DNA Polymerase Chain Reaction Testing Research Articles

245 Outcome of early infant diagnosis in exposed infants in Northern Nigeria

Background: The rate of vertical transmission of HIV-1 in the Northern Nigeria was 35%. This prompted the intervention of PMTCT and Early Infant Diagnosis program in Northern Nigeria. Aim Evaluating the outcome of early infant diagnosis of HIV in exposed infants in Northern Nigeria and the rate of mother to child transmission of HIV in the region. Method: A total of 1236 HIV exposed infants from the Northern Nigeria, (Kano, Kaduna, Katsina, Jigawa, and Sokoto states of Nigeria) who had Dry Blood Spot (DBS) HIV-1 DNA Polymerase Chain Reaction test were enrolled for the study. The age of enrollment was between 6 weeks and 18 months with mean age of 7 months. Roche HIV 1 DNA version 1.5 method was used for analysis. Result: Total number of women who had PMTCT was 1,036 (83.8%), 182 (14.7%) had no PMTCT and 18 (1.5%) women had unknown PMTCT status. 124 (10%) children were HV-1 positive and 1112 (90%) were HIV-1 negative. 109 (87.9%) of those whose mothers had PMTCT were HIV-1 positive and those whose mother did not have PMTCT had 15 (12.1)% HIV-1 positive results. 520 (42%) children were not breast fed NBF and 495 (95.6%) of this children were HIV-1 DNA PCR negative and 25 (4.4%) children were HIV-1 DNA PCR positive. 437 (84%) of the NBF had PMTCT(PMTCT +) while 70 (16%) had no PMTCT (PMTCT−). 345 ceased breast feeding (BF Ceased) before 6weeks and 18 months and after their babies were tested for HIV. 285 (82.8%) children from this BF ceased mothers were tested HIV-1 DNA PCR negative and 60 (17.2%) was HIV-1 DNA PCR positive. Conclusion: Through the PMTCT and EID interventions, HIV-1 prevalence rate among the children of exposed mothers was dropped from 35% to 10% in Northern Nigeria. Attitude of women to absolute breast feeding in the Northern Nigeria have been changed for the benefit of the children. Through the EID program, children's status are known and early ARV intervention is instituted. These result in decline mortality rate and more and more children are living meaningful lives in this part of the world.

Routine offering of HIV testing to hospitalized pediatric patients at university teaching hospital, Lusaka, Zambia: acceptability and feasibility.

The difficulties diagnosing infants and children with HIV infection have been cited as barriers to increasing the number of children receiving antiretroviral therapy worldwide. We implemented routine HIV antibody counseling and testing for pediatric patients hospitalized at the University Teaching Hospital, a national reference center, in Lusaka, Zambia. We also introduced HIV DNA polymerase chain reaction (PCR) testing for early infant diagnosis. Caregivers/parents of children admitted to the hospital wards were routinely offered HIV counseling and testing for their children. HIV antibody positive (HIV+) children <18 months of age were tested with PCR for HIV DNA. From January 1, 2006, to June 30, 2007, among 15,670 children with unknown HIV status, 13,239 (84.5%) received counseling and 11,571 (87.4%) of those counseled were tested. Overall, 3373 (29.2%) of those tested were seropositive. Seropositivity was associated with younger age: 69.6% of those testing HIV antibody positive were <18 months of age. The proportion of counseled children who were tested increased each quarter from 76.0% in January to March 2006 to 88.2% in April to June 2007 (P < 0.001). From April 2006 to June 2007, 1276 PCR tests were done; 806 (63.2%) were positive. The rate of PCR positivity increased with age from 22% in children <6 weeks of age to 61% at 3-6 months and to 85% at 12-18 months (P < 0.001). Routine counseling and antibody testing of pediatric inpatients can identify large numbers of HIV-seropositive children in high prevalence settings. The high rate of HIV infection in hospitalized infants and young children also underscores the urgent need for early infant diagnostic capacity in high prevalence settings.

