Detection of Simkania negevensis by culture, PCR, and serology in respiratory tract infection in Cornwall, UK.

Respiratory tract infections are often treated empirically without investigation to detect the aetiological agent, which may be a virus or a bacterium, including atypical pathogens such as Chlamydophila pneumoniae or Mycoplasma pneumoniae. Recently, several types Chlamydia-like intracellular bacteria have been detected in environmental samples and clinical specimens. Little is known of their geographical distribution and potential pathogenicity. We describe the detection, by PCR and isolation in cell culture, of Simkania negevensis in nasopharyngeal aspirates of paediatric patients with bronchiolitis in Cornwall, UK. We also present serological evidence of exposure to the organism in 62% of adult patients and 46% of a sample of pregnant women. Empirical treatment of serious respiratory tract infection should consider the possible contribution of these organisms.

Serological evidence of acute infection with the Chlamydia-like microorganism Simkania negevensis (Z) in acute exacerbation of chronic obstructive pulmonary disease.

The aims of this study were twofold: (i) to test for possible associations between serological evidence of acute Simkania negevensis (Sn) infection and acute exacerbation of chronic obstructive pulmonary disease and (ii) to examine the prevalence of past infections with Sn in patients with chronic obstructive pulmonary disease. In 120 patients (63%) there was serological evidence of past infection with Sn, which was not significantly different from the rate in a control population. In five hospitalizations serological evidence existed of acute infection with Sn around the time of the exacerbation of chronic obstructive pulmonary disease. In four of these cases, there was serological evidence of acute infection with at least one other respiratory pathogen. It is concluded that Sn can be associated serologically with exacerbation of chronic obstructive pulmonary disease, in most cases together with other respiratory pathogens. The implications of these findings should be investigated further.

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis survival within amoebal cysts.

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.

Infectious etiologies in acute exacerbation of COPD.

Acute exacerbation (AE) is a frequent episode during the prolonged chronic course of chronic obstructive pulmonary disease (COPD), which entails significant morbidity and mortality. The purpose of this study was to determine the frequency distribution of infectious etiologies in these episodes. Two hundred forty hospitalizations for AECOPD were included in a prospective, purely serologically based study. Paired sera were obtained for each of the hospitalizations and were tested using immunofluorescence or EIA methods to identify 13 different pathogens. Only significant changes in antibody titers were considered diagnostic. The mean age ( +/- SD) of the patients was 66.8 +/- 9.0 years and 179 (84%) were males. In 175 (72.9%) hospitalizations at least one infectious etiology was identified. In 117 (48.8%) hospitalizations at least one of 7 viral etiologies was identified. In 72 (30.0%) hospitalizations at least one of the following atypical bacteria was identified: Legionella spp. in 40 (16.7%), Mycoplasma pneumoniae in 34 (14.2%), and Coxiella burnetii in a single hospitalization. In 58 (24.2%) hospitalizations at least one classic bacterial etiology was found: Streptococcus pneumoniae in 48 (20.0%), Hemophilus influenzae in 10 (4.2%) and Moraxella catarrhalis in 9 (3.8%). More than one etiology was found in 72 (30.0%) hospitalizations. There were no significant differences in the etiologic distribution when the patients were classified by severity of airway obstruction or the clinical type of the exacerbation. We conclude that in most cases of hospitalization due to AECOPD the infectious etiology is viral or atypical bacteria and is classic bacteria in only a minority of cases. More than one etiologic cause can be identified in a third of the cases. The frequency distribution of the etiologies is not associated with the severity of airway obstruction or the clinical type of the exacerbation. The results of our study suggest that atypical bacteria should be covered in antibiotic regimens recommended for AECOPD. This issue should be addressed in future studies.

An unspliced group I intron in 23S rRNA links Chlamydiales, chloroplasts, and mitochondria.

Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simkania negevensis Z(T) and Parachlamydia acanthamoebae strains. The present study of Chlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU. 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function.

Simkania negevensis strain ZT: growth, antigenic and genome characteristics.

Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order Chlamydiales. In the current study, growth, antigenic and genomic characteristics of this intracellular bacterium were investigated and compared to those of members of the family Chlamydiaceae. Growth of the organism, as assessed by infectivity assays, reached a plateau in 2-3 d although by light microscopy the cytopathic effect on the host cells increased for 12 or more days after infection. S. negevensis growth was unaffected by sulfadiazine. Cells infected by S. negevensis strain ZT were not recognized by either of two monoclonal antibodies specific for Chlamydiaceae LPS and several specific Chlamydiaceae ompA primers were unable to PCR amplify a S. negevensis gene. The S. negevensis genome contained one copy of the ribosomal operon. The genome size of S. negevensis strain ZT was determined by PFGE to be 1.7 Mbp, and the G + C content was 42.5 mol%. These data, taken together with other published data, are consistent with the proposal that S. negevensis belongs to a distinct family in the order Chlamydiales.

