Evaluation of real time polymerase chain reaction targeting mpb64 gene for diagnosis of extrapulmonary tuberculosis

BackgroundPaucibacillary nature of extrapulmonary tuberculosis (EPTB) has paved way for molecular methods increasingly being used for diagnosis. We undertook a study for evaluation of sensitivity and specificity of real-time polymerase chain reaction (RT-PCR) targeting mpb64 gene for diagnosis of EPTB.MethodsA total of 152 clinical samples from suspected cases of EPTB were included in this study. All samples were extracted using spin column based commercial DNA extraction kit and were subjected to RT-PCR targeting mpb64 and IS6110. Smear and culture was also done for samples whenever quantity was sufficient. Cytology report was noted from hospital information system. Receiver operating characteristic (ROC) curve analysis was done for determining cut-off Ct value for mpb64 RT-PCR. Melt curve analysis was done for samples whose cycle threshold (Ct) value was more than 37. The sensitivity and specificity of the mpb64 RT-PCR was calculated using a composite gold standard i.e., positive for one or more of the following: microscopy (including fine needle aspiration cytology (FNAC), acid-fast bacilli positivity), culture and IS6110 RT-PCR.ResultsOut of the 152 samples, 72 (47.4%) were positive for tuberculosis by composite gold standard. Samples consisted of ascitic fluid (12), CSF (35), pus (23), lymph node aspirate (35), pleural fluid (37), synovial fluid (4), urine (1), pericardial fluid (1) and tissue bits (4). Microscopy (AFB smear including lymph node aspirate) was done for 124 samples of which 43 (34.7%) were positive. Culture results were available for 79 samples, 25 (31.6%) of which were positive and 42 (27.6%) of the 152 samples were positive by IS6110 PCR. Based on ROC and melt curve analysis, mpb64 RT-PCR was able to detect 38 (52.8%) of the 72 positive samples. In comparison to IS6110 RT PCR, 4 additional cases were detected by mpb64 RT-PCR. Compared to composite gold standard mpb64 showed overall sensitivity of 52.8%.ConclusionThe mpb64 RT-PCR is highly specific or MTB and can be used as a supplemental test for diagnosis of EPTB along with other diagnostic tests. However the overall sensitivity of mpb64 RT-PCR is too low to be used as an independent test for diagnosis of EPTB. Combining the results of IS6110 RT PCR and mpb64 RT PCR improved the overall sensitivity and hence mpb64 can be used as an additional target for diagnosis of EPTB.     

Sexually transmitted infections during pregnancy

Two million of the 15 million (13.3%) new cases of sexually transmitted infections (STIs) among persons 15 to 49 years old occur in pregnant women. Access to care and a provider’s ability to assess risk, screen, and treat STIs are critical factors in preventing adverse pregnancy outcomes. Significant variations in provider STI screening and treatment practices exist despite recommended guidelines. This article reviews issues related to screening and management of common STIs during pregnancy, with emphasis on the new 2006 US Centers for Disease Control and Prevention Sexually Transmitted Diseases Treatment Guidelines and recently revised recommendations for HIV testing of pregnant women in healthcare settings.     

Predicting Discordance Between Self-reports of Sexual Behavior and Incident Sexually Transmitted Infections with African American Female Adolescents: Results from a 4-city Study

This study examined correlates of the discordance between sexual behavior self-reports and Incident Sexually Transmitted Infections. African American adolescent females (N = 964) from four U.S. cities were recruited for an HIV/STI prevention trial. Self-reported sexual behaviors, demographics, and hypothesized psychosocial antecedents of sexual risk behavior were collected at baseline, 6-, 12-, and 18-month follow-up assessments. Urine specimens were collected and tested for three prevalent STIs (chlamydia, gonorrhea, trichomonas) at each assessment. Seventeen percent of participants with a laboratory-confirmed STI reported either lifetime abstinence or recent abstinence from vaginal sex (discordant self-report). Lower STI knowledge, belief that fewer peers were engaging in sex, and belief that more peers will wait until marriage to have sex were associated with discordant reports. Discordance between self-reported abstinence and incident STIs was marked among African American female adolescents. Lack of STI knowledge and sexual behavior peer norms may result in underreporting of sexual behaviors.     

