Published Ahead of Print 27 November 2013. 10.1128/JCM.02739-13. 2014, 52(2):483. DOI:J. Clin. Microbiol.  Litvintseva Bethany Abrams, S. Arunmozhi Balajee and Anastasia P. Christina M. Scheel, Yitian Zhou, Raquel C. Theodoro,   in Clinical Samples Detection of Histoplasma capsulatum DNA Isothermal Amplification Method for Development of a Loop-Mediated http://jcm.asm.org/content/52/2/483 Updated information and services can be found at: These include: REFERENCES http://jcm.asm.org/content/52/2/483#ref-list-1at: This article cites 38 articles, 13 of which can be accessed free CONTENT ALERTS more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to: on O ctober 13, 2014 by U N E S P - U niversidade E stadual P aulista http://jcm .asm .org/ D ow nloaded from on O ctober 13, 2014 by U N E S P - U niversidade E stadual P aulista http://jcm .asm .org/ D ow nloaded from http://jcm.asm.org/content/52/2/483 http://jcm.asm.org/content/52/2/483#ref-list-1 http://jcm.asm.org/cgi/alerts http://journals.asm.org/site/misc/reprints.xhtml http://journals.asm.org/site/subscriptions/ http://jcm.asm.org/ http://jcm.asm.org/ Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples Christina M. Scheel,a Yitian Zhou,a* Raquel C. Theodoro,b Bethany Abrams,a* S. Arunmozhi Balajee,c Anastasia P. Litvintsevaa Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USAa; Universidade Estadual Paulista, Campus de Botucatu-UNESP, San Paulo, Brazilb; Division of Global Disease Detection and Emergency Response, Centers for Disease Control and Prevention, Atlanta, Georgia, USAc Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the or- ganism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n � 6) collected from HIV-positive individuals with culture- and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n � 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was <6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of an- tigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. cap- sulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to de- tect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted. Histoplasma capsulatum is a dimorphic fungus that causes his- toplasmosis. In immunocompromised persons, H. capsula- tum can disseminate throughout the body, causing progressive disseminated histoplasmosis (PDH), which is characterized by fe- ver, weight loss, and hepatosplenomegaly. Without early diagno- sis and antifungal intervention, PDH can cause death. Timely detection of PDH is problematic in resource-chal- lenged countries, since few rapid assays exist for this disease (1) and its symptoms are vague and often confused with those of mycobacterial or leishmanial infections (2, 3). Many laboratories in resource-limited areas in which the disease is endemic rely on sterile-site cultures for diagnosis of PDH; however, H. capsulatum grows slowly and may take several weeks for identification in cul- tures. The AccuProbe H. capsulatum culture identification test (Gen-Probe) can be used for rapid molecular identification of H. capsulatum in cultures, but this test is expensive and is not readily available in developing countries. Several additional molecular assays for detection of H. capsulatum have been developed (1, 4, 5), but none has been subjected to large-scale interlaboratory eval- uation. These assays rely on PCR methodology and require expen- sive reagents and equipment, which may be unsustainable in lab- oratories with limited funding. Here we describe the development of a loop-mediated isother- mal amplification (LAMP) assay for histoplasmosis, which pro- vides an affordable method of molecular identification that can be performed and interpreted without costly equipment in resource- challenged regions. Briefly, LAMP is a nucleic acid amplification technique that utilizes a polymerase with helicase activity, Bst from Bacillus stearothermophilus. The helicase activity allows for amplification of DNA at a constant temperature and is facilitated by four primers, 2 with cDNA, that form stem-loop