Evaluating preanalytical sample storage parameters for nucleic acid-based detection of Neisseria gonorrhoeae.

OBJECTIVES: To ensure accurate diagnosis of infectious diseases, preanalytical factors should be considered when assessing specimen quality and subsequent test result. Accordingly, we aimed to systematically assess the effect of storage time, temperature and buffer on the analytical sensitivity of detecting the sexually transmitted pathogen, Neisseria gonorrhoeae across multiple molecular diagnostic platforms. METHODS: Cultured N. gonorrhoeae was spiked into generic and commercial storage buffers and stored at four temperatures and five time points, ranging from -20°C to 37°C, over 30 days. Samples were processed using the Alinity m STI, Xpert CT/NG and Aptima Combo 2 nucleic acid amplification assays and an in-house quantitative PCR assay. A reduction in analytical sensitivity was defined as a significant (p<0.05) increase in cycle threshold (Ct) value relative to control samples. RESULTS: In total, 2756 samples were processed, with N. gonorrhoeae detected in 99.2% of samples. With respect to time, analytical sensitivity was maintained from day 2 (113/120; 94.2%) up to day 30 (110/120; 91.7%) relative to baseline samples. With respect to temperature, analytical sensitivity was maintained from -20°C (147/150; 98.0%) up to 37°C (136/150; 90.7%) relative to baseline samples. Generic buffers, Viral Transport Medium and Amies Liquid Media showed a reduction in analytical sensitivity compared with their commercial counterparts, Aptima Multitest Swab Transport Media and Abbott Alinity transport buffer using select diagnostic assays; this reduction appeared temperature dependent, with the largest differences in median Ct values observed at 37°C (p<0.05). CONCLUSIONS: Increased prevalence of sample self-collection for sexually transmitted infections (STIs) warrants an evaluation of preanalytical sample storage variables on diagnostic testing performance. Here, across a range of time points, temperatures and storage buffers, N. gonorrhoeae was successfully detected, supporting flexibility in sample storage, and by extension the feasibility of analysing self-collected samples to improve access to STI testing.

Treponema pallidum Detection at Asymptomatic Oral, Anal, and Vaginal Sites in Adults Reporting Sexual Contact with Persons with Syphilis.

We investigated Treponema pallidum PCR positivity at mucosal sites (oral, anal, and vaginal sites) among adults who had sexual contact with a person with syphilis (syphilis contacts). All syphilis contacts had oral rinse and swab samples collected for testing. Men who have sex with men had anal swab and women had vaginal swab samples collected for testing, regardless of the presence of lesions. Of 407 persons tested, 42 (10%) had early syphilis diagnosed; of those, 19 (45%) tested positive by PCR from any anatomic site and had a positive serologic test. T. pallidum was positive from vaginal samples in 3 women, anal samples in 3 men, and oral cavity samples in 2 women and 3 men, without symptoms at those sites. Three women had no prior syphilis serologic test. T. pallidum detection at asymptomatic mucosal sites suggests early syphilis infections, particularly in cases that would conventionally be staged as latent syphilis of unknown duration.

Treponema pallidum PCR screening at mucosal sites of asymptomatic men who have sex with men taking HIV pre-exposure prophylaxis.

