Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes

Objective To detect malaria DNA in mosquitoes.Methods A nested polymerase chain reaction (nested PCR) procedure which amplifies a 121 bp DNA of a SSUrRNA gene specific to Plasmodium vivax was used.Results In laboratory-infected mosquitoes, nested PCR could detect as few as 3 sporozoites or 1 infected mosquito mixed in a group of 99 normal ones. Furthermore, no specific 121?bp band was seen with DNA templates from other malaria parasites or negative mosquitoes.Conclusion Sensitivity and specificity obtained indicated an advantage of the nested PCR over DNA probes or direct PCR for the detection of Plasmodium vivax sporozoites in mosquitoes with low-grade parasitic infections.

Diagnosis of Helicobacter pylori : What should be the gold standard?

Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages. Noninvasive tests such as serology, 13C urea breath test (UBT) and stool antigen tests are usually preferred by the clinicians. Serology has its own limitation especially in endemic areas while 13C UBT is technically very demanding. The stool antigen detection method, although specific, is usually associated with poor sensitivity. The 13C UBT is believed to be specific, but with present revelation of the fact that stomach is colonized by many other urease producing bacteria makes it questionable. Histology, culture, rapid urease test and polymerase chain reaction (PCR) are the tests which are carried out on antral biopsies collected by invasive means. Histology has been proposed to be very sensitive and specific but the question is how by simply looking the morphology of the bacteria in the microscope, one can claim that the curved bacterium is exclusively H. pylori. Rapid urease test (RUT), the doctor’s test, is also challenged because the presence of other urease producing bacteria in the stomach cannot be denied. Moreover, RUT has been reported with poor sensitivity specially, when density of the bacterium is low. Isolation of H. pylori is essential to investigate its growth requirements, antibiotic susceptibility testing, studying virulence factor to develop vaccine and many more explorations. It has also got several disadvantages i.e., special condition for transporting, media, incubation and few days waiting for the colonies to appear, apart from the speed essentially needed to process the specimens. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. The option left is PCR methods to detect H. pylori’s DNA in gastric mucosa, gastric juice, saliva, dental plaques and environmental specimens. There are speculations for false positivity due to detection of non-pylori Helicobacters due to genetic sharing; and false negativity due to low bacterial counts and presence of PCR inhibitors. However, specimen collection, transportation and processing do not require speed and special conditions. PCR based diagnosis may be considered as gold standard by designing primers extremely specific to H. pylori and targeting at least more than one conserved genes. Similarly specificity of PCR may be improved by use of internal Primers. Further, nested PCR will take care of false negatives by countering the effect of PCR inhibitors and low bacterial counts. Therefore, nested PCR based methods if performed properly, may be proposed as gold standard test.

Update on occult hepatitis B virus infection

The event of mutations in the surface antigen gene of hepatitis B virus(HBV) results in undetectable hepatitis B surface antigen with positive/negative anti-hepatitis B core(anti-HBc) antibody status in serum and this phenomenon is named occult hepatitis B infection(OBI). The presence of anti-HBc antibody in serum is an important key for OBI tracking, although about 20% of OBI cases are negative for anti-HBc antibody. The diagnosis of OBI is mainly based on polymerase chain reaction(PCR) and real-time PCR assays. However, real-time PCR is a more reliable method than PCR. OBI is a great issue for the public health problem and a challenge for the clinical entity worldwide. The persistence of OBI may lead to the development of cirrhosis and hepatocellular carcinoma. With regard to OBI complications, the screening of HBV DNA by the highly sensitive molecular means should be implemented for:(1) patients with a previous history of chronic or acute HBV infection;(2) patients co-infected with hepatitis C virus/human immunodeficiency virus;(3) patients undergoing chemotherapy or anti-CD20 therapy;(4) recipients of organ transplant;(5) blood donors;(6) organ transplant donors;(7) thalassemia and hemophilia patients;(8) health care workers;(9) patients with liver related disease(cryptogenic);(10) hemodialysis patients;(11) patients undergoing lamivudine or interferon therapy; and(12) children in time of HBV vaccination especially in highly endemic areas of HBV. Active HBV vaccination should be implemented for the close relatives of patients who are negative for OBI markers. Thus, the goal of this review is to evaluate the rate of OBI with a focus on status of high risk groups in different regions of the world.

