Isolation and identification of mycobacteria responsible for tuberculosis of dromedaries in Algeria. / Isolement et identification de mycobactéries responsables de la tuberculose du dromadaire en Algérie.

Tuberculosis in dromedaries in Algeria has been little studied to date. The purpose of this study is to determine the prevalence of tuberculosis in dromedaries in three Algerian slaughterhouses using samples from suspected tuberculosis lesions, which were detected on carcasses during a post-mortem visual inspection. The study also uses laboratory diagnosis to isolate and identify the agents responsible for the infection. Between 2016 and 2018, 102 carcasses (3.05%; with a confidence interval [CI] of 95% from 2.05 to 3.69) were suspected of tuberculosis out of a total of 3,342 dromedary carcasses inspected. The lesions were located as follows in the carcasses: 64 of 102 were in the lungs, 37 in the liver and 1 in a bronchial ganglion. Five and six samples respectively of suspected tuberculosis lesions were found to be positive by bacilloscopy (4.9%; with a CI of 95% from 1.61 to 11.1) and in culture (5.88%; with a CI of 95% from 2.19 to 12.36). The concordance between bacilloscopy and culture was good (kappa coefficient of 0.71) and the probability of finding a positive culture was 184 times greater when the bacilloscopy was positive (value of p = 0.01). Molecular characterisation by polymerase chain reaction of extracts of DNA gave a positive signal, indicating that the isolated strains belonged to a mycobacterium genus. An enzymatic restriction on DNA extracts indicated the presence of mycobacterium DNA belonging to the Mycobacterium tuberculosis complex. Spoligotyping on the same DNA extracts confirmed the presence of four strains of Mycobacterium bovis with the same spoligotype SB0941 and another strain with spoligotype SB2562, a newly described profile in this study, which is phylogenetically close to the previous profile. Using suspected tuberculosis lesions in dromedaries, a non-tuberculosis mycobacterium was identified as Mycobacterium virginiense MO-233 (sequence ID: Nr149186) using the sequencing technique on the region 16SrDNA. Having demonstrated the presence of tuberculosis with M. bovis in the Algerian dromedary population, it is now necessary to implement measures to control it in order to reduce transmission between animals and humans.

À ce jour, la tuberculose a été peu étudiée chez les camélidés en Algérie. L'objectif de cette étude est de déterminer la prévalence de la tuberculose cameline dans trois abattoirs algériens à partir de prélèvements de lésions suspectes de cette maladie qui ont été détectées sur des carcasses lors de l'examen à l'inspection visuelle post-mortem. L'étude vise aussi à isoler et à identifier les agents responsables de cette infection par un diagnostic de laboratoire. Durant la période de 2016 à 2018, 102 carcasses (3,05 % ; avec un intervalle de confiance [IC] à 95 % de 2,05 à 3,69) ont été déclarées suspectes de tuberculose sur un total de 3 342 carcasses de dromadaires inspectées. Concernant la localisation des lésions sur ces 102 carcasses, 64 présentaient des lésions aux poumons, 37 au niveau du foie et 1 dans un ganglion bronchique. Respectivement cinq et six échantillons de lésions suspectes de tuberculose cameline ont été trouvés positifs à la bacilloscopie (4,90 % ; avec un IC à 95 % de 1,61 à 11,10) et en culture (5,88 % ; avec un IC à 95 % de 2,19 à 12,36). La concordance entre la bacilloscopie et la culture était bonne (coefficient kappa de 0,71) et la probabilité de trouver une culture positive était 184 fois plus élevée lorsque la bacilloscopie était positive (valeur de p = 0,01). La caractérisation moléculaire par réaction de polymérisation en chaîne (PCR) des extraits d'ADN a montré un signal positif, signifiant que les souches isolées appartenaient au genre mycobactérien. Une restriction enzymatique réalisée sur des extraits d'ADN a indiqué la présence d'ADN d'une mycobactérie appartenant au complexe Mycobacterium tuberculosis. Une technique de spoligotypage réalisée sur les mêmes extraits d'ADN a permis de confirmer la présence de quatre souches de Mycobacterium bovis avec un même spoligotype SB0941 et d'une autre souche avec un spoligotype SB2562, un profil nouvellement décrit dans cette étude et qui est phylogénétiquement proche du profil précédent. Au départ de lésions suspectes de tuberculose chez le dromadaire, une souche de mycobactérie non tuberculeuse a été identifiée comme étant un Mycobacterium virginiense MO-233 (séquence ID : Nr149186) par la technique de séquençage de la région 16SrDNA. La présence de la tuberculose à M. bovis ayant été démontrée dans la population cameline algérienne, il est dès lors nécessaire de mettre en oeuvre des mesures visant à la contrôler en vue de réduire la transmission entre l'animal et l'homme.

