Maternal colonization with group B Streptococcus and antibiotic resistance in China: systematic review and meta-analyses.

BACKGROUND: Maternal rectovaginal colonization with group B Streptococcus (GBS) or Streptococcus agalactiae is the most common pathway for this disease during the perinatal period. This meta-analysis aimed to summarize existing data regarding maternal colonization, serotype profiles, and antibiotic resistance in China. METHODS: Systematic literature reviews were conducted after searching 6 databases. Meta-analysis was applied to analyze colonization rate, serotype, and antimicrobial susceptibility of GBS clinical isolates in different regions of China. Summary estimates are presented using tables, funnel plots, forest plots, histograms, violin plots, and line plots. RESULTS: The dataset regarding colonization included 52 articles and 195 303 pregnant women. Our estimate for maternal GBS colonization in China was 8.1% (95% confidence interval [CI] 7.2%-8.9%). Serotypes Ia, Ib, III, and V account for 95.9% of identified isolates. Serotype III, which is frequently associated with the hypervirulent clonal complex, accounts for 46.4%. Among the maternal GBS isolates using multilocus sequence typing (MLST), ST19 (25.7%, 289/1126) and ST10 (25.1%, 283/1126) were most common, followed by ST12 (12.4%, 140/1126), ST17 (4.8%, 54/1126), and ST651 (3.7%, 42/1126). GBS was highly resistant to tetracycline (75.1% [95% CI 74.0-76.3%]) and erythromycin (65.4% [95% CI 64.5-66.3%]) and generally susceptible to penicillin, ampicillin, vancomycin, ceftriaxone, and linezolid. Resistance rates of GBS to clindamycin and levofloxacin varied greatly (1.0-99.2% and 10.3-72.9%, respectively). A summary analysis of the bacterial drug resistance reports released by the China Antimicrobial Resistance Surveillance System (CARSS) in the past 5 years showed that the drug resistance rate of GBS to erythromycin, clindamycin, and levofloxacin decreased slowly from 2018 to 2020. However, the resistance rates of GBS to all 3 antibiotics increased slightly in 2021. CONCLUSIONS: The overall colonization rate in China was much lower than the global colonization rate (17.4%). Consistent with many original and review reports in other parts of the world, GBS was highly resistant to tetracycline. However, the resistance of GBS isolates in China to erythromycin and clindamycin was greater than in other countries. This paper provides important epidemiological information, to assist with prevention and treatment of GBS colonization in these women.

Lonicerae flos polysaccharides improve nonalcoholic fatty liver disease by activating the adenosine 5'-monophosphate-activated protein kinase pathway and reshaping gut microbiota.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver cirrhosis and cancer. Lonicerae flos polysaccharides (LPs) have been shown to be effective in treating metabolic diseases; however, the therapeutic effects and underlying molecular mechanisms of LPs in NAFLD remain unclear. PURPOSE: The objective of this study was to investigate the morphological characterization of Lonicerae flos polysaccharides (LPs) and the mechanism of LPs in relieving NAFLD. METHODS: The morphology of LPs was observed using atomic force microscopy (AFM), X-ray diffraction (XRD), thermal weight (TG), and thermal weight derivative (DTG); NAFLD mice were treated with LPs at the same time as they were induced with a Western diet, and then the indexes related to glycolipid metabolism, fibrosis, inflammation, and autophagy in the serum and liver of the mice were detected. RESULTS: The atomic force microscope analysis results indicated that the LPs displayed sugar-chain aggregates, exhibited an amorphous structure, and were relatively stable in thermal cracking at 150 °C. It was also found that LPs exerted therapeutic effects in NAFLD. The LPs prevented high-fat and -cholesterol diet-induced NAFLD progression by regulating glucose metabolism dysregulation, insulin resistance, lipid accumulation, inflammation, fibrosis, and autophagy. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor compound C abrogated LP-induced hepatoprotection in mice with NAFLD. The LPs further treated NAFLD by reshaping the structure of the gut microbiota, in which Desulfovibrio bacteria plays a key roles. CONCLUSION: Lonicerae flos polysaccharides exert protective effects against NAFLD in mice by improving the structure of the intestinal flora and activating the AMPK signaling pathway. © 2023 Society of Chemical Industry.

