Intrahost Monkeypox Virus Genome Variation in Patient with Early Infection, Finland, 2022.

Monkeypox virus was imported into Finland during late May-early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3-specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme-mediated evolution or co-infection.

The burden of cytomegalovirus infection remains high in high-risk kidney transplant recipients despite six-month valganciclovir prophylaxis.

Cytomegalovirus continues to be a concern after transplantation despite prophylaxis regimens. Our aim was to analyse post-prophylaxis primary cytomegalovirus infections among kidney transplant recipients after 6-month valganciclovir prophylaxis and to determine the usefulness of surveillance after prophylaxis. Data from all cytomegalovirus D+/R- kidney transplant recipients from January 2004 to October 2018 at our center who received 6-month prophylaxis with valganciclovir were retrospectively analysed (N = 481). Detailed analyses were performed for 136 patients who were monitored every 2-4 weeks for DNAemia after the discontinuation of prophylaxis. Post-prophylaxis primary cytomegalovirus infection occurred in 182/481 (38%) patients median 264 days after transplantation (IQR: 226-367) and median 84 days after the end of prophylaxis (IQR: 46-187). In 49% patients, cytomegalovirus infection occurred over 3 months after the end of prophylaxis. Cytomegalovirus infection was not associated with lower patient or graft survival and no independent risk factors for infection were found. From patients monitored closely, 71/136 (52%) patients developed post-prophylaxis primary cytomegalovirus infection. Altogether, 52/136 (38%) patients were diagnosed with probable post-prophylaxis cytomegalovirus disease and 19/136 (14%) patients had asymptomatic CMV infection. Recurrent infection occurred in 38/71 (39%) patients. The incidence of post-prophylaxis primary cytomegalovirus infection among D+/R- kidney transplant recipients remains high despite 6-month prophylaxis. Surveillance after prophylaxis was challenging as a considerable portion of the infections occurred late and already symptomatic.

Evaluation of commercial and automated SARS-CoV-2 IgG and IgA ELISAs using coronavirus disease (COVID-19) patient samples.

Antibody-screening methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be validated. We evaluated SARS-CoV-2 IgG and IgA ELISAs in conjunction with the EUROLabworkstation (Euroimmun, Lübeck, Germany). Overall specificities were 91.9% and 73.0% for IgG and IgA ELISAs, respectively. Of 39 coronavirus disease patients, 13 were IgG and IgA positive and 11 IgA alone at sampling. IgGs and IgAs were respectively detected at a median of 12 and 11 days after symptom onset.

Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland, January to February 2020.

The first case of coronavirus disease (COVID-19) in Finland was confirmed on 29 January 2020. No secondary cases were detected. We describe the clinical picture and laboratory findings 3-23 days since the first symptoms. The SARS-CoV-2/Finland/1/2020 virus strain was isolated, the genome showing a single nucleotide substitution to the reference strain from Wuhan. Neutralising antibody response appeared within 9 days along with specific IgM and IgG response, targeting particularly nucleocapsid and spike proteins.

Mycoplasma pneumoniae outbreak, Southeastern Finland, 2017-2018: molecular epidemiology and laboratory diagnostic lessons.

This study characterizes a large Mycoplasma pneumoniae outbreak observed in Kymenlaakso in Southeastern Finland during August 2017-January 2018. The first part of the investigation included 327 patients, who sought healthcare consultation at local GPs or hospitals due to clinical symptoms, and were tested for M. pneumoniae antibodies (Patient cohort). The second part of the investigation, conducted approximately 4 weeks after the peak of the outbreak, consisted of school screening of pupils (N = 239) in three different school buildings by PCR on respiratory specimens and questionnaires (Screening cohort). PCR positive respiratory specimens were subsequently utilized for molecular typing. The outbreak peaked in late October 2017. Of the Patient cohort, 9/106 (8.5%) respiratory specimens were PCR positive. In contrast, 3/182 (1.6%) of the Screening cohort were PCR positive. Asymptomatic carriage was observed. Multiple-locus variable-number tandem-repeat analysis (MLVA) identified two distinct MLVA types. All typed M. pneumoniae strains belonged to P1 type 1. No mutations leading to macrolide resistance were observed. In total, 61/327 (19%) of the Patient cohort had a serological indication of recent infection. The IgM test reactivity at the time of a negative PCR test result varied from a completely non-reactive value up to very strong reactivity, highlighting the difficulty in a single specimen serodiagnosis.

