Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR-RFLP

Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions that leads to economic losses in these animals. A nested PCR–restriction fragment length polymorphism (RFLP) was carried out to identification Theileria species in sheep in some area in western half of Iran (Sari, Rasht, Urmia, Ilam, and Ahvaz). Two hundred and fifty blood samples were taken from sheep during tick activating season (summer of 2008). Microscopic examination revealed that 9.2% (23/250) sheep were infected by Theileria spp. piroplasms. Parasitemia ranged from 0.011% to 0.015%. In nested PCR assessment of DNA samples, 32.8% (82/250) sheep were positive. The negative samples were confirmed by amplifying of ovine beta-actin gene as an internal control. The differentiation of Theileria species was based on RFLP patterns using three restriction enzymes: HpaII, Rsa1, and Bsh 1285I. Out of 82 positive samples, 54.8% (45/82) and 40.2% (33/82) were positive for Theileria lestoquardi and Theileria ovis respectively. Mixed infection was detected in 4.8% (4/82) cases. Based on their PCR product digestion pattern with HpaII (1178, 900, 278, and 106 bp), it seemed to be mixture of Theileria annulata and T. lestoquardi. The presence of T. annulata was supported by sequence analysis. This is the first report of naturally infected sheep with T. annulata in Iran. Geographical distribution of Theileria species in sheep is shown according to the result of microscopy and nested PCR and RFLP data.     

Detection of haemotropic Mycoplasma (Haemobartonella) using multiplex PCR and its relationship with epidemiological factors in dogs

The aim of the present study was to conduct multiplex PCR for detection of two haemotropic Mycoplasma species in dogs from central Iran and study their relationship with epidemiological factors. In order to achieve this, blood samples from 100 Esfahan dogs were analysed by PCR to evaluate haemoplasma infection status. Thirteen were infected with an organism closely related to Mycoplasma haemocanis, and ten were infected with Candidatus Mycoplasma haematoparvum, whilst three dogs were contaminated with both organisms. The results showed a significant difference between young (1–5 years old) and ectoparasite-infected dogs for the presence of haemotropic Mycoplasma. Our survey indicates that multiplex PCR assay is able to successfully detect haemotropic Mycoplasma in dogs.     

Detection of Theileria orientalis in Iran by semi-nested PCR

In order to identify and differentiate Theileria orientalis in cattle which may be infected with Theileria annulata simultaneously, a semi-nested PCR was performed. Thus, 160 blood samples were collected from apparently healthy native cattle in Golestan province of northern Iran, during 2009 to 2011. The Tbs-S/Tbs-A primer set derived from the 18S rRNA encoding gene was used for first PCR amplification, and the amplified sequence weight by this primer set for Theileria sp. was 426–430 bp. Then, DNA solution from purified PCR product was used for the semi-nested PCR analysis. The first PCR product amplified using T. orientalis primer set (To-S/Tbs-A) derived from the 18SrRNA encoding gene, and this specific primer weight was 235 bp. Also, the first PCR product amplified using T. annulata primer set (Ta-S/Tbs-A) derived from the 18SrRNA encoding gene and this specific primer weight was 193 bp. Having extracted DNA of each sample, using Tbs-S/Tbs-A primer set for PCR and analyzing the PCR products on the 2% agarose gel electrophoresis, 13 out of 160 blood samples (8.12%) were positive for Theileria sp. Meanwhile, performing semi-nested PCR with T. orientalis-specific primers, 9 out of 13 blood samples (5.62%) were positive and performing semi-nested PCR with T. annulata-specific primers, 12 out of 13 blood samples (7.5%) were also positive. This molecular assay approves the presence of T. orientalis in the native cattle of northern parts of Iran for the first time. In addition, this procedure will detect the concurrent infection of T. orientalis and T. annulata in the cattle too.     

Changes in serum iron concentration and hepatic enzyme activities in cattle infected with Theileria annulata and Babesia bigemina

Analyses for alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities, and iron concentration were carried out in infected and uninfected cattle in order to determine the degree of hepatic damage and blood iron status caused by Theileria annulata and Babesia bigemina. Blood samples from 30 infected and 20 uninfected cattle with T. annulata and also from 40 infected and 20 uninfected cattle with B. bigemina were taken. The biochemical analyses revealed increased serum AST and ALP activities in infected cattle with T. annulata and B. bigemina compared with uninfected cattle (P?<?0.05). Significant decreases in iron concentration were observed in cattle infected with T. annulata and B. bigemina when compared with uninfected cattle (P?<?0.05) although the level of serum iron concentration in infected cattle was within the normal reference values for cattle. It was concluded that iron deficiency anemia is not an important factor in T. annulata- and even in B. bigemina-infected cattle with severe intravascular hemolysis. The increased activities of serum enzymes indicated the hepatic injuries associated with infection with T. annulata and B. bigemina.     