Genetic Diversity of Listeria monocytogenes Isolates from Invasive Listeriosis in China.

Listeria monocytogenes is a deadly foodborne pathogen, and infections can result in meningoencephalitis and sepsis with mortality rates of up to 30%. In this study, we performed comparative whole-genome analysis of 30 clinical isolates sequenced together with 32 previously sequenced clinical and food isolates from China. The data indicate that L. monocytogenes isolates belonging to the clonal complexes (CC) -1, -8, -9, -87, -121, and -155 are present in human clinical cases. The majority of isolates are from CC-87, 9, and 8 and overlap with those CCs previously reported on the basis of multilocus sequence typing for isolates from Chinese food products. Detailed genome analysis of isolates, representative of CCs in clinical and food products, revealed strong similarities both in their core- and accessory genomes indicating that they are highly related. When compared to genome sequences of isolates of a given CC worldwide, clinical isolates of China were distinct and clustered in unified clades. Our data indicate that epidemic clones of L. monocytogenes (CC-87, 9, and 8) with unusually high occurrence of plasmids are unique to China and suggest that common populations of L. monocytogenes clones are present in both clinical and food products in China.

Identification of oligopeptide-binding protein (OppA) and its role in the virulence of Streptococcus suis serotype 2.

The oligopeptide permease (Opp) cassette, an oligopeptide transport system belongs to the superfamily of ATP-binding cassette (ABC) transporter, is widely distributed in bacteria, including Streptococcus suis (S. suis). It is encoded by the opp operon containing oppA, oppB, oppC, oppD, and oppF. In addition to the uptake of peptide, the oppA gene also plays an important role in virulence of many pathogens. In this study, an oppA homologue from the highly virulent S. suis serotype 2 (S. suis 2) strain 05ZYH33 was identified. Flow cytometry and Western blot confirmed that OppA is a surface immunogenic protein and is expressed during S. suis 2 infection. To explore the role of oppA in S. suis 2 growth and pathogenicity, an isogenic 05ZYH33 mutant of oppA (△oppA) was obtained by homologous recombination. Although the complementary strain was not obtained due to the △oppA strain is not transformable, the current data revealed that deletion of the oppA gene in S. suis 2 has greatly affected its growth and virulence. Our data revealed that the growth rate is significantly slow for the △oppA. Adherence of the △oppA strain to human epithelial cells is greatly reduced comparing to the wild strain. Mouse infection experiment showed that inactivation of oppA greatly attenuated the high pathogenicity of S. suis 2. The observed results suggest that OppA is a surface-exposed protein and plays important roles in the growth and pathogenicity of S. suis 2.

Rapid visual detection of highly pathogenic Streptococcus suis serotype 2 isolates by use of loop-mediated isothermal amplification.

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that causes considerable economic losses to the pig industry and significantly threatens public health worldwide. The highly pathogenic S. suis 2, which contains the 89K pathogenicity island (PAI), has caused large-scale outbreaks of infections in humans, resulting in high mortality rates. In this study, we established two loop-mediated isothermal amplification (LAMP)-based assays that can rapidly detect S. suis 2 and the 89K PAI and can be performed simultaneously under the same conditions. Further, based on the findings of these two LAMP assays and using the same set of serially diluted DNA samples, we compared the sensitivities of different LAMP product detection methods, including SYBR green detection, gel electrophoresis, turbidimetry, calcein assays, and hydroxynaphthol blue detection. The results suggest that target genes can be amplified and detected within 48 min under 63°C isothermal conditions. The sensitivity of tests for S. suis 2 detection varies between detection methods and reaction systems, indicating that for each LAMP reaction system, multiple detection methods should be performed to select the optimal one. The sensitivities of the optimized methods (7.16 copies/reaction) in the present study were identical to those of the real-time PCR assay, and the test results for reference strains and clinical samples showed that these LAMP systems have high specificities. Thus, since the LAMP systems established in this study are simple, fast, and sensitive, they may have good clinical potential for detecting the highly pathogenic S. suis 2.

