Slit Skin Smear Samples Research Articles

Ofloxacin resistance in multibacillary new leprosy cases from Purulia, West Bengal: a threat to effective secondary line treatment for rifampicin-resistant leprosy cases

Purulia is one of the high-endemic districts for leprosy in West Bengal (the eastern part of India). The annual new case detection rate (ANCDR) of leprosy in West Bengal is 6.04/100000 (DGHS 2019-20). Our earlier report provided evidence of secondary drug resistance in relapse cases of leprosy. The aim of the current study was to observe primary drug resistance patterns for dapsone, rifampicin, and ofloxacin amongst new leprosy patients from Purulia, West Bengal in order to better understand the emergence of primary resistance to these drugs. In the present study, slit-skin smear samples were collected from 145 newly diagnosed leprosy cases from The Leprosy Mission (TLM) Purulia hospital between 2017 and 2018. DNA was extracted from these samples and the Mycobacterium leprae genome was analyzed for genes associated with drug resistance by polymerase chain reaction (PCR), followed by Sanger sequencing. Wild-type strain (Thai-53) and mouse footpad-derived drug-resistant strain (Z-4) were used as reference strains. Of 145 cases, 25 cases showed mutations in genes associated with resistance to rifampicin, dapsone, and ofloxacin (as described by the World Health Organization, rpoB, folP, and gyrA, respectively) through Sanger sequencing. Of these 25 cases, 16 cases showed mutations in ofloxacin, two cases showed mutations in combinations of ofloxacin and rifampicin, four cases showed a mutation only in rifampicin, one case showed mutations in combinations of rifampicin and dapsone, and two cases showed mutations only in dapsone. Results from this study indicated the emergence of resistance to antileprosy drugs in new cases of leprosy. As ofloxacin is the alternate drug for the treatment of rifampicin-resistant cases, the emergence of new cases with resistance to ofloxacin indicates that ofloxacin-resistant M. leprae strains are actively circulating in this endemic region (i.e., Purulia, West Bengal), posing challenges for the effective treatment of rifampicin-resistant cases.

Mycobacterium lepromatosis as Cause of Leprosy, Colombia.

Leprosy is a granulomatous infection caused by infection with Mycobacterium leprae or M. lepromatosis. We evaluated skin biopsy and slit skin smear samples from 92 leprosy patients in Colombia by quantitative PCR. Five (5.4%) patients tested positive for M. lepromatosis, providing evidence of the presence of this pathogen in Colombia.

Molecular detection of Mycobacterium leprae using RLEP-PCR in post elimination era of leprosy.

Leprosy is considered as a contagious disease and is still a health problem in several countries including India. Diagnosis of leprosy is based either on clinical findings or on acid fast bacilli staining. Due to low sensitivity of acid fast bacilli staining most of the leprosy cases were remained undetected. The present study aims to assess the efficacy of RLEP-PCR in the field condition where majority of the patients are acid fast bacilli negative and have early disease. A total of 80 suspected leprosy cases were recruited. Slit skin smear samples were taken for microscopy and molecular experimentation. DNA was extracted and RLEP-PCR was executed for all the 80 samples. To establish the statistical correlation χ2 test and Fisher's exact test were made. To elucidate the sensitivity of the test Receiver Operating Characteristic (ROC) was drawn. These 80 leprosy patients comprised of 38 paucibacillary and 42 multibacillary leprosy cases. Of 80 leprosy patients 18 (22.5%) were AFB positive while 53 (66.25%) leprosy cases were RLEP-PCR positive. The results of test of significance (P=0.0001) and Cohen's kappa coefficient(κ) (0.614) indicated that the RLEP-PCR is a better diagnostic tool over AFB microscopy in case detection of leprosy. From the findings we concluded that RLEP-PCR could be used for the definitive detection of leprosy cases in accordance with the clinical findings in the field condition in the post elimination era of leprosy.

Molecular detection of multidrug-resistant Mycobacterium leprae from Indian leprosy patients.

