Immunofluorescent Antibody Assay Research Articles

Hemorrhagic Fever with Renal Syndrome, Vietnam

Hemorrhagic Fever with Renal Syndrome, Vietnam

Indirect ELISA and Indirect Immunofluorescent Antibody Assay for Detecting the Antibody against Murine Norovirus S7 in Mice

To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.

Real-Time PCR for Diagnosis of Oculoglandular Tularemia

Real-Time PCR for Diagnosis of Oculoglandular Tularemia

Distribution of Antibodies Reactive toBorrelia lonestariandBorrelia burgdorferiin White-Tailed Deer (Odocoileus virginianus) Populations in the Eastern United States

Southern tick-associated rash illness is a Lyme-like syndrome that occurs in the southern states. Borrelia lonestari, which has been suggested as a possible causative agent of southern tick-associated rash illness, naturally infects white-tailed deer (WTD; Odocoileus virginianus) and is transmitted by the lone star tick (Amblyomma americanum). To better understand the prevalence and distribution of Borrelia exposure among WTD, we tested WTD from 21 eastern states for antibodies reactive to B. lonestari using an indirect immunofluorescent antibody assay and Borrelia burgdorferi using the IDEXX SNAP 4Dx test. A total of 107/714 (15%) had antibodies reactive to B. lonestari, and prevalence of antibodies was higher in deer from southern states (17.5%) than in deer from northern states (9.2%). Using the SNAP 4DX test, we found that 73/723 (10%) were positive for B. burgdorferi, and significantly more northern deer (23.9%) were positive compared with southern deer (3.8%). Our data demonstrate that WTD are exposed to both Borrelia species, but antibody prevalence for exposure to the two species differs regionally and distributions correlate with the presence of Ixodes scapularis and A. americanum ticks.

Specific real-time reverse transcription-polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in tissues and feces from turkeys infected with turkey coronavirus

Turkey coronavirus (TCoV) infection causes acute atrophic enteritis in the turkey poults, leading to significant economic loss in the U.S. turkey industry. Rapid detection, differentiation, and quantitation of TCoV are critical to the diagnosis and control of the disease. A specific one-step real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay for detection and quantitation of TCoV in the turkey tissues was developed using a dual-labeled fluorescent probe. The fluorogenic probe labeled with a reporter dye (FAM, 6-carboxytetramethylrhodamin) and a quencher dye (AbsoluteQuencher™) was designed to bind to a 186 base-pair fragment flanked by the two PCR primers targeting the 3′ end of spike gene of TCoV. The assay was performed on different avian viruses and bacteria to determine the specificity as well as serial dilutions of TCoV for the sensitivity. Three animal trials were conducted to further validate the assay. Ten-day-old turkey poults were inoculated orally with 100 EID 50 of TCoV. Intestinal tissues (duodenum, jejunum, ileum, cecum), feces from the cloacal swabs, or feces from the floor were collected at 12 h, 1, 2, 3, 5, 7, and/or 14 days post-inoculation (DPI). RNA was extracted from each sample and subjected to the RRT-PCR. The designed primers and probe were specific for TCoV. Other non-TCoV avian viruses and bacteria were not amplified by RRT-PCR. The assay was highly sensitive and could quantitate between 10 2 and 10 10 copies/μl of viral genome. The viral RNA in the intestine segments reached the highest level, 6 × 10 15 copies/μl, in the jejunum at 5 DPI. Eighty-four intestine segments assayed by the developed RRT-PCR and immunofluorescence antibody assay (IFA) revealed that there were 6 segments negative for TCoV by both assays, 45 positive for TCoV by IFA, and 77 positive for TCoV by RRT-PCR. Turkey coronavirus was detected in the feces from the cloacal swabs or floor 1–14 DPI; however, the viral RNA load varied among different turkey poults at different intervals from different trials. The highest amount of viral RNA, 2.8 × 10 10 copies/μl, in the feces was the one from the cloacal swab collected at 1 DPI. The average amount of TCoV RNA in the cloacal fecal samples was 10 times higher than that in the fecal droppings on the floor. Taken together, the results indicated that the developed RRT-PCR assay is rapid, sensitive, and specific for detection, differentiation, and quantitation of TCoV in the turkey tissues and should be helpful in monitoring the progression of TCoV induced acute enteritis in the turkey flocks.

Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status

BackgroundAnaplasma phagocytophilum infection in domestic ruminants is widespread in the coastal areas of southern Norway. The bacteria may persist in mammalian hosts. Several genetic variants of A. phagocytophilum exist. In the present study, we investigate whether superinfection occurs in the acute and persistent phase of the infection.MethodsFive-month-old lambs of the Norwegian Dala breed were experimentally infected with two 16S rRNA gene variants of A. phagocytophilum, i.e. A. phagocytophilum variant 1 (GenBank accession number M73220) and variant 2 (GenBank acc. no. AF336220). Eighteen lambs were used, two lambs in each group. Eight groups were experimentally inoculated with either variant 1 or 2 on day 0. Six of these groups were then challenged with the other variant on either days 7, 42 or 84, respectively. One group was left uninfected. The occurrence of A. phagocytophilum in blood samples was determined using semi-nested PCR analysis and gene sequencing. Specific antibodies were measured by an indirect immunofluorescence antibody assay (IFA).ResultsA. phagocytophilum variant 1 and 2 differed significantly with regards to clinical reaction and cross-immunity in infected lambs. Both variants were found in the blood after challenge. However, variant 1 was detected most frequently.ConclusionThe present experiment indicates that superinfection of different genotypes occurs during the acute as well as the persistent phase of an A. phagocytophilum infection, even in lambs protected against the challenged infection.

Tick-Borne Rickettsiosis in Traveler Returning from Honduras

Tick-Borne Rickettsiosis in Traveler Returning from Honduras

Levofloxacin versus tetracycline antibiotics for the treatment of scrub typhus

Scrub typhus is an important febrile disease in Southeast Asia and is caused by Orientia tsutsugamushi. Fluoroquinolones have proved to be effective for scrub typhus in an animal model. However, it is unclear whether they are also effective clinically. We retrospectively reviewed the hospital-based medical records of patients diagnosed to have scrub typhus by an indirect immunofluorescence antibody assay or PCR at a large referral hospital in Taiwan for a 6-year period (2001-2007). To determine the efficacy of levofloxacin for the treatment of scrub typhus, we divided the patients into a levofloxacin-treated group and a tetracycline antibiotics-treated group. Out of 132 patients with scrub typhus, 71 initially received levofloxacin and 61 initially received tetracycline antibiotics. There was no statistically significant difference in the effective rate between the two groups (91.5% and 95.1% cured, respectively; p=0.648). The time to defervescence in the levofloxacin-treated group was longer than in the other group (49+/-41.1 and 24+/-19.6hours, respectively; p=0.001). In the patients with higher APACHE II scores, higher mortality was found in the levofloxacin-treated group (44.4% and 0%; p=0.033). Levofloxacin is effective in patients with scrub typhus, but has a longer time to defervescence compared with tetracycline antibiotics. When levofloxacin is used for severe scrub typhus, higher mortality may be attributed to the longer time to defervescence.

KSHV seroprevalence, and blood and saliva viral loads in the HIV-infected population of south Texas