DNA struc- tures. Once formed, the stem-loop structures become the tem- plate DNA for further amplification, which occurs very rapidly (6). Nucleic acid amplification via LAMP has several cost advan- tages over PCR. First, Bst polymerase is less expensive and more robust than Taq (7–9). Further, LAMP requires no thermal cy- cling equipment, since the assay is performed at a single temper- ature, allowing the use of either a heat block or a water bath to achieve nucleic acid amplification. Reactions are carried out in single tubes, and results can be visualized under UV light. In order to facilitate rapid inexpensive molecular diagnosis of PDH, we developed a LAMP assay targeting the single-copy gene Hcp100. Hcp100 is a member of the p100 gene family and is overexpressed in H. capsulatum during macrophage invasion (10). Unlike many multicopy housekeeping genes, Hcp100 shows little sequence identity with the DNA of related organisms and is not prone to false hybridization that may lead to cross-reactivity. MATERIALS AND METHODS H. capsulatum isolates. H. capsulatum isolates (n � 91) used in this study were cultured from frozen mycelial stocks provided by Roche Molecular Received 3 October 2013 Returned for modification 11 November 2013 Accepted 19 November 2013 Published ahead of print 27 November 2013 Editor: S. A. Moser Address correspondence to Christina M. Scheel, zsr3@cdc.gov. * Present address: Yitian Zhou, University of Pennsylvania Graduate School, Philadelphia, Pennsylvania, USA; Bethany Abrams, Emory University, Atlanta, Georgia, USA. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02739-13 February 2014 Volume 52 Number 2 Journal of Clinical Microbiology p. 483– 488 jcm.asm.org 483 on O ctober 13, 2014 by U N E S P - U niversidade E stadual P aulista http://jcm .asm .org/ D ow nloaded from http://dx.doi.org/10.1128/JCM.02739-13 http://jcm.asm.org http://jcm.asm.org/ Systems (Pleasanton, CA). Mycelia were grown on brain heart infusion (BHI) agar slants and subcultured three times to ensure optimal growth and purity prior to DNA extraction. All isolates were previously identified with respect to their geographic subspecies by multilocus sequence typing (MLST) and phylogenetic analysis (11), as described by Theodoro et al. (12). The geographic subspecies of study isolates were as follows: four North American 1 (NAm 1), 65 North American 2 (NAm 2), 11 Latin American A (LAm A), five Latin American B (LAm B), two lineage H81, and one each African, Netherlands, lineage H66, and lineage H68. Fungal DNA extraction. Genomic DNA was extracted using a Qiagen DNeasy tissue kit (Qiagen, Valencia, CA), with several modifications to the manufacturer’s instructions. Briefly, a portion of the fungal mat was transferred to 5-ml polypropylene tubes containing 800 �l Qiagen ATL buffer and 60 U of proteinase K and was homogenized inside a biological safety cabinet using an Omni tissue homogenizer (Omni International, Kennesaw, GA) at slow speed for 30 s and then at high speed for 30 s, using a clean probe for each isolate. Homogenates were capped, incubated at 55°C for 1 h with frequent vortex mixing, and then cooled to room tem- perature (RT). For each homogenate, RNase A (Sigma-Aldrich Corp., St. Louis, MO) was added to a final concentration of 1 mg/ml and the mixture was incubated for 5 min at RT, followed by the addition of 900 �l Qiagen buffer AL and vortex mixing. Homogenates were incubated at 70°C for 10 min, transferred to 1.7-ml microcentrifuge tubes, and centrifuged at 15,000 � g for 10 min. Clear supernatants (1 ml each) were transferred to clean microcentrifuge tubes, and 500 �l of 200-proof genomic-grade eth- anol (Sigma-Aldrich Corp.) was added to each tube. The suspensions were vortex mixed and transferred to Qiagen DNeasy columns; the manufac- turer’s instructions were followed throughout the remainder of the pro- cedure, except that DNA was eluted with 0.01 M Tris. DNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technolo- gies, Wilmington, DE). Archived