Bacterial etiology of sexually transmitted infections at a STI clinic in Ghana; use of multiplex real time PCR

Background: Most sexually transmitted infection (STI) management efforts focus on the syndromic approach to diagnose and treat patients. However, most women with STIs have been shown to be entirely asymptomatic, or if symptoms exist, are often missed when either clinical or conventional bacteriologic diagnostic tools are employed.Methods: We assessed the performance of a multiplex real time PCR assay to describe other potential pathogens that could be missed by conventional bacteriological techniques in 200 women attending a routine STI clinic in Kumasi, Ghana.Results: Although a total 78.00% of the women were asymptomatic, 77.1% of them tested positive for at least one bacterial STI pathogen. Mycoplasma genitalium was the most commonly detectable pathogen present in 67.5% of all women. Of those testing positive, 25.0% had single infections, while 38.0% and 19.5% had double and triple infections respectively. Altogether, 86.54% and 90.91% of the symptomatic and asymptomatic women respectively tested positive for at least one pathogen (p<0.05). There were no significant associations (p<0.05) between the clinical manifestations of the symptomatic women and the pathogens detected in their samples.Conclusions: Our study confirmed the importance of complementing the syndromic approach to STI management with pathogen detection and most importantly recognise that STIs in women are asymptomatic and regular empirical testing even for both symptomatic and asymptomatic patients is critical for complete clinical treatment.Funding: EOD (Ellis Owusu-Dabo Research working group, KCCR)Keywords: Etiology, Syndromic, Sexually Transmitted Infections, Multiplex real time PC

Comparative Study of the Sensitivity of Different Diagnostic Methods for the Laboratory Diagnosis of Buruli Ulcer Disease

Background. Several diagnostic laboratory methods are available for case confirmation of Buruli ulcer disease. This study assessed the sensitivity of various diagnostic tests in relation to clinical presentation of the disease, type of diagnostic specimen, and treatment history. Methods. Swab samples, 3-mm punch biopsy tissue specimens, and surgically excised tissue specimens from 384 individuals with suspected Buruli ulcer disease were obtained at 9 different study sites in Ghana and were evaluated with dry reagent-based polymerase chain reaction (PCR), microscopic examination, culture, and histopathological analysis. The study subjects presented with nonulcerative and ulcerative lesions and were divided into 3 treatment groups: (1) previously untreated patients scheduled for antimycobacterial treatment, (2) patients treated with surgery alone, and (3) patients treated with surgery in combination with previous antimycobacterial treatment. Results. Of 384 suspected cases of Buruli ulcer disease, 268 were confirmed by at least 1 positive test result. The overall sensitivity of PCR (85%) was significantly higher than that of microscopic examination (57%) and culture (51%). After data were stratified by treatment group, type of lesion, and diagnostic specimen type, analysis revealed that PCR of 3-mm punch biopsy tissue specimens (obtained from previously untreated nonulcerative lesions) and of swab samples (obtained from previously untreated ulcers) had the highest diagnostic sensitivity (94% and 90%, respectively). Although duration of the disease did not significantly influence the sensitivity of any test, previous antimycobacterial treatment was significantly associated with decreased sensitivity of PCR and culture. Conclusions. Across all subgroups, PCR had the highest sensitivity. PCR assessment of 3-mm punch biopsy tissue specimens proved to be the best diagnostic tool for nonulcerative lesions, and PCR assessment of swab samples was the best diagnostic tool for ulcerative lesions. For monitoring of antimycobacterial treatment success within controlled trials, however, only culture is appropriat

Efficiency of Fine-Needle Aspiration Compared with Other Sampling Techniques for Laboratory Diagnosis of Buruli Ulcer Disease ▿

In accordance with recent WHO recommendations, this study evaluates the sensitivities of PCR and microscopy for fine-needle aspiration (FNA) versus techniques involving swabs and punch biopsy specimens and suggests that FNA can replace punch biopsies for nonulcerative lesions and may serve as an alternative for ulcerative lesions in cases where scarred edges prevent the collection of swabs

Antimicrobial treatment for early, limited Mycobacterium ulcerans infection: a randomised controlled trial

Background Surgical debridement was the standard treatment for Mycobacterium ulcerans infection (Buruli ulcer disease) until WHO issued provisional guidelines in 2004 recommending treatment with antimicrobial drugs (streptomycin and rifampicin) in addition to surgery. These recommendations were based on observational studies and a small pilot study with microbiological endpoints. We investigated the efficacy of two regimens of antimicrobial treatment in early-stage M ulcerans infection. Methods In this parallel, open-label, randomised trial undertaken in two sites in Ghana, patients were eligible for enrolment if they were aged 5 years or older and had early (duration Findings Four patients were lost to follow-up (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, three). Since these four participants had healed lesions at their last assessment, they were included in the analysis for the primary endpoint. 73 (96%) participants in the 8-week streptomycin group and 68 (91%) in the 4-week streptomycin plus 4-week clarithromycin group had healed lesions at 1 year (odds ratio 2.49, 95% CI 0.66 to infinity; p=0.16, one-sided Fisher's exact test). No participants had lesion recurrence at I year. Three participants had vestibulotoxic events (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, two). One participant developed an injection abscess and two participants developed an abscess close to the initial lesion, which was incised and drained (all three participants were in the 4-week streptomycin plus 4-week clarithromycin group). Interpretation Anti mycobacterial treatment for M ulcerans infection is effective in early, limited disease. 4 weeks of streptomycin and rifampicin followed by 4 weeks of rifampicin and clarithromycin has similar efficacy to 8 weeks of streptomycin and rifampicin; however, the number of injections of streptomycin can be reduced by switching to oral clarithromycin after 4 weeks