Kaposi sarcoma (KS) is the most common neoplasm in HIV-infected subjects, and is associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV). Our previous studies have shown that KSHV epidemiology in South Texas is distinct with an increased KSHV seroprevalence in blood donors and unique distribution of KSHV genotypes in KS subjects. However, KSHV epidemiology in the HIV-infected population in South Texas remains undefined. In this cross sectional study, we examined specific antibodies to KSHV latent nuclear antigen (LANA) by immunofluorescence antibody assay (IFA) and to KSHV lytic antigen ORF65 by ELISA in 25 HIV-infected KS subjects, 314 HIV-infected subjects, and 335 HIV-negative subjects. Relative antibody titers to ORF65 were estimated based on optical density values (O.D.). Blood and saliva viral loads were also determined in KSHV-seropositive subjects by quantitative real-time PCR. Antibodies to KSHV antigens were detected in 25 (100%) KS subjects, 114 (36%) HIV-infected subjects and 65 (19%) HIV-negative subjects. KS subjects had higher antibody titers to ORF65 compared to HIV-infected and -negative subjects (median O.D. 0.76 versus 0.47 and 0.27; p = 0.0017 and 0.0001, respectively). Antibody titers in HIV-infected subjects were also significantly higher than HIV-negative subjects (p = 0.0001). Among the HIV-infected subjects, males had higher KSHV seroprevalence than females (42%, 95% CI: 35.6–47.4% versus 8%, 95% CI: 0.5–15.8%, p 400 CD4+ T cell counts, those with <200 CD4+ T cell counts had higher KSHV seroprevalence (OR = 2.59, 95% CI: 1.4–4.80, p = 0.017) and higher antibody titers (median O.D. 0.48 versus 0.32; p = 0.001). KSHV blood and saliva viral loads were detected in 12 (48%) and 9 (36%) of 25 KS subjects, 16 (26%) and 12 (19%) of 62 HIV-positive KSHV-seropositive subjects, and none of the HIV-negative KSHV-seropositive subjects, respectively. Among the KS subjects, KSHV blood and saliva viral loads were detected more frequently in those with active KS than those without active KS (81.8%, 95% CI: 48.2–97.7% and 54.5%, 95% CI: 23.3–83.2% versus 18%, 95% CI: 2.2–51.7% and 45%, 95% CI: 16.7–76.6%, p = 0.004 and 0.05, respectively). Together, these results indicate that HIV infection and the status of HIV disease might modulate KSHV infection and replication, and impact the development of KS.

A Survey of Antibodies against Rickettsia rickettsii and Ehrlichia chafeensis in Domestic Animals from a Rural Area of Colombia

In a rural area of Colombia endemic for Rocky Mountain spotted fever, we performed indirect immunofluorescent antibody assays for Rickettsia spp. and Ehrlichia spp. using sera from randomly sampled dogs and horses to test the use of domestic animals as possible sentinels. Antibodies against Ehrlichia spp. were detected in 8 dogs (31.8%). Antibody titers against Rickettsia rickettsii antigen were positive in 4 dogs (18.2%) and 26 horses (16.3%). These values, albeit not directly comparable, are lower than those previously reported for humans in this region. A systemic approach to understanding dynamics of transmission is needed before implementing the use of domestic animals for disease surveillance activities.

Evaluation of the Chagas Stat-PaktmAssay for Detection of Trypanosoma cruzi Antibodies in Wildlife Reservoirs

An immunochromatographic assay (Chagas Stat-Pak) was evaluated for the detection of Trypanosoma cruzi antibodies in 4 species of wildlife reservoirs. Antibodies to T. cruzi were detected in raccoons (Procyon lotor) (naturally and experimentally infected) and degus (Octodon degu) (experimentally-infected) using the Chagas Stat-Pak. In naturally exposed wild raccoons, the Chagas Stat-Pak had a sensitivity and specificity of 66.7-80.0% and 96.3%, respectively. Compared with indirect immunofluorescent antibody assay results, seroconversion as determined by Chagas Stat-Pak was delayed for experimentally infected raccoons, but occurred sooner in experimentally infected degus. The Chagas Stat-Pak did not detect antibodies in naturally or experimentally infected Virginia opossums (Didelphis virginiana) or in experimentally infected short-tailed opossums (Monodelphis domestica). These data suggest that the Chagas Stat-Pak might be useful in field studies of raccoons and degus when samples would not be available for more-conventional serologic assays. Because this assay did not work on either species of marsupial, the applicability of the assay should be examined before it is used in other wild species.

Effect of nitazoxanide on cryptosporidiosis in experimentally infected neonatal dairy calves