fungal DNA samples used as controls were buffer exchanged into 0.01 M Tris using the Qiagen protocol for cleanup of genomic DNA (13), in order to eliminate EDTA and other additives known to interfere with subsequent applications. PCR and sequencing of the Hcp100 genetic locus. Portions of the H. capsulatum Hcp100 gene were PCR amplified using Hc I (5=-GCGTTCC GAGCCTTCCACCTCAAC-3=) and Hc II (5=-ATGTCCCATCGGGCGC CGTGTAGT-3=) primers, as described previously (14). Each 25-�l reac- tion mixture contained 0.2 �M each primer, 0.25 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate (dNTP), and 0.625 U Taq DNA poly- merase in a buffer of 10 mM Tris (pH 8.3), 50 mM KCl (Roche Carolina Inc., Florence, SC). Thermal cycling was performed in MicroAmp 96-well optical reaction plates, using a GeneAmp PCR system 9700 (Applied Bio- systems, Inc., Foster City, CA), as follows: 1 cycle of 94°C for 5 min; 40 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min; and a final cycle of 72°C for 5 min. The resulting amplicons were sized on 1.75% agarose gels, visualized with ethidium bromide under UV light, and cleaned using Exo-SAP-IT (USB Corp., Cleveland, OH), according to the manufacturer’s instructions. Amplicons were sequenced using the ampli- fication primers Hc I and Hc IV (5=-AGGAGAGAACTGTATCGGTGGC TT-3=) (14) and a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems Inc.), and reactions were analyzed with a 3730 DNA analyzer (Applied Biosystems, Inc.). Comparative sequence analysis and LAMP primer design. Consen- sus sequences among 14 isolates from geographically and genetically di- verse clades were generated from raw data using Sequencher 4.9 (Gene Codes Corp., Ann Arbor, MI) and were aligned using MUSCLE (15) in the MEGA 5.05 software package (16). Primers for LAMP were designed on the basis of conserved regions of the reverse complement of Hcp100 using Primer Explorer 4, a Web-based, open-use, primer design program (Eiken Chemical Co., Ltd., Tokyo, Japan) (http://primerexplorer.jp /elamp4.0.0/index.html). Primers used for LAMP were forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), and backward outer primer (B3) (Table 1). Clinical specimens. Six urine specimens (one specimen per person) from HIV-infected persons with symptoms consistent with PDH, positive Histoplasma urine antigen test results, and culture-confirmed H. capsula- tum infections were collected between 2004 and 2009 at the Clinica Familiar Luis Angel Garcia (Guatemala City, Guatemala) (17), under con- ditions reviewed and approved by the internal review boards of the Cen- ters for Disease Control and Prevention and the Universidad del Valle de Guatemala. Ten urine specimens were collected from healthy persons who had no indication of histoplasmosis. Two “mock-positive” samples were prepared from different pools of histoplasmosis-negative urine sam- ples, collected from healthy persons, by spiking with 1 ng H. capsulatum DNA prior to extraction. Urine DNA extraction and PCR. A QIAmp circulating nucleic acid kit (Qiagen) was used to extract DNA from urine specimens, according to the manufacturer’s instructions. Briefly, 4 ml urine was centrifuged at 16,000 � g for 10 min, and the pellet was resuspended in 0.01 M phos- phate-buffered saline (PBS) (pH 7.2). Both the supernatant and the resus- pended pellets were subjected to DNA extraction using lysis buffers ATL and ACB, according to the manufacturer’s instructions. A vacuum man- ifold designed for DNA extraction (Promega, Madison, WI) was attached to a rotary vane vacuum pump (Gast, Benton Harbor, MI) with a filter trap, and Qiagen Mini columns were attached to the vacuum using adapt- ers provided in the kit. After lysis, the urine specimens were pulled through the columns with the vacuum pump at a pressure of �85,000 pascals, bound to the resin columns, and washed. Nucleic acids were eluted in 30 �l of 0.01 M Tris-HCl by centrifugation and were quantified using a NanoDrop ND-1000 spectrophotometer. All urine specimen DNA was subjected to PCR using