Early detection and treatment of syphilis will reduce the infectious period and transmission. We aimed to determine whether screening men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP) for syphilis using Treponema pallidum polymerase chain reaction (PCR) could detect syphilis before the appearance of syphilis antibodies in serology. MSM attending 3-monthly PrEP clinic visits in Melbourne, Australia, were screened with a PCR assay targeting the polA gene of T. pallidum from an anal swab and an oral rinse between November 2019 and March 2020. Participants were serologically screened for syphilis using chemiluminescence immunoassay. A total of 309 asymptomatic participants provided an anal swab and oral rinse sample for T. pallidum PCR screening. Two syphilis cases (0.6%) were detected: one man had a positive serology only; another man had T. pallidum detected by PCR from an anal swab and a positive serology. PCR positivity was 0.3% (n = 1) for anal swabs and 0% (n = 0) for oral rinse. In this study, T. pallidum PCR screening at routine PrEP clinic visits did not identify additional cases of early syphilis over serological screening performed at these visits. IMPORTANCE With the ongoing syphilis epidemic in men who have sex with men (MSM), we investigated the role of using Treponema pallidum polymerase chain reaction (PCR) testing at the oral cavity and anus in MSM taking pre-exposure prophylaxis for the early detection of syphilis. We evaluated whether the PCR tests from these mucosal sites can detect syphilis infection early, before the development of syphilis antibodies in serology. Our study found two syphilis cases among 309 MSM, and only one syphilis case had a positive anal PCR swab, although serology was positive. We conclude that additional PCR testing is likely to be expensive and would not be cost effective for individuals who regularly screen for syphilis. However, future studies with a larger sample size are required.

Clinical and laboratory aspects of condylomata lata lesions of syphilis.

OBJECTIVES: Condylomata lata are a less common but distinctive syphilitic lesion. Variable theories as to their nature and origin exist. The aim of this study was to determine the clinical and laboratory characteristics of condylomata lata by determining (1): the most closely aligned stage of syphilis, based on the rapid plasma reagin (RPR) titre; (2) symptom duration and (3) Treponema pallidum PCR cycle threshold (CT) values, as an indicator of organism load. METHODS: This was a retrospective study of patients with T. pallidum PCR-positive condylomata lata lesions, attending a clinic in Melbourne, Australia, between 2011 and 2021. Syphilis serology was undertaken and RPR titres compared between condylomata lata, primary and secondary syphilis cases. RESULTS: 51 cases with T. pallidum PCR-positive condylomata lata were included. 41 cases were in men, 40 of whom were men who have sex with men (MSM), and 10 in women. Twelve of 51 (24%) cases were in HIV-positive MSM. Thirty-three of 51 (65%) had other mucocutaneous signs of secondary syphilis; 18 (35%) had no other signs of secondary syphilis. The median RPR titre among the 51 condylomata lata cases was 1:128, compared with the median RPR titre of primary syphilis (1:4) and of secondary syphilis (1:128). The median duration of lesions was 24 (IQR 10-60) days, with no significant difference between those with and without other signs of secondary syphilis (p=0.75). Median CT values for condylomata lata (CT=31) and primary syphilis (CT=31) were significantly lower than for other secondary syphilis lesion types (CT=33), indicating higher T. pallidum loads for condylomata lata and primary lesions compared with other secondary syphilis lesion types. DISCUSSION: These findings support condylomata lata as lesions that occur during the secondary stage of syphilis and which are likely to be highly infectious.

Anal and oral detection of Treponema pallidum in men who have sex with men with early syphilis infection.

OBJECTIVES: We aimed to characterise patterns of anal and oral detection of Treponema pallidum among men who have sex with men (MSM) with early syphilis. METHODS: 200 MSM with serologically confirmed primary, secondary and early latent syphilis were tested with T. pallidum polA PCR using an anal canal swab, oral rinse, plus swabs from any anal and oral lesions in a prospective, cross-sectional study. Anal and oral T. pallidum cycle threshold values were compared between subsets of men and according to rapid plasma reagin (RPR) titre. RESULTS: Of 200 men with early syphilis, 45 and 48 had anal and oral T. pallidum detected, respectively. Cycle threshold values were lower with anal compared with oral T. pallidum whether lesions were present or not. Among 27 and 42 men with anal and oral T. pallidum detected, respectively, and no anal or oral primary lesion, frequency of detection increased with increasing RPR titre, with 95% (25/27) and 98% (41/42) of shedding from respective sites occurring with RPR titres ≥1:16. 6.5% (13/200) of men with syphilis had concurrent detection of T. pallidum from both anal and oral sites: 9/13 with secondary syphilis, 7/9 of whom had anal lesions with a median duration of 30 days (range 7-180 days). CONCLUSIONS: These data suggest T. pallidum load at the anus is higher than at the oral cavity and that a subset of men with secondary syphilis and prolonged anal lesions may be relatively infectious. Earlier detection and treatment of syphilis, when RPR titres are lower than 1:16, could potentially reduce infectiousness from anal and oral sites.

