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Objective: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. Materials and Methods: DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.

We compared the Sanger sequencing and the commercial INNO-LiPA® HBV assay for the routine detection of precore (PC) and basal core promoter (BCP) mutations of hepatitis B virus in chronically infected patients. The overall agreement rate between assays was 94.2% and 98.8% for the detection of PC and BCP mutations, respectively.

Resume Objectifs Etudier la prevalence des papillomavirus humains (HPV) dans des cancers de localisation differente chez des patients residant dans la region ouest de l’Algerie durant une periode comprise entre 2010 et 2014. Materiel et methodes L’ADN extrait d’echantillons fixes au formol et inclus en paraffine provenait de 39 cancers de la sphere ano-genitale, 10 cancers des VADS et 36 cancers de la peau. La presence d’ADN d’HPV a ete recherchee en utilisant le test INNO-LiPA HPV Genotyping Extra®. Resultats La prevalence des HPV etait de 100 % dans les cancers du col uterin, 40 % dans les cancers du vagin, 17 % dans l’ensemble des cancers de la vulve, 33 % dans les cancers de l’anus, 0 % dans les cancers de l’amygdale et du larynx et 6,4 % dans les cancers spinocellulaires. Les HPV16 et 18 ont ete detectes seuls dans 52 % et 12 % des cancers du col uterin respectivement et associes a d’autres HPV dans 4 % et 16 % des cas respectivement. Conclusion L’ensemble de nos resultats sont globalement en accord avec ceux de la litterature. Des etudes de plus grande ampleur s’averent necessaires pour promouvoir la vaccination anti-HPV en Algerie.

Human papillomavirus (HPV) causes most cases of anal cancers. In this study, we analyzed biopsy material from 112 patients with anal cancers in Australia for the presence of HPV DNA by the INNO LiPA HPV genotyping assay. There were 82% (92) males and 18% (20) females. The mean age at diagnosis was significantly (p = 0.006) younger for males (52.5 years) than females (66 years). HIV-infected males were diagnosed at a much earlier mean age (48.2 years) than HIV negative (56.3 years) males (p = 0.05). HPV DNA was detected in 96.4% (108) of cases. HPV type 16 was the commonest, at 75% (81) of samples and being the sole genotype detected in 61% (66). Overall, 79% (85) of cases had at least one genotype targeted by the bivalent HPV (bHPV) vaccine, 90% (97) by the quadrivalent HPV (qHPV) vaccine and 96% (104) by the nonavalent HPV (nHPV) vaccine. The qHPV vaccine, which is now offered to all secondary school students in Australia, may prevent anal cancers in Australia. However, given the mean age of onset of this condition, the vaccine is unlikely to have a significant impact for several decades. Further research is necessary to prove additional protective effects of the nHPV vaccine.

Seven weeks after being kicked in the face by a cow, a 34-year-old male patient developed a posttraumatic mycobacterial lymphadenitis. A rapidly growing mycobacterial isolate cultured from a surgically drained lymphadenitis pus specimen was identified as Mycobacterium smegmatis by matrix-assisted laser desorption/ionization mass spectrometry and a combination of ITS-, hsp65-, and 16S rRNA-DNA sequence analysis, but as Mycobacterium fortuitum complex using the commercial INNO-LiPA Mycobacteria v2 line probe assay. As it is unclear if the misidentification of this strain is an exception, more research is required.

Despite the effectiveness of nucleoside/nucleotide analogues in the treatment of chronic hepatitis B (CHB), their long-term administration is associated with the emergence of resistant hepatitis B virus (HBV) mutants. In this study, mutations resulting in antiviral resistance in HBV DNA samples isolated from 23 CHB patients (nine treatment naïve and 14 treated previously) were studied using a line probe assay (INNO-LiPA HBV DR; Innogenetics) and ultradeep pyrosequencing (UDPS) methods. Whilst the INNO-LiPA HBV DR showed no resistance mutations in HBV DNA samples from treatment-naive patients, mutations mediating lamivudine resistance were detected in three samples by UDPS. Among patients who were treated previously, 19 mutations were detected in eight samples using the INNO-LiPA HBV DR and 29 mutations were detected in 12 samples using UDPS. All mutations detected by the INNO-LiPA HBV DR were also detected by UDPS. There were no mutations that could be detected by INNO-LiPA HBV DR but not by UDPS. A total of ten mutations were detected by UDPS but not by INNO-LiPA HBV DR, and the mean frequency of these mutations was 14.7 %. It was concluded that, although INNO-LiPA HBV DR is a sensitive and practical method commonly used for the detection of resistance mutations in HBV infection, UDPS may significantly increase the detection rate of genotypic resistance in HBV at an early stage.