Comparative Study of the Sensitivity of Different Diagnostic Methods for the Laboratory Diagnosis of Buruli Ulcer Disease

Background. Several diagnostic laboratory methods are available for case confirmation of Buruli ulcer disease. This study assessed the sensitivity of various diagnostic tests in relation to clinical presentation of the disease, type of diagnostic specimen, and treatment history. Methods. Swab samples, 3-mm punch biopsy tissue specimens, and surgically excised tissue specimens from 384 individuals with suspected Buruli ulcer disease were obtained at 9 different study sites in Ghana and were evaluated with dry reagent-based polymerase chain reaction (PCR), microscopic examination, culture, and histopathological analysis. The study subjects presented with nonulcerative and ulcerative lesions and were divided into 3 treatment groups: (1) previously untreated patients scheduled for antimycobacterial treatment, (2) patients treated with surgery alone, and (3) patients treated with surgery in combination with previous antimycobacterial treatment. Results. Of 384 suspected cases of Buruli ulcer disease, 268 were confirmed by at least 1 positive test result. The overall sensitivity of PCR (85%) was significantly higher than that of microscopic examination (57%) and culture (51%). After data were stratified by treatment group, type of lesion, and diagnostic specimen type, analysis revealed that PCR of 3-mm punch biopsy tissue specimens (obtained from previously untreated nonulcerative lesions) and of swab samples (obtained from previously untreated ulcers) had the highest diagnostic sensitivity (94% and 90%, respectively). Although duration of the disease did not significantly influence the sensitivity of any test, previous antimycobacterial treatment was significantly associated with decreased sensitivity of PCR and culture. Conclusions. Across all subgroups, PCR had the highest sensitivity. PCR assessment of 3-mm punch biopsy tissue specimens proved to be the best diagnostic tool for nonulcerative lesions, and PCR assessment of swab samples was the best diagnostic tool for ulcerative lesions. For monitoring of antimycobacterial treatment success within controlled trials, however, only culture is appropriate

Study participants, clinical information, and diagnostic results.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t006" target="_blank">Table 6</a> shows suspected BUD cases with ulcerative lesions enrolled in the pre-treatment cohort (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-g001" target="_blank">Figure 1</a>), clinical information, and diagnostic results. Swab samples from 24 suspected BUD cases were subjected to 16S rRNA RT/IS<i>2404</i> qPCR viability assay (swab 1 in PANTA), microscopic examination and enumeration of acid fast bacilli (AFB) following Ziehl-Neelsen staining (swab 2, direct smear), and conventional IS<i>2404</i> dry-reagent-based (DRB) PCR (swab 3 in Cell Lysis Solution [Qiagen]). 18 patients were laboratory confirmed by IS<i>2404</i> qPCR and 15 out of those were RNA positive; the quantification by IS<i>2404</i> qPCR revealed a bacillary load (1–2 bacilli per sample) below the lower limit of detection of the RNA assay for samples from three RNA negative patients. All samples from six IS<i>2404</i> qPCR negative study participants were also RNA negative. Direct correlation of AFB enumeration with IS<i>2404</i> qPCR quantification is not feasible due to inhomogeneous distribution of <i>M. ulcerans</i> in different clinical samples. NA, not applicable; Neg, negative test result; Pos, positive test result.</p>a<p>Results of the 16S rRNA RT/IS<i>2404</i> qPCR viability assay. Clinical swab samples in PANTA were directly processed at KCCR, and <i>M. ulcerans</i> DNA and cDNA were transported to DITM and subjected to qPCR.</p>b<p>Routine diagnostics were conducted following standardized procedures at KCCR <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Beissner1" target="_blank">[3]</a>.</p>c<p>No., consecutive number of study participants.</p>d<p>Yes, IS<i>2404</i> qPCR confirmed BUD patients; No, IS<i>2404</i> negative study participants.</p>e<p>Duration of disease before presentation of study participants in weeks.</p>f<p>Category of lesion according to the World Health Organization's clinical criteria <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-World1" target="_blank">[1]</a>.</p>g<p>Results of the IS<i>2404</i> qPCR with corresponding cycle threshold (Ct)-values.</p>h<p>The bacillary load in the respective swab samples (No. 2) was estimated on the basis of IS<i>2404</i> quantification given an IS<i>2404</i> copy number of 209 copies per <i>M. ulcerans</i> genome <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Fyfe1" target="_blank">[9]</a>. For bacterial numbers <10 ranges were estimated.</p>i<p>Results of the 16S rRNA RT-qPCR.</p>k<p>MIC, microscopic detection and enumeration of AFB was conducted at KCCR including external quality assurance by DITM. The following scale was applied: 0 = negative, +1 = 10–99 AFB/100 fields, +2 = 1–10 AFB/1 field, +3 = more than 10 AFB/1 field.</p>l<p>PCR, conventional, single target gel-based IS<i>2404</i> DRB PCR.</p