positive-control primers Beta 2 and Beta 3, which amplify portions of the human �-globin (BG) gene (Beta 2 [G1], 5=-GAAGAGCCAAGGACAGGTAC; Beta 3 [G2], 5=-CAACTTCATCCACGTTCACC) (18), and H. capsulatum Hc I/Hc IV primers specific for the Hcp100 genetic locus (14). The resulting amplicons were visualized on agarose gels as described above. LAMP method. Reaction mixtures were prepared according to the manufacturer’s specifications, using a Loopamp DNA amplification kit (Eiken Chemical Co., Ltd). Each 25-�l reaction mixture contained 12.5 �l 2� reaction buffer [40 mM Tris-HCl (pH 8.8), 20 mM KCl, 16 mM MgSO4, 20 mM (NH4)SO4, 1.6 M betaine, 0.2% Tween 20, 2.8 mM each DNTP], 40 pmol FIP, 40 pmol BIP, 10 pmol F3, 10 pmol B3, 1 �l Bst polymerase (Loopamp fluorescent detection reagent; Eiken Chemical Co., Ltd.), 2 �l of DNA, and 1 �l calcein-MnCl2 dye. Reaction mixtures were incubated at 63°C for 1.5 h, and the reactions were stopped at 95°C for 2 min. Amplification of H. capsulatum DNA was detected as calcein fluorescence directly visualized over a UV light box, in comparison with a lack of fluorescence in control reactions to which either non-H. capsula- tum DNA or no DNA template was added (19). LAMP assay validation. All H. capsulatum clinical isolates (n � 91) were assayed using the Hcp100 LAMP assay to determine its sensitivity. To define the limit of detection (LOD), DNA from 39 H. capsulatum isolates, including at least one representative of each geographic subspecies, was diluted 10-fold (from 1 ng/ml to 10 fg/ml) and assayed using LAMP. To test for potential cross-reactivity, archived DNA extracted from clinical fungal isolates maintained by the Mycotic Diseases Branch (n � 50) (Ta- ble 2) was assayed at concentrations of �2 ng/�l. Tenfold dilutions (50 ng/�l to 5 fg/�l) of Mycobacterium tuberculosis (ATCC 25177D-5) and TABLE 1 Primers used for LAMP of the Hcp100 genetic locus of H. capsulatum Primer Sequence (5= to 3=) FIP TCCCCGCGTCTCCCGAATACCGATCCAATGTCCGTTCACC BIP TCTGCACGGAAAACTGCGGCCTACGGCAACTCCGAAACC F3 GTAGTCGACGTTCGCAACT B3 GCCGACGTCGTTTACATCG Scheel et al. 484 jcm.asm.org Journal of Clinical Microbiology on O ctober 13, 2014 by U N E S P - U niversidade E stadual P aulista http://jcm .asm .org/ D ow nloaded from http://primerexplorer.jp/elamp4.0.0/index.html http://primerexplorer.jp/elamp4.0.0/index.html http://jcm.asm.org http://jcm.asm.org/ human (catalog no. G304A; Promega, Madison, WI) genomic DNA were also assayed. All DNA and clinical specimen extracts were tested in dupli- cate. Two negative controls (human DNA and no DNA template) and at least one positive control were included in each LAMP procedure, and assays were repeated twice, to ensure reproducibility. RESULTS LAMP assay validity with Histoplasma isolates. The Hcp100 LAMP assay was able to detect DNA of all geographically diverse H. capsulatum isolates. The LOD was strain dependent, and values fell between 10 fg/�l and 1 pg/�l (1 to 30 genomes per reaction) of H. capsulatum (Table 3), with a median of 6 genomes. Sixteen strains were detected at concentrations of �100 fg/�l (1 to 6 ge- nomes), and 19 were reactive at �10 fg/�l (one genome). When Hcp100 LAMP was compared with traditional PCR of Hcp100, the LAMP assay showed a 10-fold lower LOD (data not shown). The specificity of the LAMP assay was determined by testing the reactivity of LAMP primers against DNA of other clinically relevant yeasts and molds (Table 2), as well as human and myco- bacterial DNA. No cross-reactivity occurred with any other or- ganism tested, and the assay was 100% specific. When the assay incubation time was increased from 1.5 to 2 h, however, cross- reactivity did occur with some negative-control DNA samples. LAMP assay validity with human urine specimens. The two mock-positive samples that were prepared by spiking control urine samples with known concentrations of H. capsulatum DNA were assayed using LAMP and conventional PCR. These samples showed strong signals in the LAMP assay, and Hcp100 bands were present with PCR