Lactate dehydrogenase A promotes nasopharyngeal carcinoma progression through the TAK1/NF-κB Axis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor that originates in the nasopharyngeal mucosa and is common in China and Southeast Asian countries. Cancer cells reprogram glycolytic metabolism to promote their growth, survival and metastasis. Glycolysis plays an important role in NPC development, but the underlying mechanisms remain incompletely elucidated. Lactate dehydrogenase A (LDHA) is a crucial glycolytic enzyme, catalyzing the last step of glycolysis. This study aims to investigate the exact role of LDHA, which catalyzes the conversion of pyruvate into lactate, in NPC development. METHODS AND RESULTS: The western blot and immunohistochemical (IHC) results indicated that LDHA was significantly upregulated in NPC cells and clinical samples. LDHA knockdown by shRNA significantly inhibited NPC cell proliferation and invasion. Further knockdown of LDHA dramatically weakened the tumorigenicity of NPC cells in vivo. Mechanistic studies showed that LDHA activated TGF-ß-activated kinase 1 (TAK1) and subsequent nuclear factor κB (NF-κB) signaling to promote NPC cell proliferation and invasion. Exogenous lactate supplementation restored NPC cell proliferation and invasion inhibited by LDHA knockdown, and this restorative effect was reversed by NF-κB inhibitor (BAY 11-7082) or TAK1 inhibitor (5Z-7-oxozeaenol) treatment. Moreover, clinical sample analyses showed that LDHA expression was positively correlated with TAK1 Thr187 phosphorylation and poor prognosis. CONCLUSIONS: Our results suggest that LDHA and its major metabolite lactate drive NPC progression by regulating TAK1 and its downstream NF-κB signaling, which could become a therapeutic target in NPC.

A multi-degree-of-freedom reconfigurable ankle rehabilitation robot with adjustable workspace for post-stroke lower limb ankle rehabilitation.

Introduction: A multi-degree-of-freedom ankle rehabilitation robot with an adjustable workspace has been designed to facilitate ankle joint rehabilitation training. It features a rotation center adapted to the human body, making it suitable for patients with ankle dysfunction following a stroke. Method: In this study, a multi-degree-of-freedom reconfigurable ankle rehabilitation robot (RARR) with adaptable features, based on the principles of ergonomics, has been proposed to cater to the varying needs of patients. This robot offers an adjustable workspace, allowing for different types of ankle joint rehabilitation exercises to be performed. By adjusting the assembly of the RARR, personalized and targeted training can be provided to patients, circumventing issues of redundancy in degrees of freedom during its use. A kinematic model of the robot has been established, and finite element simulation has been employed to analyze the strength of critical components, ensuring the safety of the robot. An experimental platform has been set up to assess the smoothness of the rehabilitation process with RARR, with angle measurements conducted using an Inertial Measurement Unit (IMU). Results and discussion: In conclusion, both simulation and experimental results demonstrate that the robot offers an adjustable workspace and exhibits relatively smooth motion, thereby confirming the safety and effectiveness of the robot. These outcomes align with the intended design goals, facilitating ankle joint rehabilitation and advancing the field of reconfigurable robotics. The RARR boasts a compact structure and portability, making it suitable for various usage scenarios. It is easily deployable for at-home use by patients and holds practical application value for wider adoption in rehabilitation settings.

GFAT1-linked TAB1 glutamylation sustains p38 MAPK activation and promotes lung cancer cell survival under glucose starvation.

Reprogrammed cell metabolism is deemed as one of the hallmarks of cancer. Hexosamine biosynthesis pathway (HBP) acts as an "energy sensor" in cells to regulate metabolic fluxes. Glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of HBP, is broadly found with elevated expression in human cancers though its exact and concrete role in tumorigenesis still remains unknown and needs further investigation. P38 mitogen-activated protein kinase (MAPK) is an important component of stress-signaling pathway and plays a critical role in cell fate decision, whereas the underlying mechanism of its activation under nutrient stress also remains elusive. In this study, we show that glucose deprivation induces the interaction of GFAT1 with transforming growth factor ß-activated kinase 1 binding protein 1 (TAB1) in a TAB1 S438 phosphorylation-dependent manner. Subsequently, the binding of GFAT1 to TAB1 facilitates TTLL5-GFAT1-TAB1 complex formation, and the metabolic activity of GFAT1 for glutamate production further contributes to TTLL5-mediated TAB1 glutamylation. In consequence, TAB1 glutamylation promotes the recruitment of p38α MAPK and thus drives p38 MAPK activation. Physiologically, GFAT1-TAB1-p38 signaling promotes autophagy occurrence and thus protects tumor cell survival under glucose deficiency. Clinical analysis indicates that both GFAT1 and TAB1 S438 phosphorylation levels correlate with the poor prognosis of lung adenocarcinoma patients. These findings altogether uncover an unidentified mechanism underlying p38 MAPK signaling regulation by metabolic enzyme upon nutrient stress and provide theoretical rationality of targeting GFAT1 for cancer treatment.

