate reductase gene, dfr13, which occurred as a cassette integrated in a site-specific manner in a class 1 integron. The gene product shared 84% amino acid identity with dfr12 and exhibited a trimethoprim inhibition profile similar to that of dfr12. Gene probing experiments with an oligonucleotide probe specific for this gene showed that 12.3% (44 of 357) of the isolates which did not hybridize to probes for other dihydrofolate reductases hybridized to this probe. Immediately downstream of dfr13, a new cassette, an aminoglycoside resistance gene of the class AADA ¿ANT(3")(9)-I, which encodes streptomycin-spectinomycin resistance, was identified. This gene shares 57% identity with the consensus aadA1 (ant(3")-Ia) and has been called aadA4 (ant(3")-Id). The 3' end of the aadA4 cassette was truncated by IS26, which was contiguous with a truncated form of Tn3. On the same plasmid, pUK2381, a second copy of IS26 was associated with sul2, which suggests that both integrase and transposase activities have played major roles in the arrangement and dissemination of antibiotic resistance genes dfr13, aadA4, bla(TEM-1), and sul2.

New trimethoprim-resistant dihydrofolate reductase cassette, dfrXV, inserted in a class 1 integron.

The nucleotide sequence of a plasmid-borne trimethoprim resistance gene from a commensal fecal Escherichia coli isolate revealed a new dihydrofolate reductase gene, dfrXV, which occurred as a gene cassette integrated in a site-specific manner in a class 1 integron. The new gene shows 84% nucleotide identity and the predicted protein shows 90% amino acid identity with dfrI and DHFR type I, respectively. Genes for spectinomycin resistance, aadA1 [ant (3")-Ia], and sulfonamide resistance, sulI, were located downstream of dfrXV in a manner identical to that in pLMO229.

Pneumococcal vaccines: an update on current strategies.

Streptococcus pneumoniae is a major cause of morbidity and mortality in infants, children and the elderly. Despite the availability of excellent antimicrobial therapy and adequate health care systems, respiratory diseases and invasive infections caused by pneumococci still comprise a major health problem. The emerging resistance to penicillin and other commonly used antibiotics underscores the importance of the development of novel vaccine strategies to combat pneumococcal disease. Although the 23-valent polysaccharide (PS) vaccine is immunogenic and protective in most adults and children over 5 years of age, they fail to protect children under 2 years of age. Fortunately, the recent conjugate vaccines have shown to be highly efficacious in preventing invasive diseases in this risk group. Moreover, promising results regarding prevention of pneumonia and acute otitis media have been published. Unfortunately, protection is raised against a limited number of pneumococcal serotypes, and serotype replacement and subsequent vaccine failure have become a serious concern. Currently, several pneumococcal surface proteins are considered as alternative vaccine candidates because of their serotype-independence. Thus far, pneumococcal surface adhesin A (PsaA) has proven to be highly protective against colonization in animal models. Moreover, pneumococcal surface protein A (PspA) and pneumolysin have shown to elicit protection against invasive diseases. Future research will elucidate their true potential in protecting humans. In this paper we discuss the present knowledge on pneumococcal vaccines and the current status of novel vaccine strategies.

The prevalence of antimicrobial resistance in human faecal flora in South Africa.

Between January and March 1992, 361 faecal specimens were collected from the healthy black population in the Transvaal Province of South Africa. Each specimen was examined for the prevalence of antimicrobial resistance in commensal bacteria. Volunteers, from both rural and urban dwellings, were divided into four age groups. The overall carriage rate of resistance varied from 88.6% for ampicillin, 74.2% for trimethoprim, 52.6% for chloramphenicol, 10.2% for nalidixic acid to 7.5% for gentamicin. The carriage of resistance found to each individual antimicrobial agent was slightly higher in the rural population rather than the urban population but there was no correlation between the prevalence of antimicrobial resistance and the age group.

Mutations in ribosomal protein L16 conferring reduced susceptibility to evernimicin (SCH27899): implications for mechanism of action.