The emergence of multidrug-resistant (MDR) organisms for any infectious disease is a public health concern. Global efforts to control leprosy by intensive chemotherapy have led to a significant decrease in the number of registered patients. Currently recommended control measures for treating leprosy with multidrug therapy (MDT) were designed to prevent the spread of dapsone-resistant Mycobacterium leprae strains. Here we report the identification of MDR M. leprae from relapse leprosy patients from endemic regions in India. Resistance profiles to rifampicin, dapsone and ofloxacin of the isolated strains were confirmed by identification of mutations in genes previously shown to be associated with resistance to each drug. Between 2009-2016, slit-skin smear samples were collected from 239 relapse and 11 new leprosy cases from hospitals of The Leprosy Mission across India. DNA was extracted from the samples and was analysed by PCR targeting the rpoB, folP and gyrA genes associated with resistance to rifampicin, dapsone and ofloxacin, respectively, in M. leprae. M. leprae Thai-53 (wild-type) and Zensho-4 (MDR) were used as reference strains. Fifteen strains showed representative mutations in at least two resistance genes. Two strains showed mutations in all three genes responsible for drug resistance. Seven, seven and one strain, respectively, showed mutations in genes responsible for rifampicin and dapsone resistance, for dapsone and ofloxacin resistance and for rifampicin and ofloxacin resistance. This study showed the emergence of MDR M. leprae in MDT-treated leprosy patients from endemic regions of India.

Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples

PurposePCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacteriumleprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. MethodLeprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl–Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. ResultsRLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. ConclusionAmongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes.

Identification of primary drug resistance to rifampin in Mycobacterium leprae strains from leprosy patients in Amazonas State, Brazil.

The aim of this study was to identify polymorphisms in the folp1, gyrA, and rpoB genes in leprosy patients treated in Amazonas State, Brazil. Among 197 slit-skin smear samples from untreated or relapsed patients, we found three cases of primary resistance to rifampin and one confirmed case of multidrug resistance.

Detection of Mycobacterium leprae in saliva and the evaluation of oral sensitivity in patients with leprosy

The aim of this study was to investigate sensitivity disorders in the oral cavity related to the presence of Mycobacterium leprae in the saliva of treatment-naïve patients with leprosy in the state of Amazonas, Brazil. A cross-sectional study was conducted involving 45 subjects with leprosy. The subjects were interviewed to evaluate the sensitivity of the oral cavity. For the detection of M. leprae, saliva and slit-skin smear samples were collected. The samples were analysed using a bacteriological index (BI) protocol and the real-time quantitative polymerase chain reaction (qPCR). The results indicated that 15 of the 45 (33.3%) subjects with leprosy showed decreased oral sensitivity, which confirmed the importance of the oral cavity sensitivity evaluation. There was not a direct relationship between the presence of M. leprae in saliva and changes in oral sensitivity. Positive saliva qPCR results from six (31.6%) of 19 paucibacillary (PB) patients suggested the possibility of a new site for sample collection. Positive results using these diagnostic techniques (BI, slit-skin smear and saliva qPCR) increased to 55.5%, thus opening the possibility of combining these different techniques to increase the rate of positive diagnoses, especially in PB patients.

Detection of Mycobacterium leprae in saliva and the evaluation of oral sensitivity in patients with leprosy

The aim of this study was to investigate sensitivity disorders in the oral cavity related to the presence of Mycobacterium leprae in the saliva of treatment-naïve patients with leprosy in the state of Amazonas, Brazil. A cross-sectional study was conducted involving 45 subjects with leprosy. The subjects were interviewed to evaluate the sensitivity of the oral cavity. For the detection of M. leprae, saliva and slit-skin smear samples were collected. The samples were analysed using a bacteriological index (BI) protocol and the real-time quantitative polymerase chain reaction (qPCR). The results indicated that 15 of the 45 (33.3%) subjects with leprosy showed decreased oral sensitivity, which confirmed the importance of the oral cavity sensitivity evaluation. There was not a direct relationship between the presence of M. leprae in saliva and changes in oral sensitivity. Positive saliva qPCR results from six (31.6%) of 19 paucibacillary (PB) patients suggested the possibility of a new site for sample collection. Positive results using these diagnostic techniques (BI, slit-skin smear and saliva qPCR) increased to 55.5%, thus opening the possibility of combining these different techniques to increase the rate of positive diagnoses, especially in PB patients.