Genetic diversity and evolutionary characterization of Chinese porcine reproductive and respiratory syndrome viruses based on NSP2 and ORF5.

To more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the Nsp2 and ORF5 gene sequences of 35 representative PRRSV isolates from 2008 to 2012. Sequence analysis revealed that the Nsp2 and ORF5 genes have undergone genetic variation. Furthermore, the isolate FJLYDX04 contains five insertions at positions 599 to 603 and is the first isolate from China reported to have an insertion in Nsp2. Our results suggest that the highly pathogenic PRRSV has become the dominant strain in China and that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines.

[Capsular saliva acid of Streptococcus suis 2 influences virulence and host inflammatory responses].

OBJECTIVE: We clarified the pathogenic influence of the absence of Streptococcus suis type 2 capsular saliva acid on BLAB/c mice. METHODS: The virulence of the experimental strains were compared; the distribution of strains in vivo was determined by quantitative plating. Histopathological analysis was used to qualitatively compare the different pathogenicity of wild strain and knockout strains. ELISA was used to test the levels of cytokine in whole blood cells for the stimulation of strains. RESULTS: The virulence of mutant strains was significantly reduced, and when the genes were restored, toxicity levels were recovered to that of the wild type strain. The distribution in blood and in the brain between wild strain and knock out strains has significant difference, and Streptococcus suis type 2 strains can cause different degrees of brain damage. During the in vitro test, the mutant strains can stimulate the whole blood cells to secrete higher levels of MCP-1 and IL-6. CONCLUSION: Capsular saliva acid affects bacterial virulence and host cell inflammation response. As an important virulence factor of Streptococcus suis type 2, it can damage the blood brain barrier and cause meningitis.

Contribution of the Rgg transcription regulator to metabolism and virulence of Streptococcus suis serotype 2.

The Rgg-like regulators, a family of transcription factors commonly found in many Gram-positive bacteria, play multiple roles, especially in the control of pathogen virulence. Here, we report an rgg homologue from a Chinese isolate, 05ZYH33, of Streptococcus suis serotype 2 (SS2). Deletion of the rgg gene in SS2 increased its adhesion to Hep-2 cells and hemolytic activity in vitro. Significantly, inactivation of the rgg gene attenuated SS2 virulence in an experimental piglet infection model. Using DNA microarrays and quantitative reverse transcription-PCR, we found that the Rgg regulator affects the transcriptional profile of 15.87% (n = 345) of all of the annotated chromosomal genes, including those involved in nonglucose carbohydrate metabolism, DNA recombination, protein biosynthesis, bacterial defense mechanisms, and others. It was experimentally verified that the deletion of rgg in SS2 reduced the utilization of nonglucose carbohydrates, such as lactose and maltose. In addition, the rgg gene was found to be associated with changes in the bacterial microscopic phenotype and growth curve. These data suggested that Rgg in SS2 is a global transcriptional regulator that plays an important role in promoting SS2 bacterial survival during pathogen-host interaction.

[Construction and in vitro assay of the sortase BCD geneknock-out mutant of Streptococcus suis 2].