Evidences of increasing risk of dirofilarioses in southern Italy

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P?=?0.6 % (95 % confidence interval (CI)?=?0.12–1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26–25.28; P?<?0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.     

Detection of <Emphasis Type="Italic">Theileria annulata</Emphasis> by the PCR-RFLP in ticks (Acari,Ixodidae) collected from cattle in West and North-West Iran

The presence of potential vectors, ticks, and susceptible hosts of bovine malignant theileriosis in all parts of Iran pose a real threat to food animal industry. The present study was conducted to determine the infection rate of ticks collected from naturally occurring bovine theileriosis in West and North-West Iran. Two hundred and thirty seven cattle suspected of suffering from theileriosis were investigated for the presence of Theileria annulata in the blood smears and any tick species on their body. In this study, 402 ticks were obtained from 99 cattle. The examination of 402 ticks by polymerase chain reaction (PCR) using primers derived from the gene encoding heat shock protein70 (Hsp70) revealed that 39.9% of Hyalomma anatolicum anatolicum, 3.5% of H. asiaticum asiaticum, and 18.2% H. anatolicum excavatum, were infected with T. annulata. The results suggest that H. a. anatolicum may play a major role in transmission of T. annulata infection in Iran. Finally, digestion of the PCR products of T. annulata with two different restriction enzymes produced only a single pattern.     

Targeting tams-1 gene results in underestimation of Theileria annulata infection in diseased cattle in Egypt

Tropical theileriosis is considered one of the most economically important tick borne diseases that cause significant mortality and morbidity to livestock. In the context of epidemiological studies on livestock in Egypt, this report investigated the spread of Theileria annulata among diseased farm cows (Bos indicus) over one year. Blood samples collected from 130 cows were investigated by routine staining and 64 samples were investigated by PCR assay using two different probes targeting tams-1 gene. Microscopy revealed the infection of 33.8% of animals with Theileria while PCR detected infection in 51% of animals with one primer pair and the other primer pair detected the infection in 31% of animals. Combined PCR results indicated the infection of 68.8% of animals with T. annulata. Seasonal fluctuation of parasite infection was evident with the highest infection percentage and prevalence recorded during summer based on both microscopy and PCR data. For the first time, the current study reports the presence of two T. annulata isolates based on tams-1 gene partial sequence in Egypt. Targeting polymorphic genes for parasite detection may result in underestimation of infection and target gene diversity has to be considered. The high infection with these pathogens in the clinically ill cows necessitates implementing serious programs to minimize their economic burden on the Egyptian farming industry.     

Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in Dogs in a Village of Eastern Sudan by Using a Screening PCR and Sequencing Methodologies

Babesia and Hepatozoon infections of dogs in a village of eastern Sudan were analyzed by using a single PCR and sequencing. Among 78 dogs, 5 were infected with Babesia canis rossi and 2 others were infected with B. canis vogeli. Thirty-three dogs were positive for Hepatozoon. Hepatozoon canis was detected by sequence analysis.     

A study on the prevalence of a tick-transmitted pathogen, Theileria annulata, and hematological profile of cattle from Southern Punjab (Pakistan)

The present study was carried out to determine the prevalence of Theileria annulata in large ruminants in Southern Punjab (Pakistan). Blood samples were collected from 144 large ruminants, consisting of 105 cattle and 39 buffaloes, from six districts of Southern Punjab including Multan, Layyah, Muzaffar Garh, Bhakar, Bahawalnagar, and Vehari. Data on the characteristics of the animals and herds were collected through questionnaires. The age of animals (P = 0.02), presence of ticks on animals (P = 0.02), and presence of ticks on dogs associated with herds (P = 0.05) were among the major risk factors involved in the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa-stained slides, were compared, and it was found that PCR amplification is a more sensitive tool (19% parasite detection) as compared to smear scanning (3% parasite detection) for the detection of T. annulata. Twenty eight out of 144 animals produced the 721-bp fragment specific for T. annulata from five out of six sampling districts. Different blood (hemoglobin, glucose) and serum (ALT, AST, LDH, cholesterol) parameters of calves and cattle were measured and compared between parasite-positive and parasite-negative samples to assess the effect of T. annulata on the blood and serological profile of infected animals.     