Prevalence and risk factors of bacterial enteric pathogens in men who have sex with men: A cross-sectional study at the UK's largest sexual health service

ObjectivesOutbreaks of bacterial enteric pathogens (BEPs) in men who have sex with men (MSM) associated with antimicrobial resistance are a public health concern. We investigated the prevalence and risk factors of BEPs in MSM to inform infection control.MethodsWe conducted a cross-sectional study at a London sexual health clinic between 20/12/2017 and 06/02/2018. Residual rectal swabs from MSM attending for sexually transmitted infection (STI) testing were anonymously tested for a range of BEPs using real-time PCR. A sub-set of samples were tested for the mphA gene (a marker of azithromycin resistance). Results were linked to electronic health records.ResultsBEPs were detected in 207 of 2116 participants, giving an overall prevalence of 9.8% (95% CI 8.5%-11.1%) ranging from 0.8% (0.4%-1.2%) for Shigella to 4.9% (4.0%-5.9%) for Enteroaggregative E. coli. MSM with BEPs were more likely to have a history of bacterial STIs (p = 0.010), to report more sexual partners (p<0.001), and among HIV-negative MSM, to report current HIV pre-exposure prophylaxis use (p<0.001). Gastrointestinal symptoms were rare (1.7%) and not associated with BEPs. 41.3% of MSM with BEPs and 14.1% of those without BEPs carried mphA (p<0.001). Among the former, this was associated with a history of bacterial STIs (51.5% vs 31.1%, p = 0.003).ConclusionsOne in ten MSM had a BEP detected and most did not report symptoms. MphA carriage was common, particularly among those with BEPs. Bacterial STI treatment might contribute to selection of resistant gut organisms, emphasising the need for better antimicrobial stewardship.     

Factors associated with Trichomonas vaginalis infection in reproductive-aged women attending cervical screening in southeast of Brazil

BackgroundSexually Transmitted Infections (STIs) can be caused by viruses, bacteria, and parasites. The World Health Organization estimated more than 300 million new global cases of curable STIs among individuals of reproductive age. Infection by Trichomonas vaginalis is one of the most prevalent curable STI. Despite the current treatments available, the diagnosis of T. vaginalis can be difficult, and the resistance to the treatment increased concern for the healthcare system.ObjectivesThe aim of this study was to determine the prevalence and factors associated with Trichomonas vaginalis infection among women of reproductive age attending community-based services for cervical screening.Patients and methodsA total of 1477 reproductive-aged women attending 18 Primary Health Care Units in Botucatu, Brazil, from September to October 2012, were enrolled. A structured questionnaire was used for individual face-to-face interviews for obtaining data on sociodemographic, gynecologic, and obstetrics history, sexual and hygiene practices, among others. Cervicovaginal samples were obtained for detection of T. vaginalis by culture using Diamond's medium and microscopic vaginal microbiota classification according to Nugent. A multivariable logistic regression analysis was carried out to estimate Odds Ratios (OR) and 95% Confidence Intervals (95% CI) for the association between participants’ sociodemographic, behavioral factors, and clinical factors with T. vaginalis infection.ResultsMedian age of study participants was 33 years (ranging from 18 to 50). The overall prevalence of T. vaginalis infection was 1.3% (n = 20). Several factors were independently associated with T. vaginalis infection, such as self-reporting as black or Pardo for ethnicity (OR = 2.70; 95% CI 1.03‒7.08), smoking (OR=3.18; 95% CI 1.23‒8.24) and having bacterial vaginosis (OR = 4.01; 95%CI = 1.55–10.38) upon enrollment. A protective effect of higher educational level (having high school degree) was observed (OR = 0.16; 95% CI 0.05‒0.53).ConclusionsOur data suggest that screening programs to correctly detect T. vaginalis infection can be helpful to guide prevention strategies to the community. Our study supports an association between abnormal vaginal microbiota and T. vaginalis infection.     

