Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections.

Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites (Balamuthia mandrillaris and Acanthamoeba), six bacterial pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Mycoplasma pneumoniae, Mycobacterium tuberculosis, and Bartonella), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease.

Swab protocol for rapid laboratory diagnosis of cutaneous anthrax.

The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as culture and directly straining smears of clinical specimens. However, these methods rely on recovery of viable Bacillus anthracis cells from swabs of cutaneous lesions and often yield negative results. This study developed a rapid protocol for detection of B. anthracis on clinical swabs. Three types of swabs, flocked-nylon, rayon, and polyester, were evaluated by 3 extraction methods, the swab extraction tube system (SETS), sonication, and vortex. Swabs were spiked with virulent B. anthracis cells, and the methods were compared for their efficiency over time by culture and real-time PCR. Viability testing indicated that the SETS yielded greater recovery of B. anthracis from 1-day-old swabs; however, reduced viability was consistent for the 3 extraction methods after 7 days and nonviability was consistent by 28 days. Real-time PCR analysis showed that the PCR amplification was not impacted by time for any swab extraction method and that the SETS method provided the lowest limit of detection. When evaluated using lesion swabs from cutaneous anthrax outbreaks, the SETS yielded culture-negative, PCR-positive results. This study demonstrated that swab extraction methods differ in their efficiency of recovery of viable B. anthracis cells. Furthermore, the results indicated that culture is not reliable for isolation of B. anthracis from swabs at ≥ 7 days. Thus, we recommend the use of the SETS method with subsequent testing by culture and real-time PCR for diagnosis of cutaneous anthrax from clinical swabs of cutaneous lesions.

Clinical and virological characterization of persistent human infection with simian foamy viruses.

Persons occupationally exposed to nonhuman primates (NHPs) can be persistently infected with simian foamy virus (SFV). The clinical significance and person-to-person transmissibility of zoonotic SFV infection is unclear. Seven SFV-infected men responded to annual structured interviews and provided whole blood, oral, and urogenital specimens for study. Wives were tested for SFV infection. Proviral DNA was consistently detected by PCR in PBMCs of infected men and inconsistently in oral or urogenital samples. SFV was infrequently cultured from their PBMCs and throat swabs. Despite this and a long period of intimate exposure (median 20 years), wives were SFV negative. Most participants reported nonspecific symptoms and diseases common to aging. However, one of two persons with mild thrombocytopenia had clinically asymptomatic nonprogressive, monoclonal natural killer cell lymphocytosis of unclear relationship to SFV. All participants worked with NHPs before 1988 using mucocutaneous protection inconsistently; 57% described percutaneous injuries involving the infecting NHP species. SFV likely transmits to humans through both percutaneous and mucocutaneous exposures to NHP body fluids. Limited follow-up has not identified SFV-associated illness and secondary transmission among humans.

The epidemiology of simian immunodeficiency virus infection in a large number of wild- and captive-born chimpanzees: evidence for a recent introduction following chimpanzee divergence.

Simian immunodeficiency virus (SIVcpz) from the chimpanzee subspecies Pan troglodytes troglodytes has been linked phylogenetically to the origin of HIV-1. Related but distinct SIVcpz strains have also been found in P. t. schweinfurthii , suggesting that SIVcpz may have coevolved among the four chimpanzee subspecies. However, SIVcpz strains from P. t. verus and P. t. vellerosus have not yet been identified. To better understand the epidemiology and natural history of SIVcpz among chimpanzees, we tested serum samples from 1415 chimpanzees housed at eight U.S. research centers and six zoos. Records indicated that 264 (18.6%) of the chimpanzees were African-born. Subspecies identities for 161 chimpanzees, based on analysis of mitochondrial DNA sequences, were found to be P. t. troglodytes (n = 14), P. t. schweinfurthii (n = 3), P. t. verus (n = 143), and P. t. vellerosus (n = 1). All samples were screened for HIV/SIV antibodies by using an HIV-1/2 peptide- based enzyme immunoassay (EIA). Reactive samples were tested further by Western blot (WB). Eight sera (0.57%) were EIA reactive, but none was HIV-1/2 WB positive. Two samples were HIV-1 WB indeterminate. Both samples tested negative for SIVcpz and HIV-1 sequences by reverse transcriptase PCR, suggesting an absence of infection. We also tested sera available from 8 male sexual partners, 6 offspring, and 12 cage mates of a known SIVcpz-infected chimpanzee. All samples were negative, suggesting that SIVcpz may not be easily transmitted to close contacts. Our data show that this large population of chimpanzees is not infected with SIVcpz. The absence of SIVcpz infection in P. t. verus suggests that SIVcpz may not be endemic to this subspecies and implies that SIVcpz may have been introduced more recently into the chimpanzee subspecies following divergence.