A día de hoy, la tuberculosis está poco estudiada en los camélidos de Argelia. Los autores presentan un estudio encaminado a determinar la prevalencia de la tuberculosis de los camélidos en tres mataderos argelinos a partir de muestras de lesiones sospechosas, detectadas en las canales durante la inspección visual post-mortem. El estudio consistía pues en aislar e identificar, mediante técnicas de diagnóstico de laboratorio, a los agentes responsables de la infección. Entre 2016 y 2018, de un total de 3 342 canales de dromedarios inspeccionadas, 102 fueron declaradas sospechosas de tuberculosis (un 3,05%; intervalo de confianza [IC] del 95%: 2,05-3,69). En cuanto a la localización de las lesiones, en 64 de los 102 animales, estas se encontraban en los pulmones, en 37 de los 102 en la región del hígado y 1 de los animales presentaba una lesión en un ganglio bronquial. De las muestras de lesiones sospechosas de tuberculosis de los camélidos, cinco resultaron positivas por baciloscopia (un 4,90%; IC del 95%: 1,61-11,10) y seis en cultivo (un 5,88%; IC del 95%: 2,19-12,36). Entre la baciloscopia y el cultivo hubo una estrecha concordancia (coeficiente kappa: 0,71) y la probabilidad de encontrar un cultivo positivo fue 184 veces mayor cuando la baciloscopia era positiva (p = 0,01). La caracterización molecular de extractos de ácido desoxirribonucleico (ADN) por reacción en cadena de la polimerasa (PCR) deparó una señal positiva, lo que significa que las cepas aisladas pertenecían al género Mycobacterium. La aplicación de una técnica de restricción enzimática a extractos de ADN puso de relieve la presencia de ADN de una micobacteria perteneciente al complejo Mycobacterium tuberculosis. Aplicando una técnica de espoligotipificación a los mismos extractos de ADN se pudo confirmar la presencia de cuatro cepas de Mycobacterium bovis con un mismo espoligotipo, SB0941, y de otra cepa con el espoligotipo SB2562, patrón nuevo y filogenéticamente cercano al anterior que se describe aquí por primera vez. A partir de lesiones sospechosas de tuberculosis detectadas en dromedarios se pudo caracterizar una cepa de micobacteria no tuberculosa, identificada como Mycobacterium virginiense MO-233 (secuencia ID: Nr149186), con la técnica de secuenciación de la región 16S rDNA. Habiendo quedado demostrada la presencia de tuberculosis por M. bovis en la población de camélidos argelina, se impone la necesidad de implantar medidas para controlarla, a fin de reducir la transmisión entre los animales y el ser humano.

Large-scale cultivation of Leishmania infantum promastigotes in stirred bioreactor.

BACKGROUND & OBJECTIVES: Bioreactors are practical tools that are used for economical, time-conserving and large-scale production of biomass from cell cultivation. They provide optimal environmental conditions such as pH and temperature required for obtaining maximum amounts of biomass. However, there is no evidence in the literature on the large-scale cultivation of Leishmania infantum parasites in the bioreactor. Therefore, the present study was undertaken to develop a new approach for obtaining L. infantum biomass by using pH and temperature controllable stirred bioreactor and to compare parasitic growth kinetics with classical method within erlenmeyers. METHODS: In order to obtain parasite biomass, a newly developed pH and temperature controlled stirred bioreactor was used and its efficacy was compared with a graduated classical scale-up method. Growth kinetics of parasites within erlenmeyers and bioreactors were determined by evaluating promastigote numbers using haemocytometer. The graduated scale enlargement of culture was followed by T25 flask, T75 flask, and 1 L erlenmeyer, respectively. RESULTS: Obtained results showed a 10-fold increase in the number of promastigotes within the conventional culture performed in 700 ml medium, while parasite numbers increased approximately 15 times due to initial inoculation amounts in the bioreactor culture performed in the 3.5 l medium. Thus, there was 7.5 times more biomass collection in bioreactor compared to classical method. INTERPRETATION & CONCLUSION: It is postulated that constant culture pH and temperature in the bioreactor extends cultivation time. This could lead to significant increase in parasite numbers. Hence, pH and temperature controllable bioreactors provided acquisition of sufficient amounts of biomass in contrast to classical methods. Therefore, this type of bioreactors may substitute classical culture methods in the production of antigenic molecules for vaccine development.