The novel SPF10 real-time PCR assay allows the simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. This study aims to evaluate the performance of the SPF10 real-time PCR in combination with the LiPA assay for HPV detection and typing in cervical samples. Thirty-nine cervical samples were subjected to the SPF10 conventional PCR in combination with the LiPA assay. Subsequently, the SPF10 real-time PCR was performed to enable the comparison between the SPF10 conventional and the real-time PCR results. In case of discrepancy, the samples were subjected to the CLAR(R) HPV2 assay. As a result, 27 out of 39 samples were identified as HPV-positive by the SPF10 real-time PCR and were genotyped further by the LiPA assay. Twenty samples (74.1%) showed an absolute agreement between the conventional and real-time SPF10 PCR (concordant), three (11.1%) displayed additional or fewer types (compatible), two (7.4%) did not show any similarity between both assays (discordant) and the remaining two (7.4%) were LiPA-negative. The two assays showed an excellent strength of agreement for individual (k = 0.932) and multiple genotype detection (k = 0.834). In conclusion, the two SPF10 PCR methods are comparable. Therefore, the SPF10 real-time PCR with subsequent LiPA could be used for the detection and genotyping of HPV in cervical samples. (C) 2013 Elsevier B.V. All rights reserved.

Objective: Hepatitis B virus (HBV) genotyping and detection of resistance to drugs have become essential in epidemiological and clinical diagnosis. Our main objective was to determine the prevalence of HBV genotypes and drug-resistance mutations in chronic asymptomatic carriers and chronic HBV sufferers comparing 2 detection assays. Methods: Serum samples from 28 chronic HBV patients and 22 chronic asymptomatic carriers were analyzed. For HBV genotyping, the INNO-LIPA and TRUGENE™ HBV genotyping kits were evaluated. For drug-resistance mutations, INNO-LIPA DR v2 and INNO-LIPA DR v3 prototype and the TRUGENE™ HBV genotyping kit were evaluated. Results: In HBV genotyping, concordant results were 98% and both assays were able to detect more than one genotype. Different genotypes were detected, the most prevalent being D (46%) and A (26%). In relation to drug-resistance mutations, the sensitivity of the line probe assay was lower than TRUGENE because INNO-LIPA could not detect two mutations (S202G and V214A). Conclusions: Both assays are easy and suitable for detecting HBV genotype and drug-resistance mutations and for routine laboratory use. However, TRUGENE presented better sensitivity in both analyses and it is possible to conduct both on the same sample. This assay is also able to detect primary and secondary mutations.

Aim Two billion people around the world are exposed to the hepatitis B virus (HBV) and about 350 million are infected with chronic HBV. The infection can be acquired early (neonatal) and becomes chronic in 90%; this rate reduces to 30% between ages one and five years. There is a 25% risk of chronicity in adults. Nowadays, immunomodulatory and antiviral pegylated-interferons or oral antiviral agents are used in the treatment of chronic hepatitis B. Lamivudine is an effective oral antiviral agent which inhibits the replication of HVB by blocking reverse transcriptase enzyme. The study aims to detect the resistance of HBV to lamivudine in the community and evaluate the effectiveness and suitability of early treatment with lamivudine. Subjects and Methods One hundred patients who presented to our Faculty of Medicine Hospital Infectious Diseases and Clinical Microbiology Department and had not received any antiviral treatment were recruited. The INNO-LiPA method was applied to investigate primary lamivudine resistance in patients. Results Seventy-eight patients were HBeAg-negative and 22 patients were HBeAg-positive. A statistically significant correlation was found between HBeAg positivity, alanine aminotransferase (ALT) elevation and HBV DNA (p < 0.05). The rtM204V and L180M mutation motif was found in one patient with HBeAg positivity. Conclusions Hepatitis B virus in our region is not a lamivudine-resistant strain and early treatment with lamivudine is an effective and convenient method.