Timing of primary syphilis treatment and impact on the development of treponemal antibodies: a cross-sectional clinic-based study.

BACKGROUND: Serology is negative in a proportion of primary syphilis cases where Treponema pallidum PCR testing is positive. We aimed to identify discordant, T. pallidum PCR-positive, serology-negative primary syphilis cases and any clinical or laboratory factors associated with failure to subsequently seroconvert. METHODS: Serodiscordant primary syphilis cases that were T. pallidum PCR-positive and serology-negative (including rapid plasma reagin, T. pallidum particle agglutination, T. pallidum enzyme immunoassay or T. pallidum chemiluminescence assay) were identified from the Melbourne Sexual Health Centre electronic records between April 2011 and December 2019. Clinical and laboratory associations were examined. RESULTS: There were 814 primary syphilis cases in the study period and 38 (4.7%) were serodiscordant, 35 in men who have sex with men. Thirty-two had follow-up serology performed a median of 24 days later, of which 16 (50%) seroconverted, mostly (81%) within 6 weeks. Failure to seroconvert was significantly associated with treatment on day 1. Of the 12 cases treated on day 1, 10 (83%) failed to seroconvert compared with 6 of 20 (30%) among those who were treated after day 1. DISCUSSION: Earlier treatment of primary syphilis can prevent the development of serological markers. T. pallidum PCR can identify primary syphilis lesions before the development of serological markers and improve diagnosis of early primary syphilis lesions. Serology alone will miss a proportion of primary syphilis infections and should be repeated if a diagnosis of syphilis is being considered.

Treponema pallidum detection in lesion and non-lesion sites in men who have sex with men with early syphilis: a prospective, cross-sectional study.

BACKGROUND: Syphilis transmission is increasing, and precisely how Treponema pallidum is transmitted sexually from person to person is unclear. We aimed to determine the frequency of T pallidum shedding from potentially asymptomatic sites and the stage of infection at which shedding is most frequent in men who have sex with men (MSM), who have been disproportionately affected by syphilis. METHODS: We did a prospective, cross-sectional study in MSM recruited from Melbourne Sexual Health Centre (Melbourne, VIC, Australia). Men were eligible if they were aged 18 years or older, reported sex with men during the past 12 months, and had laboratory confirmed primary, secondary, or early latent syphilis, consistent with Australian definitions. Primary and secondary syphilis lesions were swabbed and non-lesion samples were collected via oral rinse, oral cavity swab, anal canal swab, urine, and semen. Samples were tested for T pallidum using PCR assays targeting polA (lesion and non-lesion samples) and 47 kDa (non-lesion samples only) gene targets. The primary outcome was the proportion of men with T pallidum detected from potentially asymptomatic sites-namely, the mouth, anus, urethra, and semen. FINDINGS: Between Nov 30, 2015, and May 23, 2019, 246 MSM were screened for inclusion, of whom 200 had serologically confirmed early syphilis and were included in the study: 54 (27%) of 200 had primary syphilis, 93 (47%) had secondary syphilis, and 53 (27%) had early latent syphilis. T pallidum DNA was detected in 48 (24%; 95% CI 18·3-30·5) of 200 men by oral rinse or oral lesion swab, or both, of whom 24 had no oral lesions. Oral T pallidum detection was most frequent in those with secondary syphilis compared with those at other stages of disease (41 [44%] of 93 vs seven [7%] of 107; p<0·0001), and in men with rapid plasma reagin titres of 1/64 or higher compared with those with lower titres (37 [32%] of 117 vs 11 [13%] of 83; p=0·0026). T pallidum was detected by anal canal swab or anal lesion swab, or both, in 45 (23·0%; 95% CI 17·3-29·5) of 196 men with available samples, of whom ten had no anal lesion. Furthermore, T pallidum was detected in urine samples of 12 (6·1%, 3·2-10·3) of 198 men and in semen samples from six (12·0%, 4·5-24·3) of 50 men who provided samples. Among the 93 men with secondary syphilis, 69 (74%) had T pallidum detected at any site, and 24 (26%) had detection at two or more separate sites. Among the 54 men with primary syphilis, 49 (91%) had T pallidum detected at any site, and 11 (20%) had detection at two or more separate sites. Among the 53 men with early latent syphilis, four (8%) had T pallidum detected at any site and none had T pallidum detected at two or more separate sites. INTERPRETATION: Unrecognised oral and anal shedding of T pallidum occurs in MSM with early syphilis, most frequently in those with secondary syphilis, suggesting secondary syphilis is the most infectious stage and that earlier detection and treatment of syphilis to prevent progression to the secondary stage might improve syphilis control. Future research is needed to ascertain the contribution of shedding of T pallidum from non-lesion sites to transmission of syphilis. FUNDING: Australian National Health and Medical Research Council.