Early Diagnosis of Human Immunodeficiency Virus in Infants Using Polymerase Chain Reaction on Dried Blood Spots in Botswana's National Program for Prevention of Mother-to-Child Transmission

Botswana has high antenatal human immunodeficiency virus (HIV) prevalence (33.4%). The public health system provides free services for prevention of mother to child transmission of HIV (PMTCT) and antiretroviral therapy, which can reduce vertical HIV transmission from 35% to <5%. Infant HIV diagnosis is challenging in resource-limited settings, and HIV prevalence among HIV-exposed infants in Botswana is unknown. Dried blood spot (DBS) polymerase chain reaction (PCR) provides a feasible method to assess PMTCT programs and identify HIV-infected children. We trained staff in 15 clinics and a hospital to obtain DBS on HIV-exposed infants age 6 weeks to 17 months receiving routine care. Samples were sent to the national HIV reference laboratory. Roche Amplicor 1.5 DNA PCR testing was performed. Between June-December 2005, 1931 HIV-exposed infants age 6 weeks to 17 months were tested for HIV, of whom 136 (7.0%) were HIV infected. Among infants <or=8 weeks old, 27 of 544 (5.0%) were HIV infected. Among infants tested in clinics (primarily during routine health visits), 65 of 1376 (4.7%) were infected; among infants tested in the hospital, 71 of 555 (12.8%) were infected. Collection and testing of DBS was successfully integrated into routine infant care in the public health system. HIV prevalence among infants in the Botswana PMTCT program is low. National expansion of infant DBS PCR in Botswana is planned.

Falso negativo en el diagnóstico de VIH-1

We report a case of a false negative diagnosis of HIV-1 infection in an African girl. Two HIV-1 DNA polymerase chain reaction (PCR) tests were negative at the second and fourth months of life. Because anti-HIV antibodies persisted when the patient was 18 months old, the HIV-1 RNA PCR test was performed with a positive result, confirming HIV-1 non-B subtype, recombinant A-G. The prevalence of non-B HIV-1 subtypes are increasing in Spain, which could be related to the phenomenon of immigration. Approximately one-third of HIV-infected foreigners have non-B subtypes and the percentage increases to 70 % of the African population in Spain. In non-B HIV-1 subtypes, false negative results and inconsistencies between viral load and CD4 count are more frequent. These subtypes also show a higher rate of resistance to protease inhibitors, which can have therapeutic implications.

Is There a Difference in the Efficacy of Peripartum Antiretroviral Regimens in Reducing Mother-to-Child Transmission of HIV in Africa?

There are positive reports about the ability of antiretroviral agents to prevent mother-to-child transmission (MTCT) of HIV. The authors undertook a pooled analysis of African breast-feeding mothers from clinical trials of various antiretroviral regimens to directly compare their efficacy in lowering the prevalence of MTCT 6 weeks postpartum. A total of 3465 live-born singleton infants participating in 6 randomized clinical trials made up the study population. There were 2 West African zidovudine (ZDV)/placebo trials; 2 regimens of ZDV + lamivudine (3TC) and placebo (pre-/intra-/postpartum and intra-/postpartum); nevirapine (NVP) and ZDV + 3TKC; NVP and ultra-short ZDV; and a vitamin A trial as a placebo arm. Peripartum HIV infection was diagnosed from a positive RNA or DNA polymerase chain reaction test before 2 months of age. In multivariable logistic regression analysis using placebo for comparison and adjusting for numerous factors (maternal CD4 cell count, breast feeding, low birth weight, geographic area), the lowest rates of MTCT 6 weeks postpartum were observed with ZDV + 3TC pre-/intra-/postpartum with an adjusted odds ratio (AOR) of 0.23. Next most effective, in declining order, were ZDV + 3TC intra-/postpartum (AOR, 0.49); antenatal ZDV short (AOR, 0.55); and NVP (AOR, 0.60). Compared with NVP, only the longest regimen of ZDV + 3TC was significantly more effective (AOR, 0.39). The risk of MTCT was not influenced by cesarean delivery or gender on univariate analysis. In multivariable analysis, the 6-week rate of MTCT was significantly associated with the maternal CD4 cell count. These findings are consistent with current World Health Organization guidelines that consider single-dose NVP and short-course ZDV to be equivalent regimens. Also evident from this study is that the regimen of ZDV + 3TC is more effective than any single antiretroviral agent.