Pooling of urine samples for molecular detection of Chlamydia trachomatis,Neisseria gonorrhoeae and Mycoplasma genitalium as a screening strategy among young adults in Catalonia

IntroductionBacterial sexually transmitted infections (STIs) have an important impact on reproductive health, highlighting the increase in Chlamydia trachomatis infection rates among young people. To reduce the costs of STI detection, the pooling strategy is beneficial for high-throughput tests in low-prevalence populations using non-invasive samples.Objectives(1) To describe the performance of a 7-STI PCR assay using the pooling of three urine samples to detect C. trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium; (2) to estimate the cost saving of the pooling strategy; (3) to describe the prevalence, risk factors and coinfections of C. trachomatis, N. gonorrhoeae and M. genitalium in young people ≤25 years in Catalonia.Methodscross-sectional prevalence study conducted in 2016 among young people ≤25 years of age seen in sexual and reproductive health centres throughout Catalonia from pools of three urine samples. A standardized questionnaire was used to collect clinical-epidemiological and behavioural variables.Results1032 young people were tested. The prevalence of C. trachomatis, N. gonorrhoeae and M. genitalium was 8.5%, 0.6% and 3.5%, respectively. The pooling strategy provided a 33% savings in reagent costs.ConclusionsThe pooling strategy implemented for epidemiological studies in our context provides a savings that has an impact on the viability of STI detection programmes. In the same way, this study shows that C. trachomatis prevalence continues to increase in this population and, for the first time in Catalonia, the prevalence of M. genitalium in young people is shown.     

Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia

BACKGROUND:Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.METHODS:Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.RESULTS:Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.CONCLUSION:PJ real-time PCR improved detection of PJ in immunocompromised patients.     

A simple multiplex PCR for assessing prevalence of extended-spectrum β-lactamases producing Klebsiella pneumoniae in Intensive Care Units of a referral hospital in Shiraz,Iran

ObjectiveTo identify three common genes (bla_TEM, bla_SHV and bla_CTX-M) responsible for ESBL production in Klebsiella pneumoniae (K. pneumoniae)isolated from Intensive Care Units of Namazi Hospital, Shiraz, Iran.MethodsA total of 60 non-repetitive nosocomial isolates from 60 patients were selected during 2009-2010. The phenotypic identification of ESBL production was confirmed by Double Disk Synergy Test (DDST) according to CLSI guidelines. The ESBL's genotype was then analyzed by multiplex PCR of bla_TEM, bla_SHV and bla_CTX-M genes and DNA sequencing.ResultsThe primary susceptibility tests of K. pneumoniae showed that among 10 examined antibiotics, the most resistant and susceptible antibiotics identified in this study were ampicillin and imipenem, respectively. The phenotypic determination of ESBL by DDST showed that 60% (n=36) of isolates produced ESBL. Multiplex PCR of genes among K. pneumoniae isolates showed that 39% (n=18) of them have TEM, 39% (n=18) of them have both CTX-M and TEM and 13% (n=8) of them have TEM, SHV, CTX-M.ConclusionsOur findings reveal the high prevalence (60%) of ESBL producing K. pneumoniae from ICU patients along with a new pattern of bla_TEMdistribution differ from other countries.     

Identification of Streptococcus pneumoniae lytA,plyA and psaA genes in pleural fluid by multiplex real-time PCR

Introduction

The aim was to evaluate the utility of a multiplex real-time PCR to detect Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid (PF).

Detection of Hepatitis B Virus M204V Mutation Quantitatively via Real-time PCR

Background and AimsDrug-resistant DNA mutations of the hepatitis B virus (HBV) affect treatment response in chronic hepatitis B patients. We have established a new, sensitive, specific, accurate and convenient real-time PCR method to detect HBV mutations quantitatively.MethodsBlood samples were collected from patients showing viral breakthrough, primary nonresponse, or poor response during treatment, and mutations were detected via direct sequencing to assess our method. A plasmid containing the M204V mutation was synthesized and standard curves plotted.ResultsThe determination coefficient for linear correlation between Ct and log plasmid copy numbers was 0.996, where Ct value was −3.723log (DNA concentration) +48.647. Coefficients of variation indicated good reproducibility. Correctness was within tolerable bias. Limit of detection was 10³ copies/mL. Specificity, accuracy, positive predictive value and negative predictive value were 92.86%, 100%, 96.88%, 100% and 94.74%, respectively.ConclusionsThese results show that our method can be used to detect HBV M204V mutations with the advantages of sensitivity, specificity and efficiency, providing a new choice for monitoring drug resistance.     

