Evaluation of an in-house immunoperoxidase staining assay for histodiagnosis of human pythiosis.

Pythiosis, a life-threatening infectious disease of humans and animals in tropical and subtropical countries, is caused by the fungus-like organism Pythium insidiosum. As diagnosis of pythiosis is difficult, delayed diagnosis of pythiosis leads to poor prognosis. We developed an immunoperoxidase staining assay using rabbit anti-P. insidiosum antibodies to detect P. insidiosum directly in infected tissues of 19 patients with vascular (n = 11), ocular (n = 7) or cutaneous (n = 1) pythiosis. Tissue sections from 31 patients with various fungal infections were included as controls. Tissue sections from all pythiosis patients and 2 patients with Fusarium infections were stained positive, whereas the other 29 control sections were stained negative. Sensitivity and specificity of the assay was 100% and 94%, respectively. Based on the prevalence of human pythiosis (2%), calculated positive predictive value and negative predictive value was 24% and 100%, respectively. Thus, the diagnostic value of this assay is for ruling out pythiosis. The assay requires routine laboratory equipments and can easily be performed by pathologists in rural hospitals where the disease is more prevalent.

One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis.

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.

Evaluation of polymerase chain reaction and restriction enzyme analysis for routine identification of mycobacteria: accuracy, rapidity, and cost analysis.

Polymerase chain reaction and restriction enzyme analysis (PCR-REA) of the hsp65 gene was evaluated for use as a routine identification method for identifying mycobacteria. The accuracy, rapidity, and cost were assessed compared with the conventional biochemical method. Five hundred and forty-one mycobacterial clinical isolates obtained from the Department of Microbiology, Faculty of Medicine at Siriraj Hospital, Mahidol University, were submitted for PCR-REA and biochemical identification. PCR-REA showed high concordant result with 100, 96.2, and 94.1% for identification of Mycobacterium tuberculosis, rapid- and slow-growing mycobacteria, respectively. Discordant results were obtained from 24 (4.4%) out of 541 isolates, consisting of 9 rapid growers (6 M. chelonae, 2 M. abscessus, and 1 M. fortuitum) and 15 slow growers (9 M. scrofulaceum, 2 M. gordonae, 1 M. avium, 1 M. kansasii, 1 M. malmoense, and 1 M. terrae complex). PCR-REA demonstrated not only accurate results but was also less expensive (2.1 US dollars/sample). This method was rapid with a turn-around time of 30 hours compared with 2-4 weeks for the conventional method.

Eleven-year experience on anti-TB drugs direct susceptibility testing from Siriraj Hospital, Thailand.

The early detection of drug-resistant tuberculosis (TB) strains is of utmost importance for patient management and effective TB control programs. This study aimed to demonstrate the performance of direct drug susceptibility testing (DST) performed in our laboratory in the past 11 years. The direct DST was performed on Middlebrook 7H10 medium using isoniazid (INH) and rifampicin (RIF), and the results were compared with those obtained from indirect DST (gold standard). The direct DST was performed with 15,598 smear-positive sputum samples, of which 11,284 (72%) yielded reportable results. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated and revealed 89%, 99%, 95%, and 99%, respectively, for RIF and 90%, 98%, 93%, and 97%, respectively, for INH. Direct DST results could be reported within 1 month after sample processing. This method was also shown to be suitable for use in resource-limited countries, particularly in settings with high numbers of TB cases.

Hemagglutination test for rapid serodiagnosis of human pythiosis.

Human pythiosis is an emerging, life-threatening infectious disease, caused by the oomycete Pythium insidiosum. Thailand is an area where human pythiosis is endemic and the genetic blood disorder thalassemia is a predisposing factor. Patients with pythiosis present with arterial occlusions of the lower extremities, corneal ulcers, or chronic cutaneous infections. Diagnosis relies on time-consuming, relatively insensitive tests such as culture identification and immunodiffusion assay. Most patients undergo surgical removal of infected organs, and many die from the infection. Delayed diagnosis results in a poor prognosis. Here, we describe a hemagglutination (HA) test for rapid diagnosis of human pythiosis. Sheep red blood cells were coated with P. insidiosum protein extract and used in duplicated detection assays using serum samples from 33 patients with vascular (n = 27), cutaneous (n = 2), or ocular (n = 4) pythiosis and serum samples from 289 control patients with other infectious diseases (n = 77), with highly positive antinuclear antibody (n = 5), with thalassemia (n = 21), or with no known disorder (i.e., healthy blood donors) (n = 186). Based on receiver-operating characteristic analysis, a serum titer of 1:160 was selected as the cutoff point for the HA test. Serum samples that generated HA at the cutoff titer were read as positive, while samples that did not were read as negative. Positive results were obtained with the serum samples of all patients with vascular and cutaneous pythiosis and with two serum samples from the control group. Negative results were obtained with serum samples from all ocular pythiosis patients and the 287 remaining serum samples from the control group. Sensitivity and specificity of the HA were 88% and 99%, respectively. In conclusion, the HA test for detection of anti-Pythium antibodies is a simple, rapid, and reliable test for serodiagnosis of vascular and cutaneous pythiosis.

Transcriptome analyses extend understanding of Streptococcus pyogenes regulatory mechanisms and behavior toward immunomodulatory substances.

The recent genome sequencing of several Streptococcus pyogenes serotype strains allowed the design of corresponding DNA microarrays and their usage for specific transcriptome analyses. In the present study, we employed transcriptomics together with functional tests to investigate the impact of the CiaH sensor gene of the CiaRH two-component regulator on gene expression and virulence traits of serotype M49 S. pyogenes strains CS101 and 591. In parallel, we studied the effects of the immunostimulatory substance Luivac on the serotype M49 S. pyogenes transcriptome and several biological features of serotype M1, M2, M3, M6, M18, and M49 S. pyogenes strains. Overall, the transcriptome analyses allowed a swift identification of differences in transcript abundance apparently associated with the observed strain-specific changes in matrix protein binding, eukaryotic cell interactions, or biofilm formation of the ciaH mutants and of wild-type strains exposed to a commercially available substance used for preventing upper respiratory tract infections.

Distribution of hsp65 PCR-restriction enzyme analysis patterns among Mycobacterium avium complex isolates in Thailand.

A total of 227 clinical Mycobacterium avium complex isolates from Thailand were differentiated into species and types by using PCR-restriction enzyme analysis of hsp65. The distribution of types showed the predominance of M. avium I (77%) in blood specimens, whereas M. intracellulare I was more commonly found in pulmonary specimens (44.2%). In addition, infections with M. avium were more likely to be found in younger adults (20 to 39 years old), while infections with M. intracellulare were more likely to be found in older adults (> or =60 years old). Our results provide the useful epidemiological information that some particular types have more invasive and virulent characters than others.