Enteric pathogen infection and consequences for child growth in young Aboriginal Australian children: a cross-sectional study.

BACKGROUND: To determine the prevalence of enteric infections in Aboriginal children aged 0-2 years using conventional and molecular diagnostic techniques and to explore associations between the presence of pathogens and child growth. METHODS: Cross-sectional analysis of Aboriginal children (n = 62) residing in a remote community in Northern Australia, conducted from July 24th - October 30th 2017. Stool samples were analysed for organisms by microscopy (directly in the field and following fixation and storage in sodium-acetate formalin), and by qualitative PCR for viruses, bacteria and parasites and serology for Strongyloides-specific IgG. Child growth (height and weight) was measured and z scores calculated according to WHO growth standards. RESULTS: Nearly 60% of children had evidence for at least one enteric pathogen in their stool (37/62). The highest burden of infection was with adenovirus/sapovirus (22.9%), followed by astrovirus (9.8%) and Cryptosporidium hominis/parvum (8.2%). Non-pathogenic organisms were detected in 22.5% of children. Ten percent of children had diarrhea at the time of stool collection. Infection with two or more pathogens was negatively associated with height for age z scores (- 1.34, 95% CI - 2.61 to - 0.07), as was carriage of the non-pathogen Blastocystis hominis (- 2.05, 95% CI - 3.55 to - 0.54). CONCLUSIONS: Infants and toddlers living in this remote Northern Australian Aboriginal community had a high burden of enteric pathogens and non-pathogens. The association between carriage of pathogens/non-pathogens with impaired child growth in the critical first 1000 days of life has implications for healthy child growth and development and warrants further investigation. These findings have relevance for many other First Nations Communities that face many of the same challenges with regard to poverty, infections, and malnutrition.

Incursions of Candida auris into Australia, 2018.

Candida auris is an emerging global healthcare-associated pathogen. During July-December 2018, four patients with C. auris were identified in Victoria, Australia, all with previous overseas hospitalization. Phylogenetic analysis revealed putative transmission between 2 patients and suspected overseas acquisition in the others. Vigilant screening of at-risk patients is required.

Painful and multiple anogenital lesions are common in men with Treponema pallidum PCR-positive primary syphilis without herpes simplex virus coinfection: a cross-sectional clinic-based study.