Sexual transmission of giardiasis: A neglected route of spread?

Sexually transmitted infections (STIs) are often discussed in the context of syphilis, gonorrhea, herpes, chlamydiasis and AIDS. However, since the past 30 years of the last century, epidemiology and natural history studies have led to improved understanding of giardiasis as a STI, as a result of oral–anal sexual contact. Studies suggest that Giardia is an increasingly recognized infection that may be underdiagnosed under the STI context. Health care providers should maintain a high index of suspicion for Giardia, obtain suitable diagnostic tests to identify and screen those at high risk for this infection, institute appropriate therapy, counsel patients regarding treatment compliance, follow-up, encourage partner notification and teach strategies for preventing the transmission of this disease, including the discussion of the risk of enteric infections after oral–anal sexual contact. We summarize some data concerning the research and clinical literature on Giardia infection as a STI and identify the specific recommendations for control of giardiasis as STI that available evidence indicates can reduce its transmission.     

Prevalence,incidence and recurrence of sexually transmitted infections in HIV-negative adult women in a rural South African setting

Objective

Sexually transmitted infections (STIs), including syphilis, chlamydia, gonorrhoea and trichomoniasis, are of global public health concern. While STI incidence rates in sub-Saharan Africa are high, longitudinal data on incidence and recurrence of STIs are scarce, particularly in rural areas. We determined the incidence rates of curable STIs in HIV-negative women during 96 weeks in a rural South African setting.

Methods

We prospectively followed participants enrolled in a randomised controlled trial to evaluate the safety and efficacy of a dapivirine-containing vaginal ring for HIV prevention in Limpopo province, South Africa. Participants were included if they were female, aged 18–45, sexually active, not pregnant and HIV-negative. Twelve-weekly laboratory STI testing was performed during 96 weeks of follow-up. Treatment was provided based on vaginal discharge by physical examination or after a laboratory-confirmed STI.

Results

A total of 119 women were included in the study. Prevalence of one or more STIs at baseline was 35.3%. Over 182 person-years at risk (PYAR), a total of 149 incident STIs were diagnosed in 75 (65.2%) women with incidence rates of 45.6 events/PYAR for chlamydia, 27.4 events/100 PYAR for gonorrhoea and 8.2 events/100 PYAR for trichomoniasis. Forty-four women developed ≥2 incident STIs. Risk factors for incident STI were in a relationship ≤3 years (adjusted hazard ratio [aHR]: 1.86; 95% confidece interval [CI]: 1.04–2.65) and having an STI at baseline (aHR: 1.66; 95% CI: 1.17–2.96). Sensitivity and specificity of vaginal discharge for laboratory-confirmed STI were 23.6% and 87.7%, respectively.

Conclusion

This study demonstrates high STI incidence in HIV-negative women in rural South Africa. Sensitivity of vaginal discharge was poor and STI recurrence rates were high, highlighting the shortcomings of syndromic management in the face of asymptomatic STIs in this setting.     

Comparative evaluation of microscopy,OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner,India

