Impact of drug-resistant tuberculosis on the survival of HIV-infected patients.

OBJECTIVE: To determine the effect of drug-resistant tuberculosis (TB) on the survival of human immunodeficiency virus (HIV) infected patients in an area with a high prevalence of TB. DESIGN: Retrospective cohort study. RESULTS: Of 225 HIV-TB patients with a mean age of 35.8 years, 72.4% were male. The median CD4 cell count at TB diagnosis was 44 cells/mm3. Sixty per cent presented with extra-pulmonary TB (EPTB). Sixty-three (28%) patients were infected with Mycobacterium tuberculosis resistant to at least one drug; respectively 16.4%, 9.3%, 5.3% and 12.9% were resistant to isoniazid (INH), rifampicin (RMP), ethambutol and streptomycin, and 14 (6.2%) had multidrug-resistant TB (MDR-TB). During a median follow-up of 11.5 months, 4% died. From Kaplan-Meier analysis, INH resistance, RMP resistance and MDR-TB were associated with shorter survival (log-rank test, P < 0.005). Cox's proportional hazard model showed that MDR-TB (hazard ratio [HR] 11.7; 95% CI 2.1-64.9), not receiving antiretroviral therapy (ART) (HR 7.9; 95% CI 1.5-43.1) and EPTB (HR 5.1; 95% CI 1.9-25.9) were significant risk factors for death. CONCLUSION: MDR-TB and EPTB substantially reduce survival among patients co-infected with HIV and TB. Early detection and optimal treatment of MDR-TB are crucial. ART significantly prolongs survival and should be initiated in HIV-TB co-infected patients.     

Mutations in the gyrA and gyrB genes of fluoroquinolone-resistant Mycobacterium tuberculosis from TB patients in Thailand

Among fluoroquinolone-resistant Mycobacterium tuberculosis (FQr-MTB) isolates, mutation at positions 90, 91, and 94 in gyrA gene and at positions 495, 516, and 533 in gyrB gene have been frequently reported. In this study, 35 isolates of FQr-MTB were collected from Siriraj Hospital and Chest Disease Institute. The quinolone-resistance-determining regions (QRDR) of gyrA and gyrB genes in all 35 FQr-MTB isolates and from the H37Ra MTB strain were amplified using polymerase chain reaction (PCR). DNA-sequencing and single-strand conformation polymorphism (SSCP) were further utilized for characterization of the mutations in the QRDR of gyrA and gyrB genes and mutation screening, respectively. From DNA-sequencing, 21 of 35 (60%) exhibited single-point mutations in different positions, at Ala90Val, Ser91Pro, and Asp94(Gly/Ala/His/Asn); and one novel mutation position at Gly88Cys in the gyrA gene and Asp495Asn in the gyrB gene. These positions were previously frequently reported to be responsible for FQr-MTB. The other 14 FQr-MTB isolates (40%) had no mutation. This study is the first report of mutation occurring only in the QRDR of the gyrB gene, without prior mutation in the gyrA QRDR among FQr-MTB isolates. By SSCP analysis for screening of the mutant FQr-MTB, the SSCP patterns of mutated FQr-MTB isolates were clearly differentiated from the SSCP patterns of FQs-MTB.     