BK polyomavirus viremia and antibody responses of pediatric kidney transplant recipients in Finland.

BACKGROUND: BKPyV is an important cause of premature graft failure after KT. Most clinical studies describe BKPyV infection in adult KT patients. We studied the prevalence of post-transplant BKPyV viremia, serology, and graft function in pediatric KT recipients. METHODS: Forty-six pediatric patients transplanted between 2009 and 2014 were followed up for BKPyV DNAemia by plasma PCR for median 2.3 (range: 1-6) years. BKPyV-specific antibodies were retrospectively analyzed using virus-like particle ELISA. GFR was measured annually by 51 Cr-EDTA clearance, and serum samples were screened for DSAs by Luminex assay. RESULTS: BKPyV viremia was demonstrated in nine patients at a median of 6 months post-KT. Early BKPyV viremia at 3 months post-KT associated with decreased concomitant GFR and tendency for decreased subsequent graft function. Three of nine patients with BKPyV viremia developed DSA, all against class II antigens. PyVAN developed to four patients and responded to judicious reduction in IS. One graft was lost later due to ABMR. BKPyV-IgG was found in 18 of 31 patients (58%) tested at transplantation, and seven recipients seroconverted after transplantation with a significant increase in IgG levels with IgM. Finally, BKPyV-IgG was detectable in 31 of 40 patients (78%) at the end of the study. CONCLUSIONS: Post-transplant BKPyV viremia in pediatric KT patients may alter graft function and contribute to progression of chronic allograft injury. BKPyV-IgG predicts past exposure. Low or absent BKPyV-specific antibody levels were seen pretransplant in 42% of tested patients, but were not predictive of prolonged replication or poor outcome.

Single-Molecule Sequencing Revealing the Presence of Distinct JC Polyomavirus Populations in Patients With Progressive Multifocal Leukoencephalopathy.

Background: Progressive multifocal leukoencephalopathy (PML) is a fatal disease caused by reactivation of JC polyomavirus (JCPyV) in immunosuppressed individuals and lytic infection by neurotropic JCPyV in glial cells. The exact content of neurotropic mutations within individual JCPyV strains has not been studied to our knowledge. Methods: We exploited the capacity of single-molecule real-time sequencing technology to determine the sequence of complete JCPyV genomes in single reads. The method was used to precisely characterize individual neurotropic JCPyV strains of 3 patients with PML without the bias caused by assembly of short sequence reads. Results: In the cerebrospinal fluid sample of a 73-year-old woman with rapid PML onset, 3 distinct JCPyV populations could be identified. All viral populations were characterized by rearrangements within the noncoding regulatory region (NCCR) and 1 point mutation, S267L in the VP1 gene, suggestive of neurotropic strains. One patient with PML had a single neurotropic strain with rearranged NCCR, and 1 patient had a single strain with small NCCR alterations. Conclusions: We report here, for the first time, full characterization of individual neurotropic JCPyV strains in the cerebrospinal fluid of patients with PML. It remains to be established whether PML pathogenesis is driven by one or several neurotropic strains in an individual.

Human herpes virus 6 infection in pediatric organ transplant patients.

Transplant patients need lifelong immunosuppressive medication, but this reduces their defense mechanisms, making them prone to viral infections and reactivations. We aimed to clarify the prevalence and clinical manifestations of the human herpes virus 6 (HHV-6) infection in children after pediatric solid organ transplants. Clinical findings and viral loads were compared between primary HHV-6 infections and reactivations. The study comprised 47 kidney, 25 liver, and 12 heart transplant patients who underwent surgery from 2009 to 2014. HHV-6 antibodies were analyzed before surgery, and HHV-6 DNAemia tests were regularly carried out after the transplant using a real-time quantitative polymerase chain reaction method. We found the primary HHV-6 infection in 19 of 22 (86%) seronegative patients, and it was more common in patients under 3 years of age (79%) than over 3 (38%, P=.0002). Post-transplant HHV-6 DNAemia affected 48 of 84 (57%) patients and was significantly higher in primary infections than reactivations (P=.001), and 17 of 48 (35%) patients had symptoms when it was detected at a median of 2 weeks post-transplant. The HHV-6 infection was common after solid organ transplants, especially under 3 years of age, and it typically started 2 weeks after surgery. Testing for HHV-6 DNAemia is recommended shortly after transplantation, especially in patients with fever, diarrhea, rash, seizures, or abnormal liver enzyme tests.