The detection of micrometastases in the peripheral blood of patients with breast cancer for hSBEM mRNA and CD44V6 mRNA

Objective： Successful treatment of breast cancer greatly depends on the early detection of its metastasis, therefore a sensitive and specific biomarker for detecting dissemination of the cancer cells will help to achieve this goal. This study was to evaluate the prognostic significance of human small breast epithelial mucin （hSBEM） and CD44V6 in breast cancer. Methods： The expressions of hSBEM mRNA and CD44V6 mRNA were detected with nested reverse transcription polymerase chain reaction （nested RT-PCR） in 67 samples of breast cancer and adjacent normal breast tissue, 16 samples of breast benign lesions tissue, and 67 specimens of peripheral blood from patients with breast cancer, 16 specimens of benign breast lesions, 20 specimens of healthy volunteers, and 25 （each five cases） other carcinomas tissue samples, including those of gastric carcinoma, colorectal carcinoma, esophageal carcinoma, lung carcinoma, and ovary carcinoma, were analyzed for hSBEM mRNA expression by nested RT-PCR. Results： hSBEM mRNA expression was observed in 62/67 （92.54%） of breast cancer, 14/16 （87.50%） of breast benign lesions and 59/67 （88.05%） of normal breast tissue, with no significant differences between them （P 〉 0.05）. None of the samples from other cancer tissues were positive. In peripheral blood the expression of hSBEM mRNA was detected in 34/67 （50.75%） from patients with breast cancer, with significant increasing （P 〈 0.05） in the cases of metastatic disease （stage Ⅳ） and those with lymph node metastasis compared with localized disease （stage Ⅰ, Ⅱ and Ⅲ） and without lymph node metastasis, but its expression was not found in peripheral blood of patients with benign breast lesions or healthy volunteers. Although CD44V6 mRNA was significantly higher in breast cancer than in benign breast lesions tissue and normal breast tissue, its expression in peripheral blood show no significant difference （P 〉 0.05） in the patients with breast cancer （82.09%）, benign breast lesion （75.00%）, or healthy volunteers （70.00%）. The expressions of hSBEM mRNA and CD44V6 mRNA had no correlation with the age of the patients, size of primary tumor, histological type and estrogen or progestin receptor status （P 〉 0.05）. Conclusion： hSBEM mRNA, as assessed by nested RT-PCR, shows a mammary-specific and mammary-sensitive expression, and is a sensitive indicator of hematogeneous spread of breast cancer cell, while CD44V6 shows low sensitivity and specificity in detecting dissemination of breast cancer cell in peripheral blood, hSBEM mRNA is a promising molecular biomarker for detecting breast cancer micrometastases.

RELATIONSHIP BETWEEN HUMAN PARVOVIRUS B19 INFECTION AND APLASTIC ANEMIA

Objective. To explore the relationship between human parvovirus B19 (HPV B19) infection and aplastic anemia (AA) and to investigate the role of HPV B19 in the occurrence of AA. Methods. The presence of HPV B19 DNA was detected in the peripheral blood samples of 60 patients with AA (children 38 and adults 22) by nested polymerase chain reaction (PCR) assay, and 30 healthy persons were selected as control. Results. Sixteen (26.7％ ) of 60 AA cases were HPV B19 DNA positive, while all the samples in the control group were negative for HPV B19 (P = 0.000914). Among the case group, the positive rates of HPV B19 DNA were 21.4％ (6 / 28), 30.0％ (3 / 10), 20.0％ (1 / 5) and 35.3％ (6 / 17) in children acute AA (AAA), children chronic AA (CAA), adults AAA and adults CAA patients respectively, which were significantly higher than that in the control group. Furthermore, there was no remarkable difference between children AA and adults AA in the 16 HPV B19 DNA positive patients; neither was there between AAA and CAA. Conclusions. HPV B19 infection is not only correlated with the occurrence of children AAA and CAA, but also with adults AAA and CAA, and might be an important viral cause for AA in humans.

Clinical and laboratory features of PCR-confirmed periocular tuberculosis in China

Experts lack knowledge of periocular tuberculosis（TB） in China. Nested polymerase chain reaction（PCR） shows advantages in diagnosis of extrapulmonary TB. Our study aims to explore the clinical and laboratory features of PCR-confirmed periocular TB. We retrospectively reviewed medical records of presumptive periocular TB and performed nested PCR test to confirm diagnosis. Nine cases were recruited. Clinical symptoms were chronic and insidious. Eight cases achieved favorable visual acuity, while one underwent enucleation due to fungalTB panophthalmitis. Sensitivity of caseous necrosis, acidfast bacilli（AFB） staining and interferon γ release assay（T-SPOT） test are 33.3%, 44.4% and 85.7% respectively. Low lymphocyte percentage（P=0.019） and high monocytelymphocyte ratio（P=0.042） positively correlate with AFB staining. Male gender（P=0.048） and Langhans giant cell（P=0.048） positively correlate with caseous necrosis. To conclude, traditional TB ancillary tests are not as sensitive as nested PCR technique. Several factors facilitate diagnosis including male gender, decreased lymphocytes, and typical Langhans giant cells.

