Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

CTX-M extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae in a Thai university hospital.

This study presents updates on molecular epidemiology of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Enterobacteriaceae from Srinagarind Hospital, Khon Kaen University, Thailand. All isolates were screened for the presence of ESBL genes, bla(TEM), bla(SHV), bla(VEB) and bla(CTX-M), using PCR followed by nucleotide sequence determination. The results revealed that beta-lactamase genes among 48 isolates collected between 1998 and 1999 were bla(SHV) (79%), bla(CTX-M-9) (52%), bla(TEM-1) (48%) and bla(VEB) (33%), whereas those found in 52 isolates collected in 2003 were bla(TEM-1) (79%), bla(CTX-M-15) (44%), bla(SHV) (36%), bla(VEB) (36%), bla(CTX -M-14) (11%) and bla(CTX-M-9) (10%). In addition, 45 isolates carried at least two different ESBL genes. Using PCR, part of insertion sequence ISEcpl was found in the upstream regions of bla(CTX-M-14) and bla(CTX-M-15). ERIC-PCR analysis revealed that most ESBL-producing isolates were of different strains. This is the first report of CTX-M-9, CTX-M-14 and CTX-M-15 beta-lactamase genes in Enterobacteriaceae in Thailand.

Multi-drug resistant HIV-1 reverse transcriptase genotype in children treated with dual nucleoside reverse transcriptase inhibitors (NRTIs).

BACKGROUND: Multi-drug resistant HIV mutants have been reported after prolonged dual antiretroviral therapy. OBJECTIVE: To evaluate the prevalence and resistance pattern in HIV-infected children treated with dual NRTIs. MATERIAL AND METHOD: Records of HIV-infected children treated with dual NRTIs at Srinagarind Hospital, Khon Kaen University, Thailand, were reviewed for baseline data and their consensually-stored plasma were checked for the occurrence of HIV mutants by genotyping. RESULTS: Fifty-seven HIV-infected children were treated with dual NRTI regimens (27 males; 30 females). The median age and median CD4+ T-lymphocyte at genotypic testing were 83.5 months and 10.9%, respectively. The median duration of ARV therapy was 22 months. More than half the children (42) were on zidovudine and didanosine. A set of three or more nucleoside analog mutations (NAMs), conferring multi-dideoxynucleoside resistance, was found in 60% of the cases. CONCLUSION: High percentages of NAMs were found in HIV-infected children previously on dual ARV therapy for long periods. Genotypic testing was helpful in designing the second antiretroviral regimen.

Therapeutic potential of chloroquine added to zidovudine plus didanosine for HIV-1 infected children.

OBJECTIVE: To evaluate the efficacy and safety of CHQ in a combination treatment with ZDV/ddI in HIV-1-infected children. MATERIAL AND METHOD: Fifty five HIV-infected children were randomly enrolled into 3 treatment groups: (I) ZDV + ddI (n = 25); and (II) ZDV + ddI + CHQ (n = 21); and (III) ZDV + ddI experienced children were non-randomly added CHQ (n = 9). Weight, CD4+ T-lymphocytes and plasma HIV-RNA were measured at weeks 0, 8 and 24. RESULTS: Fifteen, 16 and 8 children from Groups I, II and III were evaluated. No significant improvement in the mean Z-score for weight in groups I and II, but a decrease occurred in group III after 6 months of therapy. In group I, II and III, the respective change in the mean CD4+ T-lymphocyte percentage was +6.7, +4.0 and -0.6. The decrease in the plasma HIV-RNA log was 0.9, 1.1 and 0.7, respectively. There was a trend for more nausea/vomiting in group II/III and more opportunistic infections in group III. CONCLUSION: 1. The addition of chloroquine in ZDV/ddI regimen provided no significant improvement in clinical, immunological and virological parameters. 2. Chloroquine induced immunosuppression and nausea complicated its use.

Etiologies and treatment outcomes for out-patients with community-acquired pneumonia (CAP) at Srinagarind Hospital, Khon Kaen, Thailand.

Most patients with community-acquired pneumonia are treated as out-patients with empirical therapy, since initially the etiologic agent is unknown. We prospectively assessed the etiologies and treatment outcomes of pneumonia from February 2003 to 2004 at ambulatory clinics. Forty-four patients were included with a mean age of 49.2 (SD 18.2) years. The male to female ratio was 1:1.4. The incubation period was 6.9 (SD 4.4) days. Half of the patients were healthy. Asthma and COPD were common in patients with underlying diseases. The etiologic diagnosis was determined by a sputum culture and a serology test of paired serum samples. Hemo-culture produced no growth in any patients. Atypical pathogens and H. influenzae were the most common finding, each occurring in 31.8% of the patients followed by S. pneumoniae and H. parainfluenzae (27.3% each). Twenty-two patients were infected with multiple pathogens. C. pneumoniae was the most common co-infecting pathogen. Two of 12 S. pneumoniae isolates were penicillin resistant. Nine of 14 H. influenzae isolates were cotrimoxazole resistant and 8 of 14 were not sensitive to erythromycin. For H. parainfluenzae, 11 of 12 isolates were not sensitive to erythromycin, and 7 of 12 were not sensitive to cotrimoxazole. Oral antibiotics were prescribed as out-patient treatment. Forty patients (90.9%) improved, with symptoms-score improvement averaging 6.4 days. Four patients got worse and needed a change of antibiotics, the symptoms usually worsen within 3-5 days. We conclude that, antibiotics for CAP out-patients should cover atypical pathogens, H. influenzae, S. pneumoniae and H. parainfluenzae. If the clinical symptoms do not respond after 3-5 days of out-patient treatment, resistance or an unusual organism (eg B. pseudomallei) should be considered.