Identification of hard ticks collected from sheep naturally infected with Anaplasma ovis in Isfahan province, central Iran

Attached ticks and blood samples were collected from 150 sheep in Isfahan province, central part of Iran. Blood samples were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Of the 150 sheep examined, 50 (33.33%) were found positive for Anaplasma ovis by PCR-RFLP. Of 50 sheep naturally infected with A. ovis, 553 ixodid ticks were collected. The ticks were identified (three species belonging to two genera) as follows: Rhipicephalus sanguineus (53.9%), Hyalomma marginatum marginatum (27.5%), and Hyalomma anatolicum anatolicum (18.6%). All A. ovis-infected sheep were infested with R. sanguineus. This suggests that R. sanguineus may be one of the main vectors of sheep anaplasmosis in the central part of Iran.     

Copro-DNA test for diagnosis of canine echinococcosis

Echinococcosis is one of the most important parasitic zoonotic diseases in the world. The life cycle of Echinococcus granulosus is dependent to the dog–sheep cycle and is actively transmitted in all pastoral regions where sheep, cattle and camels predominate. DNA approaches are now being used routinely for accurate identification of Echinococcus and Taenia species, subspecies and strains. In this study, faecal samples were collected from 50 stray dogs from Mashhad city in the northeast of Iran during June 2011. All samples were frozen at least for 1 week in ?80°C. The embryophore layer of the eggs was broken by freezing–thawing method after egg concentration by the formalin-ether method. DNA was extracted using a DNA isolation kit (MBST, Germany/Iran) according to the manufacturer’s instructions. After PCR, by the primers expressed in the following it became, clear that about 20 % of stray dogs are infected with E. granulosus. In this study, we describe a modified method for DNA isolation from faeces for coprodiagnosis of Echinococcus spp.     

Development of a PCR technique specific for Demodex injai in biological specimens

The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex–host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies.     

Seroprevalence of tick-borne diseases among cattle in the Sudan

This study was carried out to determine the prevalence of Theileria annulata, Theileria mutans, Babesia bigemina, and Anaplasma marginale antibodies among cattle in the Sudan. A total of 600 serum samples were collected from indigenous (zebu) and crossbred cattle (zebu × Friesian) of both sex and different age groups. Indirect enzyme-linked immunosorbent assay was used to assess antibodies against tick-borne diseases in apparently healthy cattle. The overall prevalence rates of T. annulata, T. mutans, B. bigemina, and A. marginale antibodies were found to be 30.8%, 6.1%, 10.7%, and 38.9%, respectively. The highest seroprevalence of T. annulata was reported in Atbara and El Damer, Northern Sudan. There were no significant associations for the seroprevalence of all tick-borne diseases reported among different age groups. Although there were no significant differences between the two breeds of cattle examined for T. annulata, T. mutans, and B. bigemina antibodies, there was a significant difference for prevalence of antibodies against A. marginale, with highest percentages of antibodies in indigenous cattle. Six different combinations of mixed infection were detected. This is the first report in which antibodies against A. marginale among cattle in Northern Sudan is reported. The findings imply that antibodies to tick-borne infections are widely distributed in the region. The need for further investigations using more advanced techniques is recommended.     

Clinicopathological findings of a natural outbreak of Theileria annulata in cattle: an emerging disease in southern Iran

Theileriosis is an economically important hemoprotozoal disease with high morbidity and mortality in cattle. The present study reported the pathological features of a natural outbreak of tropical bovine theileriosis due to Theileria annulata in Fars Province, southern Iran. T. annulata was confirmed by the presence of T. annulata piroplasms in the blood smears and also by polymerase chain reaction test. On necropsy, pale mucous membranes and petechial and ecchymotic hemorrhages in the mucosal and serosal surfaces together with lymphadenopathy were observed. The liver was friable, yellowish, and larger than normal. Hemorrhages and punched-out ulcers were observed in the abomasal mucous membrane. Severe petechial hemorrhages were seen in the skin particularly in the hairless areas. Pulmonary edema and emphysema with petechial and ecchymotic hemorrhagic foci in the lungs were evident. The main histological changes were proliferation of lymphocytes in the lymph nodes and proliferation of macrophages, lymphocytes, and plasma cells in the spleen, Peyer's patches, portal tracts of the liver, and interstitial tissue of the kidneys. The mucous membrane of the abomasum showed numerous multifocal areas of necrosis and ulceration, and the submucosal area and lamina propria adjacent to these lesions showed hyperemia and hemorrhages, with mononuclear cell infiltration. The skin showed multifocal necrotic changes, petechial and ecchymotic hemorrhages, and chronic dermatitis. The schizonts of Theileria were evident in the cytoplasm of the lymphocytes and macrophages of the lymph nodes, spleen, and skin. Molecular examination revealed that these animals were infected with T. annulata. The present study describes the clinicopathological findings of bovine tropical theileriosis in an unpredictable weather condition.     