[Rapid diagnosis of diarrhea viruses]. / Ripulivirusten pikadiagnostiikka.

Viral diagnosis is required mainly in the analysis of outbreaks of diarrhea, cases of gastroenteritis in infants and in the exploration of the cause of diarrhea in severely ill patients. Antigens of rotaviruses and adenoviruses can be detected in the feces of the patient, and the rapid tests applied have proven to possess sufficient sensitivity. Sensitivities of the tests intended for norovirus antigen detection have instead remained poor. In addition to antigen detection tests, a real-time PCR test based on the,detection of norovirus nucleic acids has come onto the market, being both easy to use and substantially more sensitive. In the future, multiplex PCR tests allowing simultaneous detection of several different diarrhea-causing microorganisms are expected to become more common.

Comparison of two quantitative real-time CMV-PCR tests calibrated against the 1st WHO international standard for viral load monitoring of renal transplant patients.

Cytomegalovirus (CMV) replication in organ transplant recipients is commonly diagnosed by quantitative PCR methods. However, there has been a poor inter-laboratory correlation of viral load values due to the lack of an international reference standard. In a recent study, the COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) CMV test calibrated to the 1st WHO CMV standard, showed good reproducibility in CMV load values across multiple laboratories. Fifty-seven follow-up plasma specimens from 10 kidney transplant recipients with CMV replication were examined using the new quantitative CAP/CTM CMV test and the "in-house" quantitative CMV real-time PCR method, also calibrated against the 1st WHO CMV standard for their clinical applicability for monitoring CMV load in renal transplant patients. By CAP/CTM CMV test 49/57 specimens were CMV-DNA positive compared to 44/57 by the "in-house" PCR test. The "in-house" PCR and CAP/CTM CMV test correlated well in monitoring individual kidney transplant patients. Conversion of the CMV-DNA copies to IUs made the results of the "in-house" PCR and CAP/CTM CMV test less uniform in analysis of the patient samples. In specimens of one patient, significant underquantification of CMV load with "in-house" PCR emerged during follow-up due to a point mutation in the "in-house" PCR primer sequence. The CAP/CTM CMV test was found suitable for diagnosing and monitoring CMV replication in renal transplant patients. Multicenter studies are needed to provide more information of the commutability of the 1st WHO CMV standard and to define the clinical thresholds.

Differentiation of highly pathogenic strains of human JC polyomavirus in neurological patients by next generation sequencing.

BACKGROUND: JC polyomavirus (JCPyV) persists asymptomatic in more than half of the human population. Immunocompromising conditions may cause reactivation and acquisition of neurotropic rearrangements in the viral genome, especially in the non-coding control region (NCCR). Such rearranged JCPyV strains are strongly associated with the development of progressive multifocal leukoencephalopathy (PML). METHODS: Using next-generation sequencing (NGS) and bioinformatics tools, the NCCR was characterized in cerebrospinal fluid (CSF; N = 21) and brain tissue (N = 16) samples from PML patients (N = 25), urine specimens from systemic lupus erythematosus patients (N = 2), brain tissue samples from control individuals (N = 2) and waste-water samples (N = 5). Quantitative PCR was run in parallel for diagnostic PML samples. RESULTS: Archetype NCCR (i.e. ABCDEF block structure) and archetype-like NCCR harboring minor mutations were detected in two CSF samples and in one CSF sample and in one tissue sample, respectively. Among samples from PML patients, rearranged NCCRs were found in 8 out of 21 CSF samples and in 14 out of 16 brain tissue samples. Complete or partial deletion of the C and D blocks was characteristic of most rearranged JCPyV strains. From ten CSF samples and one tissue sample NCCR could not be amplified. CONCLUSIONS: Rearranged NCCRs are predominant in brain tissue and common in CSF from PML patients. Extremely sensitive detection and identification of neurotropic viral populations in CSF or brain tissue by NGS may contribute to early and accurate diagnosis, timely intervention and improved patient care.

Evaluation of the Novel CE-IVD-Marked Multiplex PCR QIAstat-Dx Meningitis/Encephalitis Panel.