Molecular epidemiology of leprosy based on VNTR typing in Thailand

Recently about 500 new cases of leprosy have been reported each year in Thailand. In addition to a steady rate of new case detection, Thailand is in Southeast Asia where leprosy is endemic in neighbouring countries; therefore, strain differentiation could be useful in tracing origins and routes of infection, and general leprosy surveillance. To identify suitable markers for differentiation of M. leprae strains in different global geographic regions and to determine the applicability of a systematic genotyping method for tracing leprosy transmission, variable nucleotide tandem repeats (VNTRs) of 14 loci were evaluated using DNA extracts from a total of 97 skin biopsies and slit skin smear samples. The alleles per locus ranged from 2-26 providing adequate strain differentiation. Microsatellite loci (GAA)21, (AT)17 are highly polymorphic followed by (GTA)9, (AC)8a, (AC)8b, and (AC)9. The minisatellites 6-7, 21-3 and 27-5 exhibited a limited number of alleles. The repeat of 23-3 showed no polymorphism. Overall, the strain types can be divided into two distinct Thai groups, according to the alleles at the (GGT)5 and 21-3 loci. However, there are no obvious geographical patterns of distribution of VNTR strain types. Closely matched VNTR profiles found in household members of two multi-case families suggested infection through a common source.

Associação do teste sorológico ML Flow com a baciloscopia

A descriptive, exploratory study was conducted analyzing the association of covariables in the results of the ML Flow serological test and slit skin smear. A total of 60 leprosy cases diagnosed at the state Sanitary Dermatology Referral Center were investigated. Slit skin smear samples were collected from four sites and the results were expressed by the bacillary index. ML Flow was registered in both qualitative and semi-quantitative terms. Cohen's kappa coefficient was used to study the agreement with Landis and Koch's observer criteria for interpretation. For statistical analysis, the logistic regression model and Kruskal-Wallis test were used. ML Flow showed a strong association with slit skin smear results, since a gradual increase in BI was accompanied by a semi-quantitative rise in antibody levels measured by ML Flow, with 100% positivity in cases presenting a positive slit skin smear. Given its strong correlation to slit skin smear, the results of this study provide evidence that the ML Flow test could be a valuable auxiliary tool in the classification and treatment of leprosy patients.

Detection of Mycobacterium leprae infection by PCR.

PCR amplification of the 531-bp fragment of the Mycobacterium leprae pra gene in fresh biopsy and slit skin smear samples was evaluated for its usefulness in the detection of leprosy bacilli in patients in Thailand. In multibacillary patients, 87.1% (27 of 31) of biopsy specimens and 41.9% (13 of 31) of slit skin smear specimens were positive by PCR, whereas in paucibacillary patients, 36.4% (8 of 22) of biopsy specimens and 18.2% (4 of 22) of slit skin smear specimens yielded detectable PCR amplification. Compared with other diagnostic procedures, PCR showed a clear advantage over both microscopic examination of slit skin smears and serologic detection of anti-phenolic glycolipid 1 antibody, especially in paucibacillary patients when bacterial indexes were 0 and seropositivity was only 6.25%. PCR was also evaluated for its potential to help monitor bacterial clearance in some of these patients during chemotherapeutic treatment. The PCR results on slit skin smear samples at 1, 3, and 6 months of chemotherapy showed that the number of PCR-positive cases of both multibacillary and paucibacillary types decreased sequentially. The results of this study are encouraging. However, investigation of a larger number of clinical specimens with an improvement in PCR methods, especially on slit skin smears, needs to be done before PCR can be established as a diagnostic procedure for leprosy patients and subclinical cases or as a tool for drug assessment.