Hsi Liu

New Tests for Syphilis: Rational Design of a PCR Method for Detection of Treponema pallidum in Clinical Specimens Using Unique Regions of the DNA Polymerase I Gene

ABSTRACT A sensitive and specific PCR method to detectTreponema pallidum in clinical specimens was developed. PCR primers were designed based on two unique features of the DNA polymerase I gene (polA). The first distinctive characteristic is that the region codes for a high cysteine content and has low homology with similar regions of DNA polymerase I gene from known microorganisms. The second unique feature is the presence of four insertions in the gene. PCR tests using primers designed on the basis these regions reacted with various pathogenic T. pallidum subspecies but did not react with nonpathogenic treponemal species or other spirochetes. An additional 59 species of bacteria and viruses, including those that cause genital ulcers, tested negative. This PCR method is extremely robust and sensitive. The detection limit is about 10 to 25 organisms when analyzed on gel. However, the analytic sensitivity can be increased by at least 1 log, to a detection limit of a single organism, when the ABI 310 Prism Genetic Analyzer is used to detect fluorescence-labeled amplicons. We further used this test in a clinical setting and compared the results with results from a previously reported multiplex-PCR test (forT. pallidum, Haemophilus ducreyi, and herpes simplex virus). We tested 112 genital ulcer specimens by the polAPCR, obtaining a sensitivity of 95.8% and a specificity of 95.7%. These results suggest that the polA PCR is applicable as a routine clinical diagnostic test for syphilis.

Molecular Subtyping of Treponema pallidum in an Arizona County with Increasing Syphilis Morbidity: Use of Specimens from Ulcers and Blood

A molecular-based subtyping system for Treponema pallidum was used during an investigation of increasing syphilis in Maricopa County, Arizona. Genital ulcer or whole blood specimens from patients with syphilis were assayed by a polymerase chain reaction (PCR) amplification of a T. pallidum DNA polymerase I gene. Positive specimens were typed on the basis of PCR amplification of 2 variable genes. In all, 41 (93%) of 44 of ulcer specimens and 4 (27%) of 15 blood specimens yielded typeable T. pallidum DNA. Twenty-four (53%) of 45 specimens were subtype 14f; other subtypes identified included 4f, 4i, 5f, 12a, 12f, 14a, 14d, 14e, and 14i. Only 2 specimens were from epidemiologically linked patients. This investigation demonstrates that multiple subtypes of T. pallidum can be found in an area with high syphilis morbidity, although 1 subtype (14f) was predominant. Four typeable specimens were from blood, a newly identified specimen source for subtyping.

Molecular subtyping of Treponema pallidum from North and South Carolina.

ABSTRACT Patients from five clinics in North and South Carolina who had lesions suggestive of primary or secondary syphilis were evaluated using molecular techniques that allow the differentiation of Treponema pallidum strains on the basis of two variable genes, tpr and arp. Lesion samples were screened for the presence of T. pallidum DNA using PCR for polA, which represents a segment of the polymerase I gene that is unique to the spirochete. Twenty-seven of 154 lesion samples were found to contain T. pallidum, 23 of which had typeable DNA. Seven molecular subtypes were found (10f, 12f, 13f, 14f, 14g, 15f, and 16f); one to four subtypes were identified at each clinic. Subtype 14f was found in 52% of the typeable specimens and was distributed in four of the five clinics. Subtype 16f was found in 22% of specimens and was concentrated at one clinic. Further data are needed to define the role of this technique in examining the epidemiology of syphilis.

The sequence of the acidic repeat protein (arp) gene differentiates venereal from nonvenereal Treponema pallidum subspecies, and the gene has evolved under strong positive selection in the subspecies that causes syphilis

Despite the completion of the Treponema pallidum genome project, only minor genetic differences have been found between the subspecies that cause venereal syphilis (ssp. pallidum) and the nonvenereal diseases yaws (ssp. pertenue) and bejel (ssp. endemicum). In this paper, we describe sequence variation in the arp gene which allows straightforward differentiation of ssp. pallidum from the nonvenereal subspecies. We also present evidence that this region is subject to positive selection in ssp. pallidum, consistent with pressure from the immune system. Finally, the presence of multiple, but distinct, repeat motifs in both ssp. pallidum and Treponema paraluiscuniculi (the pathogen responsible for rabbit syphilis) suggests that a diverse repertoire of repeat motifs is associated with sexual transmission. This study suggests that variations in the number and sequence of repeat motifs in the arp gene have clinical, epidemiological, and evolutionary significance.

