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J Clin Microbiol. Nov 2011; 49(11): 4013–4015.
PMCID: PMC3209121

Laboratory-Confirmed Case of Yaws in a 10-Year-Old Boy from the Republic of the Congo [down-pointing small open triangle]

Abstract

We report a case of yaws in a patient with puritic cutaneous eruption who was initially suspected of infection with monkeypox. The diagnosis was established by real-time PCR and sequencing of specific treponemal DNA sequences. This is the first report describing the use of DNA sequencing to identify Treponema pallidum subsp. pertenue-specific sequences in a patient with active yaws.

CASE REPORT

A 10-year-old pygmy boy presented with fever and maculopapular and pustular lesions over his entire body, including the soles of his feet. He was admitted to a hospital in Bétou, Republic of the Congo, on 25 October 2010. The patient reported onset of illness 1 week prior to admission. Symptoms included itchy lesions, nausea and vomiting, inguinal adenopathy, chills or sweats, sore throat, and sensitivity to light. Clinical examination revealed numerous lesions on the face, thorax, upper and lower extremities, and soles of the feet. The patient had no history of smallpox vaccination or varicella. He reported no history of illness associated with a rash within the previous 6 months and did not come into contact with anyone with similar symptoms in the 3-week period prior to the onset of his symptoms. In addition, no animal contact occurred 3 weeks prior to the onset of his symptoms. The differential diagnosis included monkeypox and yaws with a codiagnosis of malaria. Swabs and crusts of lesions and blood samples were collected. All specimens were sent to the Centers for Disease Control and Prevention in Atlanta, GA, for testing. The patient was treated for malaria (artesunate-amodiaquine combination therapy) and given acetaminophen (500 mg) and benzathine penicillin (600,000 IU intramuscularly). In addition, gentian violet (1% solution in water) was applied topically to his skin eruptions. The patient made a full recovery.

DNA was extracted from the swab, crust, and blood samples using a tissue kit for the BioRobot EZ1 Workstation (Qiagen, Valencia, CA). Real-time PCR testing for non-variola Orthopoxvirus performed in the Poxvirus and Rabies Branch of the Division of High-Consequence Pathogens and Pathology yielded negative results (8). Subsequently, DNA extracted from the swab and a blood sample was sent to the Laboratory Reference and Research Branch of the Division of STD Prevention for molecular testing and syphilis serology, respectively. The blood sample was heavily hemolyzed and therefore not suitable for a qualitative rapid plasma reagin (RPR) test. However, in the quantitative RPR test, the amount of hemolysis in the serum did not interfere with the endpoint reading of 1:128. The blood sample tested positive for treponemal antibodies using the TREP-SURE enzyme immunoassay (EIA; Phoenix Bio-Tech Corp., Oakville, Ontario, Canada). A real-time PCR which is specific for pathogenic treponemes and targets the polA gene (tp0105) was performed on the extracted DNA sample as described previously, yielding a positive result (3). To distinguish between the pathogenic treponemes that cause yaws (Treponema pallidum subsp. pertenue), syphilis (T. pallidum subsp. pallidum), and bejel (T. pallidum subsp. endemicum), (i) an intergenic spacer (IGR19) between the fliG gene (tp0026) and a putative hemolysin gene of unknown function (tp0027) and a (ii) specific region of the tprI gene (tp0620) present in all three subspecies were PCR amplified and sequenced (2, 6).

PCR primers for tprI (sense, 5′-AAGCACGCGTGTACATCC; antisense, 5′-ATCCCTCGCCTGTAAACTGA) corresponding to nucleotide positions 1427 to 1444 and 74 to 93 of tp0620 and the gene for a hypothetical protein (tp0619), respectively, in the T. pallidum subsp. pallidum Nichols genome were used to amplify a 493-bp region of the gene (2). PCR amplifications were performed using 4 μl of DNA sample and a 50-μl reaction mixture containing: 0.1 μl of a deoxynucleoside triphosphate mixture (100 mM dATP, dCTP, dGTP, and dTTP); 3 μl of MgCl2 (25 mM); 0.4 μM each primer; 2.5 U of AmpliTaq Gold polymerase; 5 μl of 10× PCR buffer (all from Applied Biosystems). Thermocycling was performed in an Applied Biosystems 9700 thermocycler as follows: an initial hold at 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 7 min. PCR products were analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The PCR product of interest was purified using an E-Gel CloneWell System (Invitrogen Corporation, Carlsbad, CA), and both DNA strands were directly sequenced using the BigDye Terminator v3.1 cycle sequencing kit and an ABI 3130-XL sequencer.

PCR primers for IGR19 (sense, 5′-GCCACGAGGAGATTCGGTTCTATTC; antisense, 5′-CCGCAGCAGAGAACAACATGGA) corresponding to nucleotide positions 997 to 1021 and 7 to 28 in tp0026 and tp0027, respectively, in the T. pallidum subsp. pallidum Nichols genome were used to amplify a 280-bp fragment. PCR amplification was performed as described previously, except that 1.5 μM MgCl2 was used and primer extension was done at 72°C for 1 min (6). Amplicons were purified using the QIAquick PCR purification kit (Qiagen Inc., Valencia, CA). Sequencing was performed as described above, except that sequencing buffer was not used for cycle sequencing and initial denaturation was done at 96°C for 10 min. Sequences were assembled and aligned with Lasergene software (DNASTAR, Inc., Madison, WI).