A clinical isolate of Streptococcus pneumoniae (SP#5) that showed decreased susceptibility to evernimicin (MIC, 1.5 microgram/ml) was investigated. A 4,255-bp EcoRI fragment cloned from SP#5 was identified by its ability to transform evernimicin-susceptible S. pneumoniae R6 (MIC, 0.03 microgram/ml) such that the evernimicin MIC was 1.5 microgram/ml. Nucleotide sequence analysis of this fragment revealed that it contained portions of the S10-spc ribosomal protein operons. The nucleotide sequences of resistant and susceptible isolates were compared, and a point mutation (thymine to guanine) that causes an Ile52-Ser substitution in ribosomal protein L16 was identified. The role of this mutation in decreasing susceptibility to evernimicin was confirmed by direct transformation of the altered L16 gene. The presence of the L16 mutation in the resistant strain suggests that evernimicin is an inhibitor of protein synthesis. This was confirmed by inhibition studies using radiolabeled substrates, which showed that the addition of evernimicin at sub-MIC levels resulted in a rapid decrease in the incorporation of radiolabeled isoleucine in a susceptible isolate (SP#3) but was much less effective against SP#5. The incorporation of isoleucine showed a linear response to the dose level of evernimicin. The incorporation of other classes of labeled substrates was unaffected or much delayed, indicating that these were secondary effects.

Evernimicin (SCH27899) inhibits a novel ribosome target site: analysis of 23S ribosomal DNA mutants.

Spontaneous mutants of susceptible clinical and laboratory isolates of Streptococcus pneumoniae exhibiting reduced susceptibility to evernimicin (SCH27899; MIC, 0.5 to 4.0 mg/liter) were selected on plates containing evernimicin. Four isolates that did not harbor mutations in rplP (which encodes ribosomal protein L16) were further analyzed. Whole chromosomal DNA or PCR products of the 23S ribosomal DNA (rDNA) operons from these mutants could be used to transform the susceptible S. pneumoniae strain R6 to resistance at frequencies of 10(-5) and 10(-4), respectively, rates 10- to 100-fold lower than that for a single-allele chromosomal marker. The transformants appeared slowly (48 to 72 h) on selective medium, and primary transformants passaged on nonselective medium produced single colonies that displayed heterogeneous susceptibilities to evernimicin. A single passage on selective medium of colonies derived from a single primary transformant homogenized the resistance phenotype. Sequence analysis of the 23S rDNA and rRNA from the resistant mutants revealed single, unique mutations in each isolate at the equivalent Escherichia coli positions 2469 (A --> C), 2480 (C --> T), 2535 (G --> A), and 2536 (G --> C). The mutations map within two different stems of the peptidyltransferase region of domain V. Because multiple copies of rDNA are present in the chromosome, gene conversion between mutant and wild-type 23S rDNA alleles may be necessary for stable resistance. Additionally, none of the characterized mutants showed cross-resistance to any of a spectrum of protein synthesis inhibitors, suggesting that the target site of evernimicin may be unique.

The Ami-AliA/AliB permease of Streptococcus pneumoniae is involved in nasopharyngeal colonization but not in invasive disease.

The Ami-AliA/AliB oligopeptide permease is an ATP-binding cassette transporter which is found in Streptococcus pneumoniae and which is involved in nutrient uptake. We investigated the role of the three paralogous oligopeptide-binding lipoproteins AmiA, AliA, and AliB by using murine models of pneumococcal colonization and invasive disease. A series of mutants lacking aliA, aliB, and amiA either alone or in combination as double or triple mutations were used. Inoculation of the nasopharynx with a mixture of the obl (oligopeptide-binding lipoprotein-negative) triple-mutant and wild-type (D39) bacteria resulted in significantly smaller numbers of obl bacteria colonizing the nasopharynx. The use of a mixture of individual mutants and wild-type pneumococci revealed that AmiA, AliA, and AliB were all required for successful colonization of the nasopharynx. The obl mutant was more attenuated than the aliB mutant but not the aliA or amiA mutant. Therefore, there is some redundancy in the Ami-AliA/AliB complex in terms of nasopharyngeal colonization, with AliA and AmiA being able to compensate for the removal of AliB. Animals with invasive disease caused by these mutants had survival times, bacterial loads, and inflammatory cytokine production levels similar to those of animals infected with wild-type pneumococci. Our results show that although the Ami-AliA/AliB complex is not required for virulence during pneumococcal pneumonia, it does play a role in colonization of the nasopharynx.