Treponemal infection in nonhuman primates as possible reservoir for human yaws.

To the Editor: In 2012, the World Health Organization launched plans for a second campaign to eradicate the neglected tropical disease, yaws (1). The first campaign, conducted during the mid-20th century, was tremendously successful in terms of treatment and reduced the number of cases by 95%. However, it failed to eradicate the disease, and when local efforts to prevent new cases proved insufficient, yaws resurged in some areas. Comments on the new yaws eradication campaign have emphasized the need for sustained support and resources. Here we draw attention to an additional concern that could impede yaws eradication efforts. The success of any eradication campaign depends on the absence of a nonhuman reservoir. Smallpox had no known animal reservoir, and polio and dracunculiasis (guinea worm disease), which are currently the focus of the World Health Organization eradication campaigns, also have none. By contrast, compelling evidence suggests that yaws exists in wild nonhuman primate populations residing in regions where humans are also infected (Figure). Figure Geographic proximity between human yaws and endemic syphilis, as estimated by the World Health Organization, and locations in which treponemal infection has been identified in nonhuman primates (NHPs), Africa, 1990s. Red dots indicate infection in NHPs ... The subspecies of the bacterium Treponema pallidum that cause the non–sexually transmitted diseases yaws (subsp. pertenue infection) and endemic syphilis (subsp. endemicum infection) and the sexually transmitted infection syphilis (subsp. pallidum) are close relatives. The 3 diseases cannot be distinguished serologically. Instead, the diseases they cause are usually differentiated by clinical characteristics and geographic distribution. Whereas syphilis is a venereal disease with a worldwide distribution, yaws primarily affects children in hot and humid areas of Africa and Asia, and endemic syphilis occurs in arid regions. Because methods available to differentiate between the T. pallidum subspecies were unavailable in the past, prevalence data for yaws were sometimes vague and inaccurate. Recently, molecular tests capable of distinguishing between the subspecies by using single nucleotide polymorphisms have been developed (2,3). These tests have enabled us to learn more about the T. pallidum strains that infect wild nonhuman primates. During the 1960s, researchers reported that many baboons in West Africa were seropositive for treponemal infection (4). Since then, high levels of infection have been documented in other monkey species in West Africa and in great apes (5). Recently, we documented T. pallidum infection in olive baboons (Papio anubis) at Lake Manyara National Park in Tanzania (6). In West Africa, clinical signs of infection in nonhuman primates are usually mild, if present at all, consisting of small lesions around the muzzle, eyelids, and armpits (4). A recent survey in 2013 at Parc National du Niokolo-Koba, Senegal, revealed T. pallidum antibodies in Guinea baboons (P. papio) with no signs of infection (S. Knauf et al, unpub. data). By contrast, severe manifestations resembling tertiary-stage yaws have been reported in wild gorillas (5). In terms of genetic distance, studies thus far indicate that the organisms infecting baboons in West and East Africa closely resemble T. pallidum subsp. pertenue, the agent responsible for yaws in humans (2,7). In fact, the genome sequence of a T. pallidum strain collected from a baboon in Guinea indicates that it should be considered a T. pallidum subsp. pertenue strain (8). Infection has been confirmed by serologic tests in a variety of nonhuman primate species in the yaws belt of Africa and by PCR in baboons from East and West Africa (Figure). The high prevalence of nonhuman primate infection in areas of tropical Africa where yaws is common in humans (Figure) suggests that cross-species infection may occur. Decades ago, researchers reported that the Fribourg-Blanc simian strain, collected in Guinea, can cause sustained infection in humans after inoculation (9). Such experiments are ethically questionable and the details given are scant, but this work suggests that simian strains have zoonotic potential. Additional research is needed to determine whether interspecies transmission of T. pallidum occurs under natural conditions. Bush meat preparation is common in many African countries and a major source of zoonotic infection. It involves frequent skin-to-skin contact, which is the preferred mode of transmission for yaws. Insects also have been proposed to be vectors of infection, although this has not been documented (10). If evidence of interspecies yaws transmission, either direct or by vector, is discovered, then nonhuman primates may be a major reservoir of infection for humans. Additional studies comparing human and simian strains may show whether zoonotic transmission of T. pallidum occurs frequently, an important consideration with regard to disease eradication and the conservation of great apes and other endangered nonhuman primates. To eradicate yaws, all host species and any possible reservoirs need to be taken into account. We, like the rest of the world, want the second yaws eradication campaign to succeed and hope that nonhuman primate infection will be evaluated as a factor in disease transmission. Technical Appendix: Additional references cited in the Figure. Click here to view.(131K, pdf)