Cryptosporidium is a zoonotic protozoan that is most often diagnosed in association with diarrhea in 1- to 3-wk-old dairy calves. There are neither consistently effective nor approved antimicrobial drugs for treatment in animals. The objective of this study was to test nitazoxanide (NTZ) as a treatment for cryptosporidiosis in experimentally infected dairy calves. A randomized, controlled, and blinded trial was performed using Holstein bull calves obtained from a large commercial dairy. All births were attended by study personnel and calves were fed 4L of heat-treated colostrum within 1h of birth. Calves were randomly assigned to treatment or placebo group and maintained for a 32-feeding (16 d) study period. Twenty-three calves were enrolled with 3 lost to follow up. Thirteen calves were assigned to the treatment group and 7 calves to the placebo group. All calves were inoculated with 1×106 viable Cryptosporidium parvum oocysts at feeding 3. Treatment was a commercially available NTZ product and the placebo was the carrier of the same product. Nitazoxanide was administered at 1.5g twice per day for 5 d. Nitazoxanide or placebo treatment began after feeding 10 and when the fecal score was greater than 1 out of 3. Outcome measurements included twice-daily fecal and health scores and a once-daily oocyst count by an immunofluorescent antibody assay. Data were analyzed by nonparametric and time-to-event methods. Measures of passive transfer of antibodies, initial body weight, and onset of oocyst shedding were not different between treatment and control calves. Eighty-five percent of the NTZ-treated calves stopped shedding oocysts by the end of the observation period whereas only 15% of the placebo group stopped shedding. The median number of feedings with a fecal score equal to 3 was 2 in the NTZ group while it was 6 in the placebo group. Calves receiving NTZ were 0.13 times as likely to have severe and sustained diarrhea than control calves (95% confidence interval, 0.02–0.98). Treating calves with NTZ reduced the duration of oocyst shedding and improved fecal consistency.

The CB 2 cannabinoid receptor regulates human sperm cell motility

To analyze the expression and distribution of cannabinoid receptors in human sperm cells and evaluate the effects of activation of receptors by specific agonists and antagonists, with a special emphasis on the CB(2) receptor. We performed expression assays for CB(1) and CB(2) by reverse transcriptase PCR, Western blot, and immunofluorescence techniques in spermatozoa and performed motility analysis after incubation of semen samples with cannabinoid agonists and CB(2) antagonist SR144528. Academic research laboratory. Semen from 50 normozoospermic, healthy human donors. Spermatozoa isolated from semen by two consecutive swim-ups were used for all techniques. Reverse transcriptase PCR amplification gels, immunoblots, indirect immunofluorescence antibody assays, and percentage of motile sperm. We have verified the presence of CB(1) and CB(2) receptors in human spermatozoa. The distribution of both of these receptors was distinct. Incubation with selective cannabinoid receptor agonists induced a significant reduction in the proportion of rapidly progressive motile spermatozoa, and whereas the CB(1) agonist increased the proportion of immobile sperm cells, the CB(2) receptor agonist increased the slow/sluggish progressive sperm cell population. The effect of the CB(2) agonist was antagonized by the CB(2)-specific antagonist. The functional CB(2) cannabinoid receptor is present in human spermatozoa and regulates the sperm motility in a more distinct manner than CB(1).

Recombinant K39 Immunochromatographic Test for Diagnosis of Human Leishmaniasis

A new recombinant K39 immunochromatographic test (ICT) was compared with the immunofluorescent antibody assay (IFA) for the rapid serological diagnosis of visceral leishmaniasis (VL) in Apulia, Southern Italy. A total of 264 individuals were tested, including 19 patients with VL (three of which were HIV positive), 67 individuals with suspected VL, 40 healthy controls and 138 patients with other diseases. The ICT was positive in all 19 patients with VL and negative in sera from the remaining individuals. Both the sensitivity and specificity of ICT was 100%. The ICT also worked well in HIV-Leishmania co-infected patients. Antibodies to Leishmania detected by the IFA and ICT remained at detectable levels for up to 12-24 months. A positive reaction by the ICT was detectable at a serum dilution of up to 1:20,480, indicating that a strong immunoresponse is mounted against the recombinant K39 antigen. In conclusion, the ICT is highly sensitive, specific, rapid, noninvasive and cost effective (euro8.43 for ICT and euro12 for IFA) in the diagnosis of VL in areas of low VL endemicity.