[Treatment of Graves Hyperthyroidism by Jiakangling Capsule Combined with Reduction of 131I: an Efficacy Observation].

OBJECTIVE: To observe the clinical efficacy of Jiakangling Capsule (JC) combined with reduction of 1311 in treatment of Graves hyperthyroidism. METHODS: Totally 387 Graves hyperthyroidism patients were randomly assigned to the treatment group (200 cases) and the control group (187 cases). Patients in the treatment group took JC combined with reduction of 131I. The 131I dosage per gram of thyroid tissue was 50-80 microCi. They additionally took JC one week after taking 1311 for one consecutive month. Patients in the control group took 131 routinely as one disposable treatment. The 131I dosage per gram of thyroid tissue was 70-120 microCi, without using JC or other anti-thyroid drugs. All patients were reexamined after 24-month treatment. Whether hyperthyroidism was cured, incurred, or permanent was observed. Efficacies of thyroglobulin antibody (TGAb) and thyroid microsome antibody (TMAb) were compared between the two groups. RESULTS: Compared with the control group, the incurred ratio increased in the treatment group [3.2% (6/187) vs. 16.0% (32/200), P < 0.01], the incurred ratio of strong positive TGAb and TMAb patients increased [3.5% (2/57) vs. 27.1% (16/59), P < 0.01], the permanent hypothyroidism ratio decreased [21.1% (12/57) vs. 3.4% (2/59), P < 0.05 ]. CONCLUSION: JC combined with reduction of 1311 was superior in treating Graves hyperthyroidism induced permanent hypothyroidism than routine 1311 treatment, especially for strong positive TGAb and TMAb patients.

[Effect of nesprin-1 in the differentiation of mouse embryonic stem cells into cardiomyocytes].

OBJECTIVE: To investigate the role of nesprin-1 in mouse embryonic stem cells differentiation into cardiomyocyte. METHODS: Hanging drop-suspension-adherence method was applied for the differentiation of mouse embryonic stem cells into cardiomyocytes under the inducing of salvia miltorrhiza and 5-azacytidine. Changes in nesprin-1 gene expression were detected by using Western blotting and immunofluorescent assay. RNA interference was used to reduce nesprin-1 protein levels to further investigate the importance of nesprin-1 in mouse embryonic stem cells differentiation, group I (target sequence AAAGCCAAGCACGCAACTA), group II (target sequence GGGAACCAACAGTGAGATT), group III (target sequence ACCAGGACATTGCGTACTA), and group IV (control group). RESULTS: The nesprin-1 isoform profile was altered in mouse embryonic stem cells differentiation. The rates of differentiation of the four groups were (17.78 +/- 1.92)%, (36.67 +/- 3.34)%, (44.42 +/- 5.08)%, (77.78 +/- 1.92)%; The rate of differentiation of group IV was higher than RNAi groups and the difference was significant (P < 0.05). In addition, compared with the control group, myosin in RNAi groups were dramatically reduced. CONCLUSION: Nesprin-1 played important roles in mouse embryonic stem cells differentiation into cardiomyocyte. Nesprin-1 isoforms might perform different functions in the process of mouse embryonic stem cells differentiation.

Effects of Salvia miltorrhiza in neural differentiation of rat mesenchymal stem cells with optimized protocol.

