Cryptococcus gattii serotype-C strains isolated in Bangalore, Karnataka, India.

During a retrospective study on cryptococcosis carried out in Bangalore, Karnataka, India, four Cryptococcus gattii strains were isolated from one HIV-positive and three HIV-negative patients, two of which had unknown predisposing conditions. Serotyping and genotyping showed that the isolates were C. gattii serotype C, mating-type α and genotype VGIV. All the isolates were identical by multilocus sequence typing, but presented a low similarity compared with a set of 17 C. gattii global control strains. The comparison with a larger number of previously reported C. gattii strains, including African isolates, revealed a close relationship between Indian and African serotype-C isolates.

Nerve dysfunction following surgical treatment of cervical non-tuberculous mycobacterial lymphadenitis in children.

AIM: To present our experience of nerve dysfunction following surgical treatment among 126 children with microbiologically verified non-tuberculous mycobacterial (NTM) lymphadenitis. METHODS: We retrieved data from medical records, and a questionnaire with an invitation to a clinical follow-up was returned by 88 families. RESULTS: The time from onset of symptoms to diagnosis was more than 3 months in 24% of subjects. Mycobacterium avium complex was isolated from 105, Mycobacterium malmoense from 12 and Mycobacterium scrofulaceum from one cervical lymph node. A total of 89% of the children underwent surgery and were examined in particular with regard to cranial motor nerve functions. Major persisting nerve dysfunction occurred in 3/51 (6%) children who underwent radical surgery, and minor dysfunction in seven (14%). In nine children, the marginal mandibular branch of the facial nerve was affected, and the accessory nerve was affected in one child. There were no neurological signs in 25 children treated with incision and drainage alone or in 12 followed with observation alone. Healing took >6 months in 2/76 (3%) surgically treated and 3/12 (25%) non-surgically treated children. CONCLUSION: Considering the risk of nerve dysfunction following extirpation, incision with drainage and observation alone should both be included among the management options for cervical NTM lymphadenitis in children.

Late-onset posttraumatic skin and soft-tissue infections caused by rapid-growing mycobacteria in tsunami survivors.

BACKGROUND: In the tsunami catastrophe in Thailand in 2004, several thousand Swedish tourists were injured, with contaminated crush trauma of the legs being the main cause of injury among the survivors. METHODS: Patient and laboratory data for those who received hospital care in Stockholm and Gothenburg and contracted late-onset infections due to rapid-growing mycobacteria were reviewed retrospectively. Also, concomitant infections were recorded. RESULTS: Fifteen patients with late-onset skin and soft-tissue infections due to rapid-growing mycobacteria are described here. Mycobacterium abscessus was isolated in 7 cases, Mycobacterium fortuitum was isolated in 6 cases, and Mycobacterium peregrinum and Mycobacterium mageritense were isolated in 1 case each. The infections appeared after a delay of 20-105 days (median, 60 days) after the trauma, targeting undamaged skin located near primary sutured wounds or skin grafts. Antimycobacterial drugs were given to 9 (60%) of the patients. The course of infection was protracted, but all infections due to rapid-growing mycobacteria healed within 12 months. Concomitant subcutaneous infections due to other microorganisms, such as Burkholderia pseudomallei or Cladophialophora bantiana, appeared early or late after the trauma. CONCLUSIONS: Repeated cultures of abscess and wound specimens for Mycobacterium species may be needed to find the etiologic agents causing contaminated skin and soft-tissue infections, such as those that developed after traumas that occurred during the tsunami. These cultures are especially necessary when symptoms appear late and when conventional bacterial culture results are negative. A biopsy is recommended for the best yield and for complementary histopathological examination.

Molecular epidemiology of drug-resistant tuberculosis in Sweden.

Drug-resistant tuberculosis (TB), including the more severe forms of multidrug- and extensively drug-resistant forms, is an increasing public health concern globally. In Sweden the majority of patients with TB are immigrants from countries with a high incidence of TB including the drug-resistant forms. In this study, the spread of resistant TB in Sweden was investigated by molecular fingerprinting. Isolates resistant to at least one of the drugs, isoniazid, rifampicin, ethambutol or streptomycin, from 400 patients collected between 1994 and 2005, were studied by restriction fragment length polymorphism (RFLP) and by spoligotyping. Thirty-five clusters of patients infected with strains with identical RFLP and spoligotyping patterns (2-96 patients per cluster), comprising a total of 203 patients, were found. One large outbreak of isoniazid resistant tuberculosis was identified, involving 96 patients, mainly from the Horn of Africa. To identify chains of transmission, molecular epidemiological characterization of TB isolates should, if possible, be performed on isolates from all new TB patients.