Lateral flow assay for rapid differentiation of Mycobacterium tuberculosis complex and 97 species of mycobacteria other than tuberculosis grown in Löwenstein-Jensen and TK-SLC medium.

BACKGROUND: Mycobacterial antigen MPB64 is a secretory protein specific for Mycobacterium tuberculosis complex. A lateral flow immunochromatographic assay (ICA) is a method used for the rapid differentiation of M. tuberculosis complex. AIM: We aimed to evaluate the performance of ICA in rapid differentiation of M. tuberculosis complex from 97 Mycobacterium species other than tuberculosis (MOTT), which are grown in Lφwenstein-Jensen and TK-selective (SLC) medium. MATERIALS AND METHODS: The study was performed in our laboratory between January 2009 and January 2010. A total of 394 isolates consisting of reference strains of 34 M. tuberculosis from World Health Organization (WHO) collection, 97 different MOTT bacilli, 7 Mycobacterium bovis BCG substrains and total 256 clinical Mycobacterium isolates were tested by ICA, which is based on anti-MPB64 monoclonal antibodies. All the strains were inoculated onto a TK-SLC (selective) medium and Lowenstein-Jensen medium. TK-SLC is a new rapid mycobacterial culture medium that indicates mycobacterial growth by colour change. RESULTS: The growth of mycobacterial strains was observed in 10-12 days on TK-SLC medium. ICA test was performed in 15 minutes. All strains belonging to M. tuberculosis complex group were found positive and all MOTT species were found negative on ICA slides. The results were confirmed with nucleic acid amplification by polymerase chain reaction (PCR) using primers specific for M. tuberculosis complex. CONCLUSION: With the additive effect of growth on TK-SLC medium in 10-12 days, the mycobacterial antigen MPB64 is a very useful and specific tool in rapid differentiation of M. tuberculosis and MOTT grown in culture.

Rapid detection of bacterial atypical pneumonia agents by multiplex PCR.

Approximately one third of community acquired pneumonia cases are caused by atypical pneumonia agents, Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydophila pneumoniae (formerly Chlamydia pneumoniae). The laboratory diagnosis of these organisms is difficult and time-consuming by conventional microbiological techniques. Polymerase chain reaction (PCR) is one of the important tools which can circumvent this problem. A multiplex PCR assay was developed to achieve the diagnosis of these three organisms in a single tube. Primers used in PCR were selected in a way that they amplified different length DNA fragments from different agents but they all worked at the same amplification conditions. Therefore the organisms could be diagnosed according to the length of amplified products by agarose gel electrophoresis without using any hybridization probes. After development of the multiplex PCR method, totally 309 clinical samples which were sent to our laboratory for single-agent PCR, were also evaluated by this technique. The results showed that the multiplex PCR assay is a sensitive, useful, cheap, and rapid diagnostic tool for the management of pneumonia patients.

Antimycobacterial activity of lipodepsipeptides produced by Pseudomonas syringae pv syringae B359.

Some lipodepsipeptides produced by Pseudomonas syringae pv. syringae showed strong antimycobacterial activity towards Mycobacterium smegmatis. MIC values found were between 1.5-3.2 microg/ml, which is comparable to some primary drugs for tuberculosis. Among the lipodepsipeptides, Syringomycin E (SRE) appears to be the most potent antimycobacterial agent.

Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis.

Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for pulmonary deterioration of cystic fibrosis patients. The purpose of this study was to type the P. aeruginosa isolates collected sequentially from cystic fibrosis patients, chronically colonized with P. aeruginosa, by random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR). Sequential P. aeruginosa isolates (n: 130) that had been collected from 20 CF patients over at least 9 years were investigated. The isolates were analyzed by RAPD-PCR using two arbitrary primers. Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method. RAPD-PCR typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype. Some CF patients were colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype. However, some patients may be colonized with more than one genotype. The results also demonstrated that there might be a risk of cross-colonization between CF patients followed-up at the same center.

Examination of mycosis fungoides for the presence of Epstein-Barr virus and human herpesvirus-6 by polymerase chain reaction.

BACKGROUND: The aetiology of cutaneous T-cell lymphoma (CTCL) remains unknown despite numerous investigations. In recent years, retroviruses and human herpesviruses have been implicated to play a causal part in CTCL. OBJECTIVE: The aim of this study was to elucidate the possible aetiopathogenetic role of human herpesviruses (HHV) in mycosis fungoides (MF). METHODS: Polymerase chain reaction was used to study formalin-fixed, paraffin-embedded lesional skin biopsies from 92 subjects with MF to evidence possible presence of Epstein-Barr virus (EBV) and HHV-6. RESULTS: Biopsy specimens from nine subjects (9.8%) evidenced EBV DNA, whereas all except one of the subjects (1.1%) lacked HHV-6 DNA. CONCLUSIONS: Although these findings do not support a primary aetiological role for EBV and HHV-6 in classical CTCL, the possibility remains that both viruses, particularly EBV, may act as potential cofactors in the development of CTCL.

HHV-8 is not a cofactor in the pathogenesis of environmentally induced malignant pleural mesothelioma.

After the recognition of human herpes virus 8 (HHV-8) in Kaposi's sarcoma lesions, this new virus has been shown to be associated with various types of malignancy. One of them, body cavity-based lymphoma, is a high grade B-cell lymphoma arising from the body cavities. Similarly, mesothelioma is a tumour that originates from the serosal linings of the pleural, pericardial and peritoneal cavities. One of the striking characteristics of mesothelioma cells is the secretion of interleukin-6 (IL-6). Also, it is known that HHV-8 upregulates the levels of IL-6, and this virally originated IL-6 is a well-established growth factor for HHV-8-associated lesions. Therefore, it was hypothesized that HHV-8 may have a role in the pathogenesis of malignant mesothelioma. Twenty-nine pleural biopsy specimens from environmentally induced malignant mesothelioma patients were investigated for the presence of HHV-8 deoxyribonucleic acid (DNA) using the polymerase chain reaction (PCR). Control pleural samples were collected from 15 biopsy specimens from patients with tuberculosis. From all samples, a segment of the beta-globulin gene was amplified in order to make sure that the DNA was extracted properly and did not contain any inhibitors. The specificity of the PCR amplification was confirmed by means of restriction enzyme analysis using Providencia stuartii I. PCR did not reveal HHV-8 DNA in any of the mesothelioma patients or in the control group. It was possible to amplify a segment of the human beta-globulin gene from all the samples of the patient and control groups. HHV-8 DNA was amplified in the control sample, which was a tissue biopsy specimen from a Kaposi's sarcoma lesion, and it was confirmed that the amplified DNA belonged to HHV-8 by restriction enzyme analysis. Malignant mesothelioma continues to be a public health problem in rural parts of Anatolia, Turkey. The major causal factor of the disease is exposure to asbestos and fibrous zeolite (erionite). It seems that there must be some aetiological factors other than exposure to these minerals as not all patients exposed to asbestos develop the disease and the disease is not always associated with any known exposure. From the present study, it was concluded that human herpes virus 8 does not seem to be associated with environmentally induced malignant mesothelioma in Turkey. Other possible causal factors of malignant mesothelioma should be sought.

Simian virus 40 is not a cofactor in the pathogenesis of environmentally induced malignant pleural mesothelioma in Turkey.