Central nervous system (CNS) infections such as meningitis and encephalitis are life-threatening conditions that demand hospital care and prompt identification of the causative agent. Since 2015, there has been only one CE-IVD-marked rapid multiplexed diagnostic assay in cassette format for bacterial and viral detection from cerebrospinal fluid (CSF): the BioFire FilmArray meningitis/encephalitis (ME) panel. In the beginning of 2022, Qiagen introduced the QIAstat-Dx meningitis/encephalitis panel. It is a CE-IVD-marked multiplex PCR cassette test intended for the identification of suspected infectious meningitis, encephalitis, or meningoencephalitis caused by bacterial, viral, or fungal pathogens. In this study, we evaluated patient and quality control samples using the QIAstat-Dx meningitis/encephalitis panel and compared the results to those of the BioFire FilmArray meningitis/encephalitis panel and reference methods (current routine analysis methods in our laboratory, PCR, or cultivation). The combined positive percent agreement between the two panel assays was 100%, and the negative percent agreement was 94%. We further compared specifically herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) dilution series using six commercial herpesvirus assays, including the two cassette tests. The results suggested that real-time PCR methods (with separate extraction) were the most sensitive methods. When comparing the cassette tests, the BioFire FilmArray meningitis/encephalitis panel produced more positive results than the QIAstat-Dx meningitis/encephalitis panel in the herpesvirus analyses. IMPORTANCE The diagnosis of infectious meningitis and encephalitis relies mostly on specific PCR and culturing methods, but commercial syndromic panel assays are bringing a change in diagnostics. With multiplexed analysis, the identification of the pathogen is potentially faster, and less sample material is needed. The novel QIAstat-Dx meningitis/encephalitis panel assay is intended for the rapid identification of pathogens from cerebrospinal fluid for suspected central nervous system (CNS) infection, which is a life-threatening condition and difficult to diagnose. We studied the performance of this panel assay using patient samples and dilution series of selected viruses. The evaluation data for this novel meningitis/encephalitis panel assay are useful for other clinical laboratories and organizations using or considering using this test.

BK virus viremia in a well-HLA-matched kidney transplant population mainly on low-dose cyclosporine-based immunosuppression.

The incidence and clinical course of polyomavirus-associated nephropathy (PyVAN) in our well-HLA-matched kidney transplant population mainly on low-dose cyclosporine-based triple-drug immunosuppression has not been described in detail. We aimed to characterize our patients with PyVAN and BK virus (BKV) viremia. Among 166 kidney transplantations between January 2007 and February 2011 followed up at Helsinki University Hospital nephrology clinic, 136 were screened for BKV viremia by quantitative analysis of BKV DNA in plasma. PyVAN was diagnosed by biopsy histopathology and SV40 T-antigen detection. BKV viremia or PyVAN were treated by reducing immunosuppression. BKV viremia was detected in 12 (9%) patients. PyVAN was diagnosed in six patients (4%). In the six patients with no PyVAN, four had low-level viremia (<10,000 copies/mL) of short duration (<2 months), one had high-level viremia, and one had sustained low-level viremia. After reduction of immunosuppression, all except one patient were able to clear viremia. No grafts were lost due to PyVAN. Even in a low-risk population, BKV viremia and PyVAN occur, highlighting the importance of monitoring viral loads. Reduction of immunosuppression was successful, and no grafts were lost due to PyVAN.

JC polyomavirus DNA detection in clinical practice.

BACKGROUND: There are limited data about the use and clinical value of JC polyomavirus (JCPyV) DNA detection in various clinical indications. METHODS: We reviewed the clinical records of 410 patients from whom cerebrospinal fluid (CSF), plasma, urine, or tissue samples had been collected for JCPyV DNA polymerase chain reaction (PCR) between 2012 and 2018. RESULTS: JCPyV DNA was analyzed in 224 plasma, 190 CSF-, 32 urine and 10 tissue samples. 240 patients had a history of hematopoietic stem cell or solid organ transplantation, 159 had nephrological disease, 90 had hematologic malignancies, 58 had neurological disease, 37 had infectious disease and 23 had AIDS/HIV as underlying disease. Six patients had no underlying disease. The main reasons to take CSF or plasma samples were neurological symptoms of unknown etiology. Most urine samples were taken to monitor kidney transplantation patients. JCPyV DNA PCR contributed to the diagnosis of progressive multifocal leukoencephalopathy in eight patients (2.0%), of which seven had hematologic malignancy as an underlying disease. CONCLUSIONS: JCPyV PCR is most informative among immunosuppressed patients with neurologic symptoms. CSF and brain biopsy are useful when there is clinical suspicion of PML, whereas plasma samples are not useful. The value of plasma samples is a matter of dispute in the screening of JCPyV-associated nephropathy, as BK polyomavirus is the causative agent in most polyomavirus-associated nephropathy cases. JCPyV detection is valuable in case the patient has past, current or planned treatment with immunosuppressive drugs.