Pyrosequencing of a short hypervariable 16S rDNA fragment for the identification of nontuberculous mycobacteria--a comparison with conventional 16S rDNA sequencing and phenotyping.

Conventional methods for identification of nontuberculous mycobacteria (NTM) are often inexact and time consuming. Sequencing of bacterial 16S rDNA is accurate, rapid and effective. We have retrospectively evaluated the discriminative power of pyrosequencing of a short hypervariable 16S rDNA fragment as a simple and rapid tool for NTM characterization. A series of 312 clinical NTM isolates, excluding the M. avium/intracellulare complex, was investigated. When species could not be resolved by sequencing alone, growth rate and pigment production were also examined. 54% (170/312) of the isolates were unambiguously identified by both methods. An additional 14% (45/312) were directly identified to species by conventional 16S rDNA sequencing but needed complementary phenotypic analysis when examined by pyrosequencing. The remaining 31% (97/312) needed additional phenotypic analysis for both sequencing methods. We consider the pyrosequencing procedure to be a useful alternative for the identification of several NTM species, and a versatile tool for the characterization of clinical NTM isolates. At times it requires additional tests for definite species diagnosis and correct identification.

Mycobacterium abscessus: an emerging rapid-growing potential pathogen.

Mycobacterium abscessus is the most pathogenic and chemotherapy-resistant rapid-growing mycobacterium. It is commonly associated with contaminated traumatic skin wounds and with post-surgical soft tissue infections. It is also one of the mycobacteria that are most often isolated from cystic fibrosis patients. It is essential to differentiate this species from the formerly indistinct "M. chelonae-complex", as chemotherapy is especially difficult in M. abscessussenso strictu. Clarithromycin or azithromycin are the only regular oral antimycobacterial agents with an effect on M. abscessus, and should preferably be supplemented with other drugs since long-term monotherapy may cause resistance. Amikacin is a major parenteral drug against M. abscessus that should also be given in combination with another drug. The recently introduced drug tigecycline may prove to be an important addition to chemotherapy, but has yet to be fully clinically evaluated as an antimycobacterial agent. Surgery can be curative, or at least helpful, in the healing of M. abscessus infection, and if conducted, it should include the removal of all foreign or necrotic material. There is increasing awareness of M. abscessus as an emerging pathogen.

Genetic diversity in clinical isolates of Mycobacterium avium complex from Guinea-Bissau, West Africa.

Isolates of Mycobacterium avium complex (MAC) were cultured from sputum samples obtained from patients in Guinea-Bissau, West Africa. Twenty-eight isolates hybridising with MAC probe (AccuProbe) were further characterised by different molecular techniques: hybridisation with species-specific probes (AccuProbe) for M. avium and M. intracellulare, partial sequencing of 16S rRNA gene and PCR detection of the DT1-DT6 sequences and the macrophage-induced gene (mig). Only one of the 28 isolates reacted with the M. avium probe and four with the M. intracellulare probe. Two isolates expressed the DT1 sequence, and three the DT6. The mig was detected in 18 (64%) of the isolates. Sequencing of 16S rRNA had the greatest discriminative power of the typing methods applied, without strong correlation with any other technique. Clinical MAC isolates from Guinea-Bissau demonstrated a wide genetic diversity among the members of M. avium complex that might reflect on biotope variation.

Increased sensitivity of Mycobacterium tuberculosis Cobas Amplicor PCR following brief incubation of tissue samples on Löwenstein-Jensen substrate.