Seroprevalence of IgG antibodies to the chlamydia-like microorganism 'Simkania Z' by ELISA.

The newly described microorganism 'Simkania Z', related to the Chlamydiae, has been shown to be associated with bronchiolitis in infants and community acquired pneumonia in adults. The prevalence of infection in the general population is unknown. A simple ELISA assay for the detection of serum IgG antibodies to 'Simkania Z' was used to determine the prevalence of such antibodies in several population samples in southern Israel (the Negev). The groups tested included 94 medical and nursing students, 100 unselected blood donors, 106 adult members of a Negev kibbutz (communal agricultural settlement), and 45 adult Bedouin, residents of the Negev. IgG antibodies to 'Simkania Z' were found in 55-80% of these presumably healthy individuals, independently of antibodies to Chlamydia trachomatis and Chlamydia pneumoniae. The Bedouin had a seropositivity rate of 80%, while all other groups had rates of between 55 and 64%. These results indicate that 'Simkania Z' infection is probably common in southern Israel.

High prevalence of "Simkania Z," a novel Chlamydia-like bacterium, in infants with acute bronchiolitis.

The newly described microorganism "Simkania Z" ("Z"), an obligate intracellular, penicillin-resistant microorganism most closely related to the chlamydiae, has been associated with adult community-acquired pneumonia. The possible involvement of "Z" in bronchiolitis in infants was examined in a prospective study of 239 infants with bronchiolitis and 78 controls. Other potential etiologic agents sought were respiratory syncytial virus (RSV), adenovirus, and cytomegalovirus. Evidence for the presence of "Z" in nasopharyngeal wash specimens (polymerase chain reaction and/or culture) was found in 25% of infants with bronchiolitis, while controls were all negative (P < .001). A serum IgA response to "Z" infection was detected by immunoperoxidase assay in 15% of infants with bronchiolitis versus 1.3% of controls (P < .001). Clinical findings were not different for infants with bronchiolitis associated with RSV alone, "Z" alone, or RSV and "Z" together. The high prevalence of "Z" in infants with bronchiolitis, often accompanied by an immune response, suggests a possible etiologic role of this agent in the disease.

Pneumonia with serological evidence of acute infection with the Chlamydia-like microorganism "Z".

"Z" is a recently discovered microorganism that may belong to a new genus in the family Chlamydiaceae. Using an ELISA test we developed, we measured levels of serum antibody against "Z" for 308 paired sera obtained from adult patients hospitalized with community-acquired pneumonia (CAP). In 114 patients (37%), serological evidence of past infection with "Z" was found. In eight patients (2.6%) there was serological evidence of acute infection with this pathogen. In four of these eight patients, no other pathogen for CAP was identified despite an intensive serological investigation encompassing 13 etiological agents. The four patients were about 30 yr old, and three of them had no history of chronic illness. Their illness was characterized by high fever, a nonproductive cough, gastrointestinal symptoms, a shift to the left in the white blood cell count, and a prompt, dramatic response to erythromycin therapy. We conclude that the microorganism "Z", or a close variant, is infectious for humans, in some cases causing CAP. In these cases the disease is mild and responds quickly to treatment with erythromycin.

Multiple pathogens in adult patients admitted with community-acquired pneumonia: a one year prospective study of 346 consecutive patients.

BACKGROUND: The purpose of this study was to assess the causes of community-acquired pneumonia in adult patients admitted to hospital. METHODS: A prospective study was performed on 346 consecutive adult patients (54% men) of mean (SD) 49.3 (19.5) years (range 17-94) admitted to a university affiliated regional hospital in southern Israel with community-acquired pneumonia over a period of one year. Convalescent serum samples were obtained from 308 patients (89%). The aetiological diagnosis for community-acquired pneumonia was based on positive blood cultures and/or significant changes in antibody titres to Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, respiratory viruses, Coxiella burnetii, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella sp. RESULTS: The aetiology of community-acquired pneumonia was identified in 279 patients (80.6%). The distribution of causal agents was as follows: S pneumoniae, 148 patients (42.8%); M pneumoniae, 101 (29.2%); C pneumoniae, 62 (17.9%); Legionella sp, 56 (16.2%); respiratory viruses, 35 (10.1%); C burnetii, 20 (5.8%); H influenzae 19 (5.5%); and other causes, 21 patients (6.0%). In patients above the age of 55 years C pneumoniae was the second most frequent aetiological agent (25.5%). In 133 patients (38.4%) more than one causal agent was found. CONCLUSIONS: The causal agents for community-acquired pneumonia in Israel are different from those described in other parts of the world. In many of the patients more than one causal agent was found. In all these patients treatment should include a macrolide antibiotic, at least in the first stage of their illness.