BACKGROUND: Chancres, the hallmark of primary syphilis, are classically described as single, painless ulcers at the site of Treponema pallidum inoculation. We aimed to determine the frequency of painful or multiple anogenital lesions of primary syphilis among men, whether there was concurrent herpes simplex virus (HSV) infection and whether HIV status altered clinical presentations. METHODS: This study was conducted among men with T. pallidum PCR-positive lesions, attending a clinic in Melbourne, Australia, between 2009 and 2014. Lesions were also tested with HSV PCR, and syphilis serology undertaken. RESULTS: 183 men with T. pallidum PCR-positive primary anogenital lesions were included. 89% were men who have sex with men, and 10.9% were heterosexual. 38 men (20.8%) were HIV positive. Anal lesions were more common in HIV-positive men (34.2%) than in HIV-negative men (11.6%). Primary lesions were frequently painful (49.2%) or multiple (37.7%), and infrequently associated with HSV (2.7%). Of 37 men with both painful and multiple primary lesions, only 8% had concurrent HSV. Presentation was not significantly altered by HIV status. CONCLUSIONS: Primary syphilis lesions are often painful and/or multiple in the absence of herpes coinfection, and may be clinically misdiagnosed.

Molecular diagnosis of Pneumocystis jirovecii in patients with malignancy: clinical significance of quantitative polymerase chain reaction.

Pneumocystis jirovecii pneumonia (PJP) is increasingly seen in association with the use of new and potent immunosuppressive therapies in populations not infected with human immunodeficiency virus. Today, molecular methods are widely used to improve diagnostic yield; however, the relationship between clinical findings and quantitative polymerase chain reaction (qPCR) results is undefined. Our objective was to describe characteristics of PJP in patients with malignancies and determine if qPCR results were correlated with clinical findings. From 2007 to 2012, all patients at the Peter MacCallum Cancer Centre with positive Pneumocystis PCR were identified from a microbiology database. Clinical, radiological, and microbiological records were reviewed. PJP was defined as the presence of positive PCR for Pneumocystis on a respiratory specimen, radiological abnormalities consistent with a pneumonic process, and receipt of targeted PJP treatment. qPCR was performed on all diagnostic specimens, and values were reported according to clinical findings. Forty-five patients fulfilled inclusion criteria: 44.4% had underlying solid organ tumors and 55.6% had hematological malignancies. Nonsmall cell lung carcinoma and lymphoma were the most frequent predispositions. Shortness of breath, cough, and fever were reported in 64.4%, 48.9%, and 42.2% of the patients, respectively. Admission to the intensive care unit and mortality rates were lower than in previous reports. Overall, a relationship between other clinical features and qPCR results was not identified. In the era of routine molecular diagnostics, patients with malignancy and PJP have improved outcomes. However, there was no demonstrable relationship between qPCR results and clinical features or PCR data and outcomes.

Molecular subtyping of Treponema pallidum during a local syphilis epidemic in men who have sex with men in Melbourne, Australia.

Treponema pallidum is the causative agent of syphilis, a sexually transmitted infection of significant public health importance. Since 2000 there has been a marked increase in the number of cases of syphilis infections notified in Victoria, Australia, with the majority of cases occurring in men who have sex with men (MSM) and the highest incidence being in HIV-infected MSM. The molecular subtyping method described by Pillay et al. (A. Pillay et al., Sex. Transm. Dis. 25:408-414, 1998) has been used in this study to determine the diversity of T. pallidum subtypes circulating locally and to look for any relationship between T. pallidum subtypes and HIV status over a 6-year period (2004 to 2009). Treponema pallidum DNA was detected in 303 patient specimens (n = 3,652), and full subtyping profiles were obtained from 90 of these (from 88 patients). A total of 11 T. pallidum subtypes were identified: types 14e (28, 31.1%), 14d (15, 16.7%), 14k (13, 14.4%), 14p (12, 13.3%), 14i (7, 7.8%) 14b (6, 6.7%), 14l (5, 5.6%), and 12i, 13b, 13i, and 13e (1 each, 1.1%). This study showed a similar level of variation among circulating T. pallidum strains compared with that in other studies using the same methodology. A different mix of strains and different predominating strains have been found at each geographical study location, with type 14e emerging as the predominant local strain in Victoria. There was no detectable trend between T. pallidum subtypes and the specimen collection site or stage of syphilis (where known), nor was there any relationship between particular strains and HIV status.