AIM OF THE STUDY: The present study was aimed to explore the effects of Salvia miltorrhiza in inducing rMSCs to differentiate into functional neurons. MATERIALS AND METHODS: rMSCs were isolated and cultured in vitro, then Salvia miltorrhiza was added to induce rMSCs to differentiate repeatedly for 5 times with an optimized protocol, and neurophysiological functions such as action potential, endocytosis and exocytosis of the induced cells were investigated. RESULTS: About 98% of rMSCs expressed markers related to neural stem cells after treatment with preinduction medium, but they remained fibroform, the classical morphological state of MSCs, after exposure to induction medium for 2h, and the induced cells showed a neural shape. Next, fetal bovine serum (FBS) was added into the induction medium, transforming the neuron-like cells into fibroform cells. Finally, after exposure to induction medium, the cells could be transformed into neuron-like cells again. After the procedure was repeated 5 times, the induced cells displayed a classical neural shape and more than 95% of them expressed neural markers, including TUJ-1, NF and synaptophysin. Furthermore, the induced cells displayed neurophysiological functions, as characterized by action potential, endocytosis and exocytosis in response to a solution with a high concentration of potassium (K(+)). CONCLUSION: Salvia miltorrhiza can induce rMSCs to differentiate into neurons with neurophysiological functions efficiently by an optimized protocol.

In vivo MR imaging of magnetically labeled mesenchymal stem cells transplanted into rat liver through hepatic arterial injection.

OBJECTIVE: The aim of this study was to evaluate in vivo magnetic resonance imaging (MRI) for tracking the magnetically labeled mesenchymal stem cells (MSCs) transplanted into rat liver through hepatic arterial injection. MATERIALS AND METHODS: MSCs, harvested from the bone marrow of Wistar rats and expanded by the adhesion method, were labeled with both Feridex and 4',6-diamidino-2-phenylindole (DAPI). Cell transplantation was performed by injection of 1 x 10(6) labeled cells (n = 20) or unlabeled cells (n = 10) via hepatic artery into rat livers treated with 2% carbon tetrachloride to induce acute liver necrosis. MR imaging was performed on a clinical 1.5 T MR scanner with a T(2)*-weighted gradient-echo sequence immediately before and at 1 h, 3 days, 7 days and 14 days after transplantation, and the signal-to-noise ratios (SNRs) were measured in liver, spleen, kidney and muscle. After MR examination, the animals were sacrificed, and the liver, kidney, lung and muscle were prepared for fluorescence observation and Prussian Blue staining. RESULTS: In the group treated with labeled cells, the SNR of the liver after cell transplantation was 3.12 +/- 0.43 at 1 h, 7.98 +/- 1.05 at 3 days and 11.46 +/- 1.41 at 7 days. These values were significantly lower than the pre-transplantation SNR (14.40 +/- 0.37). In the group treated with unlabeled cells, no significant difference could be found between after and before transplantation liver SNRs. Prussian Blue staining showed iron particles, contained within the cytoplasm and distributed in the liver parenchyma, which corresponded to the DAPI-stained fluorescent nuclei under the fluorescence microscope. CONCLUSION: The magnetically labeled MSCs transplanted into rat liver through hepatic arterial injection can be detected and monitored in vivo with a 1.5 T clinical MR scanner for up to 7 days after cell transplantation.

[Electron microscopic analysis of biofilm on tracheal tubes removed from intubated neonates and the relationship between bilofilm and lower respiratory infection].

