Evaluation of the LightCycler methicillin-resistant Staphylococcus aureus (MRSA) advanced test for detection of MRSA nasal colonization.

Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization is crucial for the prevention and control of MRSA infections in health care settings. The LightCycler MRSA Advanced Test (Roche Diagnostics) is a commercially available real-time PCR assay for direct detection of MRSA nasal colonization by targeting of the staphylococcal cassette chromosome mec (SCCmec)-orfX junction. The diagnostic performance of the assay was compared with that of ChromID MRSA agar (bioMérieux) culture and an in-house duplex real-time PCR assay. Among 1,246 nasal swab specimens collected from 2 general hospitals in Hong Kong, 174 (14%) were considered true positive for MRSA. Chromogenic culture and the in-house real-time PCR assay identified 147 (84.5%) and 133 (76.4%) true-positive cases with specificities of 100% and 98.6%, respectively. Based on the target melting temperature (Tm) values (57.0 to 62.0 °C) defined by the manufacturer, the LightCycler MRSA Advanced Test identified only 85 (48.9%) true-positive specimens. Interestingly, an additional 60 (34.5%) true-positive specimens were detected despite atypical Tm values of 55 °C, providing overall sensitivity and specificity values of 83.3% and 99%, respectively. Among isolates with Tm values of 55 °C, most were typed as clonal complex 45 (CC45). By sequence analysis of the SCCmec-orfX junction, characteristic single-nucleotide polymorphisms (SNPs) were identified only in isolates with Tm values of 55°C and not in those with typical Tm values. It is conceivable that those SNPs were located inside the target region of the proprietary hybridization probes, which resulted in a Tm shift in the melting curve analysis. Our study highlights the importance of a global evaluation of commercial kits so that the interpretation algorithm covers different lineages of MRSA clones prevalent in various geographical regions.

Complete sequencing of the FII plasmid pHK01, encoding CTX-M-14, and molecular analysis of its variants among Escherichia coli from Hong Kong.

OBJECTIVES: We characterized plasmids encoding CTX-M-14 ß-lactamase originating from Escherichia coli isolates recovered from patients with uncomplicated cystitis or individuals with faecal colonization in Hong Kong from 2002 to 2004. METHODS: Plasmids carrying CTX-M-14 were studied by conjugation, replicon typing, S1 nuclease-PFGE and plasmid PCR-restriction fragment length polymorphism (RFLP). The complete sequence of pHK01, a 70 kb plasmid encoding CTX-M-14 from an E. coli strain, was determined and the results compared with reference plasmids and aligned with GenBank data. RESULTS: The blaCTX-M-14 plasmids could be transferred in 23 of 44 E. coli strains tested. Among the 23 transconjugants, the replicon types of the CTX-M-14-encoding plasmid were FII (n=13), I1-Iγ (n=4), F1B (n=2), FII and I1-Iγ (n=1), K (80 kb, n=1) and undetermined (n=2). Plasmid pHK01 (FII replicon) shares a high degree of homology with R100 except mainly for a 11 kb variable region containing blaCTX-M-14 (with an upstream ISEcp1 and a downstream truncated IS903), an iron transport system, an outer membrane protein (malB, maltoporin) and a putative toxin-antitoxin plasmid stability system (yacABC). It was highly related to blaCTX-M-14 (pKF3-70) and blaCTX-M-24 (pEG356) plasmids reported from mainland China in 2006 and Vietnam in 2007, respectively. Subtyping by a plasmid PCR-RFLP scheme showed that 10 of the 13 FII plasmids originating from isolates collected by multiple laboratories exhibited either identical or highly similar profiles. CONCLUSIONS: This study showed that narrow host-range FII plasmids play important roles in the dissemination of CTX-M-14. FII plasmids closely related to pHK01 have disseminated widely in the Hong Kong community.

Severe community-acquired pneumonia caused by macrolide-resistant Mycoplasma pneumoniae in a 6-year-old boy.

