Paroxysmal cold hemoglobinuria (PCH) is a rare diagnosis of acquired hemolytic anemia in children, which is caused by a specific cold antibody named Donath-Landsteiner hemolysin. Although various bacteria or viruses were reported as triggering factor of PCH, childhood PCH related to Chlamydia pneumoniae infection is uncommon. The authors report a case of childhood PCH which is related with suspicious Chlamydia pneumoniae infection, with a review of pertinent literature.
Anemia, Hemolytic ; Bacteria ; Child ; Chlamydia Infections ; Chlamydophila pneumoniae ; Diagnosis ; Hemoglobinuria, Paroxysmal ; Humans

OBJECTIVEThis paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumoniae) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPVD2-VD3) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae.

METHODSMOMPVD2-VD3 were overexpressed in Escherichia coli and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors).

RESULTSIn Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively.

CONCLUSIONThe novel MOMPVD2-VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

Animals ; Antibodies, Monoclonal ; Bacterial Outer Membrane Proteins ; immunology ; Chlamydophila Infections ; diagnosis ; microbiology ; Chlamydophila pneumoniae ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Mice ; Protein Structure, Tertiary

The purified elementary bodies of C. pneumoniae TW-183 were used for immunization of male BALB/c mice, the spleen cells of these mice were fused with SP2/0 cells and the hybrid cells were cloned by limiting dilution. One clone that secreted the C. pneumoniae monoclonal antibody (Cpn-McAb) stably was obtained finally. The Cpn-McAb belonged to IgG2b class and anti-Cpn-MOMP; the outcome of micro-immunofluorescence showed its weak cross reaction with the C. psittaci elementary body but it has no cross reaction with C. trachoma elementary body. It has the same speciality of the imported Cpn-McAb. For the evaluation of Cpn-McAb, the peripheral blood mononuclear cell specimens of 454 patients were detected by self-made Cpn-McAb and imported Cpn-McAb at the same time. The positive rates of Cpn-antigen were 53.3% for self-made Cpn-McAb and 52.6% for imported Cpn-McAb,showing high concordance between them (Kappa=0.714). The results showed that self-made Cpn-McAb has almost the same high specificity and sensitivity as imported Cpn-McAb, so the self-made Cpn-McAb may replace imported Cpn-McAb to detect Cpn specific antigen and be helpful to diagnosing and treating the clinical diseases associated with Cpn infection.
Animals ; Antibodies, Bacterial ; immunology ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Antibody Specificity ; Chlamydia Infections ; diagnosis ; microbiology ; Chlamydophila pneumoniae ; immunology ; Hybridomas ; secretion ; Male ; Mice ; Mice, Inbred BALB C

PURPOSE: Chlamydia pneumoniae is a common intracellular bacterial pathogen and plays an important role in acute respiratory infections. The purpose of this study was to investigate clinical presentations of C. pneumoniae in children with acute respiratory infections. METHODS: We examined the medical records of pediatric patients (age<18 years) admitted with acute respiratory infections of C. pneumoniae to Gachon University Gil Medical Center between March 1, 2011 and August 31, 2014. We compared the clinical features of C. pneumoniae infection with that of Mycoplasma pneumoniae infection. RESULTS: We confirmed acute respiratory infections of C. pneumoniae in 110 patients out of 2,156 patients (5.1%) admitted with acute respiratory infections. The mean age was 37.2+/-30.1 months. More than half of them (54.5%) had coinfection. C. pneumoniae infection had mild and subacute courses. The mean duration of symptoms prior to admission was 8.5+/-13.8 days. There were remarkable seasonal variations and prevalence was higher in December and April (P=0.03 and P=0.02, respectively). Although rhinorrhea and pharyngeal injection were more common in C. pneumoniae infection (P<0.05), clinical signs and symptoms were similar between C. pneumoniae and M. pneumoniae. Extrapulmonary manifestations such as skin lesion, Gastrointestinal symptoms, hepatitis, and neurologic symptoms were common (41.0%) in C. pneumoniae infection and, had similar incidence in M. pneumoniae infection. CONCLUSION: C. pneumoniae is an important infectious agent of acute respiratory infections in children. Clinical pictures of C. pneumoniae are similar to M. pneumoniae, even in extrapulmonary manifestations. C. pneumoniae should be taken into consideration in differential diagnosis of acute respiratory infection in children.
Child* ; Chlamydia* ; Chlamydophila pneumoniae* ; Coinfection ; Diagnosis, Differential ; Hepatitis ; Humans ; Incidence ; Medical Records ; Mycoplasma pneumoniae ; Mycoplasma* ; Neurologic Manifestations ; Pneumonia ; Pneumonia, Mycoplasma* ; Prevalence ; Respiratory Tract Infections* ; Seasons ; Skin