Ancient co-speciation of simian foamy viruses and primates.

Although parasite-host co-speciation is a long-held hypothesis, convincing evidence for long-term co-speciation remains elusive, largely because of small numbers of hosts and parasites studied and uncertainty over rates of evolutionary change. Co-speciation is especially rare in RNA viruses, in which cross-species transfer is the dominant mode of evolution. Simian foamy viruses (SFVs) are ubiquitous, non-pathogenic retroviruses that infect all primates. Here we test the co-speciation hypothesis in SFVs and their primate hosts by comparing the phylogenies of SFV polymerase and mitochondrial cytochrome oxidase subunit II from African and Asian monkeys and apes. The phylogenetic trees were remarkably congruent in both branching order and divergence times, strongly supporting co-speciation. Molecular clock calibrations revealed an extremely low rate of SFV evolution, 1.7 x 10(-8) substitutions per site per year, making it the slowest-evolving RNA virus documented so far. These results indicate that SFVs might have co-speciated with Old World primates for at least 30 million years, making them the oldest known vertebrate RNA viruses.

Naturally acquired simian retrovirus infections in central African hunters.

BACKGROUND: Hunting and butchering of wild non-human primates infected with simian immunodeficiency virus (SIV) is thought to have sparked the HIV pandemic. Although SIV and other primate retroviruses infect laboratory workers and zoo workers, zoonotic retrovirus transmission has not been documented in natural settings. We investigated zoonotic infection in individuals living in central Africa. METHODS: We obtained behavioural data, plasma samples, and peripheral blood lymphocytes from individuals living in rural villages in Cameroon. We did serological testing, PCR, and sequence analysis to obtain evidence of retrovirus infection. FINDINGS: Zoonotic infections with simian foamy virus (SFV), a retrovirus endemic in most Old World primates, were identified in people living in central African forests who reported direct contact with blood and body fluids of wild non-human primates. Ten (1%) of 1099 individuals had antibodies to SFV. Sequence analysis from these individuals revealed three geographically-independent human SFV infections, each of which was acquired from a distinct non-human primate lineage: De Brazza's guenon (Cercopithecus neglectus), mandrill (Mandrillus sphinx), and gorilla (Gorilla gorilla), two of which (De Brazza's guenon and mandrill) are naturally infected with SIV. INTERPRETATION: Our findings show that retroviruses are actively crossing into human populations, and demonstrate that people in central Africa are currently infected with SFV. Contact with non-human primates, such as happens during hunting and butchering, can play a part in the emergence of human retroviruses and the reduction of primate bushmeat hunting has the potential to decrease the frequency of disease emergence.

Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates.

The recognition that AIDS originated as a zoonosis heightens public health concerns associated with human infection by simian retroviruses endemic in nonhuman primates (NHPs). These retroviruses include simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV), simian type D retrovirus (SRV), and simian foamy virus (SFV). Although occasional infection with SIV, SRV, or SFV in persons occupationally exposed to NHPs has been reported, the characteristics and significance of these zoonotic infections are not fully defined. Surveillance for simian retroviruses at three research centers and two zoos identified no SIV, SRV, or STLV infection in 187 participants. However, 10 of 187 persons (5.3%) tested positive for SFV antibodies by Western blot (WB) analysis. Eight of the 10 were males, and 3 of the 10 worked at zoos. SFV integrase gene (int) and gag sequences were PCR amplified from the peripheral blood lymphocytes available from 9 of the 10 persons. Phylogenetic analysis showed SFV infection originating from chimpanzees (n = 8) and baboons (n = 1). SFV seropositivity for periods of 8 to 26 years (median, 22 years) was documented for six workers for whom archived serum samples were available, demonstrating long-standing SFV infection. All 10 persons reported general good health, and secondary transmission of SFV was not observed in three wives available for WB and PCR testing. Additional phylogenetic analysis of int and gag sequences provided the first direct evidence identifying the source chimpanzees of the SFV infection in two workers. This study documents more frequent infection with SFV than with other simian retroviruses in persons working with NHPs and provides important information on the natural history and species origin of these infections. Our data highlight the importance of studies to better define the public health implications of zoonotic SFV infections.