Malignant pleural mesothelioma (MPM) continues to be a public health problem in Turkey, where exposure to environmental asbestos and fibrous zeolite (erionite) is the main cause of the disease. However, less than 5% of exposed individuals develop the disease, and numerous cases of MPM are documented each year in which the patient has no known exposure to either of these minerals. Thus, additional unknown factors act independently or as co-carcinogens in the development of MPM. Simian Virus 40 (SV40) may act as a co-carcinogen with asbestos in the pathogenesis of occupationally induced MPM. To determine if SV40 plays a role in the development of MPM in Turkey, we used PCR analysis to investigate if SV40 DNA sequences were present in 29 mesothelioma specimens from patients previously exposed to asbestos or erionite. PCR analysis revealed that all 29 tissue specimens from our patients did not contain SV40 DNA. 15 specimens from patients suffering from tuberculosis pleuresy were also SV40 negative. One mesothelioma and one osteosarcoma from Italy tested positive for SV40. Our results indicate that inorganic fibers, asbestos, and erionite remain the only known causal factors of mesothelioma in Turkey. The absence of SV40 in Turkish specimens and its presence in Italian specimens may be related to the fact that SV40-contaminated vaccines were not administered in Turkey.

Evaluation of 120 mycobacterial strains isolated from clinical specimens to the species level by polymerase chain reaction-restriction enzyme analysis.

In this study, a total of 120 mycobacterial strains isolated from clinical specimens in Hacettepe University Hospital Clinical Pathology Laboratories were evaluated by polymerase chain reaction-restriction enzyme analysis (PRA), which analyses the common mycobacterial heat shock protein gene (hsp65). 95 of 120 strains (79.1%) were identified as Mycobacterium tuberculosis and 25 (20.8%) were identified as non-tuberculous mycobacteria (NTM). M. gordonae I and IV were the most common NTM species (3.3% each) followed by M. chelonae (2.5%). Other NTM species isolated were M. gordonae III, M. avium, M. peregrinum (1.6%), M. fortuitum, M. flavescens, M. malmoense and M. mucogenicum (0.8%). Four isolates had PRA patterns that did not match any patterns previously described. The patients who had NTM had underlying diseases; the most frequent clinical diagnosis among these was chronic obstructive pulmonary disease (COPD) and chronic renal failure. AIDS and pulmonary carcinoma were the other underlying diseases detected.

Induction of mycobacteremia by intravesical bacillus Calmette-Guerin instillation in an experimental animal model and detection with polymerase chain reaction.

PURPOSE: The aim of this study was to detect mycobacteremia by polymerase chain reaction (PCR), induced by the instillation of bacillus Calmette-Guerin (BCG) to guinea pig bladder. We also investigated the peak time and the effect of the dose of BCG in injured and non-injured bladder. The sensitivities of routine culture and PCR were also compared. MATERIALS AND METHODS: Five different doses (0, 0.069, 0.69, 6.9 and 69 mg.) of BCG were instilled into 5 injured and 5 non-injured bladders. Blood samples were collected at 0, 5, 15, 30 and 60 minutes following instillation for routine culture and PCR for each dose. A total of 50 female guinea pigs were used. RESULTS: Three of 5 samples (60%) obtained 30 minutes after the instillation of 69 mg. BCG into injured bladders were PCR positive. Furthermore, 4 of 5 samples (80%) were PCR positive when samples were obtained at the 60th minute following instillation. All the other samples were negative for PCR and routine culture. All the routine tuberculosis culture results were negative, including those which were PCR positive. CONCLUSIONS: Mycobacteremia was detected only in injured bladders and with high doses of BCG. PCR is a highly sensitive and rapid diagnostic method for detection of mycobacteremia.

In vitro activities of antibiotics alone and in combination against Brucella melitensis at neutral and acidic pHs.

Brucellae survive acidic pHs in phagolysosomes. Azithromycin, streptomycin, and quinolones were active against Brucella melitensis at pH 7.0 but not at pH 5.0; rifampin and doxycycline retained activity at pH 5.0. Regardless of pH, azithromycin-rifampin and ofloxacin-rifampin showed less synergy than established streptomycin-doxycycline and rifampin-doxycycline combinations.

Detection of Listeria monocytogenes in milk by the polymerase chain reaction.

A on the polymerase chain reaction (PCR) method based was developed for detection of Listeria monocytogenes in milk samples after enrichment culture. It consists of culturing samples in Listeria enrichment broth, followed by DNA extraction and detection of the organism using PCR. Dilutions of L. monocytogenes in milk were subjected to PCR amplification after enrichment culture. When determining the sensitivity of the method, it was found to be possible to detect 37 CFU (colony forming unit gl/ml) of the bacterium in milk. The method was assessed as a sensitive, specific, times-saving and practical way of detecting L. monocytogenes in milk samples.