The sensitivity of Mycobacterium tuberculosis Cobas Amplicor PCR (MTB-PCR) is considerably lower for tissue than for airway samples, depending on both lower bacterial content and presence of inhibitory substances in tissues. The aim of this study was to improve the sensitivity of MTB-PCR in inhibiting biopsy samples. A brief-culture method was applied to 33 inhibitory tissue samples out of 356 obtained in Laboratory I, and compared with 44/197 such samples treated by dilution in Laboratory II. We found that 2-3 days' incubation on Löwenstein-Jensen substrate (LJ) significantly increased the sensitivity of MTB-PCR in samples exerting PCR inhibition. Sensitivity was 63% before and 92% following brief-culture of inhibitory tissue samples in Lab I, compared to 46% and 50%, respectively, with dilution in Lab II. Thus, dilution did not significantly increase sensitivity in inhibiting samples. Specificities were 99.4/99.4 and 99.2/98.2, respectively. The higher sensitivity attained by the LJ-method was probably due to diffusion of inhibiting substances into the substrate, as well as to increase in numbers of bacteria after the brief-culture. This method adds substantially to the value of MTB-PCR of biopsy material.

[Antifungal agents can change the balance between resistant and susceptible Candida species]. / Antimykotika kan rubba balansen mellan resistenta och känsliga Candida-arter.

The use of fluconazole against yeast infections eradicates susceptible but promotes naturally resistant candida strains as C glabrata and C krusei. C albicans dominated in blood cultures taken at the Karolinska Hospital during the years 1989-2001. There was no trend towards an increase of Candida non-albicans. Restrictive use of fluconazole may explain why the balance between different Candida species with varying susceptibility to fluconazole has not shifted.

[Resistant tuberculosis is spreading in Sweden. Molecular epidemiological strain identification by "fingerprinting" can make the infection tracing easier]. / Resistent tuberkulos sprids i Sverige. Molekylärepidemiologiska "fingeravtryck" av de infekterande organismerna kan underlätta smittspårning.

Resistance of Mycobacterium tuberculosis to antibiotics is a world wide problem. A study is reported with the aim to analyse the spread of resistant isolates of M tuberculosis complex from patients with tuberculosis in Sweden. The study is based on a sample of 192 M tuberculosis complex isolates from patients with drug resistant tuberculosis during 1994-2000. All isolates resistant to at least one of the drugs streptomycin, isoniazid, ethambutol and rifampicin were included in the study. Restriction fragment length polymorphism (RFLP) was performed, using IS6110 as a probe for hybridisation. Visualised bands were analysed by Gel Compar software. The majority of the isolates were from patients born in high TB prevalence countries. During the years 1996-2000 there was one major cluster generated from 34 isolates. In 1996-1998 there were two isolates per year, in 1999 it increased to 20 isolates, and eight cases in 2000. All strains were resistant to isoniazid. All patients in this cluster were found to be from Africa. In comparing the pattern in the T-base the strains matched with strain BEA-000007341 isolated from a patient in Rwanda. The majority of patients with drug resistant tuberculosis in Sweden are immigrants from countries with high incidence of tuberculosis. Spread of disease to the Swedish born population is uncommon. However, an increasingly prevalent clone of isoniazid resistant tuberculosis was found among African immigrants, mainly living in the Stockholm area.

Unreliable alcohol testing in a shipping safety programme.

BACKGROUND: Within a maritime alcohol and drug testing programme, a case showing an unphysiological urine ethanol concentration (235 mmol/L, 10.8 g/L) was found. The sample contained low levels of the ethanol metabolites ethyl glucuronide (EtG) and ethyl sulphate (EtS) which confirmed prior drinking, but also tested positive for the fermenting yeast Candida albicans which suggested post-sampling ethanol formation. This and other questionable cases prompted investigation of the suitability of urine alcohol testing for the intended application. METHODS: Besides the routine measurements of ethanol, illicit drugs and creatinine, randomly selected ethanol-positive and ethanol-negative urines collected within the maritime programme were checked for the presence of EtG and EtS and for fungal and bacterial growth. Data on sample handling and storage was also gathered. RESULTS: Ten of 15 (67%) ethanol-positive and 4 of 9 (44%) ethanol-negative urines contained yeast and/or bacteria. Among the ethanol-positive cases, 4 (27%) were obviously false positives because EtG and EtS were not detected. Microbial action as the reason for false-high ethanol concentrations was indicated in other cases. When 17 bacteria-infected but fungi-negative urines were supplemented with glucose and stored for 1 week at 21 degrees C, ethanol was formed in 2 specimens containing Escherichia coli and E. coli plus P. aeruginosa. In these samples, EtG was also formed on storage while EtS was not. CONCLUSIONS: The routines employed for urine collection and handling within this substance abuse programme caused many false-positive identifications of alcohol use with unintended medico-legal consequences. Unpreserved urines stored without cooling should not be used for alcohol testing, given the high risk for microbial interference.