Assessment of Etest as an Alternative to Agar Dilution for Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae

ABSTRACT We studied whether the Etest can be used as an alternative to agar dilution to determine antimicrobial susceptibilities of ceftriaxone, cefixime, and cefpodoxime in Neisseria gonorrhoeae surveillance. One hundred fifteen clinical and laboratory isolates of N. gonorrhoeae were tested following the Clinical Laboratory Improvement Amendments (CLIA)-approved CLSI standard agar dilution method and, separately, by the Etest according to the manufacturers recommendations. The MICs were determined and compared. Ten laboratory-generated mutants were used to simulate substantially nonsusceptible specimens. The Etest and agar dilution methods were well correlated. Statistical tests produced regression R 2 values of 88%, 82%, and 85% and Pearson correlation coefficients of 92%, 91%, and 92% for ceftriaxone, cefixime, and cefpodoxime, respectively. When paired comparisons were made, the two tests were 88.7%, 80%, and 87% within 1 log2 dilution from each other for ceftriaxone, cefixime, and cefpodoxime, respectively. The within-2-log2 agreements were 99.1%, 98.3%, and 94.8% for ceftriaxone, cefixime, and cefpodoxime, respectively. Notwithstanding the good correlations and the within-2-log2 general agreement, the Etest results produced slightly lower MICs than the agar dilution results. In conclusion, we found that the Etest can be effectively used as an alternative to agar dilution testing to determine the susceptibility of N. gonorrhoeae to ceftriaxone, cefixime, and cefpodoxime, although we recommend further research into extremely resistant isolates. For isolates within the typical range of clinical MICs, reexamination of the Etest interpretation of susceptible and nonsusceptible categories would likely allow for successful transition from agar dilution to the Etest.

Validation of serological tests for the detection of antibodies against Treponema pallidum in nonhuman primates.

There is evidence to suggest that the yaws bacterium (Treponema pallidum ssp. pertenue) may exist in non-human primate populations residing in regions where yaws is endemic in humans. Especially in light of the fact that the World Health Organizaiton (WHO) recently launched its second yaws eradication campaign, there is a considerable need for reliable tools to identify treponemal infection in our closest relatives, African monkeys and great apes. It was hypothesized that commercially available serological tests detect simian anti-T. pallidum antibody in serum samples of baboons, with comparable sensitivity and specificity to their results on human sera. Test performances of five different treponemal tests (TTs) and two non-treponemal tests (NTTs) were evaluated using serum samples of 57 naturally T. pallidum-infected olive baboons (Papio anubis) from Lake Manyara National Park in Tanzania. The T. pallidum particle agglutination assay (TP-PA) was used as a gold standard for comparison. In addition, the overall infection status of the animals was used to further validate test performances. For most accurate results, only samples that originated from baboons of known infection status, as verified in a previous study by clinical inspection, PCR and immunohistochemistry, were included. All tests, TTs and NTTs, used in this study were able to reliably detect antibodies against T. pallidum in serum samples of infected baboons. The sensitivity of TTs ranged from 97.7-100%, while specificity was between 88.0-100.0%. The two NTTs detected anti-lipoidal antibodies in serum samples of infected baboons with a sensitivity of 83.3% whereas specificity was 100%. For screening purposes, the TT Espline TP provided the highest sensitivity and specificity and at the same time provided the most suitable format for use in the field. The enzyme immune assay Mastblot TP (IgG), however, could be considered as a confirmatory test.