Prevalence and clinical features of Chlamydia pneumoniae pneumonia at Srinagarind Hospital, Khon Kaen, Thailand.

Between October 2000 and December 2002, a prospective study was conducted among hospitalized community acquired pneumonia (CAP) patients admitted to Srinagarind Hospital, Khon Kaen, Thailand. The diagnosis of Chlamydia pneumoniae infection was based on serologic testing. The prevalence of C. pneumoniae among patients hospitalized with CAP was 8.7%; 24 cases of 276 hospitalized CAP patients. The mean age was 42.7 (range, 17-79) years and the male to female ratio was 1:2.4. More than half (54.2%) of them were without underlying disease. The mean duration of symptoms prior to admission was 5.5 (SD 3.7) days. Leukocytosis was found in 62.5% of patients. Localized patchy alveolar infiltration was the most common radiographic finding, followed by bilateral interstitial infiltration. Over half (52.4%) of the patients had a non-productive cough. Gram-positive diplococci or no organisms predominated in cases where adequate sputum was obtained. Dual infection was found in 45.8% of cases, mostly with Streptococcus spp or Klebsiella pneumoniae. Four patients (16.7%) had an initial clinical presentation of severe CAP; 3 of 4 had a dual infection. Ten patients (41.7%) received macrolides or a macrolide plus a third generation beta-lactam at the beginning of management. Two patients (8.3%) did not improve clinically and were transferred home. The average hospital stay was 11 .5 (range, 1-45) days. Parapneumonic effusions complicated 20.8% of the cases. Other complications included acute respiratory failure (16.7%), shock (8.3%), hospital-acquired pneumonia (8.3%), and acute renal failure (4.2%). We concluded that C. pneumoniae caused a wide variation of clinical presentations ranging from mild disease to severe CAP. Co-infection with other bacterial pathogens was a common finding. Use of macrolides or new fluoroquinolones as part of an initial therapeutic regimen should be considered to cover this organism.

Etiologies and treatment outcomes in patients hospitalized with community-acquired pneumonia (CAP) at Srinagarind Hospital, Khon Kaen, Thailand.

Local epidemiological data on the etiologies of in-patients who are hospitalized with CAP is needed to develop guidelines for clinical practice. This study was conducted to determine the pattern of microorganisms causing community-acquired pneumonia (CAP) in adult patients admitted to Srinagarind Hospital, Khon Kaen, Thailand, between January 2001 and December 2002. Altogether, 254 patients (124 males, 130 females) averaging 56.4 (SD 19.8) years were included. Eighty-six of them (33.8%) presented with severe CAP on initial clinical presentation. The etiologies for the CAP cases were discovered by isolating the organisms from the blood, sputum, pleural fluid, and other sterile sites. Serology for Chlamydia pneunmoniae and Mycoplasma pneumoniae were performed to diagnose current infection. The causative organisms were identified in 145 patients (57.1%). Streptococcus pneumoniae was the commonest pathogen, identified in 11.4% of the cases, followed by Burkholderia pseudomallei (11.0%) and Klebsiella pneumoniae (10.2%). The atypical pathogens, C. pneumoniae and M. pneumoniae, accounted for 8.7% and 3.9% of the isolates, respectively. Sixteen patients (6.3%) had dual infections; C. pneumoniae was the most frequent coinfecting pathogen. The average length of hospital stay was 12.9 (SD 14.0) days, with 27.9% staying more than 2 weeks. Overall, 83.9% of the patients improved with treatment, 10.2% did not improve and 5.9% died. The most common complications were acute respiratory failure (31.1%) and septic shock (20.9%). We conclude that initial antibiotic use should cover the atypical pathogens, C. pneumoniae and M. pneumoniae, in hospitalized CAP patients. B. pseudomallei is an endemic pathogen in Northeast Thailand, and should be considered in cases of severe CAP.

Diagnostic yield of adenosine deaminase in bronchoalveolar lavage.

Adenosine deaminase (ADA) activity rises in various body fluids in patients with tuberculosis. A prospective study was conducted to determine the diagnostic value of ADA activity in bronchoalveolar lavage. Between March 2001 and February 2003, 148 patients were enrolled in our study, mean age 55.6 years (SD 14.6), and a male to female ratio of 2.4:1. The mean duration of symptoms was 66.2 days. All patients were either sputum-smear negative for AFB or failed to produce sputum. The final diagnosis resulted in three patient groups: 43 with pulmonary tuberculosis, 70 malignancy, and 35 miscellaneous causes. The mean ADA activity in the bronchoalveolar lavage for the pulmonary tuberculosis, malignancy, and miscellaneous causes groups was 8.98 (95% CI, 3.79-14.17), 7.63 (95% CI, 4.12-11.14), and 11.61 U/l (95% CI, 3.59-19.62), respectively. No difference was detected in the ADA level in the pulmonary tuberculosis vs other groups (p=0.56, one-way ANOVA). A high level of ADA activity was found in non-tuberculous conditions such as bronchogenic carcinoma, pulmonary hemosiderosis, chronic pneumonia with empyema thoracis and chronic myeloid leukemia. We concluded that ADA activity in the bronchoalveolar lavage was not clearly diagnostic of smear-negative pulmonary tuberculosis. Early diagnosis required histopathology of biopsied transbronchial specimens obtained by fiberoptic bronchoscopy.