OBJECTIVE: Mechanical ventilation support is a very important method for the salvage of serious patients. However, it can result in the formation of an adherent matrix of bacteria on the surfaces of implanted materials which is termed "biofilm". Biofilm is dense bacterial communities attached to a solid surface and surrounded by an exopolysaccharide matrix. One of the most important features of bacterial biofilm is their resistance to antimicrobial agents and host immune system components. As a consequence, diseases involving biofilm are generally chronic and difficult to treat. The present study was conducted to explore the relationship between ETT-biofilm and the lower respiratory infection by observing microbial colonization and associated biofilm accumulation on the surface of endotracheal tubes (ETTs) removed from neonates treated with intubated mechanical ventilation. METHODS: Twenty neonates undergoing mechanical ventilation (from January to June in 2005) were recruited into this study. Clinical data about lower respiratory infection for each case were collected. ETTs were collected at the first time of extubation. A sterile control tube was also processed. For each ETT, a 1-cm-long cross-sectional segment was divided into two portions for both scanning electron microscopy (SEM) and aerobic/anaerobic cultures. The presence of biofilm on the surface of ETTs were examined by SEM, meanwhile, bacteria harvested from the surface of ETTs and the secretions of lower respiratory tract were isolated, identified and assessed on antimicrobial susceptibility, respectively. RESULTS: The diagnosis on admission of the twenty cases included: neonatal respiratory distress syndrome (10), meconium aspirate syndrome (2), severe asphyxia (2), pneumatothorax (2), severe pneumonia (1), scleredema neonatorum (1), inborn pulmonary hypoplasia (1) and recurrent apnea (1). Thirteen cases did not present symptoms and signs of lower respiratory infection before mechanical ventilation. However, during the mechanical ventilation process, symptoms and signs of lower respiratory infection presented and lasted until extubation. Nine of the above mentioned thirteen cases (70%) had the same duration of tube use as mechanical ventilation duration (mean: 3.6 days). Observation by SEM showed that colonization was time dependent and the incidence of microbial colonization increased when the duration of tube use exceeded one days (12/20). There were no obvious bacterial colonies except that some amorphous material was noted in 5 of 20 ETTs as early as one day of tube use. Up to 2 days of tube use (4/20), attached bacterial colonization was seen embedded in amorphous material (3/4). Up to 3 days (7/20), a layer of biofilm formation presented on ETTs (5/7). Furthermore, biofilm architecture became more mature and complex if the duration exceeded 3 days. Neither bacteria nor biofilm formation was seen on the control ETT. The results of aerobic/anaerobic cultures showed that there were 14 cultures from ETTs (normal flora grew in 4) and 7 pathogens were isolated; 13 cultures from the secretions of lower respiratory tract (normal flora grew in 1) and 10 pathogens were isolated. Seven samples had the same pathogen both on the surface of ETTs and in the secretions of lower respiratory tract, which accounted for 50% of the positive cultures from ETTs, including Xanthomonas maltophilia (2), Klebsiella pneumoniae (2), Acinetobacter lwoffii (1), Acinetobacter baumannii (1) and normal flora (1). The gram-negative bacteria isolated from the surface of ETTs and the secretions of lower respiratory tract presented multi-resistance to antibiotics. CONCLUSIONS: The ETT-biofilm develops into mature and complex form with the duration of tube use increase. This study provides evidence that there is correlation between microbial colonization, biofilm formation on the surface of ETTs and the lower respiratory infection in neonates who were intubated and ventilated for a prolonged period. ETT-Biofilm could also be a possible source of the recurrent infection. Increased attention must be paid to modification of the ETT to prevent or substantially reduce biofilm formation.

[Tracing magnetically labeled mesenchymal stem cells transplanted into rat livers by MRI].

OBJECTIVE: To trace magnetically labeled MSCs transplanted into the rat livers by magnetic resonance imaging (MRI). METHODS: Feridex and DAPI labeled rat mesenchymal stem cells (MSCs) were injected via portal veins into carbon tetrachloride treated rats. MRI was performed with a clinical 1.5 T MRI machine immediately before the MSCs injection and at h 1, d 3, d 7, and d 14 after the injection, and then the signal-to-noise ratio (SNR) was measured. MRI findings were compared with the liver histopathologies after the slides were stained with fluorescence dye and Prussian blue. RESULTS: The SNR for liver was 1.10+/-0.26 at hour 1, 8.18+/-1.55 at day 3, 11.08+/-1.30 at day 7, and 14.15+/-1.02 at day 14 respectively. Within 7 days after the MSCs transplantation, the SNRs of the livers were significantly lower than those before the transplantation (P less than 0.05). Histologically, the blue fluorescent particles under the fluorescence microscopy matched in distribution with the iron particles on the Prussian blue stained slides. CONCLUSION: The magnetically labeled MSCs transplanted into livers give rise to an obvious signal decrease, and can be tracked with a 1.5 T clinical MRI machine for up to 7 days after MSCs transplantation.

[Genotyping of major outer membrane protein gene of Chlamydia trachomatis by cleavase fragment length polymorphism analysis].