Subclasses of IgA antibodies in serum and saliva samples of newborns and infants immunized against rotavirus.

Little is known about subclass levels of IgA in serum or saliva of infants in the perinatal period. We have previously shown that very young infants are capable of responding to an experimental rotavirus vaccine with both serum and salivary IgA, and that small amounts of IgA are already detectable in cord blood of these infants. In the present study, total IgA1 and IgA2 antibodies in serum and saliva samples of some of these infants at birth, at 6 weeks of age, and at 12 weeks of age, were determined by a quantitative ELISA. Also, subclass-specific IgA antibodies to the rotavirus group A common antigen were determined by ELISA. The ratio of average serum concentrations of IgA1 to IgA2 for 14 infants at 6 weeks of age was 19:1, while in saliva it was 5:1. Between 6 and 12 weeks of age levels of serum IgA1 increased by 25%, while levels of IgA2 did not increase perceptibly. Concentrations of IgA1 were higher in infant sera than in saliva, while concentrations of IgA2 were slightly higher in saliva than in serum. When calculated as specific ELISA units per mg IgA1, more salivary IgA1 was specific for rotavirus than serum IgA1. Further studies are needed to determine when infant IgA2 levels rise to values more characteristic of children and adults. This may be of significance for infant mucosal immunizations if secretory IgA2, more resistant to bacterial proteases than IgA1, is required for efficient defence of the respiratory and intestinal tracts.

Evidence that the novel microorganism 'Z' may belong to a new genus in the family Chlamydiaceae.

The purpose of this study was to investigate possible phylogenetic relationships of a new microorganism called 'Z'. The organism was previously shown to be similar to chlamydia in its growth cycle and some of its metabolic requirements, but different in others and in its major outer membrane protein. In this study we report the sequencing of 'Z"s 16S ribosomal DNA and comparison of the sequence with that of other microorganisms, including chlamydia and rickettsiae. While chlamydial species have 95.5% sequence identity among themselves, 'Z' had 83% identity with them, and 73% identity with certain rickettsia-like organisms. Based on the sequence analyses and taking into account physiologic considerations, we believe that 'Z' may belong to a novel genus in the family Chlamydiaceae.

Serum reactivity to Chlamydia trachomatis and C. pneumoniae antigens in patients with documented infection and in healthy children by microimmunofluorescence and immunoblotting techniques.

Chlamydia trachomatis and C. pneumoniae are both important human pathogens. Antigenic cross-reactivity between the two species may complicate serologic diagnosis of infection with one or the other agent. In this study we examined sera of persons with chlamydia infections and of healthy children by microimmunofluorescence (MIF) against C. trachomatis L2 antigen and against C. pneumoniae TW-183 antigen to explore the degree of cross-reactivity found by these two methods. Likewise, the cross-reactivity seen by immunoblot with sera of rabbits immunized against one of the antigens when tested on the other was examined. While among healthy children stratified by age, MIF seropositivity to C. pneumoniae TW-183 increased with age, no such trend was observed with respect to seropositivity to C. trachomatis L2 antigens, and 81% of children seropositive to TW-183 did not react on L2 antigen. Moreover, 27% of those positive on L2 antigen were negative on TW-183. Immunoblot analysis showed much greater antibody cross-reactivity than that detected by MIF. The immunoblot cross-reactivity was probably not attributable solely to double infection since sera of rabbits immunized to only one species of chlamydia reacted strongly with both chlamydiae in immunoblot analysis. The data presented need to be taken into account in the development of serologic tests based on a small number of antigens or on partially denatured antigens.

Serum and salivary responses to oral tetravalent reassortant rotavirus vaccine in newborns.