OBJECTIVE: Streptococcus suis 2 is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. In this study, we investigated the functionality of one of Streptococcus suis 2 sortases, known as the srtBCD. METHODS: To obtain the isogenic mutant srtBCD, the competent cells of 05ZYH33 were subjected to electrotrans formation with recombinant plasmid based on the principle of homologous recombination. The resulting mutant strains was further confirmed by a series of PCR and reverse transcription PCR. To better assess the role of srtBCD gene in the virulence of 05ZYH33, cell adherence assays and experimental infection of mice was adopted. RESULTS: A SrtBCD defective mutant of 05ZYH33 was found to be associated with growth curve upon cultivation in standard laboratory used in our in vitro assays. Furthermore, abolishment of the expression of srtBCD result in impaired interactions of S. suis with human laryngeal epithelial cell line. However, there is no differences when infection mice by the WT and mutant strain. CONCLUSION: These results suggest that srtBCD are critical for the pathogen-host interaction of S. suis 2, but abolishment of srtBCD does not impair the full virulence of 05ZYH33. It is to expect that future study carried out with S. suis 2 to verification the conclusions.

Streptococcus suis enolase functions as a protective antigen displayed on the bacterial cell surface.

BACKGROUND: Streptococcus suis serotype 2 (SS2) has evolved into a highly infectious entity, posing a great threat to public health. Screening for and identification of protective antigens plays an important role in developing therapies against SS2 infections. METHODS: Multiple strategies were used to investigate a new surface protein that has the potential to be a protective antigen. These strategies included molecular cloning, biochemical and biophysical analyses, enzymatic assay, immunological approaches (eg, immunoelectron microscopy), and experimental infections of animals. RESULTS: We identified an enolase gene from SS2 and systematically characterized its protein product, enolase. Biophysical data indicated that S. suis enolase is an octameric protein. Enzymatic assays verified its ability to catalyze the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. In consideration of the strong antigenicity of enolase, an efficient enolase-based method was established for monitoring SS2 infections. Combined evidence strongly indicated that SS2 enolase can localize on the bacterial cell surface and facilitate bacterial adherence. Additionally, we found that enolase can confer complete protection against SS2 infection to mice, which suggests that enolase has potential as a vaccine candidate. CONCLUSIONS: We conclude that S. suis enolase functions as a protective antigen displayed on the bacterial cell surface and that it can be used to develop new strategies to combat SS2 infections.

Identification of a Novel Linear B Cell Epitope on the Sao Protein of Streptococcus suis Serotype 2.

Surface antigen one (Sao) protein is a bacterial surface protein identified in the important zoonotic pathogen Streptococcus suis serotype 2 (S. suis 2) during an extensive search for functional proteins. The Sao protein is anchored to the bacterial cell wall by the LPVTG motif and is widely distributed in many S. suis serotypes. In this paper, we present the immunodominant epitope peptide of the Sao protein that is recognized by BALB/c antibodies against the Sao protein: 355SEKQMPSVVNENAVTPEKQMTNKENDNIET384 (location Sao355-384). To determine the core epitope recognized by antibodies, we prepared truncation peptide libraries. Analyses of the immunoreactivity of truncation peptides with anti-Sao355-384 serum revealed that the most immunoreactive sequence was 355SEKQMPSVVNENAVTPEK372 (location Sao355-372). Moreover, we observed that this core epitope also showed good specificity based on the ratio of reactivity with serum from S. suis-positive patients compared to serum from S. suis-negative patients. Our results point to the potential of using the Sao355-372 peptide in diagnostic assays to determine S. suis infection in humans.

Inactivation of the htpsA gene affects capsule development and pathogenicity of Streptococcus suis.

STREPTOCOCCUS SUIS: serotype 2 (S. suis 2) is an important swine pathogen and also an emerging zoonotic agent. HtpsA has been reported as an immunogenic cell surface protein on the bacterium. In the present study, we constructed an isogenic mutant strain of htpsA, namely ΔhtpsA, to study its role in the development and virulence of S. suis 2. Our results showed that the mutant strain lost its typical encapsulated structure with decreased concentrations of sialic acid. Furthermore, the survival rate in whole blood, the anti-phagocytosis by RAW264.7 murine macrophage, and the adherence ability to HEp-2 cells were all significantly affected in the ΔhtpsA. In addition, the deletion of htpsA sharply attenuated the virulence of S. suis 2 in an infection model of mouse. RNA-seq analysis revealed that 126 genes were differentially expressed between the ΔhtpsA and the wild-type strains, including 28 upregulated and 98 downregulated genes. Among the downregulated genes, many were involved in carbohydrate metabolism and synthesis of virulence-associated factors. Taken together, htpsA was demonstrated to play a role in the morphological development and pathogenesis of the highly virulent S. suis 2 05ZYH33 strain.