Seroepidemiologic and Occupational Risk Survey forCoxiella burnetiiAntibodies among US Veterinarians

Little is known about the occurrence of Q fever among veterinarians in the United States. In this study, we sought to estimate the prevalence of Coxiella burnetii antibodies among veterinarians and to identify risk factors for exposure. We tested serum samples from 508 veterinarians who attended the 143rd American Veterinary Medical Association Annual Convention in 2006. Samples were screened using a Q fever IgG enzyme-linked immunosorbent assay (ELISA). Samples with positive or equivocal results of ELISA were confirmed using phase I and phase II IgG immunofluorescence antibody assays, and end point IgG titers were determined for samples with positive results. Antibodies against C. burnetii were detected in 113 (22.2%) of 508 veterinarians. Risk factors associated with seropositivity included age 46 years, routine contact with ponds, and treatment of cattle, swine, or wildlife. Veterinarians have a high level of exposure to C. burnetii, the causative organism of Q fever, especially those veterinarians who treat livestock. In this study, risk of C. burnetii seropositivity was also independently associated with contact with ponds. The role of exposure to standing bodies of water in infection is not usually considered and should be investigated in future studies. Additionally, the evidence of past infection with C. burnetii in >20% of veterinarians also highlights the need for use of appropriate personal protective equipment when treating animals that are potentially infected with C. burnetii. Physicians should consider the risk of infection with C. burnetii when treating ill veterinarians and others with potential occupational exposures.

Validity of the Weil-Felix test in the diagnosis of acute rickettsial infections in Sri Lanka

The diagnosis of rickettsial infections in developing countries is based on clinical features and a positive Weil-Felix test (WFT), as tests such as indirect immunofluorescent antibody (IFA) assays are not available for routine use. We estimated the sensitivity of the WFT in Sri Lanka using IFA testing as the gold standard. The WFT demonstrated low sensitivity (33%) in diagnosing acute rickettsial infections and low specificity, with a positive titre of 1:320 seen in 54% of healthy volunteers and 62% of non-rickettsial fever patients. Therefore, the use of the WFT should be discouraged in the diagnosis of acute rickettsial infections.

Epidemiological Study of Hantavirus Infection in the Samara Region of European Russia

European Russia is a highly endemic area of hemorrhagic fever with renal syndrome (HFRS), a rodent-borne zoonotic disease, caused by hantaviruses. In total, 145 small mammals of four species (Myodes glareolus, Apodemus flavicollis, A. agrarius, and A. uralensis) were trapped in the Samara region of European Russia in August 2005 and examined for the presence of hantavirus (HV). Anti-HV antibodies were found in six of 68 (8.8%) M. glareolus and in one of 19 (5.3%) A. flavicollis by indirect immunofluorescent antibody assay (IFA). The Puumala virus (PUUV), which is one of the hantavirus species, was detected in the lungs of seven M. glareolus by RT-PCR. The virus S-segment was extremely similar (96.2% to 99.3%) to the sequence found in a fatal case of HFRS in the Samara region. Phylogenetic analyses of S and M segments showed that the Samara PUUVs form a cluster within the Russian Volga lineage and apparently differ from other European PUUVs. Anti-PUUV antibodies were found in blood sera from seven HFRS patients and from one undiagnosed patient from the Samara region, using IFA and an enzyme-linked immunosorbent assay (ELISA). These data suggest that the bank vole M. glareolus is a primary natural reservoir and vector for PUUV, which is the main causative agent of HFRS in humans in the Samara region.

P.058 Influenza A packaging control by RNA/RNA interactions: identification of critical domains also controlling genetic reassortment

In 2006, bronchiolitis due to adenovirus nosocomial infections resulted in the closure of a pediatric department in northern Portugal.To determine the etiology of bronchiolitis in northern Portugal.It was a prospective multicenter study on the etiology of bronchiolitis during the respiratory syncytial virus (RSV) season (November–April). Children ≤24 months of age admitted for a first wheezing episode were included. Nasopharyngeal specimens were analyzed by an indirect immunofluorescent-antibody assay (IFA) for RSV, adenovirus (HAdV), parainfluenza (PIV) 1–3 and influenza (IV) A and B and by polymerase chain reaction (PCR) or reverse transcription-PCR for the same viruses and for human metapneumovirus (hMPV), bocavirus (HBoV), rhinovirus (HRV), coronaviruses (229/E; NL63; OC43; HKU1) and enterovirus.During this period, 253 children were included, 249 IFA analyses and 207 PCRs were performed. IFA detected RSV in 58.1%; PCR increased it to 66.7%. IFA detected HAdV in 3.2%, PCR 10.0%. PCR detected IV A in 5; IV B in 2; PIV 1 in 6, PIV 2 in 4 and PIV 3 in 11 cases. HBoV, as single agent in 2 cases, and HRV were positive in 8 samples and hMPV in 11. With this virus panel, 19.7% remained without etiology.The most frequent agent was RSV, followed by HAdV. PCR can be cost-effective and more accurate than IFA, which is crucial for HAdV that may be associated with significant mortality (IFA alone did not detect 2/3 of the cases).