ObjectiveTo evaluate microscopy, OptiMAL® and multiplex PCR for the identification of Plasmodium falciparumm (P. falciparum) and Plasmodium vivax (P. vivax) from the field isolates of Bikaner, Rajasthan (Northwest India).MethodsIn this study, a multiplex PCR (P. falciparum and P. vivax) was further developed with the incorporation of Plasmodium malariae (P. malariae) specific primer and also a positive control. The performance of microscopy, plasmodium lactate dehydrogenase (pLDH) based malaria rapid diagnostic test OptiMAL® and 18S rRNA gene based multiplex PCR for the diagnosis of P. falciparum and P. vivax was compared.ResultsThe three species multiplex PCR (P. falciparum, P. vivax and P. malariae) with an inbuilt positive control was developed and evaluated. In comparison with multiplex PCR, which showed the sensitivity and specificity of 99.36% (95% CI, 98.11%–100.00%) and 100.00% (95% CI, 100.00%–100.00%), the sensitivity and specificity of microscopy was 90.44% (95% CI, 88.84%–95.04%) and 99.22% (95% CI, 97.71%–100.00%), and OptiMAL® was 93.58% (95% CI, 89.75%–97.42%) and 97.69% (95% CI, 95.10%–100.00%). The efficiencies were 99.65%, 95.10% and 95.45% for multiplex PCR, microscopy and OptiMAL®, respectively.ConclusionsOur results raise concerns over the overall sensitivities of microscopy and OptiMAL®, when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites. This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.     

SYBR Green荧光定量粪检PCR法检测日本血吸虫感染的敏感性研究

目的建立快速、敏感、特异的检测日本血吸虫感染的SYBR Green荧光定量PCR法,准确评估其敏感性。方法将计数的日本血吸虫虫卵掺人健康水牛粪样中,制备人工阳性粪样。采用改良QIAamp DNA Stool Kit粪样DNA提取方法,提取人工阳性粪样DNA,进行SYBR Green荧光定量PCR,建立Ct值与粪样中克粪虫卵数（EPG）的关系;提取单独（不与牛阴性粪样混合）日本血吸虫虫卵DNA与虫卵生理盐水冲洗液DNA,行定量PCR检测,以对本方法进行严格质量控制。结果每200mg粪样仅含1个虫卵时,荧光定量PCR仍呈阳性,人工阳性粪样EPG的对数与PCR的Ct值存在线性关系。结论SYBR Green荧光定量粪检PCR法检测日本血吸虫虫卵DNA敏感性高,EPG对数与PCR的Ct值存在线性关系。     

Sexually transmitted infections and infectiousness beliefs among people living with HIV/AIDS: implications for HIV treatment as prevention

Objectives

Sexually transmitted infections (STIs) significantly impact the health of people living with HIV/AIDS, increasing HIV infectiousness and therefore transmissibility. The current study examined STIs in a community sample of 490 HIV‐positive men and women.

Methods

Assessments were performed using confidential computerized interviews in a community research setting.

Results

Fourteen per cent of the people living with HIV/AIDS in this study had been diagnosed with a new STI in a 6‐month period. Individuals with a new STI had significantly more sexual partners in that time period, including non‐HIV‐positive partners. Participants who had contracted an STI were significantly more likely to have detectable viral loads and were less likely to know their viral load than participants who did not contract an STI. Multivariate analysis showed that believing an undetectable viral load leads to lower infectiousness was associated with contracting a new STI.

Conclusions

Individuals who believed that having an undetectable viral load reduces HIV transmission risks were more likely to be infectious because of STI coinfection. Programmes that aim to use HIV treatment for HIV prevention must address infectiousness beliefs and aggressively control STIs among people living with HIV/AIDS.     

Detection of α-Thalassemia in China by Using Multiplex Ligation-Dependent Probe Amplification

The multiplex ligation-dependent probe amplification (MLPA) method was used to analyze 118 DNA samples from 90 α-thalassemia (α-thal) patients and 28 normal persons from Southern China, where the main causes of α-thal are three large deletions (?α^3.7, ?α^4.2, and ? ?^SEA) and two point mutations in the α-globin gene cluster on chromosome 16. The results, detected by the P140B HBA kit, were in complete concordance with the results detected by multiplex polmymerase chain reaction (m-PCR) and real-time PCR. The advantages and limitations of the techniques are discussed. We concluded that MLPA was a rapid and reliable method to determine the cause of both deletional and nondeletional α-thal in China.     

The burden and risk factors of Sexually Transmitted Infections and Reproductive Tract Infections among pregnant women in Zimbabwe

Background  

Sexually transmitted infections (STIs) and Reproductive tract infections (RTIs) are responsible for high morbidity among women. We aim to quantify the magnitude of the burden and risk factors of STI/RTI s among pregnant women in Zimbabwe.