Comparison of human papillomavirus DNA polymerase chain reaction with cervical cytology as a screening test for the detection of cervical intraepithelial neoplasia

Objective：To evaluate the result of the human papillomavirus (HPV) DNA polymerase chain reaction (PCR) in comparison with cervical cytology for the detection of cervical intraepithelial neoplasia (CIN) and invasive cancer, and to establish the more economic and effective screening method for CIN and invasive cancer. Methods：HPV DNA PCR test and cervical cytology were applied to 476 women, who were referred for abnormal cytology from April 2004 to March 2005 at National Health Insurance Corporation Ilsan Hospital. The results of both procedures were compared with the results of the histological diagnosis and we calculated and compared the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of cytology, HPV DNA PCR, and their combination, respectively. Results：The sensitivity, specificity, PPV and NPV for the detection of CIN or higher lesion were 98.3%, 64.4%, 82.4%, and 95.7% for HPV DNA PCR; 82.9%, 72.9%, 83.8% and 71.7% for cytology; 100%, 48.6%, 76.7%, and 100% for the combination test. The sensitivity and NPV value of cytology showed statistically significant differences (p＜0.05) compared with those of HPV PCR and combination test. The PPV value of combination test showed statistically significant difference compared with those of cytology and HPV PCR. The specificity of three tests were alike (p＞0.05). The sensitivity of three tests for the detection of CIN 2, 3 or higher lesion showed no statistically significant differences. Conclusion：The routine application of HPV DNA PCR or combination test as a screening tools for the detection of CIN and invasive cancer seems to be inappropriate in view of effectiveness. But the HPV DNA PCR can be used as a valuable adjunct to cervical cytology for the triage of women with minimal abnormality in her cytology to determine the treatment and follow-up plan.

Is there a difference in the efficacy of peripartum antiretroviral regimens in reducing mother-to-child transmission of HIV in Africa?

Peripartum antiretroviral regimens have been shown to prevent mother-to-child transmission of HIV (MTCT) in randomized clinical trials; however, direct comparison of published results is impossible given methodological and population differences. To directly compare the efficacy of different antiretroviral regimens in reducing the risk of 6-week MTCT rate in African breastfeeding populations. Pooled analysis including all mother-infant pairs from any relevant trial: West African ZDV-placebo trials, Petra ZDV+3TC [two regimens A (pre/intra/post-partum) and B (intra/post-partum), placebo from Uganda and Tanzania], SAINT (NVP and Petra arm B), HIVNET012 (NVP, ultra short ZDV pp) and the Vitamin A trial (as placebo arm in South Africa). Peripartum HIV infection was any positive RNA or DNA polymerase chain reaction test < day 60. The MTCT risk was estimated at 6 weeks for each treatment arm and compared with placebo or single-dose NVP using logistic regression adjusting for maternal CD4 cell count, breastfeeding and birthweight. Overall, 4125 singleton live-births were included; 3629 (88%) were assessed for HIV status at 6 weeks of age. In comparison with placebo, zidovudine + lamivudine (ZDV+3TC) arm A [adjusted odds ratio (AOR), 0.23; P < 0.0001], ZDV+3TC arm B (AOR, 0.49; P < 0.001), antenatal ZDV short (AOR, 0.55; P = 0.006) and nevirapine (NVP) (AOR, 0.60; P = 0.0007) significantly reduced MTCT. In comparison with NVP, only the longest regimen of ZDV+3TC (AOR, 0.39, P < 0.0005) was significantly more effective. These results are in line with current World Health Organisation guidelines suggesting equivalence of choice between single-dose NVP and short-course ZDV, and confirm the greater efficacy of ZDV+3TC than with any single antiretroviral drug.

Polymerase Chain Reaction for Diagnosis of Human Immunodeficiency Virus Infection in Infancy in Low Resource Settings

Diagnosis of human immunodeficiency virus (HIV) is essential for accessing treatment. Current HIV diagnostic protocols for infants require adaptation and validation before they can be implemented in the developing world. The timing and type of HIV assays will be dictated by country-specific circumstances and experience from similar settings. The performance of an HIV-1 DNA polymerase chain reaction (PCR) test, and in particular a single test at 6 weeks of age, in diagnosing HIV subtype C infection acquired in utero or peripartum was assessed. A retrospective review of 1825 Amplicor HIV-1 DNA PCR version 1.5 tests performed between 2000 and 2004 in 2 laboratories in Johannesburg, South Africa on 769 effectively non-breast-fed infants from 3 clinically well characterized cohorts was undertaken. The HIV status of each infant was used as the standard against which the HIV PCR results were compared. The overall sensitivity and specificity of the HIV PCR test were 99.3 and 99.5% respectively. A single test was 98.8% sensitive and 99.4% specific in the 627 infants tested at 6 weeks of age (58 HIV-infected and 569 HIV-uninfected). Repeat testing of all positive HIV PCR tests minimized false positive results. In resource-poor settings where HIV PCR testing in an environment of good laboratory practice is feasible, a single 6-week HIV DNA PCR test can increase identification of HIV-infected children substantially from current levels. Further operational research on how best to implement and monitor such a diagnostic protocol in specific local settings, especially in breast-fed infants, is necessary.