The orphan response regulator CovR: a globally negative modulator of virulence in Streptococcus suis serotype 2.

Streptococcus suis serotype 2 is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. However, the pathogenesis of S. suis serotype 2 infection is not well understood. In this study, we report that an orphan response regulator, CovR, globally regulates gene expression and negatively controls the virulence of S. suis 05ZYH33, a streptococcal toxic shock syndrome (STSS)-causing strain. A covR-defective (DeltacovR) mutant of 05ZYH33 displayed dramatic phenotypic changes, such as formation of longer chains, production of thicker capsules, and increased hemolytic activity. Adherence of the DeltacovR mutant to epithelial cells was greatly increased, and its resistance to phagocytosis and killing by neutrophils and monocytes was also significantly enhanced. More importantly, inactivation of covR increased the lethality of S. suis serotype 2 in experimental infection of piglets, and this phenotype was restored by covR complementation. Colonization experiments also showed that the DeltacovR mutant exhibited an increased ability to colonize susceptible tissues of piglets. The pleiotropic phenotype of the DeltacovR mutant is in full agreement with the large number of genes controlled by CovR as revealed by transcription profile analysis: 2 genes are positively regulated, and 193 are repressed, including many that encode known or putative virulence factors. These findings suggested that CovR is a global repressor in virulence regulation of STSS-causing S. suis serotype 2.

The involvement of sortase A in high virulence of STSS-causing Streptococcus suis serotype 2.

Sortase A (SrtA), originally identified as a transpeptidase in Staphylococcus aureus, plays key roles in full virulence of pathogenic bacteria. In silico genome-wide search suggested a srtA homologue from 05ZYH33, a Chinese human isolate of streptococcal toxic shock syndrome (STSS)-causing Streptococcus suis serotype 2 (S. suis 2, SS2). An isogenic srtA mutant (DeltasrtA) of 05ZYH33 strain was obtained by homologous recombination. Immunofluorescence analysis revealed that two known virulence-associated surface proteins featuring Leu-Pro-X-Thr-Gly motif, muramidase-released protein and surface antigen one, were absent in the DeltasrtA. Piglet infection experiments showed that deletion of srtA attenuated the full virulence of 05ZYH33 strain, and impaired its colonizing potential in specific organs. Furthermore, the DeltasrtA displayed significant reduction in adherence to human cells (Hep-2 and human umbilical vein endothelial cells). Collectively, we concluded that SrtA was involved in the virulence manifestation of STSS-causing SS2.

Identification and experimental verification of protective antigens against Streptococcus suis serotype 2 based on genome sequence analysis.

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that can cause severe disease and even death in both humans and swine. No effective vaccine is clinically available. In this study, a reverse vaccinology method was first applied to identify protective antigens against S. suis 2. As a consequence, 153 genes encoding vaccine candidates were selected from the whole genome sequence by means of bioinformatics analysis, from which 10 genes were selected based on experimental evidences arising from the study of related bacteria such as Streptococcus pneumoniae, group B streptococcus, S. suis and so on. Of 10 target genes, 8 were successfully expressed in Escherichia coli Rosetta, and expressed proteins were purified and used as the immunogens for evaluating vaccine efficacy in a mouse infection model. The results have confirmed that RTX family exoprotein A (RfeA), epidermal surface antigen (ESA), immunoglobulin G (IgG)-binding protein (IBP), and suilysin (SLY) can induce a protective response of the vaccinated animals against S. suis 2, whereas RfeA, ESA, and IBP mainly induce humoral-mediated immunity, and SLY elicits a combined pattern of both humoral- and cellular-mediated immunity. Although immunoprotection of SLY against S. suis 2 was reported previously, RfeA, ESA, and IBP were explored first in this study.