Enhancement of cytotoxic T lymphocyte activity by dendritic cells loaded with Tat-protein transduction domain-fused hepatitis B virus core antigen

The protein transduction domain (PTD) of human immunodeficiency virus-1-Tat protein has a unique potency to penetrate the cellular membranes. To synthesize the sequence of Tat-PTD47-57 and hepatitis B virus core antigen (HBcAg), we spliced these sequences and linked a fusion gene into the pMAL-c2x vector. The fusion proteins were purified by affinity chromatography and pulsed with bone marrow-derived dendritic cells (DCs), and the transduction of recombinant protein was detected by immunofluorescence antibody assay. Results showed that recombinant PTD-HBcAg could penetrate into DC cytoplasm while recombinant HBcAg was detected on the surface of cells. The percentage of DC surface molecules, such as CD80, CD86 and major histocompatibility complex II, and production of cytokine (IL-12p70) induced by recombinant PTD-HBcAg were significantly higher than those induced by recombinant HBcAg or tumor necrosis factor-alpha. DCs treated with PTD-HBcAg induced T cells to differentiate into specific cytotoxic T lymphocytes (CTLs) and enhanced the CTL killing response. In conclusion, the expressed and purified PTD-HBcAg fusion protein could penetrate into cells through the plasma membrane, promote DC maturation, and enhance T cells response to generate HBcAg-specific CTLs efficiently.

HIV Testing and Prophylaxis to Prevent Mother-to-Child Transmission in the United States

Universal HIV testing of pregnant women in the United States is the key to prevention of mother-to-child transmission of HIV. Repeat testing in the third trimester and rapid HIV testing at labor and delivery are additional strategies to further reduce the rate of perinatal HIV transmission. Prevention of mother-to-child transmission of HIV is most effective when antiretroviral drugs are received by the mother during her pregnancy and continued through delivery and then administered to the infant after birth. Antiretroviral drugs are effective in reducing the risk of mother-to-child transmission of HIV even when prophylaxis is started for the infant soon after birth. New rapid testing methods allow identification of HIV-infected women or HIV-exposed infants in 20 to 60 minutes. The American Academy of Pediatrics recommends documented, routine HIV testing for all pregnant women in the United States after notifying the patient that testing will be performed, unless the patient declines HIV testing ("opt-out" consent or "right of refusal"). For women in labor with undocumented HIV-infection status during the current pregnancy, immediate maternal HIV testing with opt-out consent, using a rapid HIV antibody test, is recommended. Positive HIV antibody screening test results should be confirmed with immunofluorescent antibody or Western blot assay. For women with a positive rapid HIV antibody test result, antiretroviral prophylaxis should be administered promptly to the mother and newborn infant on the basis of the positive result of the rapid antibody test without waiting for results of confirmatory HIV testing. If the confirmatory test result is negative, then prophylaxis should be discontinued. For a newborn infant whose mother's HIV serostatus is unknown, the health care professional should perform rapid HIV antibody testing on the mother or on the newborn infant, with results reported to the health care professional no later than 12 hours after the infant's birth. If the rapid HIV antibody test result is positive, antiretroviral prophylaxis should be instituted as soon as possible after birth but certainly by 12 hours after delivery, pending completion of confirmatory HIV testing. The mother should be counseled not to breastfeed the infant. Assistance with immediate initiation of hand and pump expression to stimulate milk production should be offered to the mother, given the possibility that the confirmatory test result may be negative. If the confirmatory test result is negative, then prophylaxis should be stopped and breastfeeding may be initiated. If the confirmatory test result is positive, infants should receive antiretroviral prophylaxis for 6 weeks after birth, and the mother should not breastfeed the infant.