OBJECTIVE: To establish a methed of cleavage fragment length polymorphism (CFLP) analysis with a primer labeled at the 5'-end with digoxigenin for genotyping of Chlamydia trachomatis (Ct). The methods for detection of Ct by major outer membrane protein (MOMP) gene (ompl) with nested polymerase chain reaction (ompl-nPCR) were studied. The incidence of Ct infection in pregnant women, the common genotypes and vertical transmission rate of Ct in Chongqing area during the past one year was also investigated. METHODS: The samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates from April 2003 to Feb. 2004 in Chongqing Women and Children's Health Care Institute. Totally 300 pairs (605 specimens) were detected by using ompl-nPCR, ompl-PCR (inside pair of primers was used directly) and plasmid-PCR. The results were judged by the modified gold standard (MGS). The ompl-nPCR amplified DNA was purified by recovery of DNA from agarose gel electroelution into dialysis bags. The DNA amplified from ompl-nPCR was sequenced by ABI PRISM 377 DNA sequencer. CFLP assay with a primer labeled at the 5'-end with digoxigenin was created for genotyping of Ct, and was primarily applied. RESULTS: The minimum detectable levels of ompl-nPCR and ompl-PCR corresponded to 2.5 elementary body (EB) and 25 EB, respectively. The sensitivity of ompl-nPCR was 10 times that of ompl-PCR. The positive rate of Ct in the samples from the pregnant women was 11% (33/300). The vertical transmission rate of Ct from mothers to their infants was 24.2% (8/33). The rate of Ct isolated from nasopharyngeal swabs 5 - 10 days after birth was 38.9% (7/18), which was significantly greater than that [3.0% (1/33)] detected within 24 hours after birth (chi(c)(2) = 8.79, P < 0.01). Of the 33 Ct-positive samples from pregnant women, 9 had vaginal delivery and 24 had caesarean section. The vertical transmission rates in vaginal delivery group and caesarean section group were 66.7% (6/9) and 8.3% (2/24), respectively (chi(c)(2) = 9.16, P < 0.01). Incidence of premature rupture of membrane in Ct-positive group was 30.3% (10/33), which was greater than that of Ct-negative groups (13.5%, 36/267, chi(2) = 6.40, P < 0.05). Four different patterns were observed in the 16 Ct-positive samples from 8 pregnant women and 8 matched maternal-infants by using CFLP, which were confirmed by DNA sequencing later. They were type E (3 pairs), type F (2 pairs), type H (2 pairs) and type D (1 pair). Each pair of matched maternal-infantile samples presented identical CFLP pattern. CONCLUSIONS: This study revealed the infection rate of Ct in pregnant women, vertical transmission rate of Ct and the common genotypes of Ct in Chongqing Women and Children's Health Care Institute. The CFLP assay by using a primer labeled at the 5'-end with digoxigenin was first used for genotyping of Ct. The assay showed a good sensitivity and reproducibility, no radioactive contamination, and is simple. Therefore the assay is a potential new method for Ct genotyping.

[Comparison of the effect of ambroxol and dexamethasone on the expression of pulmonary surfactant proteins in the fetal rat lungs].

OBJECTIVE: To investigate the effects of maternally administered dexamethasone and ambroxol on the mRNA levels of surfactant proteins (SP-A, SP-B and SP-C) expression in fetal rat lungs at gestational age day 19. METHODS: A 19-day fetal rat lung model was employed. In situ hybridization was used to detect the expression of SP-B mRNA in alveolar type II cell, and the levels of SP-A, SP-B and SP-C mRNAs were detected by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: (1) SP-B mRNA was detected in situ in alveolar type II cells in fetal rat lung of day 19 gestational age; (2) In the late developmental period of fetal rat lungs, alveolar type II cells were also found around bronchus; (3) Comparing to beta-actin mRNA, the relative values of SP-A, SP-B and SP-C mRNAs were 0.81 +/- 0.26, 0.97 +/- 0.20 and 0.88 +/- 0.11 in fetal lung in the control group. The relative values of mRNAs of SP-A, SP-B and SP-C to beta-actin were 1.04 +/- 0.16, 1.28 +/- 0.29, 1.09 +/- 0.25 in fetal lungs of the ambroxol injected rats, and were 1.08 +/- 0.25, 1.23 +/- 0.35, 1.21 +/- 0.25 in fetal lungs of the dexamethasone injected rats, respectively. Both ambroxol and dexamethasone-treated rats had significantly higher mRNA expression of surfactant proteins compared to the control saline injected animals (P < 0.05). (4) There were no significant differences between ambroxol and dexamethasone in the effects of increasing expressions of surfactant protein mRNAs (P > 0.05). CONCLUSION: Antepartum administration of both ambroxol and dexamethasone can significantly increase fetal lung SP-A, SP-B and SP-C mRNAs expression.

[Study on mesenchymal stem cells entering the brain through the blood-brain barrier].