amplification (data not shown). In addition, we tested six urine samples from persons with HIV infection and proven histoplasmosis (Table 4). Four of these sam- ples (67%) showed strong signals in the Hcp100 LAMP assay, three with fluorescence in both the pellet and supernatant fractions and one with fluorescence in the pellet fraction only (Table 4). None of the 6 samples showed Hcp100 bands when DNA was amplified using traditional PCR and visualized using ethidium bromide (data not shown). Two samples failed to show amplification with the human �-globin (BG) primers; one of these samples was neg- ative by both the HCP LAMP and BG PCR assays, while one sam- ple was positive by BG PCR but negative by HCP LAMP. Further- more, one sample that was positive by the LAMP assay failed to show amplification with BG primers. Conversely, no fluorescence was observed in the pellet or supernatant fractions of urine sam- ples from healthy individuals. LAMP amplification products from positive urine samples and isolate DNA were visualized on agarose gels and produced similar characteristic patterns (Fig. 1). DISCUSSION The ability to provide laboratory diagnoses in resource-poor re- gions remains challenging. Many institutions in the developing world do not have the resources to detect and to identify infectious agents rapidly and precisely (20). In the case of PDH associated with HIV, the need for straightforward and inexpensive labora- tory diagnostic tools remains important, because PDH can cause death in 95% of cases (21) within months if it is undiagnosed or TABLE 2 Fungal isolates (n � 50) tested in the LAMP assay for detection of H. capsulatum Isolate Genus and species CDC B8815 Lichtheimia corymbifera CDC B8816 Lichtheimia corymbifera CDC B7759 Apophysomyces elegans CDC B7460 Apophysomyces elegans NRRL 485 Aspergillus flavus IFI 03 0139 Aspergillus flavus CDC B6077 Aspergillus fumigatus SRH 21 Aspergillus fumigatus ATCC 1015 Aspergillus niger IBT 14590 Aspergillus terreus UC141 Aspergillus terreus CDC B8899 Aspergillus versicolor CDC B8900 Aspergillus versicolor UC24457 Blastomyces dermatitidis CDC B3591 Blastomyces dermatitidis CAS 4016 Candida albicans CAS 4011 Candida dubliniensis CAS 3998 Candida glabrata CAS 4015 Candida krusei CAS 4031 Candida lusitaniae CAS 4000 Candida parapsilosis CAS 4012 Candida tropicalis 2010-18016 Coccidioides immitis 2011-02345 Coccidioides posadasii CDC B9302 Cryptococcus gattii CDC B7233 Cryptococcus gattii CDC B9031 Cryptococcus neoformans CDC B9039 Cryptococcus neoformans CDC B8703 Fusarium incarnatum-equiseti CDC B8704 Fusarium incarnatum-equiseti CDC B8723 Fusarium oxysporum CDC B8724 Fusarium oxysporum CDC B8601 Fusarium solani CDC B8602 Fusarium solani CDC B2699 Microsporum equinum CDC B7558 Mucor circinelloides CDC B7402 Mucor indicus CDC B9043 Penicillium sp., not marneffei CDD B9044 Penicillium spp., not marneffei UC202 Pneumocystis jirovecii UC246 Pneumocystis jirovecii UC267 Pneumocystis spp. CDC B7658 Rhizopus oryzae CDC B7662 Rhizopus oryzae CDC B3604 Sporothrix schenckii CDC B3759 Sporothrix schenckii CDC B3772 Sporothrix schenckii CDC B3909 Trichophyton mentagrophytes CDC B9131 Trichosporon asahii CDC B9132 Trichosporon asahii TABLE 3 LOD of the LAMP assay for detection of isolates of different geographic subspecies (n � 39) Geographic subspecies LOD (no. of genomes)a Median Range NAm 1 (4 isolates) �6 1–30 NAm 2 (13 isolates) �1 1–6 LAm A (11 isolates) �6 1–30 LAm B (5 isolates) �6 1–30 Other (6 isolates) �6 1–300 Avg �6 1–30 a The number of genomes was calculated by titration of DNA, based on the estimated genome size of H. capsulatum of 33 Mb. LAMP Assay for H. capsulatum February 2014 Volume 52 Number 2 jcm.asm.org 485 on O ctober 13, 2014 by U N E S P - U niversidade E stadual P aulista http://jcm .asm .org/ D ow nloaded from http://jcm.asm.org http://jcm.asm.org/ misdiagnosed. Delayed antifungal treatment of PDH results in mortality rates between 30 and 42% (22–26) in regions where the disease is endemic and where there are underserved