DNA diagnostics for reliable and universal identification of Helicobacter pylori

Reliable diagnostics are a major challenge for the detection and treatment of Helicobacter pylori(H.pylori)infection.Currently at the forefront are non-invasive urea breath test(UBT)and stool antigen test(SAT).Polymerase chain reaction(PCR)is not endorsed due to nonspecific primers and the threat of false-positives.The specificity of DNA amplification can be achieved by nested PCR(NPCR),which involves two rounds of PCR.If the primers are properly designed for the variable regions of the 16S rRNA gene,it is not difficult to develop an NPCR assay for the unambiguous identification of H.pylori.Elaborate NPCR for a 454 bp amplicon was validated on 81 clinical biopsy,stool,and saliva samples,each from the same individuals,and compared with available H.pylori assays,namely histology,rapid urease test,SAT,and 13C-UBT.The assay was much more sensitive than simple PCR,and it was equally sensitive in biopsy samples as the 13CUBT test,which is considered the gold standard.In addition,it is sufficiently specific because sequencing of the PCR products exclusively confirmed the presence of H.pylori-specific DNA.However,due to the threshold and lower abundance,the sensitivity was much lower in amplifications from stool or saliva.Reliable detection in saliva also complicates the ability of H.pylori to survive in the oral cavity aside from and independent of the stomach.The reason for the lower sensitivity in stool is DNA degradation;therefore,a new NPCR assay was developed to obtain a shorter 148 bp 16S rRNA amplicon.The assay was validated on stool samples from 208 gastroenterological patients and compared to SAT results.Surprisingly,this NPCR revealed the presence of H.pylori in twice the number of samples as SAT,indicating that many patients are misdiagnosed,not treated by antibiotics,and their problems are interpreted as chronic.Thus,it is unclear how to properly diagnose H.pylori in practice.In the first approach,SAT or UBT is sufficient.If samples are negative,the 148 bp amplicon NPCR assay should be performed.If problems persist,patients should not be considered negative,but due to threshold H.pylori abundance,they should be periodically tested.The advantage of NPCR over UBT is that it can be used universally,including questionable samples taken from patients with achlorhydria,receiving proton pump inhibitors,antibiotics,bismuth compound,intestinal metaplasia,or gastric ulcer bleeding.

Y Specific Sequence Gene Analysis of Single Fetal Nucleated Erythroblasts from the Peripheral Blood of Pregnant Women

The single cell isolation technique was used to detect fetal nucleated erythroblasts at a single cell level from the peripheral blood of pregnant women in order to investigate the feasibility of this method for noninvasive prenatal diagnosis. Single fetal nucleated erythroblasts were isolated from the peripheral blood samples from 51 pregnant women by micromanipulation techniques after density gradient centrifugation. Nested polymerase chain reaction method was used to amplify the SRY gene. It was found that the concordance rate of amplification results with real fetal sex was 82.61 %. The sensitivity and specificity were 80 % and 87.50 % respectively. It was suggested that it is feasible and promising in non invasive prenatal diagnosis to detect fetal nucleated erythroblasts at a single cell level by using micromanipulation techniques.

The clinical significance of hMAM mRNA expression in peripheral blood of patients with breast cancer

Objective: To study the clinical significance of human mammaglobin (hMAM) mRNA in peripheral blood of patients with breast cancer. Methods: The expression of hMAM mRNA was detected in peripheral blood from patients with breast cancer, breast benign lesions, healthy volunteers by nested reverse transcription polymerase chain reaction (nested-RT-PCR) method. The possible correlations of hMAM mRNA expression with clinico-pathological parameters and related molecular markers such as p53, PCNA and HER-2 were analyzed. The hMAM mRNA changed in peripheral blood after chemotherapy was observed. Results: The positive rate of hMAM mRNA was 34.0% in peripheral blood of breast cancer, hMAM mRNA was not expressed in peripheral blood from other cancers, breast benign lesions or healthy volunteers. The expression of hMAM mRNA in peripheral blood was correlated with lymph nodes status and TNM stage. After 2-3 cycles adjuvant chemotherapy, In 13 of 17 cases, the positive hMAM mRNA turned to be negative, and there was statistical difference between pre-chemo- therapy and post-chemotherapy. Conclusion: hMAM mRNA has specific expression in breast cancer, the positive expression of hMAM mRNA in peripheral blood might predict hematogenous metastatic spreading of tumor cells, and hMAM mRNA may be potential biological marker for detecting breast cancer micrometastasis.