Identification in gelada baboons (Theropithecus gelada) of a distinct simian T-cell lymphotropic virus type 3 with a broad range of Western blot reactivity.

Antibodies to simian T-cell lymphotropic virus (STLV) were found in serum or plasma from 12 of 23 (52.2 %) gelada baboons (Theropithecus gelada) captive in US zoos. A variety of Western blot (WB) profiles was seen in the 12 seroreactive samples, including human T-cell lymphotropic virus (HTLV)-1-like (n=5, 41.7 %), HTLV-2-like (n=1, 8.3 %), HTLV-untypable (n=4, 33.3 %) and indeterminate (n=2, 16.6 %) profiles. Phylogenetic analysis of tax or env sequences that had been PCR amplified from peripheral blood lymphocyte DNA available from nine seropositive geladas showed that four were infected with identical STLV-1s; these sequences clustered with STLV-1 from Celebes macaques and probably represent recent cross-species infections. The tax sequences from the five remaining geladas were also identical and clustered with STLV-3. Analysis of the complete STLV-3 genome (8917 bp) from one gelada, TGE-2117, revealed that it is unique, sharing only 62 % similarity with HTLV-1/ATK and HTLV-2/Mo. STLV-3/TGE-2117 was closest genetically to STLV-3 from an Eritrean baboon (STLV-3/PH969, 95.6 %) but more distant from STLV-3s from red-capped mangabeys from Cameroon and Nigeria (STLV-3/CTO-604, 87.7 %, and STLV-3/CTO-NG409, 87.2 %, respectively) and Senegalese baboons (STLV-3/PPA-F3, 88.4 %). The genetic relatedness of STLV-3/TGE-2117 to STLV-3 was confirmed by phylogenetic analysis of a concatenated gag-pol-env-tax sequence (6795 bp). An ancient origin of 73 628-109 809 years ago for STLV-3 was estimated by molecular clock analysis of third-codon positions of gag-pol-env-tax sequences. LTR sequences from five STLV-3-positive geladas were >99 % identical and clustered with that from a Papio anubisxP. hamadryas hybrid Ethiopian baboon, suggesting a common source of STLV-3 in these sympatric animals. LTR sequences obtained 20 years apart from a mother-infant pair were identical, providing evidence of both mother-to-offspring transmission and a high genetic stability of STLV-3. Since STLV-3-infected primates show a range of HTLV-like WB profiles and have an ancient origin, further studies using STLV-3-specific testing are required to determine whether STLV-3 infects humans, especially in regions of Africa where STLV-3 is endemic.

Screening for simian foamy virus infection by using a combined antigen Western blot assay: evidence for a wide distribution among Old World primates and identification of four new divergent viruses.

Simian foamy viruses (SFVs) belong to a genetically and antigenically diverse class of retroviruses that naturally infect a wide range of nonhuman primates (NHPs) and can also be transmitted to humans occupationally exposed to NHPs. Current serologic detection of SFV infection requires separate Western blot (WB) testing by using two different SFV antigens [SFV(AGM) (African green monkey) and SFV(CPZ) (chimpanzee)]. However, this method is labor intensive and validation is limited to only small numbers of NHPs. To facilitate serologic SFV testing, we developed a WB assay that combines antigens from both SFV(AGM) and SFV(CPZ). The combined-antigen WB (CA-WB) assay was validated with 145 serum samples from 129 NHPs (32 African and Asian species) and 16 humans, all with known SFV infection status determined by PCR. Concordant CA-WB results were obtained for all 145 PCR-positive or -negative primate and human specimens, giving the assay a 100% sensitivity and specificity. In addition, no reactivity was observed in sera from persons positive for human immunodeficiency virus or human T cell lymphotropic virus (HIV/HTLV) (n = 25) or HIV/HTLV-negative U.S. blood donors (n = 100). Using the CA-WB assay, we screened 360 sera from 43 Old World primate species and found an SFV prevalence of about 68% in both African and Asian primates. We also isolated SFV from the blood of four seropositive primates (Allenopithecus nigroviridis, Trachypithecus françoisi, Hylobates pileatus, and H. leucogenys) not previously known to be infected with SFV. Phylogenetic analysis of integrase sequences from these isolates confirmed that all four SFVs represent new, distinct, and highly divergent lineages. These results demonstrate the ability of the CA-WB assay to detect infection in a large number of NHP species, including previously uncharacterized infections with divergent SFVs.