High-Throughput Molecular Testing of Ticks Using a Liquid-Handling Robot

To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 ± 0.04 μg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-μl volumes with a reproducibility of ±0.038 μl when dispensing 1.0 μl. By using this process, 96 samples can be extracted and tested while reducing human labor to ≈30 min.

High-Throughput Molecular Testing of Ticks Using a Liquid-Handling Robot

To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 +/- 0.04 microg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-microl volumes with a reproducibility of +/- 0.038 microl when dispensing 1.0 microl. By using this process, 96 samples can be extracted and tested while reducing human labor to approximately 30 min.

Dried Blood Spots Improve Access to HIV Diagnosis and Care for Infants in Low-Resource Settings

Effective health care delivery to the majority of perinatally exposed infants worldwide, including those enrolled in prevention of mother-to-child transmission programs, is hampered by lack of access to an HIV diagnosis in infancy. Dried blood spot collection from young infants for centralized HIV polymerase chain reaction (PCR) testing is attainable in low-resource settings, provided PCR methodology suitable for routine laboratory service is available. The accuracy of the Roche Amplicor HIV-1 DNA test version 1.5 (Branchburg, NJ) performed on dried blood spots collected prospectively on ordinary Whatman filter paper from a cohort of 300 6-week-old infants born to HIV-infected women in Johannesburg, South Africa, was assessed. Anonymous analysis of the blood spots using a unique DNA extraction procedure was performed in a routine diagnostic laboratory and the results compared with HIV DNA and RNA PCR liquid blood tests at age 6 weeks, and the HIV status of the infant. Dried blood spots were available for 288 infants (96%) of whom 25 (8.7%) were HIV infected. The Roche Amplicor assay yielded a sensitivity of 100% and a specificity of 99.6%. HIV DNA PCR tests on dried blood spots have the potential to improve health care delivery to HIV-affected children in low-resource settings right now.

Failure of Serial Human Immunodeficiency Virus Type 1 DNA Polymerase Chain Reactions to Identify Human Immunodeficiency Virus Type 1 Clade A/G

The most commonly used test to screen for human immunodeficiency virus type 1 (HIV-1) infection in HIV-exposed infants in the United States is HIV-1 qualitative DNA polymerase chain reaction (PCR). However, the commercially available HIV-1 DNA PCR lack optimal sensitivity to detect non-subtype B subtypes of HIV-1. We report here HIV-1 infection in a West African infant that went undetected by serial HIV-1 DNA PCR tests.

Rational testing of the HIV-exposed infant.

The objectives of this study were 1) to evaluate testing regimens of human immunodeficiency virus (HIV)-exposed infants and 2) to determine optimal methods of follow-up by enzyme-linked immunosorbent assay (ELISA) testing. We reviewed the results from 742 HIV-exposed infants in the state of North Carolina; 2474 samples were tested for HIV by DNA polymerase chain reaction (PCR) at the University of North Carolina Retrovirology Core Laboratory. We then reviewed the utility and costs of ELISA testing of all HIV-exposed infants who were seen at the Duke University Pediatric Infectious Disease Clinic between January 1, 1993, and May 5, 1998. We used likelihood ratios to model probability of HIV infection given 3 negative DNA (PCR) tests and to provide recommendations on the use of ELISA follow-up. The overall sensitivity of the DNA PCR was 87.1%, and its specificity was 99.9%. We evaluated 224 HIV-exposed infants who were seen at Duke University and who had at least 3 negative diagnostic tests using either DNA PCR tests or HIV blood cultures. All 178 infants who subsequently underwent ELISA testing ultimately demonstrated seroreversion. The Duke University Pediatric Infectious Disease Clinic transferred the care of 65 patients to primary care physicians before ELISA testing and retained the care of the remaining 159 patients. Children who remained in Duke's care were more likely to have documentation of seroreversion (158 of 159 vs 20 of 65). We reviewed costs of travel, physician appointment, and HIV antibody testing in a tertiary care setting. Given 3 negative PCR tests, the expected cost per case of HIV detected by a positive ELISA assay is $23.8 million. Documentation of seroreversion in this cohort was nearly complete in the multidisciplinary subspecialty clinic but not when such responsibility was left to the primary care physician. Given the low probability of disease in patients who have had 3 negative PCR tests, documentation of a negative ELISA may not be an appropriate use of medical resources.

Cost-effectiveness of cytomegalovirus (CMV) disease prevention in patients with AIDS: oral ganciclovir and CMV polymerase chain reaction testing.