Validation and diagnostic application of NS and HA gene-specific real-time reverse transcription-PCR assays for detection of 2009 pandemic influenza A (H1N1) viruses in clinical specimens.

Real-time reverse transcription-PCR assays specific for the nonstructural (NS) and hemagglutinin (HA) genes of the 2009 pandemic influenza A (H1N1) virus were developed and evaluated with clinical samples from infected patients. The tests are characterized by high sensitivity and specificity and performed well throughout the first year of the 2009 pandemic.

Analysis of Chlamydia pneumoniae infection in mononuclear cells by reverse transcription-PCR targeted to chlamydial gene transcripts.

Chlamydia pneumoniae (C. pneumoniae) is an important etiological agent of respiratory infections including pneumonia. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that monocytes can assist the spread of infection to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells, we have established an in vitro model in the human Mono Mac 6 cell line. In the present study, we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real-time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells. Furthermore, our study shows that genes related to secretion are transcribed, and secreted bacterial proteins are also translated during infection of monocytes, creating novel opportunities for the management of chlamydial infection of monocytes.

Comparison of Two Commercial Platforms and a Laboratory-Developed Test for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA.

Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing.

Real-life clinical sensitivity of SARS-CoV-2 RT-PCR test in symptomatic patients.

BACKGROUND: Understanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has implications for patient management. Our aim was to determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR. METHODS: This population-based retrospective study was conducted in March-April 2020 in the Helsinki Capital Region, Finland. Adults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, with sufficient data in their medical records for grading of clinical suspicion were eligible. In addition to examining the first RT-PCR test of repeat-tested individuals, we also used high clinical suspicion for COVID-19 as the reference standard for calculating the sensitivity of SARS-CoV-2 RT-PCR. RESULTS: All 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women) admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals (mean [SD] age, 45.4 [17.2] years; 69.1% women) was sampled from epidemiological line lists by systematic quasi-random sampling. The sensitivity (95% CI) for laboratory confirmed cases (repeat-tested patients) was 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the sensitivity (95% CI) was: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all. CONCLUSIONS: The clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations, and when using RT-PCR as a reference for other tests.

SARS-CoV-2 sample-to-answer nucleic acid testing in a tertiary care emergency department: evaluation and utility.

BACKGROUND: Rapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. OBJECTIVES: We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert® Xpress SARS-CoV-2 and Mobidiag Novodiag® Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag® test in tertiary care emergency departments was assessed. RESULTS: In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert®, whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag®. Rapid SARS-CoV-2 testing with Novodiag® was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag® and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag® and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag®, but positive with the reference method with late Ct values. On average, a test result using Novodiag® was available nearly 8 hours earlier than that obtained with the large-scale PCR tests. CONCLUSIONS: While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.

Transcriptional Expression of the ompA, cpaf, tarp, and tox Genes of Chlamydia trachomatis Clinical Isolates at Different Stages of the Developmental Cycle.

The transcriptional gene expression patterns of Chlamydia trachomatis have mainly been studied using reference strains propagated in cultured cells. Here, using five low-passage-number C. trachomatis clinical isolates that originated from asymptomatic or symptomatic female patients, the in vitro expression of the ompA, cpaf, tarp, and tox genes was studied with reverse transcriptase real-time PCR during the chlamydial developmental cycle. We observed dissimilarities in the gene expression patterns between the low-passage-number clinical isolates and the reference strains. The expression of ompA and the peak of the tox expression were observed earlier in the reference strains than in most of the clinical isolates. The expression of cpaf was high in the reference strains compared with the clinical isolates at the mid-phase (6-24 hours post infection) of the developmental cycle. All of the strains had a rather similar tarp expression profile. Four out of five clinical isolates exhibited slower growth kinetics compared with the reference strains. The use of low-passage-number C. trachomatis clinical isolates instead of reference strains in the studies might better reflect the situation in human infection.