Comparison of three different primer pairs for the detection of Mycobacterium tuberculosis by polymerase chain reaction in paraffin-embedded tissues.

SETTING: More than five different primer pairs have been used for the detection of Mycobacterium tuberculosis deoxyribonucleic acid (DNA) with the polymerase chain reaction (PCR). OBJECTIVE: The sensitivity and specificity of PCR were evaluated using three different primer pairs in the detection of M. tuberculosis in paraffin-embedded tissues. DESIGN: Thirty-eight tissue specimens from 23 patients were studied. Eighteen samples were obtained from 10 tuberculosis patients, and 20 samples obtained from 13 patients with other diseases were used as negative controls. DNA extracted from paraffin-embedded tissues was used directly for PCR amplification using primers IS1 and IS2 to amplify a 123 base pair (bp) region of IS6110, sjMT3 and sjMTr2 to amplify a 281 bp region of protein antigen b, and INS1 and INS2 to amplify a 245 bp region of IS986. Each amplification was performed double-blinded and repeated three times including positive and negative control samples. RESULTS: IS1 and IS2 gave a positive result in each of the double samples obtained from eight tuberculosis patients and in the single samples obtained in the two others, sjMT3 and sjMTr2 detected 13 of the 18 tuberculosis samples, and INS1 and INS2 detected only three of the 18. CONCLUSION: These results highlight the importance of selecting appropriate primers to obtain high sensitivity in detecting M. tuberculosis in paraffin-embedded tissues by PCR.

Activity of amoxicillin/clavulanate in patients with tuberculosis.

Some beta-lactam antibiotics are active in vitro against Mycobacterium tuberculosis. There are anecdotal reports of successful treatment of tuberculosis caused by multiple-drug-resistant strains of M. tuberculosis with regimens that included amoxicillin/clavulanate. Reduction of M. tuberculosis in the sputum of patients with pulmonary tuberculosis during administration of amoxicillin/clavulanate was measured by a quantitative culture method to determine the activity in vivo. Patients were randomized to receive isoniazid, ofloxacin, or amoxicillin/clavulanate for 7 days. Isoniazid was the most effective agent, reducing M. tuberculosis after 2 days at a mean rate (+/- standard deviation) of 0.60 +/- 0.30 log10 cfu/mL per day, compared with 0.32 +/- 0.05 and 0.34 +/- 0.03 for ofloxacin and amoxicillin/clavulanate, respectively. The early bactericidal activity of amoxicillin/clavulanate was comparable to that reported for antituberculous agents other than isoniazid. Further studies of beta-lactam antibiotics with in vitro activity against M. tuberculosis are warranted to define their role in treatment of tuberculosis.

Typing of urinary JC virus DNA offers a novel means of tracing human migrations.

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra.

To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml, and high-level resistance (i.e., MIC, > and = 5 micrograms/ml) was associated with two gyrase mutations. Mutations in gyrB and possibly altered levels of intracellular accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance. Because sparfloxacin is more active in vitro and selection of resistance appears to be less likely to occur, it may have important advantage over ofloxacin or ciprofloxacin for the treatment of tuberculosis.

Quantitative culture of Mycobacterium tuberculosis from clinical sputum specimens and dilution endpoint of its detection by the Amplicor PCR assay.

The minimum number of Mycobacterium tuberculosis CFU detectable in clinical sputum specimens by the Amplicor PCR test was estimated by performing the test on duplicate samples of quantitatively cultured serial dilutions of sputum. Positive PCR test results were obtained for all samples that contained 42 CFU of M. tuberculosis. The detection limits of the PCR assay for decontaminated (N-acetyl-L-cysteine [NALC]-NaOH) and nondecontaminated (NALC only) specimens were equivalent, even though the number of CFU cultured from decontaminated samples was only 11 to 20% of the number cultured from nondecontaminated samples. Thus, the 42 CFU that could be detected in nondecontaminated specimens by the Amplicor PCR test correspond to the approximately 8 CFU (0.20 x 42) that could be recovered in culture after decontamination with NALC-NaOH.