BACKGROUND: Standardized and validated methods for the specific detection and identification of a spectrum of high-risk (hr) HPV genotypes will be necessary if HPV genotyping gains an important role in the clinical management of HPV-related precancerous lesions and cancers. OBJECTIVES: The first comparative evaluation of novel HPV genotyping Digene HPV Genotyping RH Test RUO (Qiagen, Hilden, Germany) with standard INNO-LiPA HPV Genotyping Extra CE assay (Innogenetics, Gent, Belgium). STUDY DESIGN: Seventy hr-HPV positive samples were tested in parallel with both genotyping assays. The results were interpreted taking into account 15 hr-HPV and 3 probable hr-HPV genotypes that can be identified by both assays (assay-common genotypes). RESULTS: Concordant results (a complete match of assay-common genotypes or negative using both assays) and compatible results (at least one genotype in common) were obtained in 42 (60.0%) and 28 (40.0%) samples, respectively. No discordant results for assay-common genotypes were obtained. Of 42 samples with compatible results, the presence of at least one assay-common genotype was detected in 37 samples, while no HPV was detected in two samples by both assays and only a single low-risk HPV was detected by INNO-LiPA in three samples. CONCLUSIONS: A novel Digene test is suitable for the detection of hr-HPV genotypes in clinical samples and it provides comparable results to the well established INNO-LiPA assay. Although INNO-LiPA identified significantly more samples with multiple HPV genotypes than the Digene test, the clinical benefit of such a difference is at present unclear.

International audience; The study was aimed to evaluate the feasibility of detecting human papillomavirus (HPV) in women with normal or abnormal cervical smears using the Roche Amplicor MWP HPV Test. We compared by AMPLICOR Test and Hybrid Capture II (HCII) Test, the prevalence of HR-HPV in 470 cervical samples including 55 samples with WNL cytology, 208 ASC-US, 193 LGSIL and 14 HGSIL. Samples with discordant results were retested with INNO-LiPA Genotyping HPV Test v2. The HR-HPV positivity in WNL cytology samples was similar (21.8%) by AMPLICOR and HCII. In ASC-US, the HPV positivity was 42.3% by both tests. In LGSIL, HPV positivity was 66.3% and 66.8% by AMPLICOR and HCII, respectively. In HGSIL, 92.8% of samples were positive by AMPLICOR and 85.7% by HCII. The agreement of both tests was 96.2% with a Kappa value of 0.92. Eighteen cases were discordant: 9 HCII positive/AMPLICOR negative and 9 HCII negative/AMPLICOR positive. The INNO-LiPA test revealed HPV positivity in every case. Interestingly, all HCII+/AMPLICOR- samples were found to harbour HPV53. As for the HCII-/AMPLICOR+ samples, 8 demonstrated a multiple infection with HR 16- and/or 18- and/or 56-phylogenetically related HPV types. Moreover, two of these samples were co-infected with HPV6 and two other with HPV54. By using consensus HR-HPV as our reference HPV positivity, the sensitivity (96.6%) and specificity (100%) of AMPLICOR was similar to that of HCII Test. The AMPLICOR HPV Test is sensitive, specific, feasible and appropriate for routine HPV detection.

We validated the implementation of the INNO-LiPA Rif.TB line probe assay, a diagnostic test for rapid detection of multidrug-resistant tuberculosis (MDR-TB), in Rwanda. No substantial difference was found between results obtained in Rwanda and results obtained in Belgium with the same samples. This rapid diagnostic test for MDR-TB can therefore be reliably implemented in a resource-poor setting.

