Rama Murthy Sakamuri

Fonsecaea species cause chromoblastomycosis and phaeohyphomycosis, but links between environmental exposure and infection are poorly defined. We describe a 4-year-old boy with finger swelling after oak tree laceration in Houston, Texas. Broad-range polymerase chain reaction identified F. pedrosoi or F. monophora, and metagenomic sequencing of tree bark and soil detected Fonsecaea DNA.

Background: Intrafamily homology has impeded correlation of expression of individual PE_PGRS proteins with stage of tuberculosis (TB). We investigated the in vivo expression of PE_PGRS51, which has 3 unique regions. Methods: Sera from patients across the spectrum of TB were used to screen peptide arrays spanning PE_PGRS51. Results: Antibodies against a subset of conserved “core epitopes” within PE/PGRS domains are elicited during early TB. The epitope repertoire expands to adjacent regions with disease progression. Antiunique region antibodies appear only during cavitary TB. Conclusions: Elicitation of antiunique region antibodies can serve as markers for in vivo expression of PE_PGRS proteins.

Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-based optical biosensor. We apply an ultra-sensitive detection strategy developed by our team, termed lipoprotein capture, that exploits the pull-down of high-density lipoprotein (HDL) nanodiscs from cattle blood that allows for the recovery and detection of associated LM. We also profile the change in the expression of these TB biomarkers as a function of time from a small set of samples collected from studies of bovine TB-infected cattle. We demonstrate for the first time the direct detection of bovine LM in serum, and clearly show that the biomarker is expressed in detectable concentrations during the entire course of the infection.

An active search for Mycobacterium leprae drug resistance was carried out, 243 multibacillary patients from endemic regions of Colombia were included from 2004 to 2013 in a surveillance program. This program was a World Health Organization initiative for drug resistance surveillance in leprosy, where Colombia is a sentinel country. M. leprae DNA from slit skin smear and/or skin biopsy samples was amplified and sequenced to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. Three isolates exhibited mutations in the DRDR rpoB gene (Asp441Tyr, Ser456Leu, Ser458Met), two in the DRDR folP1 gene (Thr53Ala, Pro55Leu), and one isolate exhibited mutations in both DRDR rpoB (Ser456Met) and DRDR folP1 (Pro55Leu), suggesting multidrug resistance. One isolate had a double mutation in folP1 (Thr53Ala and Thr88Pro). Also, we detected mutations outside of DRDR that required in vivo evaluation of their association or not with drug resistance: rpoB Arg505Trp, folP1 Asp91His, Arg94Trp, and Thr88Pro, and gyrA Ala107Leu. Seventy percent of M. leprae mutations were related to drug resistance and were isolated from relapsed patients; the likelihood of relapse was significantly associated with the presence of confirmed resistance mutations (OR range 20.1–88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. In summary, the current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapse in leprosy patients. A high frequency of DRDR mutations for rifampicin was seen in a region where dapsone monotherapy was used extensively. Mycobacterium leprae drug resistance is cause of surveillance due to the increase of leprosy relapsed cases. World Health Organization initiative for drug resistance surveillance in leprosy included Colombia, a country considered in post-elimination stage, as a sentinel country. During 10 years (2004–20013) an active search for M. leprae drug resistance was carried out in volunteer patients (each of whom signed a consent form) recruited from a convenience sample of patients diagnosed with leprosy in fourteen departments of Colombia: Amazonas, Antioquia, Atlántico, Bolívar, Caquetá, Cesar, Cundinamarca, Chocó, Huila, Magdalena, Norte de Santander, Santander, Tolima, and Valle. 243 multibacillary patients in various stages of multi drug therapy (MDT) were enrolled with the aim to search for primary and secondary M. leprae drug resistance: 33 new patients before treatment, 136 currently undergoing MDT with not clinical improvement of lesions after three months or more of MDT, 36 post-MDT with positive bacillary index (BI) persistence, 4 non-adherent to MDT, and 34 relapsed cases. M. leprae DNA from patient´s samples was tested to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. We obtained isolates that exhibited mutations in the DRDR, three in rpoB gene, two in folP1 gene, and one isolate exhibited mutations in both rpoB and folP1, suggesting multidrug resistance. One isolate had a double mutation in folP1. Also, we detected mutations outside of DRDR that required in vivo test validation. 70% of M. leprae confirmed resistance mutations were isolated from relapsed patients (OR range 20.1–88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. The current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapsed in leprosy patients.

Although bio-detection strategies have significantly evolved in the past decade, they still suffer from many disadvantages. For one, current approaches still require confirmation of pathogen viability by culture, which is the ‘gold-standard’ method, and can take several days to result. Second, current methods typically target protein and nucleic acid signatures and cannot be applied to other biochemical categories of biomarkers (e.g.; lipidated sugars). Lipidated sugars (e.g.; lipopolysaccharide, lipoarabinomannan) are bacterial virulence factors that are significant to pathogenicity. Herein, we present two different optical strategies for biodetection to address these two limitations. We have exploited bacterial iron sequestration mechanisms to develop a simple, specific assay for the selective detection of viable bacteria, without the need for culture. We are currently working on the use of this technology for the differential detection of two different bacteria, using siderophores. Second, we have developed a novel strategy termed ‘membrane insertion’ for the detection of amphiphilic biomarkers (e.g. lipidated glycans) that cannot be detected by conventional approaches. We have extended this technology to the detection of small molecule amphiphilic virulence factors, such as phenolic glycolipid-1 from leprosy, which could not be directly detected before. Together, these strategies address two critical limitations in current biodetection approaches. We are currently working on the optimization of these methods, and their extension to real-world clinical samples.

Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.

Drug resistance surveillance identified six untreated leprosy patients in the Philippines withMycobacterium lepraefolP1mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked byM. lepraevariable-number tandem repeat (VNTR) strain types. In India,folP1mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target generpoB. These findings indicate that dapsone resistance is being transmitted.