OBJECTIVE: Neonatal hypoxic-ischemic encephalopathy (HIE) harms the lives and health of newborn infants and children severely. The prognosis is not satisfied, especially of the severe HIE. Mesenchymal stem cells (MSCs) can secrete a series of growth factors and neurotrophic factors. As well they have the potential ability to differentiate to the neural cells in vitro and in vivo. Therefore MSCs transplantation has been employed as a source of progenitor cells for cell therapy in patients with HIE in order to promote recovery of brain function and reduce the sequelae. Studies have shown that MSCs could enter the cerebral parenchyma and differentiate to neural cells through systemic infusion, but most of the researches applied adult stroke animal models. This study used neonatal HIE models to test the hypothesis that MSCs could enter the brain of newborn Wistar rats through the blood-brain barrier (BBB) by intraperitoneal infusion followed by observing the characteristics of the distribution and differentiation of MSCs in brain tissues, and exploring the effects of hypoxic-ischemic brain damage to the penetration and differentiation of MSCs. METHODS: Isolation and purification of MSCs were established from the whole bone marrow of juvenile Wistar rats by removing the nonadherent cells in primary and passage cultures. For cellular identification, MSCs of three to five passages were continuously pre-labeled with 5-bromo-2-deoxyuridine (BrdU) for 72 hours before transplantation. Animal models of HIE were built in 7-day-postnatal Wistar rats according to the method described by Rice. Two hours after hypoxia-ischemia, rats in HIE group (n = 8) were intraperitoneally infused with MSCs (4 x 10(6), 0.5 ml). In control group (n = 8), 7-day-postnatal normal Wistar rats were intraperitoneally infused with the same amount of MSCs. All rats were sacrificed and their cerebra were sectioned by cryomicrotome 14 days after transplantation. Immunohistochemical staining with chromogen diaminobenzidine (DAB) was used to detect and measure the cells derived from MSCs, and study the characteristics of distribution. To determine the differentiation of the BrdU positive cells entering the brains, immunofluorescence double labeling for BrdU and neural cells specific antigens was performed. RESULTS: MSCs were distributed throughout the cerebra in both groups at the 14th day after transplantation. The number of MSCs detected was 2415 +/- 226 in the control group, and 3626 +/- 461 in HIE group, respectively (t = 6.68, P < 0.05). More BrdU reactive cells were observed in the right ischemic hemisphere (1904 +/- 267) than in the contralateral hemisphere (1723 +/- 204), (t = 4.47, P < 0.05). No significant difference was found while comparing both cerebral hemispheres of the control group (t = 0.31, P > 0.05). In the HIE group, MSCs distributed more extensively, and some focal aggregations of MSCs were noticed. A few MSCs expressed Nestin-protein marker of neural progenitor cells, and almost none of the MSCs which expressed proteins characteristic of neuron (e.g. NSE) and astrocyte (e.g. GFAP) was detected at the 14th day after transplantation. CONCLUSION: 1. MSCs could enter the cerebral parenchyma through BBB and migrate throughout the brain by intraperitoneal infusion. 2. More MSCs injected intraperitoneally were localized and directed to the sites of hypoxic-ischemic brain damage. 3. Transplanted MSCs could not differentiate to neuron and astrocyte without other interventions during 14 days after transplantation.

[Development of a gap ligase chain reaction for detection of Chlamydia trachomatis in newborn infants].

OBJECTIVE: To establish a gap ligase chain reaction (G-LCR) assay for the detection of Chlamydia trachomatis (Ct) in neonates with pneumonia. METHODS: A G-LCR DNA amplification assay that targeted the outer major membrane protein gene (omp1) of Ct was developed to detect Ct. The sensitivity and specificity of the G-LCR test was examined by the use of highly purified elementary bodies (EBs). Nasopharyngeal swabs taken from 328 neonates with pneumonia were analyzed by Gap-LCR and cell culture. RESULTS: The detection limit of G-LCR was 2 EBs. G-LCR could detect five species of Ct and was not cross-reacted with C psittaci and other bacteria. The prevalence of Ct in 328 neonates with pneumonia, using an expanded gold standard of a positive cell culture or two confirmed positive non-culture tests, was 21% (69/328). After analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive values for the G-LCR were 98.6%, 100%, 100% and 99.6%, respectively; whereas those for culture were 86.9%, 100%, 100% and 96.6%, respectively. CONCLUSION: This study demonstrated that the G-LCR was a highly sensitive nonculture technique and good alternative test for the detection of chlamydial infections.