Two commercial HPV tests target the same 65 bp fragment of the human papillomavirus genome (designated SPF10): the original HPV SPF10 PCR-DEIA-LiPA25 system, version 1, (LiPA25) and the INNO-LiPA HPV Genotyping Extra (INNO-LiPA). The original SPF10 LiPA25 system was designed to have high analytical sensitivity and applied in HPV vaccine and epidemiology studies worldwide. But due to apparent similarities, this test can be easily confused with INNO-LiPA, a more recent assay of which the intended use, i.e., epidemiological or clinical, is currently unclear. The aim was to compare the analytical sensitivity of SPF10 LiPA25 to that of INNO-LiPA on the level of general HPV detection and genotyping. HPV testing by both assays was performed on the same DNA isolated from cervical swab (n = 365) and biopsy (n = 42) specimens. In cervical swabs, SPF10 LiPA25 and INNO-LiPA identified 35.3% and 29.3% multiple infections, 52.6% and 51.5% single infections, and no HPV type in 12.1% and 19.2%, respectively. Genotyping results were 64.7% identical, 26.0% compatible and 9.3% discordant between both methods. SPF10 LiPA25 detected significantly more genotypes (p < 0.001). The higher analytical sensitivity of SPF10 LiPA25 was confirmed by the MPTS123 genotyping assay. HPV positivity by the general probes in SPF10 DEIA was significantly higher (87.9%) than by those on INNO-LiPA (77.0%) (kappa = 0.592, p < 0.001). In cervical biopsies, SPF10 LiPA25 and INNO-LiPA identified 21.4% and 9.5% multiple types, 76.2% and 81.0% single types, and no type in 2.4% and 9.5%, respectively. Between both tests, the identification of genotypes was 76.3% identical, 14.3% compatible and 9.5% discordant. Overall, significantly more genotypes were detected by SPF10 LiPA25 (kappa = 0.853, p = 0.022). HPV positivity was higher by the SPF10 DEIA (97.6%) than by the INNO-LiPA strip (92.9%). These results demonstrate that SPF10 LiPA25 is more suitable for HPV genotyping in epidemiologic and vaccine-related studies, due to its higher analytical sensitivity.

The INNO-LiPA Mycobacteria Test (Innogenetics, N.V., Belgium) is a PCR-based reverse hybridization assay for the simultaneous identification of several mycobacterial species. We evaluated two simplified lysis methods for mycobacterial DNA release for application in the INNO-LiPA Mycobacteria Test. The two methods were based on either (i) heat treatment or (ii) sonication. Both methods were performed directly on 45 positive liquid cultures (MB-BacT, BioMérieux, Marcy l'Etoile, France) containing 17 different mycobacterial species. These two simple lysis procedures demonstrated similar effectiveness (100%) to that recommended by the manufacturer. They also significantly shortened the time required for mycobacterial DNA release.

Comparative evaluation of Abbott RealTime and Innogenetics INNO-LiPA on alternately processed formalin-fixed, paraffin-embedded specimens of 31 cervical cancers and 31 uterine myomas showed complete agreement in the detection of 14 assay-common HPV genotypes and partial genotyping of HPV-16 and HPV-18. The tissue preparation protocol was shown to be sample-to-sample contamination safe.

A highly sensitive SPF10 real-time PCR was developed to achieve simultaneous amplification and detection of the human papillomavirus (HPV) target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. Here, we describe in detail a SYBR Green I-based real-time PCR assay based on SPF10 primers using the LightCycler(®) 480 system to generate and detect HPV amplicons, which are compatible with the LiPA assay.

Persistent infection with oncogenic human papillomavirus (HPV) genotypes has been shown to be necessary for the development of cervical cancer. The recently developed AdvanSure HPV GenoBlot assay (AdvanSure assay) is based on real-time PCR amplification and reverse line-blot hybridization.We first evaluated the performance of the AdvanSure assay compared to the INNO-LiPA HPV Genotyping assay (INNO-LiPA assay) for the molecular genotyping of HPV.A total of 200 cervical swab specimens were genotyped by the AdvanSure and INNO-LiPA assays. The AdvanSure and INNO-LiPA assays can detect 35 and 28 HPV genotypes, respectively. Samples that showed discrepancy were genotyped using sequencing.The percent agreement between the two assays ranged from 95.4 to 100% according to genotype. Analyzing the kappa values showed almost perfect agreement (0.9) for high-risk HPV genotypes 16, 18 and 33 and substantial agreement (0.724) for HPV genotype 31. The positive rates of the two assays for frequent HPV genotypes (16, 18, 31, 33, 53, 58, 66, 68, and 70) were not statistically different, but the INNO-LiPA assay showed higher positive rates than the AdvanSure assay for HPV genotypes 51, 52, and 56 (P0.05). All HPV genotypes that can be detected by only the AdvanSure assay (HPV genotypes 32, 34, 42, 55, 61, 62, and 81) were confirmed by sequencing.The AdvanSure assay was comparable with the well-established INNO-LiPA assay. The AdvanSure assay is a new alternative test for HPV genotyping in clinical laboratories.