populations or limited diagnostic resources. Molecular diagnostic tests have the potential to detect very small amounts of DNA with high spec- ificity, and the LAMP method is both rapid and inexpensive. We developed a LAMP assay to assist in rapid molecular identification of cultured H. capsulatum isolates, as well as to detect H. capsula- tum DNA in urine from patients with disseminated disease. Using DNA from cultured isolates, we showed that the Hcp100 LAMP assay could detect less than 30 Histoplasma genomes, a sensitivity 10-fold greater than that of traditional PCR assays. Further, the LAMP assay did not show cross-reactions with DNA of other fun- gal organisms or with mycobacteria; the latter cause disseminated infections with symptoms nearly identical to those of PDH in persons with HIV/AIDS. This novel LAMP assay may have two potential applications for the diagnosis of histoplasmosis in limited-resource settings. First, in a pilot validation study, we found that the LAMP assay was more sensitive than traditional PCR assays in detecting Hcp100 DNA in urine, and the LAMP assay showed no cross- reactions with DNA isolated from urine specimens from healthy persons, suggesting that this assay can be useful for direct detec- tion of H. capsulatum DNA in clinical samples. Second, our data demonstrate that the LAMP assay can be used for rapid confirma- tion of H. capsulatum in culture when the AccuProbe test is not available. The advantages of and caveats for each application are discussed below. The LAMP method has proven successful in detecting fungal DNA in specimen types in which whole intact organisms are lo- calized. Pneumocystis species DNA has been detected in sputum and bronchiolar lavage (BAL) fluid specimens from patients with pneumonia (27), Paracoccidioides brasiliensis DNA has been de- tected in sputum specimens (28), and Penicillium marneffei DNA has been detected in formalin-fixed, paraffin-embedded (FFPE) tissue specimens (29). In our study, we targeted residual intracel- lular (in leukocyte debris) and free circulating fungal DNA in urine. Obvious advantages of urine are that sample collection is noninvasive and large quantities can easily be obtained from in- dividual patients. Examination of urine sediment reveals a variety of cells including phagocytes (30), in which H. capsulatum sur- vives by avoidance of lytic digestion (31). Additionally, DNA re- leased from dying fungal cells is known to cross the renal barrier and is subsequently excreted in urine as cell-free DNA in lengths suitable for detection using PCR (32). In fact, urine is now com- monly utilized in molecular diagnostic testing, and many organ- isms that cause systemic infections are detected in this bodily fluid (33–37). We tested urine specimens collected from persons with HIV infection who had culture-proven PDH and positive antigenuria to determine LAMP assay sensitivity. Although an obvious caveat of our study is that only a small number of culture-positive urine TABLE 4 Detection of H. capsulatum in human urine specimens using the LAMP assay Urine specimen no. Health statusa ELISAb antigenuria (ng/�l) LAMP resultc Human �-globin (PCR) 208 HIV infection, culture-proven histoplasmosis 25.3 � S, P � 209 HIV infection, culture-proven histoplasmosis 12.4 � � 260 HIV infection, culture-proven histoplasmosis 13.8 � P � 258 HIV infection, culture-proven histoplasmosis 12.9 � � 256 HIV infection, culture-proven histoplasmosis 13.1 � S, P � 548 HIV infection, culture-proven histoplasmosis 12.6 � S, P � C5 Healthy 0.0 � � C6 Healthy 0.0 � � C7 Healthy 0.0 � � C12 Healthy 0.0 � � C13 Healthy 0.0 � � C15 Healthy 0.0 � � C16 Healthy 0.0 � � C17 Healthy 0.0 � � UP1 Healthy 0.0 � � UP2 Healthy 0.0 � � a Urine specimens were collected from persons with HIV infection and histoplasmosis (n � 6) and from healthy control subjects (n � 10). b ELISA, enzyme-linked immunosorbent assay. c S, supernatant fraction; P, pellet fraction. FIG 1 Hcp100 LAMP assay of DNA from human urine specimens and cul- tured