Drug resistance and genotypic analysis of Mycobacterium tuberculosis strains from Thai tuberculosis patients.

The aim of this study was the molecular characterization of primary drug-resistant Mycobacterium tuberculosis strains in Thailand. We examined a group of M. tuberculosis isolates from newly registered tuberculosis (TB) cases, collected at the largest university hospital, the Siriraj Hospital, in Thailand. Of 76 selected drug-resistant M. tuberculosis strains recovered from previously untreated pulmonary TB patients whose sputum samples were sent to this hospital, 29 (38%) were single-drug resistant, 26(34%) multidrug resistant and two (2.6%) extensively drug resistant. Fifty (66%) strains belonged to Beijing genotype. The study demonstrated a severe problem of drug resistance among recently detected TB patients, and two large clusters of genetically similar strains indicated ongoing transmission of drug-resistant TB.

Non-tuberculous mycobacteria in China.

Infection with non-tuberculous mycobacteria (NTM) is relatively rare in areas that are high-endemic for tuberculosis. We identified 126 strains of NTM in respiratory specimens collected by the Chinese Nationwide Survey in 2000, the Beijing Tuberculosis and Thoracic Tumour Institution, and the Guangzhou Thoracic Hospital. Species diagnosis was attained by sequencing of 16S-rDNA complemented by phenotypic characterization when necessary. Mycobacterium avium-intracellulare-complex - well known as a potential pulmonary pathogen - was diagnosed in all 3 surveys. Potentially pathogenic rapid growing strains of M. abscessus, M. chelonae and M. fortuitum were also detected. Moreover, species non-pathogenic in the airways such as M. nonchromogenicum, M. terrae and M. gordonae were encountered. Also, single strains of rare NTM species were isolated. Thus, several potentially pathogenic species of NTM were isolated from pulmonary specimens in China, and molecular techniques made possible exact species diagnosis.

Non-tuberculous mycobacterial infections.

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms in contrast to those belonging to the M. tuberculosi complex (MTB). NTM infects and causes disease only in hosts with local or general predisposing factors. Lung infection following inhalation of NTM is the most common NTM disease but soft tissue infections may occur in connection with contaminated trauma or surgery. Microbiological diagnosis is obtained by microscopy for acid-fast bacteria (AFB) on secretions or biopsies, and by culture on special media. With the high specificity of MTB- polymerase chain reaction (PCR), a positive AFB smear combined with negative MTB-PCR denotes infection with NTM. Sophisticated species diagnosis of cultured NTM is attained by various molecular methods, where 16S rDNA-sequencing remains the gold standard. The panorama of infection with different rapidly growing (RGM) or slowly growing mycobacteria (SGM) in Sweden is described. Sensitivity testing in vitro to antimycobacterial drugs against NTM does not always preclude the in vivo efficacy. Standard antimycobacterial treatment regimens have been defined for infection with several NTM species. Sensitivity testing should be performed in selected cases only, as in case of relapse or suspected development of resistance of the NTM strain. The spectrum of disease caused by NTM species that display a very low pathogenic potential is likely to widen over time as severe immunosuppression will continue to be prevalent in several patient categories.

Endogenous ethanol fermentation in a child with short bowel syndrome.

UNLABELLED: The term "auto-brewery syndrome" has been frequently used to describe patients who show features of alcohol intoxication because of abnormal yeast proliferation after ingesting carbohydrate-rich meals. We present a case of a 3-y-old girl with short bowel syndrome (SBS) who demonstrated signs of alcohol intoxication on repeated occasions. A blood test indicated an ethanol concentration of 15 mmol/l, and cultures from gastric fluid and faeces showed the presence of Candida kefyr. An association was found between the introduction of a carbohydrate-rich fruit drink and the occurrence of symptoms. CONCLUSION: The possibility of endogenous ethanol fermentation should be considered in patients with SBS and the diagnosis of auto-brewery syndrome added to the differential diagnosis list for D-lactic acidosis. Management includes both antifungal treatment and special diet modification.