The SPF10 PCR targets a conserved 65 bp region of the HPV L1 gene for broad-spectrum amplification. The LiPA assay allows subsequent genotyping of the HPV amplicons. This study aims to develop a SPF10 real-time PCR to achieve simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. The real-time PCR shows an analytical sensitivity of 29.7 copies for HPV 6, 16, 18 and 31 and an HPV-specific melting peak. Thirty-one HPV DNA plasmids were genotyped correctly using the SPF10 real-time PCR in combination with the LiPA. Here, the LiPA assay was performed at an increased hybridisation temperature (49.5 degrees C) in combination with a reduced amplicon volume (1 mu l) to avoid cross-reactivity. In conclusion, the SPF10 real-time PCR proves to be very sensitive and generates amplicons, which are compatible with the LiPA. (C) 2012 Elsevier B.V. All rights reserved.

INNO-LiPA MYCOBACTERIA (Innogenetics, Ghent, Belgium) is a reverse hybridization DNA probe assay that has been recently improved by increasing the number of identifiable mycobacterial species to 16. Our assessment, performed with 197 mycobacteria belonging to 81 taxa, revealed 100% specificity and sensitivity for 20 out of 23 probes. The probes specific for Mycobacterium fortuitum complex, for the Mycobacterium avium-intracellulare-scrofulaceum group, and for Mycobacterium intracellulare type 2 cross-reacted with several mycobacteria rarely isolated from clinical specimens. The overall sensitivity was 100%, and the overall specificity was 94.4%.

In order to investigate the influence of DNA extraction on two PCR-based HPV genotyping tests (Linear Array, Roche and INNO-LiPA Extra, Innogenetics), three different procedures were used to purify DNA from 28 cervico-vaginal samples tested previously by the Hybrid Capture 2: the AmpliLute Liquid Media Extraction kit (Roche), the QIAamp DNA Blood mini kit (QIAGEN), and the NucliSENS EasyMAG automated platform (bioMerieux). All HC2-positive samples were found positive by both assays, independently of the extract used. Type-specific concordance (i.e., identical HPV type-specific profile in all the extracts of the same sample) was observed in 55% and 75% of the cases testing samples by the Linear Array and the INNO-LiPA, respectively. Using the DNA extracted with the two manual methods the results were concordant in 75% of the cases both for the Linear Array and the INNO-LiPA. When comparing the Linear Array results obtained on either of the two manual extracts with those obtained following automated extraction, 65% of the samples showed type-specific concordance in both cases. The INNO-LiPA results were concordant in 80% of the cases comparing the AmpliLute versus the automated extract, while concordant results were observed in 90% of the cases when comparing the QIAGEN versus the automated extract. In conclusion, the Linear Array and INNO-LiPA results are affected by the method of DNA extraction. Consequently, different HPV type-specific profiles may be observed using different extracts of the same sample. The use of consistent protocols for DNA purification is a priority to guarantee intra-assay reproducibility over time.

Objective: It was our aim to compare the INNO-LiPA HPV Genotyping Extra (LiPA; Innogenetics) versus the Linear Array test (LA; Roche) in cervical samples. Study Design: One hundred cervical samples were selected, obtained from a cancer prevention center. All samples were human papillomavirus (HPV) DNA positive by Hybrid Capture 2 HPV test and genotyped by LA HPV genotyping test. All data were analyzed using Cohen’s ĸ tests. ĸ values ranged from poor, fair, moderate and good to very good agreement strength. Results: Detection of multiple infection and HPV genotypes per sample was higher by LA than by LiPA (61.5 vs. 52.1 and 2.2 vs. 1.7%, respectively). There were concordant results in 65 samples and compatible results in 33 samples. Only 2 samples were considered as discordant. In 21 samples, additional types were detected by LA, and in 13 samples, additional types were detected by LiPA. Analyzing the ĸ values, we have found very good agreement for 14 genotypes (6, 16, 26, 31, 33, 35, 45, 51, 52, 53, 58, 66, 68 and 70). Conclusions: We considered that the new LiPA is highly comparable with other methods and suitable for clinical and epidemiological studies.