Rapid Diagnosis of Cholera Caused by Vibrio cholerae O139

Hybridomas secreting specific monoclonal antibodies (MAbs) to Vibrio cholerae serogroup O139 were produced. Six monoclones (hybridomas) secreting MAbs specific only to lipopolysaccharide of V. cholerae O139 strains and which did not cross-react to 137 strains of other enteric microorganisms were obtained. These clones were designated 12F5-G11, 12F5-G2, 15F5-H5, 5B9-F8, 14C9-D2, and 6D2-D8. The immunoglobulin (Ig) heavy chain isotypes secreted by these clones were IgG2b, IgG2b, IgG2b, IgM, IgG2b, and IgG3, respectively. Clone 12F5-G11 was selected for mass production of MAb, which was used as a detection reagent in the antigen detection assay for diagnosis of cholera caused by V. cholerae O139, and this assay was compared to the conventional bacterial isolation method. Five batches of rectal swab cultures in alkaline-peptone water were collected from 6,497 patients with watery diarrhea. These were 6,310 patients admitted to Bamrasnaradura Infectious Diseases Hospital, 16 patients from Krung Thon Hospital, 78 patients from Bangkok Children’s Hospital, 19 patients from Karen refugee camps, and 74 Indian patients from the National Institute of Cholera and Enteric Diseases, Calcutta, India. The V. cholerae O139 isolations from the rectal swab cultures and the antigen detection assays (i.e., the MAb-based dot-blot ELISA) were performed by different persons of different laboratories, and the results were revealed after all specimens had been tested. Of the 6,497 samples tested, the dot-blot ELISA correctly identified 42 of 42 V. cholerae O139-positive samples and gave a result of positive for three samples which were culture negative for V. cholerae O139. The diagnostic sensitivity, specificity, and efficacy of the dot-blot ELISA were 100, 99.95, and 99.26%, respectively. The ELISA is easy to perform and relatively inexpensive. It can test multiple samples at a single time, does not require special equipment, and does not produce great quantities of contaminated waste. Most of all, it reduces the diagnostic time from at least 2 days for the bacterial isolation to less than 90 min. The assay is recommended as a rapid screening test of cholera cases caused by V. cholerae O139.     

Molecular Typing of Penicillium marneffei Isolates from Thailand by NotI Macrorestriction and Pulsed-Field Gel Electrophoresis

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.     

Comparison of Cryptosporidium parvum development in various cell lines for screening in vitro drug testing

This study describes the development of Cryptosporidium parvum in MDCK, MA-104, Hep-2 and Vero cell lines. Differences in susceptibility, infectivity, and the methodology of excystation were determined. Various solutions were considered to determine the factors which enhanced the excystation (eg with and without sodium hypochlorite, trypsin or sodium taurocholate). It was shown that the sporozoites could be excysted in media either with or without trypsin and sodium taurocholate, but the number of sporozoites in the latter solution was less than the former one. Only oocysts digested by sodium hypochlorite and trypsin can enter the culture cells. Numerous meronts and oocysts were demonstrated and persisted for 9 days. Asexual stages were not observed in MA-104. Only few oocysts could be detected 1-3 days post-inoculation. There was a significant difference between the number of oocysts, which invaded MDCK, MA-104, and Hep-2 cells. MDCK gave the highest susceptibility to oocyst invasion among the three cell lines and asexual stages were also found. Among the 25 isolates, which had been cultivated, 23 isolates could infect MDCK and Hep-2. Only 2 isolates could not infect the MDCK cell. These 2 isolates could infect the Vero cell and yielded high numbers of trophozoites. Praziquantel (PZQ), doxycycline, and paromomycin (PRM) were tested on the infecting parasites. The drugs were added either with the inoculum or 24 hours after inoculation. None of them was effective, including PRM, which had been previously reported as effective.     

Causes of chronic diarrhea in patients with AIDS in Thailand: A prospective clinical and microbiological study

A prospective study was designed to investigate the causes of chronic diarrhea in AIDS patients in Thailand. Forty-five patients from Bamrasnaradura Infectious Diseases Hospital were enrolled. Extensive investigations included multiple stool examinations for ova and parasites, using the stool formalin-ether concentration method, stool culture, stool acid-fast bacilli (AFB) stain, stool modified AFB stain, esophago-gastroduoscopy with duodenal aspirate and biopsy, and colonoscopy with biopsy. Biopsied specimens were examined with H&E, Giemsa, Gram, Periodic acid Schiff, and AFB stains. Definitive causes were found in 29 patients (64.4%). Of these 29, 7 patients were found to habor more than 1 pathogen (15.5%). The most commonly found enteric pathogen wasCryptosporidium parvum (20.0%). Less frequently found pathogens wereMycobacterium tuberculosis (17.8%),Salmonella spp. (15.5%),Cytomegalovirus (11.1%),Mycobacterium avium intracellulare (6.6%), Strongyloides stercoralis (4.4%),Giardia lamblia (4.4%),Cryptococcus neoformans (2.2%),Histoplasma capsulatum (2.2%),Campylobacter jejun (2.2%), andCyclospora cayetanensis (2.2%).Salmonella spp., Mycobacterium tuberculosis, andMycobacterium avium intracellulare infections were shown to be more common in Thailand than in African countries.     