Mycoplasma pneumoniae is the commonest agent causing atypical pneumonia in children. Macrolides have long been used in the treatment of community-acquired pneumonia not responsive to beta-lactams alone. In this report, we describe the first locally acquired paediatric patient with severe community-acquired pneumonia caused by macrolide-resistant Mycoplasma pneumoniae, possessing an A-to-G transition at position 2063 of the 23s rRNA gene. In addition, we have detected two more strains of macrolide-resistant Mycoplasma pneumoniae out of a total of 10 cases with chest infection that were confirmed by polymerase chain reaction. Therefore macrolide-resistant Mycoplasma pneumoniae accounted for 33% (3 out of 10 patients) of the polymerase chain reaction-confirmed cases.

Outbreak of intestinal infection due to Rhizopus microsporus.

Sinopulmonary and rhinocerebral zygomycosis has been increasingly found in patients with hematological malignancies and bone marrow transplantation, but intestinal zygomycosis remains very rare in the literature. We investigated an outbreak of intestinal infection due to Rhizopus microsporus in 12 patients on treatment for hematological malignancies over a period of 6 months in a teaching hospital. The intake of allopurinol during hospitalization (P < 0.001) and that of commercially packaged ready-to-eat food items in the preceding 2 weeks (P < 0.001) were found to be independently significant risk factors for the development of intestinal zygomycosis. A total of 709 specimens, including 378 environmental and air samples, 181 food samples, and 150 drug samples, were taken for fungal culture. Among them, 16 samples of allopurinol tablets, 3 prepackaged ready-to-eat food items, and 1 pair of wooden chopsticks were positive for Rhizopus microsporus, which was confirmed by ITS1-5.8S-ITS2 rRNA gene cluster (internal transcribed spacer [ITS]) sequencing. The mean viable fungal counts of allopurinol obtained from wards and pharmacy were 4.22 x 10(3) CFU/g of tablet (range, 3.07 x 10(3) to 5.48 x 10(3)) and 3.24 x 10(3) CFU/g of tablet (range, 2.68 x 10(3) to 3.72 x 10(3)), respectively, which were much higher than the mean count of 2 x 10(2) CFU/g of food. Phylogenetic analysis by ITS sequencing showed multiple clones from isolates of contaminated allopurinol tablets and ready-to-eat food, of which some were identical to patients' isolates, and with one isolate in the cornstarch used as an excipient for manufacture of this drug. We attempted to type the isolates by random amplification of polymorphic DNA analysis, with limited evidence of clonal distribution. The primary source of the contaminating fungus was likely to be the cornstarch used in the manufacturing of allopurinol tablets or ready-to-eat food. Rhizopus microsporus is thermotolerant and can multiply even at 50 degrees C. The long holding time of the intermediates during the manufacturing process of allopurinol amplified the fungal load. Microbiological monitoring of drugs manufactured for highly immunosuppressed patients should be considered.

Distribution of integron-associated trimethoprim-sulfamethoxazole resistance determinants among Escherichia coli from humans and food-producing animals.

AIMS: To compare the distribution of integrons and trimethoprim-sulfamethoxazole resistance genes among Escherichia coli isolates from humans and food-producing animals. METHODS AND RESULTS: A collection of 174 multidrug-resistant E. coli isolates obtained from faecal samples of food-producing animals (n = 64) and humans (n = 59), and patients with urinary tract infections (n = 51) in Hong Kong during 2002-2004 were studied. The strains were analysed for their phylogenetic groups, the presence of sul genes (sul1 and sul2), integrons (intl1 and intl2) and class 1 integron-associated dfr cassette genes by PCR, restriction enzyme analysis and sequencing. Integrons were identified in 110 (63.2%) isolates. The prevalence of integrons was significantly different according to the specimen sources (animal faecal 84.4%, human faecal 67.8% and human urinary 31.4%) and phylogenetic groups (B1 80.8%, A 77.6%, D 54.1% and B2 11.5%). Faecal isolates (both human and animal) are more likely to belong to group A and B1. In contrast, most urinary isolates were either groups B2 and D. Among dfr containing isolates, dfrA1 and dfrA12 were almost exclusively found in strains of phylogenetic groups A and B1; and were present in animal and human faecal isolates. In contrast, dfrA17 was found in both faecal and urinary isolates and comprised strains from all phylogenetic groups. The sul1 and sul2 genes were equally prevalent among the isolates irrespective of the specimen source and phylogenetic group status. Pulsed-field gel electrophoresis analysis of isolates with identical cassette genes showed that they were genetically diverse. CONCLUSIONS: More animal faecal isolates carry class 1 integrons than human faecal and human urinary isolates, and the distribution of phylogenetic groups is common across animal and human faecal isolates but different from human urinary isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Commensal isolates from food-producing animals are an important reservoir for integrons carrying antibiotic resistance genes.