Diagnosis of extrapulmonary tuberculosis (TB) is often missed or delayed because of nonspecific clinical and laboratory findings. Novel detection methods, such as polymerase chain reaction and QuantiFERON-TB Gold In Tube, can aid in the diagnosis of active extrapulmonary TB. Here, we demonstrate a case of epididymo-orchitis as the sole presentation of TB in a 32-year-old man.

Fifty-seven isolates of mycobacteria comprising 10 reference strains, 47 field isolates and one non-Mycobacterium isolate were screened using commercial INNO-LiPA v2 assay kits. All mycobacteria isolates tested hybridized with the Mycobacterium genus probe on the LiPA strip. All M. marinum, M. fortuitum and M. chelonae reference and field strains and three out of the four M. gordonae isolates hybridized to their corresponding species- or complex-specific probes. Two cultures (a type strain and a field isolate) yielded mixed growth of two mycobacterial species, i.e. M. chelonae and M. fortuitum. A Mycobacterium isolate from one of these cultures was subsequently purified and correctly identified with the kit. However, sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region of various mycobacteria isolates revealed a misidentification of M. shottsii and M. pseudoshottsii with the kit because these isolates reacted with the M. marinum/M. ulcerans probe. Moreover, nine of the 13 field isolates presumed to be M. fortuitum from the results of the kit had closer ITS sequence homology with M. conceptionense, a species which, to our knowledge, has never been reported in fish. These findings highlight the need to redesign the M. fortuitum-M. peregrinum probe included in the INNO-LiPA assay and to introduce additional complex-specific probes into the kit. Nevertheless, the kit proved to be a rapid and reliable method for identifying mycobacteria in the aquatic environment and would be particularly useful in laboratories without sequencing facilities.

In Chad, during a study on tuberculosis in humans and cattle, 52 non-tuberculous mycobacteria (NTM) strains were isolated. By means of INNO-LiPA, PRA-hsp65 amplification and sequencing of 16S rDNA, NTM species of 25/52 isolates were identified. M. fortuitum complex (8) was the most frequent species, followed by M. nonchromogenicum (4) and M. avium complex (4). PRA method could identify M. fortuitum 3rd variant among isolates derived from cattle specimens. This finding could confirm the existence of farcy in the Chadian cattle population as M. fortuitum 3rd variant and putitative pathogen M. farcinogenes can't be distinguished by the methods used in this study. Half of the NTM isolates could not be specified and we considered them as contaminants from the environment.

Using 106 clinical isolates of mycobacteria, we showed that INNO-LIPA Mycobacteria assay is an excellent tool to rapidly identify the most frequently isolated nontuberculous mycobacteria, in one procedure. It may be used as an additional technique to AccuProbe assay, which remains the fastest and the cheapest tool for a rapid and accurate identification of the M. tuberculosis complex.

We report a rare mesenteric localized Mycobacterium genavense infection in a severely immunocompromised human immunodeficiency virus-infected patient. An INNO-LiPA MYCOBACTERIA v2 test was performed directly on biopsy samples. This new molecular tool could be used for simultaneous identification of mycobacterium species from human specimens, but other studies are needed to validate our first results.

To the Editor: Lamivudine is a registered nucleoside analogue which has been found to suppress hepatitis B virus (HBV) replication. In chronic HBV carriers the decrease in serum HBV DNA levels is followed by improvement in liver histology [1]. The emergence of viral resistance however may compromise this. A 54-year-old woman with treatment naive chronic HBV infection (biopsy stage cirrhose, HBeAg negative, HBV DNA 1.53 E7 geq/ml (Digene Hybrid Capture II assay), ALT 48 IU/l (normal ,31 IU/l) was treated with Lamivudine therapy for 95 weeks. Six weeks before therapy no variant viruses could be found. After 77 weeks of treatment HBV DNA-level levels increased 1.48 E7 geq/ml. Ten weeks later the ALT level increased to 106 IU/l. DNA sequence analyses were performed (Inno Lipa HBVDR, Innogenetics Ghent Belgium) starting from week 89. Analyses of the YMDD motif of the HBV polymerase gene showed a methionine-toisoleucine substitution; rtM204I (YIDD variant). After 95 weeks of treatment with Lamivudine treatment was ceased. HBV DNA level at that time was 2.85 E8 geq/ml and ALT level 44 IU/l. Patient was closely monitored and HBV DNA, ALT and DNA sequence analyses were repeated (Fig. 1). Although Lamivudine was withdrawn, while at week 18 post-dosing a mixture of wild type virus and YIDD variants were detected, at week 41 post-dosing a mixture of wild type-virus, YIDD and YVDD (rtM204V), was still detectable. Mutant detection dropped below the 5% range of the assay detection limit at week 54 post-dosing. Viral resistance to Lamivudine with the emergence of YMDD variants during long-term Lamivudine therapy is well described and considered to be a problem for future optimal therapy [2–4]. Also described is replacement of variant viruses by wild type virus 3–6 months after cessation of Lamivudine therapy [2,3]. This is however the first report of persistence of YMDD variants for at least 41 weeks. Our Letters to the Editor 304