A reactive RPR screening test and TREP-SURE treponemal EIA confirmed treponemal infection; however, serological testing cannot distinguish among the infections caused by the three pathogenic T. pallidum subspecies. Real-time PCR amplification of the polA gene of T. pallidum in the swab specimen yielded a positive result. This assay is specific for pathogenic treponemes; therefore, it detects all three T. pallidum subspecies. In order to determine which of the three T. pallidum subspecies was present in the DNA sample, sequence analysis of IGR19 (fliG-tp0027) and tprI (tp0620) was performed. Sequence analysis of IGR19 showed that the clinical specimen (ROC1) had a genetic signature (CCCTCC) that is homologous to that found in T. pallidum subsp. pertenue strain CDC 2575 and T. pallidum subsp. endemicum strain Bosnia A, while the T. pallidum subsp. pallidum Nichols strain (causes syphilis) has a homonucleotide tandem repeat (CCCCCCCCC) at the same locus (Fig. 1 A). The tandem repeat within IGR19 is present only in T. pallidum subsp. pallidum strains and has been shown to vary in number (6). Sequence analysis of the region between nucleotide positions 1740 and 1766 in the tprI gene (Fig. 1B) revealed a genetic signature that is unique to T. pallidum subsp. pallidum and pertenue strains which was also present in ROC1 (2). In addition, only T. pallidum subsp. endemicum strains have a BsrDI site (GGCATTGC) at this locus (produces restriction fragments of 334 and 159 bp based on a 493-bp tprI amplicon), which distinguishes this subspecies from T. pallidum subsp. pallidum and pertenue strains (2). Based on the analysis of both the IGR19 (rules out T. pallidum subsp. pallidum) and tprI (rules out T. pallidum subsp. endemicum) sequences, ROC1 was most likely T. pallidum subsp. pertenue, the causative agent of yaws. The molecular approach used to characterize the specimen is shown in Fig. 2.

Fig. 1.
Partial sequences of the 280-bp IGR19 (A) and 493-bp tprI amplicons (B). (A) IGR19 is located between tp0026 (fliG gene) and tp0027 (putative hemolysin gene of unknown function) in the T. pallidum genome. The T. pallidum subsp. pallidum Nichols strain ...
Fig. 2.
Flow diagram of the molecular diagnosis of yaws. The asterisk indicates the number of C residues within IGR19 of the T. pallidum subsp. pallidum Nichols strain. Various numbers of repeats have been found in T. pallidum subsp. pallidum strains.

Yaws is a nonvenereal endemic treponematosis caused by the bacterium T. pallidum subsp. pertenue. It is often referred to as a disease of the poor and generally affects children under 15 years old. Active cases of the disease are commonly reported in Africa (Ghana, Republic of the Congo, Democratic Republic of the Congo, Ivory Coast), Southeast Asia (Indonesia, Timor-Leste, Papua New Guinea), and India (5, 12). A recent community-based survey conducted in the Democratic Republic of the Congo reported an overall disease prevalence of 4.7% (5). Clinical manifestations of yaws occur in three distinct stages, a primary phase characterized by a single “raspberry like” lesion or mother yaw at the site of inoculation approximately 3 to 4 weeks after infection, a secondary phase which consists of widespread dissemination of smaller skin lesions that contain high numbers of treponemes, and a tertiary phase which occurs in approximately 10% of untreated persons about 5 to 10 years after the initial infection. Tertiary yaws is characterized by destructive cutaneous lesions and bone and joint deformities (1, 4). The infectious stages of the disease occur in individuals with primary or secondary lesions who are able to transmit the disease mainly through direct skin-to-skin contact. Although reported infrequently in the Republic of the Congo, monkeypox is endemic in neighboring regions of the Democratic Republic of the Congo (7, 11). The illness is characterized by a febrile prodrome and disseminated vesiculopustular rash often involving the palms of the hands and the soles of the feet, which may have contributed to the initial suspicion of monkeypox in this instance.

Existing diagnostic PCR tests and syphilis serology cannot distinguish among the subspecies of T. pallidum (9, 10). Centurion-Lara et al. (2) used PCR-restriction fragment length polymorphism analysis to differentiate among the etiologic agents of yaws, syphilis, and bejel. However, their basis was a single nucleotide polymorphism (SNP) resulting in an Eco47III restriction site within the putative 5′-methylthioadenosine/5′-adenosylhomocysteine nucleosidase protein gene, a BsrDI restriction site within tprI, and a BsrDI or BsiEI site within the tprC (tp0117) gene. We differentiated among the subspecies using sequence analysis of a genetic signature within an intergenic spacer (IGR19) and a specific region within tprI. We also sequenced the tprC locus (data not shown), and while these data suggest that ROC1 was a T. pallidum subsp. pertenue strain, some T. pallidum subsp. pallidum strains differ from T. pallidum subsp. pertenue strains by one SNP (results in a BsiEI restriction site) in this locus, which might complicate subspecies differentiation. This is the first report describing the use of DNA sequencing to confirm a diagnosis of yaws in a patient with active skin lesions. A diagnosis of yaws could have been made on the basis of clinicoepidemiological features and reactive RPR and treponemal serological testing; however, such tests were not performed while the patient was in the hospital. Although DNA sequencing proved to be useful for identifying the unknown organism as T. pallidum subsp. pertenue, a diagnostic real-time PCR assay that can distinguish among the three subspecies of T. pallidum in a single assay is needed—especially in cases in children and because syphilis no longer occurs in areas geographically distinct from those where nonvenereal endemic treponematoses occur. In addition, yaws might be confused with other skin conditions, such as eczema, psoriasis, idiopathic keratoderma calluses, infected bites, excoriated chronic scabies, tungiasis, sarcoidosis, verrucae, and vitamin deficiencies (4).

Nucleotide sequence accession numbers.

The sequences obtained in this study were deposited in GenBank under accession no. JN411088 and JN582336 to JN582339.

Acknowledgments

We are grateful to Heather Jost for performing TREP-SURE EIA testing.

The findings and conclusions in this report are ours and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 September 2011.

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