Serum and salivary responses of 95 infants to either a standard (4 x 10(4) plaque-forming units (PFU), 47 neonates) or a high dose (4 x 10(5) PFU, 48 neonates) of tetravalent reassortant rhesus rotavirus vaccine (administered at 2 days and at 6 weeks of age) were evaluated in a double-blind clinical trial. Serum and salivary IgA antibodies to the rotavirus group A common antigen were determined by ELISA and radioimmunoassay (RIA). Serum neutralizing antibodies to rhesus rotavirus were determined by fluorescent focus reduction assay. No significant differences in responses to the high versus standard dose were noted in serum or saliva. Response was influenced by cord blood antibodies. All infants who were cord blood-negative for rhesus rotavirus neutralizing antibodies (nine who received the standard dose and 20 who received the higher dose) had serum responses, compared with 42-70% of those who were cord blood-positive. The serum response rate recorded for babies with cord blood neutralizing titres > 1000 was 44%. Infants being bottle fed had a higher serum response rate than did babies being breast fed exclusively. If serum and salivary responses were combined, the response rate reached 80% for bottle fed infants. Thus, determination of serum responses alone underestimates vaccine 'take' in infants, and more so in highly endemic areas than in areas subject only to sporadic outbreaks. However, determination of salivary responses in newborn breastfed infants may be inaccurate, due to possible persistence of antibodies derived from colostrum or breast milk.

Description and partial characterization of a new Chlamydia-like microorganism.

A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture contaminant of unknown origin. The organism grew in a variety of cultured cells with a 5-7-day developmental cycle, within cytoplasmic phagosomes, similarly to Chlamydia and some Rickettsia spp. Two alternating developmental forms (elementary bodies and reticulate bodies) were observed by electron microscopy. SDS-PAGE electrophoresis, immunoblotting with chlamydia-specific antibodies, and polymerase chain reaction using chlamydial genus specific primers provided evidence that our bacterium differs significantly from chlamydiae. Further characterization of 'Z' including determination of 16S ribosomal RNA sequences will allow its taxonomic position to be established.

Reversibility of heat shock in Chlamydia trachomatis.

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.

Antibodies in human tears during and after infection.

The tear content of antibodies specific for various infectious agents has recently begun to be investigated. Important parameters of tear analysis with respect to antibody content are the method of tear collection and the laboratory techniques used to detect specific antibodies in the lacrimal fluid. Normal tears contain antibodies directed against both bacteria and viruses, and the antibody response in lacrimal fluid during immunization of animals and humans has been studied to some extent. This response has also been analyzed in humans during and after natural infection with certain viral and bacterial pathogens. It has become clear that local antibody synthesis takes place in the lacrimal gland, but at least some of these antibodies appear in tears because of lymphocyte sensitization in the common mucosal immune system. A certain degree of transudation of serum antibodies to tears is also often encountered, especially in severely inflamed eyes. Much of the data currently available needs to be confirmed, and more extensive studies need to be carried out for many pathogens. Potential benefits of such studies include development of new diagnostic techniques as well as a better understanding of when and how antibodies confer protection or may be potentially damaging.

Virus-specific IgA in serum, saliva, and tears of children with measles.

Measles-specific IgA antibody titres were determined by radioimmunoassay (RIA) for serial serum, saliva and tear samples obtained from 21 children with measles infection, from onset of rash until up to 14 months later. Serum IgA titres rose rapidly after onset of illness and remained detectable throughout the follow-up period. Virus-specific salivary IgA titres peaked at 4 to 7 days after onset of rash and decreased thereafter. Measles-specific lacrimal fluid IgA antibodies remained elevated for long periods of time; however, secretory component-bearing measles-specific antibodies in tears became for the most part undetectable by 1 month after onset of rash. These data raise anew the question of whether some form of viral latency is associated with the presence of virus-specific IgA antibody, or whether such antibody is simply a reflection of immune memory.

Two sequential outbreaks of rotavirus gastroenteritis: evidence for symptomatic and asymptomatic reinfections.

In two sequential outbreaks of rotavirus gastroenteritis that occurred in a kibbutz in southern Israel (the Negev), 32 persons (9% of the population) were ill in the first and 45 (13% of the population) in the second. Excretion of virus, changes in titers of rotavirus-specific serum IgG, or both implicated rotavirus in 72% of the illnesses in outbreak 1 and in 56% of the illnesses in outbreak 2. In both outbreaks the age-specific morbidity rate decreased with increasing age. Half (six of 12) of the children six to 27 months of age who were ill with rotavirus in outbreak 1 were ill with rotavirus again in outbreak 2, whereas two were asymptomatically infected; older children who were ill in outbreak 1 were not ill in outbreak 2. Serotype determination by enzyme-linked immunosorbent assay using monoclonal antibodies to VP7 implicated a serotype 3 virus in outbreak 1 and a serotype 1 virus in outbreak 2.