Colonization factors associated with enterotoxigenic Escherichia coli isolated in Thailand.

Eighty-six percent (72 of 84) of heat-labile and heat-stable, none of 141 heat-labile, and 24% (27 of 111) heat-stable enterotoxigenic Escherichia coli isolates from Thailand aggregated in less than 1 M (NH4)2SO4, hemagglutinated human group A and bovine erythrocytes in 1% D-mannose, and possessed either colonization factor I or colonization factor II. No other colonization factors were identified by these two methods.     

Bacterial causes of AIDS-associated diarrhea in Thailand

The incidence of bacterial diarrhea in AIDS patients has increased steadily and has led to enormous medical and public health problems. In this study, the clinical data together with 350 rectal swab samples each from AIDS patients with diarrhea (APD) and non-AIDS patients with diarrhea (NAPD), were collected and examined for bacterial enteropathogens at the Bamrasnaradura Infectious Diseases Hospital (BIDH), Nonthaburi, Thailand from May to December 1996. Patients were matched by age and sex. The majority of these patients were male (79%, 554/700), aged between 15 and 34 years (70.9%). The study found that the isolation rates of bacterial enteropathogens causing diarrhea in APD (18%, 62/350) were considerably lower than those in NAPD (43%, 152/350) (p<0.05). The infection rate with Salmonella group B (19.7%, 12/61) in APD was found to be significantly higher than that in NAPD (14.3%, 2/14) (p<0.05). Vibrio parahaemolyticus (53.3%, 81/152), Plesiomonas shigelloides (27%, 41/152), Aeromonas spp (19.1%, 29/152) and V. cholerae O1 (15.1%, 23/152), were more frequently detected in NAPD than in APD (p<0.05). Only nine Escherichia coli strains were isolated from APD, of which six were enteroinvasive E. coli, two enterotoxigenic E. coli and one enterohemorrhagic E. coli (non O157) possessing both vtl and vt2. No V. cholerae strains were detected in APD. The least effective antibiotics were ampicillin, tetracycline and cotrimoxazole. Antibiotic resistant patterns of the isolated organisms were similar from both groups. The results from this study might be useful in Thailand in the diagnosis and management of clinical cases of bacterial diarrhea, especially APD.     

Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3:K6 and O4:K68) in Thailand and India

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.     

Phenotypic switching and genetic diversity of Cryptococcus neoformans

Niger seed agar was used as a primary plating medium for the isolation of Cryptococcus neoformans from cerebrospinal fluid specimens from AIDS patients with untreated primary cryptococcosis. The medium was used as the primary means to detect variations in the colony morphology of the yeast. To search for phenotypic and genetic variations, nine patients individually harboring two or three types of colony morphology were studied. Intraindividual isolates from nine patients had minor variations in the API 20C profile, and the MICs of one or more antifungal agents (amphotericin B, fluconazole, and itraconazole) for isolates from three patients were significantly different. Intraindividual isolates from three patients had minor karyotype differences, and one showed a dramatic chromosomal length polymorphism. In addition, three serial isolates from a patient with two episodes of infection showed similar karyotypes, confirming persistent infection by the same strain. Random amplified polymorphic DNA products were identical for all isolates (including three isolates from a relapse case). Our results provided evidence suggesting that (i) in humans, C. neoformans may undergo phenotypic and genetic changes during early infection prior to antifungal agent administration; (ii) dramatic variations in electrophoretic karyotypes and in phenotypes, as demonstrated during the early infection of one patient, may be due to infection by different strains; and (iii) the use of niger seed agar as a primary plating medium is useful for studying antifungal susceptibility, phenotypic switching, genetic diversity, and multiple-strain infections.