We read with great interest the recent article published by Mercier et al. (4) on the overestimation of mixed infections as a result of erroneous genotype H detection by use of the new version of the INNO-LiPA HBV genotyping assay. In this study, the INNO-LiPA assay identified 28 mixed infections in a population of 200 HBsAg- and anti-HBcAg-hepatitis B virus (HBV)-DNA-positive French blood donors. Nine of the 28 mixed infections included genotype H as part of a double or triple infection. Sequencing after molecular cloning did not confirm the presence of genotype H in these samples; therefore, the authors concluded that the high proportion of genotype H infection observed with INNO-LiPA is erroneous and contributes to an overestimation of mixed infections. As already stated by the authors, current available data indicate that genotype H is restricted to South and Central America (1) and that very few coinfections with genotype H are described in the

Background : The prevalence of multidrug resistant tuberculosis (MDR-TB), resistant to isoniazid (INH) and rifampin (RFP), was 5.3% worldwide in 1995 and its increment has raised important public health problems. Resistance to RFP, one of the key drugs in the treatment of tuberculosis, results in grim clinical outcome. Recently rapid detection of RFP-resistant mutations in rpoB gene based on PCR method has become available. This study evaluated the prevalence of RFP resistance in first diagnosed, treatment failure, and recurred patients using INNO-LiPA test, and compared the results of INNO-LiPA with those of conventional mycobacterial drug susceptibility test. Methods : Forty-six patients, who were diagnosed of pulmonary tuberculosis and had revealed positive sputum AFB smear, were enrolled in this study from 1998 to 2002. The cases were classified as one three groups; first diagnosed, treatment failure, or recurred. RFP resistance was studied using an INNO-LiPA Rif. TB kit and compared with that obtained from drug susceptibility based on M. tuberculosis culture study. Results : Twenty-one out of 46 patients were enrolled under first diagnosis of pulmonary tuberculosis

I have read with interest the recent paper of Suffys et al. (1) concerning the identification of 157 mycobacterium strains by using the INNO LiPA Mycobacteria (LiPA) assay. The same journal had published, 1 year ago, a similar paper, which I coauthored, on 238 strains (2), the results of which are now substantially confirmed. I feel, however, that Suffys et al. did not take it into due account; in fact, they quote it cursorily on two occasions and inappropriately. In the first case, the authors state that their results are in contrast with ours as their Mycobacterium abscessus strains reacted with LiPA probes MCH-1 and MCH-2 and as all of their Mycobacterium kansasii strains that were MKA-1-positive belonged to PCR restriction enzyme analysis (PRA) group I. I do not think, on the contrary, that there is any contrast; in fact, as the LiPA system does not make any distinction between Mycobacterium chelonae and M. abscessus, we behaved likewise. Consequently, the fact that among our M. chelonae sensu lato strains there were strains reacting with all of the MCH LiPA probes does not imply that M. chelonae sensu stricto strains reacted with MCH-2 and all the more so since no strain labeled as M. abscessus was present in our panel. Furthermore, regarding M. kansasii, our paper did not made any mention of PRA; it compared the results of LiPA with those of the widely used AccuProbe assay and highlighted an interesting correlation between the reactivity of the first- and second-generation AccuProbe assays and the different M. kansasii-specific LiPA probes. In the second case, the authors do not seem to realize that our discrepant case, a strain identified as Mycobacterium avium complex (MAC) intermediate with LiPA and as Mycobacterium intracellulare with the AccuProbe assay, fits exactly with their two strains, MAC intermediate with LiPA and M. intracellulare PRA group I.