The status of Linguatula serrata infection of stray dogs in Shiraz, Iran

Linguatula serrata is one of the parasitic zoonoses causing visceral and nasopharyngeal linguatulosis in human beings. Recently, L. serrata found a more public health importance, and new cases of human infection is reported from different parts of Iran including the Fars Province. Infected vegetables, fruits, and water resources with eggs of the mature parasite excreted via carnivores’ (especially stray dogs) nasopharyngeal secretions or feces is the main source of infecting human beings. However, consumption of infected improperly cooked viscera of the intermediate hosts including sheep, goats, cattle, or other herbivores containing the larval stages of this parasite is the other potential source of infection of human beings. Therefore, the present study was undertaken to investigate the prevalence of this parasite in stray dogs of Shiraz, the capital city of the Fars Province in Southern part of Iran. In a cross-sectional study, 85 stray dogs including 48 males and 37 females were captured at different parts of Shiraz city and the nasopharyngeal area, nasal turbinates, sinuses, eusthasian tubes, and brain were examined for L. serrata. The adult parasites were collected and fixed, cleared, and stained using formalin, alcohol, azocarmine, and lactophenol. A total of 65 (76.5%) dogs were infected with L. serrata. Age, sex, weight, and geographical locations did not have significant effects in the prevalence rate of this parasite. The number of parasites recovered from each dog ranged from 1 to 19 with an average of 4.06 per infected dog. The maximum length and width of the mature Linguatula were 80 and 10 mm for female parasites and 20 and 3 mm for the male ones, respectively. From the above results, it could be concluded that the rate of infection in dogs and possibly other carnivores, herbivores, and man is high in this locality, and strict control measures should be conducted to overcome the risk of infection with this zoonotic disease.     

Comparison of selected biochemical parameters between naturally infected and non-infected goats with Anaplasma ovis

There are few extensive studies about biochemical profiles of caprine anaplasmosis caused by Anaplasma ovis. For detection of A. ovis and its effect on serum biochemical parameters in goats of north and northeast Iran, blood samples were collected from 84 goats of different ages and of both sexes from ten suspected herds to anaplasmosis. Forty-seven out of 84 samples were positive (infected), and 37 samples were negative (uninfected) for A. ovis by PCR method. Biochemical analysis of infected goats indicated a significant (P?<?0.05) elevation of serum aspartate aminotransferase, γ-glutamyl transferase, urea, creatinine, total bilirubin, and direct bilirubin, in addition to a nonsignificant (P?>?0.05) increase of total protein, albumin, and triglyceride, when compared to the uninfected goats. The cholesterol concentration in the infected goats was lower than that in the uninfected goats, but this difference was not statistically significant (P?>?0.05). The result of this study indicated that A. ovis infection in goats could be associated with marked alterations in serum biochemical parameters which could be helpful in the diagnosis of caprine anaplasmosis.     

The role of acute phase cytokines in the recovery and disease progress of Theileria annulata-infected cattle

The present study was conducted to evaluate the cytokine response following natural infection of Theileria annulata in cattle. Initial survey included 173 crossbred cattle which were examined for the presence of Theileria piroplasms. The investigated cattle were clinically and parasitologically examined. Blood samples were collected from all examined cattle for microscopic examination, PCR assays (using primers of Theileria spp., Babesia spp., and T. annulata), and cytokines measurement. It was found that 38 cattle were positive for the presence of at least one species of Theileria; meanwhile Babesia piroplasms were not detected either by microscopy or PCR assay. When T. Annulata-specific primers were used, 33 gave positive results. Twenty-two out of 33 T. annulata-infected cattle were only included in this study together with contemporaneous controls (n?=?10). According to the severity of clinical signs, T. annulata-infected cattle were categorized into two groups; group 1 included ten cattle with mild clinical signs and group 2 included 12 cattle with overt clinical signs. Biochemically, tumor necrosis factor alpha, interleukin (IL)-1β, IL-6, IL-12, haptoglobin, and Fb were significantly higher (p?T. annulata infection provide a valuable and quantitative assessment of the response to infection. It seems likely that the pro-inflammatory cytokines play an important role in the host response to T. annulata. Our findings suggest that the pro-inflammatory cytokines are suitable markers of inflammatory reactions in T. annulata-infected cattle.     