Molecular epidemiology of community-associated methicillin-resistant Staphylococcus aureus.

1. Community-associated methicillin-resistant Staphylococcus aureus (MRSA) strains with diverse genetic backgrounds are emerging in Hong Kong. 2. Intra-familial spread of community-associated MRSA is common.

Oral bacterial flora of the Chinese cobra (Naja atra) and bamboo pit viper (Trimeresurus albolabris) in Hong Kong SAR, China.

OBJECTIVE: To determine the oral bacterial flora associated with two common local venomous snakes in Hong Kong, namely the Chinese cobra (Naja atra) and the bamboo pit viper (Trimeresurus albolabris). DESIGN: Cross-sectional study. SETTING: A non-government organisation and a regional hospital in Hong Kong. SUBJECTS: Thirty-two Chinese cobras and seven bamboo pit vipers. MAIN OUTCOME MEASURES: Species identification of bacteria in the oral cavity of both snakes and their antibiotic susceptibilities. RESULTS: The oral cavity of Chinese cobra harbour a wide range of pathogenic bacteria, including: Gram-negative bacterial species like Morganella morganii, Aeromonas hydrophila and Proteus, and Gram-positive bacteria like Enterococcus faecalis, coagulase-negative Staphylococcus as well as anaerobic species (clostridia). The oral cavity of the Chinese cobra is more likely than that of the bamboo pit viper to harbour pathogenic bacteria associated with snakebite infection (P<0.001). The median number of pathogenic bacteria per snake was significantly higher in the Chinese cobra (P<0.001). All pathogenic Gram-negative bacteria isolated were susceptible to levofloxacin. Amoxicillin/clavulanate provided good coverage against pathogenic Gram-positive bacteria (Enterococcus faecalis) and anaerobes. CONCLUSION: 'Prophylactic' antibiotic treatment for Chinese cobra bites may be beneficial, owing to the multiple pathogenic bacteria in its oral cavity and the higher risk of ensuing necrosis. The regimen of levofloxacin plus amoxicillin/clavulanate appears promising for this purpose, but further study is required to confirm its clinical utility in patients.

Community-associated methicillin-resistant Staphylococcus aureus skin and soft tissue infections in Hong Kong.

Community-associated methicillin resistant Staphylococcus aureus is an emerging cause of skin and soft tissue infections in Hong Kong, especially among certain ethnic minorities.

Effect of antibiotics on the bacterial load of meticillin-resistant Staphylococcus aureus colonisation in anterior nares.

Prevalence of hospital-acquired meticillin-resistant Staphylococcus aureus (MRSA) infection or colonisation has been associated with antimicrobial consumption. The impact of antibiotic treatment on nasal colonisation is unknown. We conducted a three-month prospective study of 116 patients with extranasal MRSA infection or colonisation, whose nasal MRSA bacterial loads were determined during and after various antibiotic courses over a period of three weeks. Environmental swabs were also taken from the near patient environment. Concomitant nasal MRSA carriage was observed in 76.7% of extranasal MRSA-colonised or -infected patients. The median nasal MRSA bacterial load increased significantly from 2.78 (range 0-6.15) to 5.30 (range 2.90-8.41) log(10) cfu per swab (cfu/swab) (P<0.001) over 21 days during beta-lactam therapy. It also increased from 0 (range 0-4.00) to 4.30 (range 0-7.46) log(10)cfu/swab (P=0.039) over 14 days during fluoroquinolone therapy. Median bacterial loads were significantly higher for beta-lactam- and fluoroquinolone-treated patients on day 7 [4.78, range 0-7.30], day 14 [4.30, range 0-7.60] and day 21 [5.30, range 2.90-8.41] than controls not receiving antibiotics (P<0.05). These loads then decreased by 2-5log(10)cfu/swab 2 weeks after discontinuation of antibiotics. The environment of patients receiving beta-lactam agents (relative risk: 3.55; 95% confidence interval: 1.30-9.62; P=0.018) or fluoroquinolones (4.32; 1.52-12.31; P=0.008) demonstrated more MRSA contamination than the environment around control patients (0.79; 0.67-0.93; P=0.002). Patients on beta-lactam or fluoroquinolone therapy have increased incidence of MRSA colonisation and higher nasal bacterial loads, and appear to spread their MRSA into the near patient environment.

