Declining prevalence of drug-resistant tuberculosis among HIV/tuberculosis co-infected patients receiving antiretroviral therapy.

BACKGROUND: Drug-resistant tuberculosis (DR-TB) is a serious threat in developing countries where the prevalence of both HIV and TB are high. Antiretroviral therapy (ART) has been more accessible in these countries. The present study aimed to determine the impact of ART on the prevalence of DR-TB among HIV/TB co-infected patients. MATERIAL AND METHOD: A retrospective cohort study was conducted among HIV-infected patients with culture-proved TB from 1999 to 2004. Susceptibilities of Mycobacterium tuberculosis to antituberculous drugs and rate ofART use were studied. RESULTS: There were 225 patients, mean age 35.8 years, 72.4% male and median CD, 44 cells/mm(3). Patients who had received ART increased from 18.5% in 1999 to 92.1% in 2004 (p<O. 001). The prevalence of DR-TB in the years 1999 and 2004 were 48% and 7.9%, respectively (p<O.001). The prevalence of isoniazid- and rifampicin-resistance significantly declined in 2004 when compared with those in 1999 (p<O. 05). CONCLUSION: The declines in the prevalence of DR-TB, INH- and RFP-resistance in HIV/TB co-infected patients are possibly attributable to the use of ART In addition to the survival benefit from ART in HIV-infected patients, increasing use of ART among HIV-infected patients may eliminate DR-TB in this population.

Multiple mutations in katG and inhA identified in Thai isoniazid-resistant Mycobacterium tuberculosis isolates.

A total of 29 Thai multi-drug-resistant/isoniazid-resistant Mycobacterium tuberculosis isolates were analyzed for mutations in katG from codons 254 to 549, inhA promoter and inhA open reading frame by DNA sequencing and single strand conformation polymorphism. Twenty-five multi-drug resistant isolates exhibited single point mutations (17 isolates at Ser315Thr plus Arg463Leu, 1 at Thr308Pro plus Arg463Leu, 7 at either Ser315Thr or Arg463Leu) while the other 4 isoniazid-resistant isolates had single point mutation only at Arg463Leu. Seven of 25 multi-drug-resistant isolates [4 at C(-15)T, 1 at T(-8)C; 1 at C(-15)T plus Ser94Ala and 1 at Ile21Val] and 2 of 4 isoniazid-resistant isolates [1 at C(-15)T, 1 at C (-15)T plus Ile21Thr] had mutations in inhA promoter and open reading frame, while the other 20 isolates had no mutation at any position. No frame shift mutation was observed in any tested isolates. This is the first report of two mutations, Trp308Pro of katG and T (-8)C of inhA in Mycobacterium tuberculosis isolates.

Antifungal susceptibilities of Cryptococcus neoformans cerebrospinal fluid isolates and clinical outcomes of cryptococcal meningitis in HIV-infected patients with/without fluconazole prophylaxis.

OBJECTIVES: To compare the MICs of FLUconazole (FLU) and amphotericin B against isolates of Cryptococcus neoformans (C. neoformans) obtained from the CerebroSpinal Fluid (CSF); and clinical outcomes of HIV-infected patients diagnosed with cryptococcal meningitis. MATERIAL AND METHOD: There were two groups including those who did not receive FLU (group A) and those who did receive either FLU 400 mg/week for primary prophylaxis cryptococosis or 200 mg/day for secondary prophylaxis cryptococosis (group B). CSF isolates of C. neoformans from group A and group B between January 2003 and October 2004 were retrospectively studied. The MICs were determined by using the standard NCCLS broth microdilution methods (M27-A). The MICs of FLU and amphotericin B, and clinical outcomes after 10 weeks of cryptococcal meningitis treatment were determined. RESULTS: There were 98 isolates; 80 in group A and 18 in group B. The patients in group B had a higher proportion of previous opportunistic infections (p = 0.008). The other baseline characteristics between the two groups were not different. The median (range) MIC of FLU was 8.0 (0.5-32) microg/ml in group A, and 6.0 (0.5-32) microg/ml in group B (p = 0.926). The median (range) MIC of amphotericin B was 0.25 (0.03-1.0) microg/ml in group A, and 0.25 (0.12-1.0) microg/ml in group B (p = 0.384). Sixty patients from group A and 14 from group B received standard treatment and continued to follow-up. After the 10-week treatment, 39/60 (65%) patients in group A and 7/14 (50%) in group B had complete recovery (p = 0.364; RR = 0.538, 95%CI = 0.166-1.742). The overall mortality rate was 14/60 (23.3%) in group A and 7/14 (50.0%) in group B (p = 0.096; RR = 3.286, 95%CI = 0.983-10.979). CONCLUSION: The MICs of FLU and amphotericin B against CSF isolates of C. neoformans and clinical outcomes between HIV-infected patients who receive or did not receive FLU prophylaxis are not different.

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.

A prevalence of Cryptosporidium infections among Thai HIV-infected patients.

The prevalence of Cryptosporidium in 156 HIV-infected Thai patients who had acute diarrheal illness at Bamrasnaradura Infectious Diseases Hospital, was studied. This cross-sectional study was performed from March to August in year 2001. The patients ranged in age from 1 month-65 years old. A stool sample from each subject was stained to find the oocysts by modified Ziehl Nelson carbolfuchsin staining. According to the present study, a diagnosis of Cryptosporidium parvum infection was found in 20 patients (11 males and 9 females). The prevalence of cryptosporidiosis in the present series was 12.8 per cent (10.0% in males and 19.1% in females). This infection rate between males and females was not significantly different. Comparing this prevalence to a report in the previous 5 years in the same hospital, the same high rate can be seen.

PCR detection and prevalence of enterotoxin (cpe) gene in Clostridium perfringens isolated from diarrhea patients.

Clostridium perfringens isolated from patients with diarrhea (n=233) were analysed by a duplex PCR assay, in order to determine the prevalence of enterotoxin (cpe) gene and various factors involved in patients with cpe-positive isolates. This duplex PCR uses two sets of primers which amplify in the same reaction two different gene fragments: the phospholipase C (plc, alpha-toxin) and the enterotoxin (cpe) genes in C. perfringens. PCR analysis of 477 colonies of fecal spore isolates, from 159 patients who had a spore count > or = 10(3) cfu/g, gave positive plc gene detection in 436 colonies. The results were consistent with those obtained by using the standard method of C. perfringens species identification. 21 of 436 colonies gave positive results for both plc and cpe genes, indicating a prevalence of 4.8 per cent of C. perfringens that carried the cpe gene in cases of diarrhea. The majority of cases with cpe-positive isolates were women over 50 years of age (71.4%). These patients had diarrhea more than 6 times per day (71.4%) with a duration of 1-3 days (100%). Furthermore, 85.7 per cent of cases developed diarrhea after food consumption, 28.6 per cent had high spore counts of more than 106/g in their feces, and 71.4 per cent were co-infected with other enteric pathogens. The spore count should be interpreted with caution because not all isolates of C. perfringens from diarrhea patients with high fecal spore count carried the cpe gene, which encodes a sporulation-associated enterotoxin. CONCLUSION: The duplex PCR assay can thus become a tool for C. perfringens species identification together with the detection of enterotoxin gene. This PCR assay is faster, less expensive and more suitable for large-scale use in epidemiological studies than conventional procedures. The authors recommend this assay to screen for enterotoxigenic C. perfringens isolates from primary fecal spore isolation cultures, particularly in elderly patients with food-borne diarrhea and non-food related diarrhea.