Prevalence and diversity of Hepatozoon canis in naturally infected dogs in Japanese islands and peninsulas

Canine hepatozoonosis is a worldwide protozoal disease caused by Hepatozoon canis and Hepatozoon americanum and is transmitted by ixodid ticks, Rhipicephalus and Amblyomma spp., respectively. H. canis infection is widespread in Africa, Europe, South America, and Asia, including Japan. The objective of this study was to study the distribution pattern and diversity of H. canis in naturally infected dogs in nine Japanese islands and peninsulas. Therefore, 196 hunting dogs were randomly sampled during the period from March to September 2011 and the ages and sexes were identified. Direct microscopy using Giemsa-stained blood smears revealed H. canis gametocytes in the peripheral blood of 45 (23.6 %) dogs. Polymerase chain reaction (PCR) was performed on EDTA-anticoagulated blood, initially with the common primer set (B18S-F and B18S-R) amplifying the 1,665-bp portion of the 18S rRNA gene, and then with the specific primer set (HepF and HepR) amplifying about 660 bp fragments of the same gene. Based on PCR, 84 (42.9 %) dogs were positive using the common primer and 81 (41.3 %) were positive using the specific primer. The current investigation indicated that all screened areas, except for Sado Island and Atsumi Peninsula, were infected. Yaku Island had the highest infection rate (84.6 % in males and 100.0 % in females), while Ishigaki Island showed the lowest infection rates (8.3 % in males and 17.7 % in females). Both sexes were infected with no significant difference. However, diversity of infection among the surveyed islands and peninsulas was significantly different (P?<?0.05). Although H. canis has previously been reported in dogs in Japan, the higher infection rate described in the current study and the diversity of infection in a wide range of islands strongly encourage prospective studies dealing with the prevention and treatment of the infection in dogs, as well as control of ticks.     

Asymptomatic domestic dogs are carriers of Leishmania infantum: possible reservoirs host for human visceral leishmaniasis in southern Iran

In the past few years, the incidence of human visceral leishmaniasis (HVL) has increased in many districts of Fars Province, southwest of Iran, particularly, among communities of nomadic tribes. Recent epidemiological reports in Leishmania infantum endemic regions of Iran indicate that more than 50–70% of seropositive dogs are asymptomatic for Leishmania infection. Between 2004 and 2006, blood samples were collected from 110 domestic dogs from nomadic and rural areas. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DATs), and for L. infantum kinetoplast deoxyribonucleic acid (kDNA), in polymerase chain reaction (PCR)-based assays. Of the 110 dogs, 5.5% (6/110) were found seropositive and 23% (25/110) PCR-positive. Four of the six seropositive (67%) and 22 of the 25 PCR-positive (88%) were asymptomatic. The rate of infection in dogs from nomadic communities was higher (27%) than dogs from rural areas (18%). Since positivity in the PCR-based assay indicated the presence of L. infantum amastigotes in the peripheral blood of 23% of the subjects, it is clear that these asymptomatic dogs (88%) are quite common in the study areas and probably act as reservoirs in the transmission of Leishmania parasites, to humans and to other dogs, by sandflies. Moreover, our study showed that application of PCR to buffy coat samples gave a better estimate of the real rate of infection in asymptomatic dogs than DAT.     

Diagnosis of Pneumocystis pneumonia by real-time PCR in patients with various underlying diseases

BackgroundPneumocystis pneumonia (PCP) is a disease caused by the opportunistic infection of the fungus Pneumocystis jirovecii. Several PCR methods have been developed to aid in the diagnosis of PCP. In this study, we evaluated the performance of a real-time PCR in the diagnosis of PCP, in patients with various underlying diseases.MethodsNinety-seven BAL samples and 94 sputum samples from 191 patients were used in the study. Patients were classified as PCP (121 patients) or non-PCP (70 patients) based on their clinical and radiological presentations.ResultsReal time PCR amplified the P. jirovecii mitochondrial large-subunit rRNA gene with a detection limit of 68 copies of DNA per reaction. Non-PCP pathogens including 32 different fungi and bacteria were also evaluated. Overall, 71.9% of the samples from PCP patients and 14.5% of those from non-PCP patients were positive for the PCR test with a C_T value of the real-time PCR below 45. The main underlying diseases of the patients were hematological or solid malignancies (47.1%) and HIV infection (8.9%). The C_T values of the test were significantly lower in BAL samples from PCP patients than those from non-PCP patients (p = 0.024). No non-PCP patient had a C_T value below 30, whereas samples from 24.8% of PCP patients with underlying diseases had a C_T value below 30.ConclusionSince false positive PCR results were obtained, perhaps due to colonization, we suggest that the diagnosis of PCP should be based on a combination of clinical symptoms, underlying diseases, and PCR results.