Acute epiglottitis in adults: a retrospective review of 106 patients in Hong Kong.

OBJECTIVES: Acute epiglottitis in adult is a potentially life-threatening condition that may be underdiagnosed. The present study reports the clinical features, management and patient outcomes in an acute hospital in Hong Kong over a seven-year period. METHOD: All adult patients aged 18 years or above admitted to Tuen Mun Hospital between July 1999 and June 2006 with the diagnosis of acute epiglottitis were included in this retrospective study. The diagnosis of acute epiglottitis was established by direct visualisation of inflamed epiglottis during laryngoscopic examination. RESULTS: 106 patients were identified. A total of 21 patients (20%) had co-morbidities, with diabetes mellitus (11%) being the most common. Five patients had a history of nasopharyngeal carcinoma and three patients had a previous history of acute epiglottitis. The majority (94%) of patients presented with sore throat as their major complaint. Blood cultures were collected from 15 patients and all were negative. A combination of cefotaxime and metronidazole was the most common empirical antibiotic regimen prescribed. Seven patients required active airway intervention (six with endotracheal intubation and one failed intubation with emergency tracheostomy performed). No mortality was reported. CONCLUSION: Acute epiglottitis in adults is not a rare entity and vigilance for this condition is needed. In general, the prognosis is good with antimicrobial therapy, close monitoring and selective airway intervention.

Review of dengue fever cases in Hong Kong during 1998 to 2005.

OBJECTIVE: To describe the epidemiology, clinical and laboratory findings, and outcomes of patients presenting locally with dengue. DESIGN: Retrospective review of case records. SETTING: Public hospitals, Hong Kong. PATIENTS: Medical records of all laboratory-confirmed dengue patients admitted to public hospitals during 1998 to 2005 were reviewed retrospectively. RESULTS: A total of 126 cases were identified, 123 (98%) being dengue fever and three (2%) dengue haemorrhagic fever. One patient who had blood transfusion-acquired dengue fever was highlighted. A total of 116 (92%) cases were 'imported', while 10 (8%) were local. Among the 56 dengue cases confirmed by reverse transcription-polymerase chain reaction, dengue virus type 1 was the most common accounting for 48% of them, followed by type 2, type 3, and type 4 responsible for 23%, 16%, and 13%, respectively. Only type 1 and type 2 were present in locally acquired infections. The median age of the patients was 38 years and the mean duration of hospitalisation was 6 days. There was no mortality, and nearly all patients (98%) presented with fever. Other symptoms at presentation included: myalgia (83%), headache (65%), fatigue (59%), and skin rash (60%). More than one third of patients had gastro-intestinal and upper respiratory complaints. Maculopapular skin rash was the most common physical finding. Thrombocytopenia, neutropenia, and lymphopenia were present in 86%, 78%, and 69% of the patients, respectively. In only 29% of the patients was dengue fever included in the initial differential diagnosis. The demographic, clinical, and laboratory findings as well as outcomes did not differ significantly among the four dengue serotypes, but the lowest lymphocyte counts of type 3 was lower than the other serotypes (P=0.004). CONCLUSION: When physicians encounter patients with a relevant travel history, presenting with fever and skin rash, and having compatible haematological findings, dengue fever should be included in the differential diagnosis.

Incidence of neonatal chlamydial conjunctivitis and its association with nasopharyngeal colonisation in a Hong Kong hospital, assessed by polymerase chain reaction.