To perform a cost-effectiveness analysis of strategies to prevent cytomegalovirus (CMV) disease. Markov model and published data. Hypothetical HIV-infected patients with CD4 cell counts < or = 50 x 10(6)/l and positive CMV serologies. Oral ganciclovir daily versus plasma CMV DNA polymerase chain reaction (PCR) testing every 3 months with oral ganciclovir for patients with positive tests. The number of CMV disease cases prevented by the interventions, life expectancy, disease-free life expectancy, and the cost to extend life by 1 year. Oral ganciclovir preventive therapy reduces the lifetime number of CMV disease cases by 50 per 1000 cohort, extends life expectancy by 5 days and disease-free life expectancy by 18 days, and costs US$ 1,762,517 per year of life extended. Periodic PCR testing reduces the lifetime number of CMV disease cases by eight per 1000 cohort, extends life expectancy by 1 day and disease-free life expectancy by 3 days, and costs US$ 495,158 per year of life extended. The prevention strategies could be acceptably cost effective only under a combination of optimistic assumptions and reduced costs. Oral ganciclovir preventive therapy and periodic plasma testing for CMV PCR with oral ganciclovir for those with positive tests result in small benefits at great cost. They are not cost-effective prevention strategies for persons with advanced HIV infection and positive CMV serologies.

Evaluation of a New HTLV‐I/II Polymerase Chain Reaction

Evaluation of a qualitative HTLV-I/II DNA polymerase chain reaction (PCR) test for the detection of HTLV-I/II DNA (Roche Diagnostic Systems, Branchburg, N.J., USA) in various panels. The panels consisted of fresh EDTA blood samples from blood donors who were anti-HTLV-I/II ELISA repeatably reactive: 53 were Western blot (WB) positive, 228 were WB indeterminate and 15 were WB negative. Elevent ELISA-negative blood donors were used as negative controls. Furthermore, specimens from 1 HTLV-II-infected intravenous drug user and from 1 HTLV-II-infected blood donor were included in the panel. Peripheral blood lymphocytes were prepared by red blood cell lysis with the Roche washing solution and stored at < -23 degrees C until processing. Amplification products were analyzed with the HTLV-I/II detection kit. All 53 anti-HTLV-I/II ELISA- and WB-positive samples and both HTLV-II-positive samples tested positively by PCR. All 228 anti-HTLV-I/II ELISA-positive and WB-indeterminate, all 15 ELISA-positive and WB-negative and all II ELISA-negative control samples tested negative by PCR. The Roche Amplicor HTLV-I/II test is a simple test, suitable for the confirmation of HTLV-I and-II infection in individuals with indeterminate or positive WB patterns.

Evaluation of a New HTLV-I/II Polymerase Chain Reaction

Aim: Evaluation of a qualitative HTLV-I/Il DNA polymerase chain reaction (PCR) test for the detection of HTLV-I/II DNA (Roche Diagnostic Systems, Branchburg, N.J., USA) in various panels. Methods: The panels consisted of fresh EDTA blood samples from blood donors who were anti-HTLV-I/II ELISA repeatably reactive: 53 were Western blot (WB) positive, 228 were WB indeterminate and 15 were WB negative. Elevent ELISA-negative blood donors were used as negative controls. Furthermore, specimens from 1 HTLV-II-infected intravenous drug user and from 1 HTLV-II-infected blood donor were included in the panel. Peripheral blood lymphocytes were prepared by red blood cell lysis with the Roche washing solution and stored at <-23 °C until processing. Amplification products were analyzed with the HTLV-I/II detection kit. Results: All 53 anti-HTLV-I/II ELISA- and WB-positive samples and both HTLV-II-positive samples tested positively by PCR. All 228 anti-HTLV-I/II ELISA-positive and WB-indeterminate, all 15 ELISA-positive and WB-negative and all 11 ELISAnegative control samples tested negative by PCR. Conclusion: The Roche Amplicor HTLV-I/II test is a simple test, suitable for the confirmation of HTLV-I and -II infection in individuals with indeterminate or positive WB patterns.