Comparing the disk-diffusion and agar dilution tests for Neisseria gonorrhoeae antimicrobial susceptibility testing

BackgroundWe assessed the validity of testing for antimicrobial susceptibility of clinical and mutant Neisseria gonorrhoeae (GC) isolates by disk diffusion in comparison to agar dilution, and Etest® (bioMerieux, France), respectively, for three third generation extended spectrum cephalosporins (ESC): ceftriaxone (CRO), cefixime (CFX), and cefpodoxime (CPD).MethodsOne hundred and five clinical isolates and ten laboratory-mutants were tested following Clinical Laboratory Standard Institute (CLSI) and manufacturer’s standards for each of the three methods. The measured diameters by the disk diffusion method were tested for correlation with the MIC values by agar dilution. In addition, comparisons with the Etest® were made. Categorical results for concordance, based on standard CLSI cutoffs, between the disk diffusion and the other two methods, respectively, were tested using the Chi-square statistics. Reproducibility was tested for CFX across a 6-month interval by repeated disk tests.ResultsAcross all 115 specimens, the disk diffusion tests produced good categorical agreements, exhibiting concordance of 93.1%, 92.1%, and 90.4% with agar dilution and 93.0%, 92.1%, and 90.4% with Etest®, for CRO, CFX, and CPD, respectively. Pearson correlations between disk-diffusion diameters and agar dilution MIC’s were -0.59, -0.67, and -0.81 for CRO, CFX, and CPD, respectively. The correlations between disk diffusion and Etest® were -0.58, -0.73, and -0.49. Pearson correlation between the CFX disk readings over a 6-month interval was 91%.ConclusionsDisk diffusion tests remain to be a useful, reliable and fast screening method for qualitative antimicrobial susceptibility testing for ceftriaxone, cefixime, and cefpodoxime.

High Prevalence of Antibodies against the Bacterium Treponema pallidum in Senegalese Guinea Baboons (Papio papio)

The bacterium Treponema pallidum is known to cause syphilis (ssp. pallidum), yaws (ssp. pertenue), and endemic syphilis (ssp. endemicum) in humans. Nonhuman primates have also been reported to be infected with the bacterium with equally versatile clinical manifestations, from severe skin ulcerations to asymptomatic. At present all simian strains are closely related to human yaws-causing strains, an important consideration for yaws eradication. We tested clinically healthy Guinea baboons (Papio papio) at Parc National Niokolo Koba in south eastern Senegal for the presence of anti-T. pallidum antibodies. Since T. pallidum infection in this species was identified 50 years ago, and there has been no attempt to treat non-human primates for infection, it was hypothesized that a large number of West African baboons are still infected with simian strains of the yaws-bacterium. All animals were without clinical signs of treponematoses, but 18 of 20 (90%) baboons tested positive for antibodies against T. pallidum based on treponemal tests. Yet, Guinea baboons seem to develop no clinical symptoms, though it must be assumed that infection is chronic or comparable to the latent stage in human yaws infection. The non-active character is supported by the low anti-T. pallidum serum titers in Guinea baboons (median = 1:2,560) versus serum titers that are found in genital-ulcerated olive baboons with active infection in Tanzania (range of medians among the groups of initial, moderate, and severe infected animals = 1:15,360 to 1:2.097e+7). Our findings provide evidence for simian infection with T. pallidum in wild Senegalese baboons. Potentially, Guinea baboons in West Africa serve as a natural reservoir for human infection, as the West African simian strain has been shown to cause sustainable yaws infection when inoculated into humans. The present study pinpoints an area where further research is needed to support the currently on-going second WHO led yaws eradication campaign with its goal to eradicate yaws by 2020.