OBJECTIVES: Using polymerase chain reactions, this study aimed to evaluate the incidence of neonatal chlamydial conjunctivitis in our region of Hong Kong and explore any association between such conjunctivitis and nasopharyngeal colonisation with Chlamydia trachomatis. DESIGN: Prospective epidemiological study. SETTING: Regional hospital, Hong Kong. PATIENTS: Consecutive patients with neonatal conjunctivitis presenting to our hospital were recruited from May 2004 to April 2005 inclusive. Both eyes were investigated separately for Chlamydia trachomatis by polymerase chain reaction, direct immunofluorescent assay, and cell culture by two assigned ophthalmologists. Neonates diagnosed to have ocular Chlamydia trachomatis infection were subjected to additional nasopharyngeal investigations. Complete sets of ocular and nasopharyngeal investigations were also undertaken 1 week after oral erythromycin treatment to confirm complete eradication of Chlamydia trachomatis. RESULTS: Of 192 patients with neonatal conjunctivitis, 24 were diagnosed to have chlamydial conjunctivitis. Fifteen of them had nasopharyngeal colonisation with Chlamydia trachomatis. Among the 20 neonatal chlamydial conjunctivitis patients that completed the follow-up study, one suffered treatment failure. None had clinically diagnosed systemic Chlamydia trachomatis infection 3 months after oral erythromycin. CONCLUSIONS: The incidence of neonatal chlamydial conjunctivitis in our region of Hong Kong was 4 in 1000 live births, of whom 63% had nasopharyngeal presence of Chlamydia trachomatis. Owing to the high rate of nasopharyngeal isolation and possibility of treatment failure, post-treatment ocular and nasopharyngeal polymerase chain reaction testing for Chlamydia trachomatis may be considered to confirm complete eradication.

Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures.

Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available identification systems also evaluated, its database needs to be expanded for accurate identification of anaerobic Gram positive bacilli.

Globicatella bacteraemia identified by 16S ribosomal RNA gene sequencing.

BACKGROUND: Globicatella are streptococcus-like organisms that have been rarely isolated from clinical specimens. Their epidemiology and clinical significance remain largely unknown. AIMS: To describe two cases of Globicatella bacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing. METHODS: Two unidentified streptococcus-like bacteria isolated from blood cultures of patients were subject to 16S rRNA gene sequencing. RESULTS: Two cases of Globicatella bacteraemia were identified by 16S rRNA gene sequencing. In the first case, a gram positive coccus was isolated from the blood culture of an 80 year old woman with diabetes mellitus and nosocomial sepsis, who died the day after developing the bacteraemia. The bacterium was unidentified by conventional phenotypic tests, the Vitek (gram positive identification) and the ATB expression (ID32 Strep) systems. In the second case, a similar bacterium was isolated from the blood culture of a 92 year old woman with polymicrobial acute pyelonephritis complicated by septic shock, who subsequently recovered after antibiotic treatment. 16S rRNA gene sequencing of the two isolates showed 0.5% nucleotide difference from that of G. sulfidifaciens and 0.7% nucleotide difference from that of G. sanguinis, indicating that they were Globicatella species. CONCLUSIONS: Because Globicatella is rarely encountered in clinical microbiology laboratories, it may have been overlooked or misidentified in these cases. 16S rRNA gene sequencing is a useful tool to better characterise the epidemiology and clinical significance of Globicatella.

Immuno-prophylaxis of babies borne to hepatitis B carrier mothers.

OBJECTIVES: To examine the efficacy of current hepatitis B immuno-prophylaxis and estimate the prevalence of S-mutant infections among local newborn babies. DESIGN: Prospective study. SETTING: Regional hospital, Hong Kong. PATIENTS: A total of 137 newborn babies delivered between the period of November 2000 and 30 June 2001 inclusive, whose mothers were chronic hepatitis B surface antigen carriers. RESULTS: Of the 121 infants who were followed up for 12 months, three were found to be chronic hepatitis B virus carriers, giving a vertical transmission rate of 2.5%. One (0.8%) was suspected to be infected by the S-mutant. All the three hepatitis B virus carrier babies were born to mothers with hepatitis B e antigen, but none to the eight mothers suspected to have S-mutants. Of 119 (98.3%) infants who developed hepatitis B surface antibody upon follow-up at 12 months, 35 were found to have hepatitis B e antigen at birth. All were born to hepatitis B e antigen-positive mothers. Only three of the 35 babies were found to be hepatitis B virus carriers. Most babies lost the hepatitis B e antigen by 6 months of age; only the infected babies had the antigen persisting at 1 year of age. The non-infected infants' hepatitis B e antigen is likely transplacental. CONCLUSIONS: Our hepatitis B virus prophylaxis programme was effective at preventing perinatal infection and the non-infected infants' hepatitis B e antigen was likely transplacental.