H. capsulatum. (A) LAMP products were visualized on a 1.75% agarose gel. Characteristic “ladder-type” banding is shown with H. capsulatum anti- gen-positive urine and cultured H. capsulatum DNA (lanes 3 and 4, respec- tively), while no DNA amplification was seen after LAMP of healthy control urine (lane 2). Lane M, molecular size marker; lane 1, no-template control. (B) Corresponding tubes were visualized under UV light; fluorescent signals in tubes with antigen-positive urine and cultured H. capsulatum DNA are shown (tubes 3 and 4, respectively). Tube 1, no-template control; tube 2, healthy control urine. Scheel et al. 486 jcm.asm.org Journal of Clinical Microbiology on O ctober 13, 2014 by U N E S P - U niversidade E stadual P aulista http://jcm .asm .org/ D ow nloaded from http://jcm.asm.org http://jcm.asm.org/ specimens were available for testing, these pilot data demon- strated that 67% of samples (4/6 samples) were positive in the Hcp100 LAMP assay. One of the two urine samples for which negative LAMP results were obtained was also negative with PCR amplification with human �-globin primers, suggesting that no PCR-amplifiable human DNA was present in that sample (Table 4). In our prior experience using FFPE tissue biopsy specimens, we were seldom able to amplify fungal DNA with PCR when the hu- man �-globin locus did not show amplification (38, 39). Human globin DNA could be amplified from a second urine sample that did not react in the LAMP assay. We assume that this sample contained insufficient fungal DNA to be detected even with the sensitive LAMP assay. We have ruled out the presence of DNA polymerase inhibitors as a cause of insensitivity, since mock-positive urine specimens amplified Hcp100 strongly in both the LAMP and PCR assays. These samples were spiked with H. capsulatum DNA and imme- diately processed for DNA extraction. All were positive, further suggesting that DNA degradation contributed to decreased sensi- tivity of LAMP detection in urine. Overall, our data suggest that LAMP can be used to detect H. capsulatum DNA in urine samples; however, a large number of urine samples will need to be tested to determine the sensitivity of this method. In addition, the difficulty of extracting high-quality fungal DNA from clinical specimens using a rapid inexpensive method poses the greatest challenge in making LAMP available as a sustainable diagnostic method for fungal infections in resource-challenged laboratories. The Hcp100 LAMP assay was highly sensitive in confirming the identification of H. capsulatum from DNA prepared from cul- tured isolates, a feature that can be helpful in countries with lim- ited resources, where culture of blood and/or bone marrow sam- ples is frequently the primary method for diagnosis of PDH. In these countries, diagnostic confirmation of cultured isolates is fre- quently made using morphological observations alone, and His- toplasma can be confused with other yeasts of similar size, such as Candida glabrata. Using the Hcp100 LAMP assay, only a small amount of yeast growth is necessary for DNA extraction and con- firmation of H. capsulatum in culture. The purpose of our study was to develop a DNA-based method for detection of disseminated histoplasmosis that could be per- formed in resource-challenged laboratories. We have shown proof of the concept that LAMP may be a valuable tool for detect- ing disseminated histoplasmosis. Further evaluation of LAMP us- ing fresh-frozen urine, serum, or whole-blood samples is re- quired, and a simpler and less expensive DNA extraction method should be evaluated for use in resource-limited countries. 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Fungal DNA extraction. PCR and sequencing of the Hcp100 genetic locus. Comparative sequence analysis and LAMP primer design. Clinical specimens. Urine DNA extraction and PCR. LAMP method. LAMP assay validation. RESULTS LAMP assay validity with Histoplasma isolates. LAMP assay validity with human urine specimens. DISCUSSION ACKNOWLEDGMENTS REFERENCES