Inactivation of dipeptidyl peptidase IV attenuates the virulence of Streptococcus suis serotype 2 that causes streptococcal toxic shock syndrome.

Di-peptidyl peptidase IV (DPP IV), originally recognized as CD26 in eukaryotic cells, is distributed widely in microbial pathogens, including Streptococcus suis (S. suis), an emerging zoonotic agent. However, the role of DPP IV in S. suis virulence remains unclear. Here, we identified a dpp IV homologue from highly invasive isolate of S. suis 2 (SS2) causing streptococcal toxic shock syndrome (STSS). Enzymatic assays reproduced its enzymatic activity of dpp IV protein product as a functional DPP IV, and ELISA analysis demonstrated that SS2 DPP IV can interact with human fibronectin. An isogenic SS2 mutant of dpp IV, Delta dpp IV, was obtained by homologous recombination. Experimental animal infection suggested that an inactivation of dpp IV attenuates greatly its high virulence of Chinese virulent strains of SS2. Functional complementation can restore this defect in SS2 pathogenicity. To our knowledge, it may confirm, for the first time, that DPP IV contributes to SS2 virulence.

[Role of Luxs gene on regulation of virulence factors in Salmonella typhimurium].

OBJECTIVE: To investigate the role of Luxs gene on the regulation of virulence factors in Salmonella typhimurium. METHODS: Luxs gene knock-out strain of Salmonella typhimurium of the line 14028S was constructed. Media with Luxs gene knock-out 14028S or wild type 14028S bacteria were added into the media of cultured human intestinal epithelial cells of the line Caco. The numbers of colony formation unit (cfu) were calculated so as to compare the invasion ability. Northern blotting was performed to examine the Luxs gene expression. RESULTS: When the 14028S cells were in the early logarithmic phase the cfu number was 590, and in the middle and late logarithmic, and stationary phases, the cfu numbers were 246, 57, and 13 respectively. The Luxs gene expression levels increased gradually along with the bacterial growth into different phases. The cfu number A600 nm = 0.25 of the Luxs gene knock-out strain was 597, significantly higher than that of the wild type (315, P < 0.05). CONCLUSION: Luxs gene may be a negative regulator on the invasion ability of Salmonella typhimurium.

Streptococcus suis DivIVA Protein Is a Substrate of Ser/Thr Kinase STK and Involved in Cell Division Regulation.

Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.

Preparation of recombinant antigen of O. tsutsugamushi Ptan strain and development of rapid diagnostic reagent for scrub typhus.

Spring scrub typhus has frequently occurred in Pingtan Island, China, since 2000. In this study, we amplified a 1352-bp DNA fragment encoding a truncated 56-kDa outer membrane protein of the Ptan strain, which was isolated from a serum sample of a patient with spring scrub typhus, and cloned it into the pET28a vector for expression. The expression product was a recombinant polypeptide containing a His-tag to facilitate purification on a Ni2+ chromatography column. The recombinant protein was further identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) and appeared to be a good diagnostic antigen candidate. A rapid colloidal gold immunochromatographic assay (CIA) for detecting serum total antibodies, IgG and IgM, which are anti-Orientia tsutsugamushi, was developed, using a mixture of the r56 of the Gilliam and Ptan strains as the diagnostic antigen. CIA performance was tested on a panel of 112 control sera from confirmed cases of scrub typhus. The detection sensitivities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.2%, 81.2%, and 94.6%, respectively, while that of IFA (using the lysate of the O. tsutsugamushi Gilliam-infected chicken yolk sac as the antigen) against IgG was 85.7%. One hundred five serum samples from healthy individuals and patients with other febrile diseases were tested with CIA as negative controls. Specificities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.1%, 100%, and 98.9%, respectively, while the specificity of IFA against IgG was 98.9%. These results indicated that CIA was a good assay and could substitute for conventional immunofluorescence assays for diagnosis of scrub typhus.