Standard curve and limit of detection of the IS<i>2404</i> qPCR.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-g004" target="_blank">Figure 4</a> shows mean Ct-values of calibration standards and clinical samples plotted versus the quantified copy number of IS<i>2404</i>. Cloned IS<i>2404</i> templates were used as standards (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t005" target="_blank">Table 5</a>). Log 10 fold serial dilutions (<i>n</i> = 8) were prepared ranging from 2E+8 to 20 copies of the IS<i>2404</i> (PCR template: 2 µl) and were subjected to the IS<i>2404</i> qPCR in quadruplicate to generate a calibration curve. The regression line was y = −3.35x+39.10 with a coefficient of correlation >0.99 and the efficiency was E = 0.97. The analytical sensitivity was determined as limit of detection (LOD) by subjecting 10 aliquots of a dilution series containing 10, 5, 4, 3, 2, or 1 copy of the IS<i>2404</i> to the assay. The LOD was 2 copies of the target sequence.</p

Specificity of 16S rRNA and IS<i>2404</i> qPCR assays.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t002" target="_blank">Table 2</a> shows DNA extracts from closely related mycobacterial species and bacteria potentially contaminating the human skin subjected to the combined 16S rRNA RT/IS<i>2404</i> qPCR viability assay and the corresponding test results. Mycobacterial species were selected according to their respective genetic contiguousness to <i>M. ulcerans</i> Agy99 (GenBank accession no. CP000325.1) within the 16S rRNA gene sequences as determined by BLASTN analysis (GenBank, NCBI) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Benson1" target="_blank">[13]</a>. <i>M.</i>, <i>Mycobacterium</i>; <i>E.</i>, <i>Escherichia</i>; <i>P., Propionibacterium</i>; <i>Staph</i>., <i>Staphylococcus</i>; <i>Str</i>., <i>Streptococcus</i>. While in-silico analysis revealed that the combined 16S rRNA RT/IS<i>2404</i> assay will also amplify mycolactone-producing mycobacteria (MPM) other than <i>M. ulcerans</i> (e.g., <i>M. pseudoshottsii</i>, <i>M. liflandii</i>, and the environmental <i>M. marinum</i> [GenBank accession No. NR_042988.1, AY500838.1, and AF456241.1, respectively]), these MPM species were not included in specificity testing.</p>a<p>DNA extracts that were not available at the DITM were provided by the National Reference Center (NRZ) for Mycobacteria, Borstel, Germany, and the Max von Pettenkofer-Institute (MVP), Ludwig-Maximilians University, Munich, Germany.</p>b<p>The respective primary patient isolates were considered as ^ppathogenic bacteria or as ^ccommensals/contaminants of clinical samples.</p>d<p>Results of the 16S rRNA RT-qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.</p>e<p>Results of the IS<i>2404</i> qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.</p

Primers and probes.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t003" target="_blank">Table 3</a> indicates primers and probes designed for the 16S rRNA RT-qPCR, the primers described by Fyfe et al., and a re-designed hydrolysis probe used for the amplification, detection, and quantification of IS<i>2404</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Fyfe1" target="_blank">[9]</a>.</p>a<p>TF, forward primer; TR, reverse primer; TP2, hydrolysis probe (TibMolBiol, Berlin, Germany).</p>b<p>16S rRNA, gene for the ribosomal 16S RNA detected as 16S cDNA; IS<i>2404</i>, insertion sequence <i>2404</i>.</p>c<p>Nucleotide positions are provided for the first (IS<i>2404</i>) or single (16S rRNA) copy of the respective amplicon in <i>M. ulcerans</i> Agy99 (GenBank accession no. CP000325.1) as determined by BLASTN analysis within GenBank (NCBI) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd.0001756-Benson1" target="_blank">[13]</a>.</p>d<p>bp, base pairs.</p>e<p>6 FAM, 6-Carboxyfluorescein fluorescent dye; BBQ, BlackBerry Quencher.</p>f<p>Primers T13 (forward) and T39 (reverse) were used for the amplification of a 935-bp region of the <i>M. ulcerans</i> 16S rRNA gene, encompassing the region amplified by qPCR primers MU16S TF and MU16S TR, to generate single copy replicates. Furthermore, these primers were used for sequencing of the <i>M. ulcerans</i> 16S rRNA gene (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001756#pntd-0001756-t001" target="_blank">Table 1</a>).</p