Diagnosis of infection with human immunodeficiency virus type 1 by a DNA polymerase chain reaction assay among infants enrolled in the Women and Infants' Transmission Study

Early diagnosis of infection with human immunodeficiency virus type 1 (HIV-1) in young infants is essential to decisions on their medical and social care. Whereas studies have suggested that polymerase chain reaction (PCR) is a sensitive and timely method of diagnosing HIV infection in children, these evaluations have been limited by the number of specimens studied. Recently, Roche Molecular Systems developed a complete HIV-1 DNA PCR testing kit (from specimen preparation to detection). In this study, use of this PCR test kit was evaluated for the detection of HIV infection in infants of seropositive mothers who were enrolled in the longitunal, multicenter Women and Infants' Transmission Study. A total of 1209 blood specimens from 483 infants were tested and analyzed. The overall sensitivity and specificity of a single PCR test in determining HIV infection status in infants more than 1 but less than 36 months of age were 95% and 97%, respectively. For infected infants 1 to 6 months of age the sensitivity of the DNA-PCR test was 90% to 100%. In a direct comparison with coculture, the Roche DNA-PCR test was significantly more sensitive than coculture in the detection of HIV-1 in infected infants and was equivalent to coculture for the diagnosis of HIV in infants when a standardized algorithm was used to define infection status. (J P EDIATR 1996;129:198-207)

Development and Application of DNA Probes and PCR Tests for Haemophilus paragallinarum

A genomic DNA library of Haemophilus paragallinarum strain Modesto was created. Screening of this library identified four clones that reacted specifically with all 56 isolates of H. paragallinarum tested and failed to react with 24 closely related bacteria from the genera Pasteurella and Actinobacillus. All four clones also failed to react with DNA extracted from one field isolate each of Mycoplasma gallisepticum and Mycoplasma synoviae. The probes based on these four clones were approximately 1.8, 2.3, 3.5, and 5.5 kb in size. The four probes were able to detect between 7.8 and 31.25 ng of purified DNA from homologous strains with no obvious correlation between probe size and sensitivity. The smallest probe, termed P601, was partially sequenced, and primers for two polymerase chain reaction (PCR) tests were designed from these sequence data. Both PCR tests, termed HPG-1 and HPG-2, were shown to be specific, all 41 isolates of H. paragallinarum tested being positive and all 26 non-H. paragallinarum isolates being negative. Both PCR tests were able to detect 1 pg DNA and between 10(2) and 10(3) cells. A method for using the HPG-2 PCR test directly on sinus swabs was developed. Using this method, there was 100% agreement between culture and the direct HPG-2 PCR for the 36 swabs processed. The DNA probes and PCR tests appear to be useful diagnostic tools for the detection of infectious coryza. The tests can be used as confirmatory tests following the isolation of a hemophilic organism. As well, the HPG-2 PCR test appears to be a potential alternative to culture.

Repeatedly positive human immunodeficiency virus type 1 DNA polymerase chain reaction in human immunodeficiency virus-exposed seroreverting infants.

Three human immunodeficiency virus type 1 (HIV-1)-exposed children who had repeatedly positive DNA polymerase chain reaction (PCR) tests for HIV in > or = 5 samples before seroreversion to HIV-negative status are reported. The children belong to a cohort of 210 infants who were born to HIV-infected mothers and were tested at intervals of 1 to 3 months by HIV viral culture, PCR, and p24 antigen; only the PCR was positive in > or = 5 samples in the children reported here. Their clinical features were indistinguishable from other seroreverters. All three children had a transient drop in CD4:CD8 ratio to < 1.0. The transiently positive DNA PCR in HIV-exposed infants may indicate either that HIV infection was eliminated by a strong host immune response or that infection was caused by an attenuated/defective strain of virus.

A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle.

This paper reviews the laboratory diagnosis of Leptospira hardjo infection in cattle. Two genotypes of L hardjo, Hardjoprajitno and Hardjobovis, have been identified in cattle, but only Hardjobovis has been isolated in Australia. There are problems with diagnosis and control of bovine leptospirosis. Infection is usually subclinical and the serological titres vary greatly in peak and duration. Leptospires may be excreted in urine for up to 18 months. Low microscopic agglutination test titres may be significant in unvaccinated herds as indicators of endemic infection. Vaccines differ in their composition, and their efficacy is difficult to evaluate. The serological response after vaccination is difficult to differentiate from the response after infection. Pregnant cows that become infected may abort, but this is usually after the serological response has peaked. Therefore, paired serum samples are of little use in diagnosing abortion caused by L hardjo. Fluorescent antibody techniques are more sensitive than dark field microscopy for detection of leptospires in urine and tissue samples. Techniques for culture have improved but are still difficult to perform and take 3 months or longer for results to be known. DNA probes and polymerase chain reaction tests are very sensitive and specific, quick to perform, and can be used on fluid and tissue samples.