BACKGROUND: Chlamydia pneumoniae has recently been established as an important cause of acute respiratory tract infections such as pneumonia and bronchitis in humans. The purpose of our study was to define the sequence of the C. pneumoniae omp1 gene. METHODS: The omp1 gene of C. pneumoniae was amplified by touchdown polymerase chain reaction (PCR) on sputum samples. The PCR product was cloned into pT7Blue T-Vector using the TA cloning technique. The nucleotide sequence of the cloned omp1 gene was determined with the Cy5TM AutoReaderTM Sequencing Kit. RESULTS: We designated the cloned PCR product as CpT-207. The sequence of CpT-207 DNA was 96%-100% identical to the omp1 gene of C. pneumoniae isolated from other countries. CONCLUSIONS: The sequence analysis of CpT-207 DNA was almost identical to the sequences of the omp1 gene of C. pneumoniae isolated from other countries. The CpT-207 can be used as a control or a probe for the molecular diagnosis of C. pneumoniae.
Base Sequence ; Bronchitis ; Chlamydia* ; Chlamydophila pneumoniae* ; Clone Cells ; Cloning, Organism ; Diagnosis ; DNA ; Humans ; Pneumonia ; Polymerase Chain Reaction ; Respiratory Tract Infections ; Sequence Analysis* ; Sputum

BACKGROUND: Chlamydia pneumoniae is a recently recognized species consisting of the strains commonly referred to as TWAR. These strains are associated with acute respiratory infections in humans, especially atypical pneumonia. So we tried to make a monoclonal antibody to Chlamydia pneumoniae. METHODS: C. pneumoniae were adapted to grow in HeLa-229 cells. The organisms were harvested and purified in a linear gradient of renograffin. BALB/c mice (female, 10weeks) were intravenously immunized with purified C. pneumoniae(TW-183).The spleen cells and SP 2/0 myeloma cells were fused with 40% polyethylene glycol (Mol.Wt.:1,450). Antibodies against C. pneumoniae were screened by an enzyme- linked immunosorbent assay (ELISA). The proteins of purified chlamydial elementary bodies were separated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blots were performed with these monoclonal antibodies. RESULTS: Two monoclonal antibodies (HYMD10, HYMG12) reacted specifically with C. pneumoniae, as measured by an ELISA and indirect immunofluorecent stain. One of the monoclonal antibody (HYMD10) reacted with 75- and 39-KDa proteins in Western blot. The other monoclonal antibody (HYMG12) reacted with 98- and 39-KDa proteins of C. pneumoniae. CONCLUSIONS: These species-specific monoclonal antibodies (HYMD10, HYMG12) to C. pneumoniae could be used for diagnosis of C. pneumoniae infections.
Animals ; Antibodies ; Antibodies, Monoclonal ; Blotting, Western ; Chlamydia* ; Chlamydophila pneumoniae* ; Diagnosis ; Diatrizoate Meglumine ; Electrophoresis ; Enzyme-Linked Immunosorbent Assay ; Humans ; Mice ; Pneumonia ; Polyethylene Glycols ; Respiratory Tract Infections ; Sodium ; Spleen

BACKGROUND: Differentiation of atypical pathogens is important for community-acquired pneumonia (CAP). In this study, we compared sputum and nasopharyngeal swabs (NPS) for use in detection of Mycoplasma pneumoniae (MP), Chlamydophila pneumoniae (CP), and Legionella pneumophila (LP), using Seeplex PneumoBacter ACE Detection Assay (PneumoBacter; Seegene). METHODS: Sputum and NPS specimens were collected from patients in 15 hospitals. DNA was extracted from sputum using QIAamp DNA Stool Mini Kit (Qiagen) and from NPS using easyMAG (bioMerieux). Both types of specimens were evaluated by multiplex PCR using PneumoBacter. To determine the diagnostic performance of this assay, sputum samples were also tested using BD ProbeTec ET Atypical Pneumonia Assay (APA; Becton Dickinson). RESULTS: Among 217 sputum and NPS, 20 (9.2%), 2 (0.9%), and 0 sputum were positive for MP, LP, and CP, respectively, whereas 8 (3.7%) NPS were positive for MP. The sputum APA test yielded 186, 206, and 204 interpretable results for MP, LP, and CP, respectively. Of these, 21 (11.3%) were positive for MP, 2 (1.0%) were positive for LP, and 0 samples were positive for CP. Compared to APA, the sensitivity and specificity of the sputum assay for MP were 95.2% and 100.0%, respectively, whereas for the NPS assay, these were 38.1% and 93.9%. Sputum testing was more sensitive than NPS testing (P=0.002). For LP and CP diagnosis, PneumoBacter and APA tests agreed 100%. CONCLUSIONS: Specimen type is crucial and sputum is preferred over NPS for simultaneous detection of MP, LP, and CP using multiplex PCR in CAP.
Chlamydophila Infections/diagnosis ; Chlamydophila pneumoniae/*genetics/isolation & purification ; Community-Acquired Infections/*diagnosis ; DNA, Bacterial/analysis/isolation & purification ; Humans ; Legionella pneumophila/*genetics/isolation & purification ; Legionnaires' Disease/diagnosis ; Multiplex Polymerase Chain Reaction ; Mycoplasma pneumoniae/*genetics/isolation & purification ; Nasopharynx/*microbiology ; Pneumonia, Mycoplasma/diagnosis ; Reagent Kits, Diagnostic ; Sputum/*microbiology