A case of Streptococcus mitis endocarditis successfully treated by linezolid.

We report the successful treatment of infective endocarditis caused by Streptococcus mitis with linezolid in a patient with pre-existing valvular heart disease. The patient had multiple allergies to conventional antibiotics. Linezolid may provide an oral alternative in the treatment of infective endocarditis in patients with adverse drug reactions to traditional antibiotic regimens.

Strongyloidiasis as a possible cause of nephrotic syndrome.

Chronic strongyloidiasis is a mild disease and has never been reported to be associated with nephrotic syndrome. Disseminated strongyloidiasis is known to have high mortality, but it frequently is not diagnosed until autopsy. We report a patient with nephrotic syndrome developing disseminated strongyloidiasis after steroid therapy. The findings in renal biopsy, the time course of the development, and resolution of nephrotic syndrome after thiabendazole treatment suggested a possible causal relationship between chronic strongyloidiasis and nephrotic syndrome. The case also demonstrated the importance of early diagnosis in disseminated strongyloidiasis and the good clinical outcome of early treatment before the development of organ failure.

Evaluation of five commercial serological tests for the detection of Helicobacter pylori infection in Chinese.

BACKGROUND: Commercial serological tests for the detection of Helicobacter pylori infection must be locally validated. We evaluated the accuracy of five commercial tests in the Chinese population. METHODS: Serum samples were collected from patients referred for upper endoscopy. Antral biopsies were taken for histological examination and culture of H. pylori. The gold standard for diagnosing H. pylori infection was positive histological staining and/or positive H. pylori culture. The serum samples were tested for H. pylori antibodies using the following tests: (i) Cobas Core Anti-H. pylori EIA; (ii) GAP IgG; (iii) GAP IgM; (iv) H. pylori microwell EIA (Quidel); and (v) Premier H. pylori. The sensitivity, specificity and accuracy of each test was calculated according to the manufacturers' instructions or according to a new cut-off value. RESULTS: A total of 158 patients were recruited amongst whom 114 (72%) were H. pylori-positive. Indeterminate results varied from 7% to 19%. The accuracy of the tests varied from 57% to 85%. By using new cut-off values, the accuracy was much improved, ranging from 73.4% to 86.7%. CONCLUSIONS: By defining new cut-off values for the Chinese population, we were able to improve the performance of some of the serology tests. This illustrates the importance of local validation.

Efficacy of enzyme immunoassay for the detection of Helicobacter pylori antigens in frozen stool specimens: local validation.

AIM: To investigate the efficacy of measurement of Helicobacter pylori stool antigen (HpSA) using stored frozen stool specimens, and to assess whether there were factors affecting efficacy in Hong Kong. METHODS: Patients undergoing upper endoscopy at Tuen Mun Hospital were recruited. Stool samples were saved for HpSA testing and questionnaires were completed. Stool samples were frozen immediately upon receipt and stored at -70 degrees C until tested. HpSA results were compared with rapid urease test and histology. RESULTS: One hundred and eighty-one patients were recruited. One hundred and seventy-eight patients were suitable for analysis. Eighty-three were H. pylori positive and 95 were H. pylori negative. The mean duration of storage of the stool samples was 120 days (range, 40-225 days). The sensitivity, specificity and positive and negative predictive values were 84.3%, 97.9%, 97.2% and 88.6%, respectively. In patients with a false negative HpSA test, there was a significant delay in collecting the stool specimen after endoscopy when compared with those with a true positive HpSA test (4.2 vs. 2.3 days; P < 0.05). However, the duration of storage of the specimens was not longer, and consumption of coffee or tea and smoking habits were similar. CONCLUSIONS: HpSA testing showed good sensitivity and specificity, even with frozen stool samples stored for up to 225 days. The efficacy was not affected by coffee, tea or smoking.